(Ele 1999)advances in protein solubilisation for 2D electrophoresis

2013诺贝尔奖】生理学奖深度解读：囊泡运输，细胞的“物流系统”Calo 2013-10-08 12:10一个细胞就好比一个人类社会。

人类社会有多复杂，细胞活动就有多精妙。

在日常生活中，我们需要进行有效率的生产生活，就必须有效率地调配生产资料与生活资源——因此，我们需要建立周密有效、安排得当的物流系统。

细胞也一样，基因的表达产物需要定位到不同的地点行使功能：膜蛋白需要奔向自己的靶位点、胰岛素需要分泌出细胞外、神经递质需要扩散到下一个神经细胞……要在正确的时间把正确的细胞货物运送到正确的目的地，细胞的物质转运机制之精妙，比无数物流师呕心沥血的杰作都更胜一筹。

而囊泡运输（vesicle trafficking），正是这一机制的重要组分。

2013年诺贝尔生理学或医学奖于10月7日颁布。

图片来源：昨天，2013年诺贝尔生理学或医学奖被授予发现囊泡转运机制的詹姆斯·罗斯曼、兰迪·谢克曼和托马斯·聚德霍夫3位科学家。

今天，让我们来看一下，大自然的物流师究竟是如何运筹于帷幄之中，决胜于细胞内外的呢？细胞中包括蛋白质在内的大多数分子都太大了，以致于不能直接穿过细胞中的膜结构。

于是，这些分子的运输需要依赖一种叫囊泡的细胞结构——这种有膜包被的小型泡状结构能够将待运输的分子包裹起来，送到目的地去释放掉。

可以想象，这种泡状的“集装箱”在运送细胞货物的过程中是极为重要的装备。

因此，在细胞中，尤其是在细胞质膜、内质网以及高尔基体中，囊泡的形成是持续不断的。

这些“集装箱”一旦被生产出来就马上投入使用，带着它们的货物奔向细胞内或细胞外的目的地。

囊泡之所以能够完成转运任务，是因为囊泡的膜与细胞质膜以及细胞内膜系统的组成成分是相似的，能够通过出芽的方式脱离转运起点、通过膜融合的方式归并到转运终点。

囊泡转运过程的第一步是膜通过出芽方式形成一个囊泡。

囊泡的外表面被蛋白包被。

通过改变膜结构的构象，这些蛋白将促使囊泡形成。

Nutrient removal in an A2O-MBR reactor with sludgereductionABSTRACTIn the present study, an advanced sewage treatment process has been developed by incorporating excess sludge reduction and phosphorous recovery in an A2O-MBR process. The A2O-MBR reactor was operated at a flux of 77 LMH over a period of 270 days. The designed flux was increased stepwise over a period of two weeks. The reactor was operated at two different MLSS range. Thermo chemical digestion of sludge was carried out at a fixed pH (11)and temperature (75℃) for 25% COD solubilisation. The released pbospborous was recovered by precipitation process and the organics was sent back to anoxic tank. The sludge digestion did not have any impact on COD and TP removal efficiency of the reactor. During the 270 days of reactor operation, the MBR maintained relatively constant transmembrane pressure. The results based on the study indicated that the proposed process configuration has potential to reduce the excess sludge production as well as it didn't detonated the treated water quality.Keywords: A2O reactor; MBR; Nutrient removal; TMP1. IntroductionExcess sludge reduction and nutrients removal are the two important problems associated with wastewater treatment plant. MBR process has been known as a process with relatively high decay rate and less sludge production due to much longer sludge age in the reactor (Wenet al., 2004). Sludge production in MBR is reduced by 28-68%, depending on the sludge age used (Xia et al.,2008). However, minimizing the sludge production by increasing sludge age is limited due to the potential adverse effect of high MLSS concentrations on membrane (Yoon et al., 2004). This problem can be solved by introducing sludge disintegration technique in MBR (Young et al., 2007). Sludge disintegration techniques have been reported to enhance the biodegradability of excess sludge (Vlyssides and Karlis, 2004). In overall, the basis for sludge reduction processes is effective combination of the methods for sludge disintegration and biodegradation of treated sludge. Advances in sludge disintegration techniques offer a few promising options including ultrasound (Guo et al., 2008), pulse power (Choi et al.,2006), ozone (Weemaes et al., 2000), thermal (Kim et al., 2003), alkaline (Li et al., 2008) acid (Kim et al., 2003) and thermo chemical(Vlyssides and Karlis, 2004). Among the various disintegration techniques, thermo chemical was reported to be simple and cost effective (Weemaes and Verstraete, 1998). In thermal-chemical hydrolysis, alkali sodium hydroxide was found to be the most effective agent in inducing cell lysis (Rocker et al., 1999). Conventionally, the nutrient removal was carried out in an A2O process. It has advantage of achieving, nutrient removal along with organic compound oxidation in a single sludge configuration using linked reactors in series (Tchobanoglous et al., 2003). The phosphoroes removal happens by subjecting phosphorous accumulating organisms (PAO) bacteria under aerobic and anaerobic conditions (Akin and Ugurlu, 2004). These operating procedures enhance predominance PAO, which are able to uptake phosphorous in excess. During the sludge pretreatment processes the bound phosphorous was solubilised and it increases the phosphorousconcentration in the effluent stream (Nishimura, 2001).So, it is necessary to remove the solubilised phosphorus before it enters into main stream. Besides, there is a growing demand for the sustainable phosphorous resources in the industrialized world. In many developed countries, researches are currently underway to recover the phosphoroes bound in the sludge's of enhanced biological phosphorus removal system (EBPR). The released phosphorous can be recovered in usable products using calcium salts precipitation method. Keeping this fact in mind, in the present study, a new advanced wastewater treatment process is developed by integrating three processes, which are: (a) thermo chemical pretreatment in MBR for excess sludge reduction (b) A2O process for biological nutrient removal (c) P recovery through calcium salt precipitation. The experimental data obtained were then used to evaluate the performance of this integrated system.2. Methods2.1. WastewaterThe synthetic domestic wastewater was used as the experimental influent. It was basically composed of a mixed carbon source, macro nutrients (N and P), an alkalinity control (NaHCO3) and a microelement solution. The composition contained (/L) 210 mg glucose, 200 mg NH4C1, 220 mg NaHCO3, 22一34 mg KH2PO4, microelement solution (0.19 mg MnCl2 4H20, 0.0018 mg ZnCl22H2O,0.022 mg CuCl22H2O, 5.6 mg MgSO47H2O, 0.88 mg FeCl36H2O,1.3 mg CaCl2·2H2O). The synthetic wastewater was prepared three times a week with concentrations of 210±1.5 mg/L chemical oxygen demand (COD), 40±1 mg/L total nitrogen (TN) and 5.5 mg/L total phosphorus (TP).2.2. A2O-MBRThe working volume of the A2O-MBR was 83.4 L. A baffle was placed inside the reactor to divide it into anaerobic (8.4 L) anoxic (25 L) and aerobic basin (50 L). The synthetic wastewater was feed into the reactor at a flow rate of 8.4 L/h (Q) using a feed pump. A liquid level sensor, planted in aerobic basin of A2O-MBR controlled the flow of influent. The HRT of anaerobic, anoxic and aerobic basins were 1, 3 and 6 h, respectively. In order to facilitate nutrient removal, the reactor was provided with two internal recycle (1R). IRl (Q= 1)connects anoxic and anaerobic and IR 2 (Q=3) was between aerobic and anoxic. Anaerobic and anoxic basins were provided with low speed mixer to keep the mixed liquid suspended solids (MLSS) in suspension. In the aerobic zone, diffusers were used to generate air bubbles for oxidation of organics and ammonia. Dissolved oxygen (DO) concentration in the aerobic basin was maintained at 3.5 mg/1 and was monitored continuously through online DO meter. The solid liquid separation happens inaerobic basin with the help of five flat sheet membranes having a pore size of 0.23 pm. The area of each membrane was 0.1 m2. They were connected together by a common tube. A peristaltic pumpwas connected in the common tube to generate suction pressure. In the common tube provision was made to accommodate pressure gauge to measure transmembrane pressure (TMP) during suction. The suction pump was operated in sequence of timing, which consists of 10 min switch on, and 2 min switch off.2.3. Thermo chemical digestion of sludgeMixed liquor from aerobic basin of MBR was withdrawn at the ratio of 1.5% of Q/day and subjected to thermo chemical digestion. Thermo chemical digestion was carried out at a fixed pH of 11(NaOH) and temperature of 75℃for 3 h. After thermo chemical digestion the supernatant and sludge were separated. The thermo-chemicallydigested sludge was amenable to further anaerobic bio-degradation (Vlyssides and Karlis, 2004), so it was sent to theanaerobic basin of the MBR2.4. Phosphorus recoveryLime was used as a precipitant to recover the phosphorous in the supernatant. After the recovery of precipitant the content was sent back to anoxic tank as a carbon source and alkalinity supelement for denitrification.2.5. Chemical analysisCOD, MLSS, TP, TN of the raw and treated wastewater were analyzed following methods detailed in (APHA, 2003). The influent and effluent ammonia concentration was measured using an ion-selective electrode (Thereto Orion, Model: 95一12). Nitrate in the sample was analyzed using cadmium reduction method (APHA, 2003).3. Results and discussionFig. 1 presents data of MLSS and yield observed during the operational period of the reactor. One of the advantages of MBR reactor was it can be operated in high MLSS concentration. The reactor was seeded with EBPR sludge from the Kiheung, sewage treatment plant, Korea. The reactor was startup with the MLSS concentration of 5700 mg/L. It starts to increase steadily with increase in period of reactor operation and reached a value of 8100 mg/L on day 38. From then onwards, MLSS concentration was maintained in the range of 7500 mg/L by withdrawing excess sludge produced and called run I. The observed yields (Yobs) for experiments without sludge digestion (run I) and with sludge digestion were calculated and given in Fig. 1. The Yobs for run I was found to be 0.12 gMLSS/g COD. It was comparatively lower than a value of 0.4 gMLSS/g CODreported for the conventional activated sludge processes (Tchoba-noglous et al., 2003). The difference in observed yield of these two systems is attributed to their working MLSS concentration. At high MLSS concentration the yield observed was found to be low (Visva-nathan et al., 2000). As a result of that MBR generated less sludge.The presently used MLSS ranges (7.5一10.5 g/L) are selected on the basis of the recommendation by Rosenberger et al. (2002). In their study, they reported that the general trend of MLSS increase on fouling in municipal applications seems to result in no impact at medium MLSS concentrations (7一12 g/L).It is evident from the data that the COD removal efficiency of A2O system remains unaffected before and after the introduction of sludge digestion practices. A test analysis showed that the differences between the period without sludge digestion (run I) and with sludge digestion (run II and III) are not statistically significant.However, it has been reported that, in wastewater treatment processes including disintegration-induced sludge degradation, the effluent water quality is slightly detonated due to the release of nondegradable substances such as soluble microbial products (Ya-sui and Shibata, 1994; Salcai et al., 1997; Yoon et al., 2004). During the study period, COD concentration in the aerobic basin of MBR was in the range of 18-38 mg/L and corresponding organic concentration in the effluent was varied from 4 to 12 mg/L. From this data it can be concluded that the membrane separation played an important role in providing the excellent and stable effluent quality.Phosphorus is the primary nutrient responsible for algal bloom and it is necessary to reduce the concentration of phosphorus in treated wastewater to prevent the algal bloom. Fortunately its growth can be inhibited at the levels of TP well below 1 mg/L (Mer-vat and Logan, 1996).Fig. 2 depicts TP removal efficiency of the A2O-MBR system during the period of study. It is clearly evident from the figure that the TP removal efficiency of A/O system was remains unaffected after the introduction of sludge reduction. In the present study, the solubilised phosphorous was recovered in the form of calcium phosphate before it enters into main stream. So, the possibility of phosphorus increase in the effluent due to sludge reduction practices has been eliminated. The influent TP concentration was in the range of 5.5 mg/L. During thefirst four weeks of operation the TP removal efficiency of the system was not efficient as the TP concentration in the effluent exceeds over 2.5 mg/L. The lower TP removal efficiency during the initial period was due to the slow growing nature of PAO organisms and other operational factors such as anaerobic condition and internal recycling. After the initial period, the TP removal efficiency in the effluent starts to increase with increase in period of operation. TP removal in A2O process is mainly through PAO organisms. These organisms are slow growing in nature and susceptible to various physicochemical factors (Carlos et al., 2008). During the study period TP removal efficiency of the system remains unaffected and was in the range of 74-82%.。

年终盘点：2016年Nature杂志重磅级突破性研究成果小福利：点击上方图片下载生物谷APP轻松获得本文中所有文献原文！年终专题系列——Nature杂志重磅研究时间过得总是很快，2016年已经步入尾声，迎接我们的将是崭新的2017年!在2016年里三大国际著名杂志Cell、Nature和Science（CNS）依然刊登了很多非常耐人寻味的研究成果，本文中谷君就盘点了2016年Nature杂志发表的一些突破性的重磅级研究，分享给大家！【1】Nature：中国首次利用CRISPR–Cas9编辑过的细胞开展人体临床试验doi：10.1038/nature.2016.20988来自中国成都市四川大学华西医院的一个研究人员团队首次将利用CRISPR–Cas9进行过基因编辑的细胞注射到一名病人体内。

《自然》期刊报道这一注射过程是在2016年10月28日发生的，而且迄今为止，这名病人表现得“还不错”。

经过基因修饰的细胞之前已被注射到人体内，但是是利用不同的技术实现的。

CRISPR-Cas9被认为是一种更加高效的方法。

在这项新的努力中，该团队从血液样品中分离出免疫细胞，然后利用CRISPR-Cas9寻找它们中的PD-1蛋白，并且让该蛋白不能发挥功能，而之前的研究已证实这会延缓免疫细胞作出的免疫反应。

人们的看法是让这种蛋白失去功能将允许免疫系统更强地抵抗肿瘤生长。

这些利用CRISPR-Cas9进行过基因编辑的细胞被放置在一个容器中，在那里，它们在体外培养后能够发生增殖---它们随后经收集后被注射到一名肺癌病人体内，其中这名病人已不能够对任何其他的疗法作出反应。

这种CRISPR-Cas9技术涉及利用一种结合特定DNA序列的向导RNA和一种能够在事先选择的位点上切割DNA链的Cas9酶，从而允许移除DNA链，或者加入新的DNA片段。

【2】Nature：实验性疫苗与免疫刺激剂组合使用有望治疗HIV doi：10.1038/nature20583在一项新的研究中，来自美国贝斯以色列女执事医疗中心、沃尔特里德陆军研究院、Janssen疫苗与预防公司（Janssen Vaccines & Prevention B.V.）和吉利德科学公司的研究人员证实将一种实验性疫苗与一种先天性免疫刺激剂结合在一起可能有助导致HIV感染者体内的病毒缓解。

利用BiFC 技术研究柔嫩艾美耳球虫顶膜抗原1结构域互作关系严 茗1,2，黄 兵22，吕 凌1,2，董 辉2（1.上海师范大学生命与环境科学学院，上海 200234；2.中国农业科学院上海兽医研究所 农业部动物寄生虫学重摘 要：为了验证柔嫩艾美耳球虫（Eimeria tenella ）顶膜抗原1结构域Ⅰ（apical membrance antigen 1 domain I ，Et AMA1-D Ⅰ）与棒状体颈部蛋白2（rhoptry neck protein 2，Et RON2）的互作关系，以柔嫩艾美耳球虫子孢子cDNA 为模板，PCR 扩增出 492 bp 的Et AMA1-D Ⅰ片段和1395 bp 的Et RON2片段，并与pGEM-T-easy 载体连接构建相应重组质粒。

获得的阳性重组质粒及双分子荧光互补技术的真核表达载体pBiFC-VN155和pBiFC-VC155用Eco R Ⅰ和BgI II 进行双酶切，将Et AMA1-D Ⅰ、Et RON2分别与pBiFC-VN155、pBiFC-VC155连接，构建真核重组质粒pBiFC-VN155-Et AMA1-D Ⅰ和pBiFC-VC155-Et RON2。

将2个真核重组质粒分别转染BHK 细胞进行表达，经间接免疫荧光鉴定，可在BHK 细胞中成功表达。

将2个真核重组质粒共转染至BHK 细胞中，同时将pBiFC-bJunVN155和pBiFC-bFos(deltaZIP)VC155、pBiFC-bJunVN155和pBiFC-bFos(delta)VC155共转染至细胞中分别作为阳性和阴性对照组。

BiFC 结果发现真核重组质粒共转染组和阳性对照组的BHK 细胞均产生绿色荧光，而阴性对照组无荧光，表明Et AMA1-D Ⅰ与Et RON2蛋白之间存在互作关系。

本研究结果为深入研究Et AMA1及Et RON2在球虫入侵过程中的功能与作用机制奠定基础。

RESEARCHA number of systems have been developed to simplify protein purifica-tion through the expression of recombinant proteins as fusions to car-rier proteins or peptides 1,2. Examples of commercially available systems include maltose binding protein 3, glutathione S -transferase 4, biotin carboxyl carrier protein 5,6, thioredoxin 6,7, and cellulose binding domain 8. Similarly, vectors for fusion to short peptide tags such as oligohistidine 9, S-peptide 10, and the FLAG tm peptide 11are also available.These systems typically allow one-step purification of a recombinant protein from cell extract by affinity chromatography, using an immobi-lized moderate-affinity ligand specific to the carrier protein 12.Although it is useful for laboratory-scale purification, affinity chro-matography on scale-up can represent a major cost of the final protein product at the preparative scale. More economical and technically sim-ple methods for purification of soluble proteins are therefore desirable.Elastin-like polypeptides (ELPs) are oligomeric repeats of the pentapeptide Val-Pro-Gly-Xaa-Gly (where the “guest residue” Xaa is any amino acid with the exception of proline) that undergo a reversible inverse temperature transition. They are highly soluble in water below the inverse transition temperature (T t ), but undergo a sharp (2–3°C range) phase transition when the temperature is raised above T t , leading to desolvation and aggregation of the polypep-tide 13–15. In previous work, McPherson et al. exploited the inverse transition to purify recombinant poly(Gly-Val-Gly-Val-Pro)polypeptides 16.Previous studies have also shown that protein conju-gates of poly(N -isopropylacrylamide), a synthetic polymer that undergoes a similar thermally reversible phase transition, also retain the transition behavior of the free polymer 17–19. Together, these observations suggested that the thermally driven inverse transition of an ELP is likely to be maintained upon incorporation in a fusion protein.We hypothesized that, upon incorporation in fusion pro-teins, ELPs would allow nonchromatographic, thermally stimulated phase separation of recombinant proteins.ResultsWe sought to design an ELP sequence with a predicted T t >37°C, so that the ELP fusion protein would remain soluble during culture in Escherichia coli,but aggregate in response to a small increase in tem-perature. The composition and chain length of guest residue(s) have been shown to affect the T t strongly 20,21. We synthesized a gene encod-ing an ELP with guest residues Val, Ala, and Gly in the ratio 5:2:3 that had a predicted T t of approximately 40°C in water for a molecular mass (M r ) greater than 50 kDa. The synthetic gene encoded 10 Val-Pro-Gly-Xaa-Gly repeats (the 10-mer or ELP10) and was oligomer-ized up to 18 times to create a library of genes encoding ELPs with pre-cisely specified molecular masses ranging from 3.9 to 70.5 kDa. T o our knowledge, these are the first examples of genetically engineered ELPs of precisely defined chain length and amino acid sequence that are designed to exhibit an inverse transition at a specified temperature.Genes for two other proteins were fused to the ELP genes. First,each ELP sequence (10-, 20-, 30-, 60-, 90-, 120-, 150-, and 180-mers) was fused to the C terminus of E. coli thioredoxin, a 109-residue protein that is commonly used as a carrier to increase the solubility of recombinant proteins 7. Second, tendamistat, a 77-residue protein inhibitor of α-amylase 22, was fused to the C terminus of a thioredoxin–ELP90 fusion, forming a ternary fusion. The ELP fusion proteins were expressed in E. coli and purified from cell lysate by either immobilized metal affinity chromatography (IMAC)23or inverse transition cycling (described below). The purified ELP fusion protein was cleaved with thrombin to release the target pro-tein from the ELP , which was separated from the target protein by an additional round of inverse transition cycling. For each construct,the purified ELP fusion protein, target protein, and ELP were char-acterized for size and purity by SDS–PAGE (results not shown).The inverse transition can be monitored by assaying solution turbidity spectrophotometrically as a function of temperature.Increase in temperature beyond a critical point results in a sharp increase in turbidity over an approximately 2°C range to a maxi-mum value (OD 350approximately 2.0), because of aggregation of the ELP . The T t , defined as the temperature at 50% maximal turbidity, is a convenient parameter to describe this process. The inverse transi-tion profiles of free ELP , thioredoxin–ELP fusion, ELP–tendamistat fusion, and ternary thioredoxin–ELP–tendamistat fusion in phos-phate-buffered saline (PBS) are shown in Figure 1A. The T t was 51°C for free ELP and 54°C for the thioredoxin fusion, showing that the T tPurification of recombinant proteins by fusion with thermally-responsivepolypeptidesDan E. Meyer and Ashutosh Chilkoti*Department of Biomedical Engineering, Box 90281, Duke University, Durham, NC 27708-0281. *Corresponding author (chilkoti@).Received 2 June 1999; accepted 30 July 1999Elastin-like polypeptides (ELPs) undergo a reversible, inverse phase transition. Below their transition temperature (T t ), ELPs are soluble in water, but when the temperature is raised above T t , phase transition occurs, leading to aggregation of the polypeptide. We demonstrate a method for purification of soluble fusion proteins incorporating an ELP tag. Advantages of this method, termed “inverse transition cycling,”include technical simplicity, low cost, ease of scale-up, and capacity for multiplexing. More broadly, the ability to environmentally modulate the physicochemical properties of recombinant proteins by fusion with ELPs will allow diverse applications in bioseparation, immunoassays, biocatalysis, and drug delivery.Keywords:elastin-like polypeptide, inverse phase transition, environmentally responsive, fusion protein, protein purification© 1999 N a t u r e A m e r i c a I n c . • h t t p ://b i o t e c h .n a t u r e .c o mRESEARCHis only slightly affected by fusion to thioredoxin. Thioredoxin–ELP produced by cleavage from the ternary tendamistat fusion had a higher T t compared with thioredoxin–ELP produced directly (60versus 54°C), presumably because of differences in the leading and trailing amino acid sequences immediately adjacent to the ELP sequence (see Fig. 3). Transition profiles of ELP–tendamistat and thioredoxin–ELP–tendamistat fusion proteins were nearly identical,with a T t of 34°C. Aggregation of the ELP fusion proteins was reversible, and the aggregates were resolubilized completely when the temperature was lowered below the T t . Thioredoxin and ten-damistat controls exhibited no change in absorbance with increasing temperature, indicating that the induced aggregation observed for the fusion proteins was due to the inverse transition of the ELP carri-er. Typically, the inverse transition of the fusion proteins was also slightly broader than that of free ELP , with small upper and lower shoulders observed in their turbidity profiles.Motivated by the studies of Urry et al.21, who observed a decrease in T t with increasing chain length, we also investigated the effect of ELP molecular mass on the inverse transition of ELP fusion proteins.The T t of a set of thioredoxin–ELP fusion proteins was determined as a function of the molecular mass of the ELP carrier, which ranged from 11.8 to 70.5 kDa (Fig. 1B). The T t values for the higher molecu-lar mass fusion proteins approached the design target temperature of 40°C, whereas the T t values for the lower molecular mass fusions were significantly greater.In addition to ELP-specific variables that affect the T t (i.e., guest residue composition and M r ), several extrinsic factors can further modulate the T t for a given ELP . Such factors include the choice of sol-vent, ELP concentration, and ionic strength 15. Controlling the ionic strength, in particular, allows the T t to be tuned over a 50°C range (Fig.1C), and thereby provides a means for isothermal triggering of the transition. The T t increases with decreasing ELP concentration, and for concentrations less than about 25 µM this effect becomes significant 22.However, this effect can be mitigated by adding salt to depress the T t ,thereby allowing recovery of ELP fusion proteins from dilute solutions.The specific activity of the thioredoxin–ELP60 fusion protein,determined by an insulin reduction assay, was identical to that of com-mercial E. coli thioredoxin (results not shown), indicating that below the T t , the ELP tag had no effect on thioredoxin activity. We also mon-itored thioredoxin activity during thermal transition cycling, in which a sample of thioredoxin–ELP60 was cycled between temperatures above and below the T t (results not shown). When resolubilized below the T t , no change in activity was observed, even after four such cycles,which confirmed that the temperature changes and resulting aggrega-tion/resolubilization had no effect on stability and function of the fused target protein. For the ternary thioredoxin–ELP–tendamistat fusion, an α-amylase inhibition assay showed that the thioredoxin–ELP90 carrier reduced the inhibitory activity of tendami-stat by twofold (results not shown). However, after thrombin cleavage and purification of tendamistat from the thioredoxin-ELP carrier, the activity of purified tendamistat was indistinguishable from recombi-nant tendamistat that was independently purified by IMAC.Six thioredoxin–ELP fusion proteins containing a C-terminal 30,60-, 90-, 120-, 150-, or 180-mer ELP tag, and the thioredoxin-ELP90–tendamistat fusion protein were purified from cell lysate by inverse transition cycling, achieved by repeated centrifugation at conditions (i.e., NaCl concentration and temperature) alternating above and below T t . Typical results are shown in Figure 2A. Figure 2B shows the corresponding thioredoxin activity and total protein at each stage of purification of the thioredoxin–ELP fusion. Before purification, the induced E. coli were harvested from culture media by centrifugation, resolubilized in a low-salt buffer, and lysed by ultrasonic disruption. After high-speed centrifugation to remove insoluble matter, polyethylenimine was added to the lysate to precip-Figure 1. Characterization of the inverse transition of ELP fusion proteins. (A) Turbidity profiles for free ELP (thrombin-cleaved and purified from thioredoxin–ELP) (v ); thioredoxin–ELP (̆); thioredoxin–ELP–tendamistat (ć); ELP–tendamistat (cleaved and purified from thioredoxin-ELP-tendamistat) (ȣ); and thioredoxin–ELP (cleaved and purified from thioredoxin–ELP–tendamistat) ( ). All fusion proteins contained the same 90-pentapeptide ELP , with 25 µM protein concentration in PBS using a 1.5°C min -1heating rate. (B) T t as a function of ELP M r for the thioredoxin–ELP fusion proteins. (C) T t as a function of NaCl concentration for the thioredoxin/60-mer ELP fusion protein (25 µM) in 50 mM phosphate buffer, pH 8.0.Figure 2. Purification by inverse transition cycling. (A)SDS–PAGE of each stage of purification for the thioredoxin–ELP90fusion (49.9 kDa, lanes 1–5) and the thioredoxin–ELP90–tendamistat (57.4 kDa,lanes 7–9). Lanes 1 and 7, soluble lysate;lanes 2 and 8, supernatant containing contaminating E. coli proteins; lanes 3 and 9, resolubilized pellet containing purified fusion protein; lane 4, second-round supernatant;lane 5, second-round pellet;lanes 6 and 10: Molecular mass markers (kDa). (B) Total protein (by bicinchonic acid assay) and thioredoxin activity (by insulin reduction assay) for each stage of purification (lanes 1–5 as in part A) of the thioredoxin–ELP90. Values were normalizedto the soluble lysate.A BA B C© 1999 N a t u r e A m e r i c a I n c . • h t t p ://b i o t e c h .n a t u r e .c o mRESEARCHitate nucleic acids, yielding soluble lysate (lanes 1 and 7). Inverse transition cycling was initiated by adding NaCl and/or increasing the solution temperature to induce the ELP inverse transition, causing the solution to become turbid as a result of aggregation of the ELP fusion protein. The aggregated fusion protein was separated from solution by centrifugation at a temperature greater than the T t , and a translucent pellet formed at the bottom of the centrifuge tube. The supernatant, containing contaminating E. coli proteins comprising approximately 80% of the total protein, was decanted and discarded(lanes 2 and 8). The low thioredoxin activity measured in the super-natant, a portion of which is contributed by native E. coli thioredox-in, confirmed that this fraction primarily contained contaminatinghost proteins and not the overexpressed thioredoxin–ELP fusionprotein. The pellet was redissolved in a buffer of low ionic strength ata temperature below T t and centrifuged at low temperature to remove any remaining insoluble matter (lanes 3 and 9). The thiore-doxin-specific activity of the resolubilized protein approached thatof commercially available thioredoxin (data not shown). Althoughadditional rounds of inverse transition cycling were undertaken(lanes 4 and 5), the level of contaminating proteins was below detec-tion after a single round of inverse transition cycling, and nodetectable increase in thioredoxin-specific activity was obtained.Protein yields for the thioredoxin fusion constructs were typical-ly >40 mg of purified fusion protein per liter of culture. We foundthat the total gravimetric yield of fusion protein decreased withincreasing ELP length, with the 30-mer averaging about 70 mg/L andthe 180-mer averaging about 40 mg/L. However, ELP length appearsto have no effect on purification efficiency, and recovery from celllysate was complete for all fusion constructs. Expression levels of sol-uble tendamistat were slightly larger for the ternarythioredoxin–ELP–tendamistat fusion (40 mg/L ternary fusion, or 6mg/L tendamistat) compared with fusion with thioredoxin only (10mg/L thioredoxin–tendamistat fusion, 4 mg/L tendamistat).DiscussionWe chose thioredoxin and tendamistat as target proteins because theyexemplify two extremes of soluble protein expression. Thioredoxin isoverexpressed at high levels and is highly soluble, and tendamistat isexpressed largely as insoluble inclusion bodies. Proteins representa-tive of the latter class, however, must exhibit some level of solubleexpression to be purified by inverse transition cycling. Althoughfusion with thioredoxin is known to promote the soluble expressionof target proteins 7, we have observed that only 5–10% of overex-pressed thioredoxin–tendamistat fusion protein is recovered as solu-ble and functionally active protein (A.C., unpublished data).The thioredoxin–ELP fusion exhibits only a small increase in T t (1–2°C) com-pared with free ELP , whereas the tendamis-tat fusion displayed a dramatic 25°C reduction in T t . This shift is identical for both the ternary (thioredoxin–ELP–ten-damistat) and binary (ELP–tendamistat)constructs, indicating that the shift in T t is associated specifically with tendamistat.We hypothesize that the observed decrease in T t is due to interactions between the ELPchain and solvent-exposed hydrophobicregions in tendamistat, whereas for the highly soluble thioredoxin, these hydrophobic interactions are negligible.Although this T t shift adds complexity to the design of ELP carriers for inverse tran-sition purification of proteins containing a large exposed hydrophobic area, for highly soluble proteins only a small perturbationof T t relative to the free ELP is likely to be introduced upon fusion with an ELP tag. In addition to surface hydrophobicity, variability in target protein size is another factor that might be expected to affect the effi-ciency of purification by inverse transition cycling. Although the results reported here are for small target proteins (<20 kDa), we have also purified immune complexes with a total molecular mass greater than 230 kDa (D.E.M. and A.C., unpublished data).The ELP M r has two important ramifications for fusion proteinpurification. In buffers of low ionic strength, the T t values of the lower M r ELPs are too high for purification and would require a high concentration of NaCl to decrease to a useful temperature.The ELP chain length is also important with respect to protein yields. In addition to the decreased total yield of fusion protein observed with increasing ELP M r , the mass percentage of target protein is further decreased. Because we have observed that purifi-cation efficiency does not appear to be reduced for lower M r ELPs,the design of our next generation ELP carriers will maximize target protein expression by minimizing the ELP M r , while retaining a target T t of about 40°C through the incorporation of a larger frac-tion of hydrophobic guest residues in the ELP sequence.Inverse transition cycling offers several advantages for purifica-tion of recombinant fusion proteins. First, the expense associated with chromatographic resins and equipment is eliminated. The potentially lower cost and ease of scale-up of this method are likely to prove attractive for large-scale (in the grams to kilograms range)purification of proteins. Second, the separation and recovery condi-tions are mild, requiring only a modest change in temperature or ionic strength. Third, the method is fast and straightforward, with only a few short centrifugation or filtration steps followed by resolu-bilization of the purified protein in a buffer of low ionic strength.Fourth, inverse transition cycling can be easily multiplexed to purify proteins simultaneously from different cell cultures. Because protein purification is frequently the limiting step in structure/function studies and in screening of proteins in pharmaceutical development,we believe that the ability to purify simultaneously a large number of proteins by inverse transition purification is likely to be extremely useful for laboratory-scale purification (in the microgram to mil-ligram range) of proteins. Finally, inverse transition purification is independent of a specific expression vector or host and is likely to be extremely useful for eukaryotic expression systems because of their ability to overexpress heterologous proteins in a soluble state 24.In addition to protein purification, a number of different appli-cations for protein-ELP conjugates can be envisioned. Enzyme–ELP fusions could be useful substitutes for immobilized enzymes inindustrial biocatalysis, by allowing easy separation of the enzymeFigure 3. Gene sequences. (A) The 10-pentapeptide ELP gene. (B) The modified pET -32b vector forproduction of thioredoxin–ELP fusions. (C) The modified pET -32a vectors for the production of thethioredoxin–ELP–tendamistat fusions with alternate thrombin cleavage sites.ABC © 1999 N a t u r e A m e r i c a I n c . • h t t p ://b i o t e c h .n a t u r e .c o mRESEARCHfrom product and recycling of the enzyme for subsequent rounds of biocatalysis 25. Similarly, fusion of ELPs with engineered antibodies could allow the capture of an analyte from biological fluids for immunoassays 26. The use of ELPs conjugated to radionuclides or protein therapeutics for precise targeting for imaging and therapy by targeted hyperthermia 27–29is also currently under investigation.Experimental protocolGene synthesis.Standard molecular biology protocols were used for gene synthesis and oligomerization 30. All clones were maintained in the XL1-Blue strain of E. coli . Colonies were screened by PCR, and the DNA sequence of all putative inserts was verified by automated fluorescent dye terminator DNA sequencing.A synthetic gene for a 10-polypentapeptide ELP was constructed from four 5′-phosphorylated, PAGE-purified synthetic oligonucleotides (Integrated DNA Technologies , Coralville, IA). The oligonucleotides were annealed to form double-stranded DNA with Eco RI- and Hin dIII-compati-ble ends (Fig. 3A), and ligated into Eco RI/Hin dIII linearized and dephospho-rylated pUC-19 (N. Engl. Biolabs, Beverly, MA). The gene sequence was selected to emphasize E. coli preferred codons while also minimizing sequence repetition. For a typical oligomerization, the vector was linearized with Pfl MI and enzymatically dephosphorylated. The insert was doubly digested with Pfl MI and Bgl I, purified by agarose gel electrophoresis (QIAquick Gel Extraction Kit, Qiagen, Valencia, CA), and ligated into the lin-earized vector.Fusion protein construction and expression . For the thioredoxin fusions,pET-32b (Novagen, Madison, WI)was modified to contain an Sfi I restric-tion site downstream of the thioredoxin gene (Fig. 3B). For the ternary ten-damistat fusion, a pET-32a-based plasmid containing a gene for a thiore-doxin–tendamistat fusion was modified to contain an Sfi I restriction site in two alternate locations, upstream or downstream of the thrombin recogni-tion site (Fig. 3C). The ELP gene segments, produced by digestion with Pfl MI and Bgl I, were then ligated into the Sfi I site of each modified expres-sion vector.Expression vectors were transformed into the expression strains BLR(DE3) (thioredoxin) or BL21-trxB(DE3) (tendamistat) (Novagen).Shaker flasks with CircleGrow media (Bio 101,La Jolla, CA), supplemented with 100 µg/ml ampicillin, were inoculated, incubated at 37°C with shaking (250 r.p.m.), and induced at an OD 600of 0.8 by the addition of isopropyl α-thiogalactopyranoside (1 mM final concentration). The cultures were incu-bated an additional 3 h, harvested by centrifugation at 4°C, resolubilized in low-ionic-strength buffer (∼1/30 culture volume), and lysed by ultrasonic disruption at 4°C. The lysate was centrifuged at ∼20,000 g at 4°C for 15 min to remove insoluble matter. Nucleic acids were precipitated by the addition of polyethylenimine (0.5% final concentration), followed by centrifugation at ∼20,000 g at 4°C for 15 min.Fusion protein purification. The thioredoxin fusions, which contained a (His)6tag, were purified by IMAC using a nickel-chelating nitrilotriacetic-derivatized resin (Novagen)23or, alternatively, by inverse transition cycling.The tendamistat fusion was purified exclusively by inverse transition cycling.For purification by inverse transition cycling, ELP fusion proteins were aggregated by increasing the temperature of the cell lysate to ≤45°C and/or by adding NaCl to a concentration ≤2 M. The aggregated fusion protein was sep-arated from solution by centrifugation at 35–45°C at 10,000–15,000 g for 15min. The supernatant was decanted and discarded, and the pellet containing the fusion protein was resolubilized by agitation in cold, low-ionic-strength buffer. The resolubilized pellet was then centrifuged at 4°C to remove any remaining insoluble matter.Characterization of ELP fusion proteins. The optical absorbance at 350nm of ELP fusion solutions were monitored in the 4–80°C range on a Cary 300 ultraviolet-visible spectrophotometer equipped with a multicell thermo-electric temperature controller (Varian Instruments, Walnut Creek, CA). The T t was determined from the midpoint of the transition-induced change at a heating or cooling rate of 1.5°C min -1. The SDS–PAGE analysis used precast Mini-PROTEAN 10–20% gradient gels (Bio-Rad, Hercules, CA) with a dis-continuous buffer system 24, stained with Coomassie brilliant blue. The con-centration of the fusion proteins was determined spectrophotometrically using calculated extinction coefficients. Total protein concentrations were determined by bicinchonic acid assay (Pierce Chemical Co., Rockford, IL).Thioredoxin activity was determined using an insulin reduction colorimet-ric assay 31. Tendamistat activity was determined by a colorimetric α-amy-lase inhibition assay (Sigma, St. Louis, MO).AcknowledgmentsWe thank R.T. Piervincenzi for the thioredoxin–tendamistat gene. This work was supported by Duke University through its provision of start-up funds to A.C., by the Whitaker Foundation, the North Carolina Biotechnology Center (ARIG no. 9605-ARG-0050), and the National Institutes of Health (1R21-GM-057373-01). We also thank the Whitaker Foundation for support of D.E.M. as a graduate fellow.1. Nilsson, B., Forsberg, G., Moks, T., Hartmanis, M. & Uhlén, M. Fusion proteins in biotechnology and structural biology. Curr. Opin. Struct. Biol.2, 569–575 (1992).2. Uhlén, M. & Moks, T. Gene fusions for purpose of expression: an introduction.Methods Enzymol.195, 129–143 (1990).3. Maina, C.V . et al. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose binding protein. Gen e 74, 365–373(1988).4. Smith, D.B. & Johnson, K.S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.Gen e 67, 31–40(1988).5. Tsao, K.W., deBarbieri, B., Hanspeter, M. & Waugh, D.W. A versatile plasmid expression vector for the production of biotinylated proteins by site-specific,enzymatic modification in Escherichia coli.Gene 169,59–64 (1996).6. Smith, P .A. et al. A plasmid expression system for quantitative in vivo biotinyla-tion of thioredoxin fusion proteins in Escherichia coli.Nucleic Acids Res.26,1414–1420 (1998).7. LaVallie, E.R. et al. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Bio/Technology 11,187–193(1993).8. Ong, E. et al. The cellulose-binding domains of cellulases: tools for biotechnolo-gy. Trends Biotechnol.7, 239–243 (1989).9. Smith, M.C., Furman, T.C., Ingolia, T.D. & Pidgeon, C. Chelating peptide-immobi-lized metal ion affinity chromatography. J. Biol. Chem.263, 7211–7215 (1988).10. Kim, J-S. & Raines, R.T. Ribonuclease S-peptide as a carrier in fusion proteins.Protein. Sci.2, 348–356 (1993).11. Su, X., Prestwood, A.K. & M cGraw, R.A. Production of recombinant porcinetumor necrosis factor alpha in a novel E. coli expression system.Biotechniques 13, 756–762 (1992).12. Nilsson, J., Ståhl, S., Lundeberg, J., Uhlén, M. & Nygren, P .Å. Affinity fusionstrategies for detection, purification, and immobilization of recombinant proteins.Protein Expr. Purif . 11, 1–16 (1997).13. Urry, D.W. Entropic elastic processes in protein mechanisms. I. Elastic structuredue to an inverse temperature transition due to internal chain dynamics. J.Protein. Chem.7, 1–34 (1988).14. Urry, D.W. Free energy transduction in polypeptides and proteins based oninverse temperature transitions. Prog. Biophys. Mol. Biol.57, 23–57 (1992).15. Urry, D.W. Physical chemistry of biological free energy transduction as demonstrat-ed by elastic protein-based polymers. J. Phys. Chem. B 101, 11007–11028 (1997).16. McPherson, D.T., Xu, J. & Urry, D.W. Product purification by reversible phase tran-sition following Escherichia coli expression of genes encoding up to 251 repeats of the elastomeric pentapeptide GVGVP . Protein Expr. Purif.7, 51–57 (1996).17. Hoffman. A.S. Applications of thermally-reversible polymers and hydrogels intherapeutics and diagnostics. J. Controlled Release 6, 297–305 (1987).18. Chen, J.P . & Hoffman A.S. Protein-polymer conjugates II. Affinity precipitationseparation of immunogammaglobulin by a poly(N -isopropylacrylamide)-protein A conjugate. Biomaterials 11, 631–634 (1990).19. Chilkoti, A., Chen, G-H., Stayton, P .S. & Hoffman, A.S. Site-specific conjugationof a temperature-sensitive polymer to a genetically-engineered protein.Bioconjugate Chem. 5, 504–507 (1994).20. Urry, D.W. et al. Temperature of polypeptide inverse temperature transitiondepends on mean residue hydrophobicity. J. Am. Chem. Soc.113,4346–4348(1991).21. Urry, D.W., Trapane, T.L. & Prasad, K.U. Phase-structure transitions of the elastinpolypentapeptide-water system within the framework of composition-tempera-ture studies. Biopolymers 24, 2345–2356 (1985).22. Vertesy, L., Oeding, V ., Bender, R., Zepf, K. & Nesemann, G. Tendamistat (HOE467), a tight-binding alpha-amylase inhibitor from Streptomyces tendae 4158.Eur. J. Biochem.141, 505–512 (1984).23. Porath, J. Immobilized metal ion affinity chromatography. Prot. Expr. Purif.3,262–282 (1992).24. Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W. & Wingfield, P .T . Currentprotocols in protein science.(John Wiley & Sons, New York; 1995).25. Hartmeier, W. Immobilized biocatalysts.(Springer-Verlag, Berlin; 1988).26.Diamandis, E.P . & Christopoulos, T.K. Immun oassay.(Academic Press, SanDiego, CA; 1996).27. Dewhirst, M.W. & Samulski, T.V . Current Concepts: Hyperthermia in the treatmentof cancer. (The UpJohn Co., Kalamazoo, MI; 1998).28. Hauck, M.L., Dewhirst, M.W., Bigner, D.D. & Zalutsky, M.R. Local hyperthermiaimproves uptake of a chimeric monoclonal antibody in a subcutaneous xenograft model. Clin. Cancer Res.3, 63–70 (1997).29. Cope, D.A., Dewhirst, M .W., Friedman, H.S., Bigner, D.D. & Zalutsky, M .R.Enhanced delivery of a monoclonal antibody F(ab ′)2 fragment to subcutaneous human glioma xenografts using local hyperthermia. Cancer Res.50, 1803–1809(1990).30. Ausubel, F .M. et al. Current protocols in molecular biology (John Wiley & Sons,New York; 1995).31. Holmgren, A. & Bjornstedt, M. Enzymatic reduction-oxidation of protein disul-fides by thioredoxin. 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ElisaRSR TM AQP4 Ab Version 2 Aquaporin-4 (AQP4) AutoantibodyELISA Version 2 Kit –Instructions for useRSR LimitedParc Ty Glas, Llanishen, CardiffCF14 5DU United KingdomTel.: +44 29 2068 9299 Fax: +44 29 2075 7770 Email: Website: EC REP Advena Ltd. Tower Business Centre, 2nd Flr., Tower Street, Swatar, BKR 4013 Malta.INTENDED USEThe RSR AQP4 Autoantibody ELISA Version 2 kit is intended for use by professional persons only, for the quantitative determination of AQP4 autoantibodies (AQP4 Ab) in human serum. Neuromyelitis optica (NMO), also known as Devic’s syndrome, is an immune-mediated neurologic disease that involves the spinal cord and optic nerves. It can be considered to be a disorder distinct from multiple sclerosis (MS). A serum immunoglobulin G autoantibody (NMO-IgG) has been shown to be a specific marker for NMO and the water channel aquaporin 4 (AQP4) has been identified as the antigen for NMO IgG. Measurement of AQP4 Ab can be of considerable value in distinguishing NMO from MS when full clinical features may not be apparent and early intervention may prevent or delay disability. REFERENCESV. A. Lennon et al.A serum autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis.Lancet 2004 364(9451): 2106 - 2112V. A. Lennon et al.IgG marker of optic-spinal multiple sclerosis bindsto the aquaporin-4 water channel.The Journal of Experimental Medicine 2005 202: 473 - 477B. G. Weinshenker et al.Neuromyelitis optica IgG predicts relapse after longitudinally extensive transverse myelitis.Annals of Neurology 2006 59: 566 - 569N. Isobe et al.Quantitative assays for anti-aquaporin-4 antibody with subclass analysis in neuromyelitis optica. Multiple Sclerosis Journal 2012 18: 1541 – 155S. Jarius et al.Testing for antibodies to human aquaporin-4 by ELISA: Sensitivity, specificity and direct comparison with immunohistochemistry.Journal of the Neurological Sciences 2012 320:32 - 37PATENTSThe following patents apply:European patent EP 1 700 120 B1, US patents US 7,101,679 B2, US 7,947,254 B2 and US 8,889,102 B2, Chinese patent ZL200480040851.3 and Japanese patent 4538464. ASSAY PRINCIPLEIn RSR’s AQP4 Ab ELISA Version 2 kit, AQP4 Ab in patient s’ sera, calibrators and controls are allowed to interact with AQP4 coated onto ELISA plate wells and liquid phase biotinylated AQP4 (AQP4-Biotin). After incubation at room temperature for 2 hours with shaking, the well contents are discarded. AQP4 Ab bound to the AQP4 coated on the well will also interact with AQP4-Biotin through the ability of AQP4 Ab in the samples to act divalently leaving AQP4-Biotin bound to the well via an AQP4 Ab bridge. The amount of AQP4-Biotin bound is then determined in a second incubation step involving addition of streptavidin peroxidase (SA-POD), which binds specifically to biotin. Excess, unbound streptavidin peroxidase is then washed away and addition of the peroxidase substrate, 3,3’,5,5’-tetramethlybenzidine (TMB), results in formation of a blue colour. This reaction is stopped by the addition of a stop solution, causing the well contents to turn yellow. The absorbance of the yellow reaction mixture at 450nm and 405nm is then read using an ELISA plate reader. A higher absorbance indicates the presence of AQP4 autoantibody in the test sample. Reading at 405nm allows quantitation of high absorbances. It is recommended that values below 10 u/mL should be measured at 450nm. If it is possible to read at only one wavelength 405nm may be used. The measuring interval is 3.0 –80 u/mL (arbitrary RSR units).STORAGE AND PREPARATION OF SERUM SAMPLESSera to be analysed should be assayed soon after separation or stored, preferably in aliquots, at or below –20o C. 100 μL is sufficient for one assay (duplicate 50 μL determinations). Repeated freeze thawing or increases in storage temperature should be avoided. Do not use lipaemic or haemolysed samples. Studies in which EDTA, citrate and heparin plasma samples were spiked with AQP4 Ab positive sera showed minor changes in signal compared with spiked serum from the same donor. In particular OD450values with spiked EDTA, citrate and heparin plasmas were 79% - 128% of spiked serum (15 samples with serum concentrations ranging from 2.6 u/mL – 30 u/mL) or 87% - 130% in terms of u/mL. When required, thaw test sera at room temperature and mix gently to ensure homogeneity. Centrifuge serum prior to assay (preferably for 5 min at 10-15,000 rpm in a microfuge) to remove particulate matter. Please do not omit this centrifugation step if sera are cloudy or contain particulates.SYMBOLSSymbol MeaningEC Declaration of Conformity IVD In Vitro Diagnostic DeviceREF Catalogue NumberLOT Lot NumberConsult InstructionsManufactured bySufficient forExpiry DateStoreNegative ControlPositive ControlMATERIALS REQUIRED AND NOT SUPPLIED Pipettes capable of dispensing 25 μL, 50 μL and 100 μL.Means of measuring various volumes to reconstitute or dilute reagents supplied.Pure water.ELISA Plate reader suitable for 96 well formats and capable of measuring at 450nm and 405nm.ELISA Plate shaker, capable of 500 shakes/min (not an orbital shaker).ELISA Plate cover.PREPARATION OF REAGENTS SUPPLIEDStore unopened kit and all kit components at 2-8o C.A AQP4 Coated Wells12 breakapart strips of 8 wells (96 in total) in a frame and sealed in foil bag. Allow foil bag to stand at room temperature (20-25o C) for 30 minutes before opening.Ensure wells are firmly fitted in the frame provided. After opening return any unused wells to the original foil bag and seal with adhesive tape. Then place foil bag in the self-seal plastic bag with desiccant provided and store at 2-8o C for up to4 months.B1-5 Calibrators1.5, 5, 20, 40, 80 u/mL (arbitrary RSR units)5 x 0.7 mLReady for useC1-2 Positive Controls I & II(see label for concentration range) 2 x 0.7 mLReady for useD Negative Control0.7 mLReady for useEAQP4–Biotin3 vialsLyophilisedImmediately before use, reconstitute withreconstitution buffer for AQP4-Biotin (F),1.5 mL per vial. When more than one vialis to be used, pool the contents of thevials and mix gently.FReconstitution Buffer for AQP4-Biotin10 mLReady for useGStreptavidin Peroxidase (SA-POD)0.8 mLConcentratedDilute 1 in 20 with diluent for diluting SA-POD (H). For example, 0.5 mL (G) + 9.5mL (H). Store for up to 16 weeks at 2-8o Cafter dilution.HDiluent for SA-POD15 mLReady for useIPeroxidase Substrate (TMB)15 mLReady for useJConcentrated Wash Solution120 mLConcentratedDilute 1 in 10 with pure water before use.Store at 2-8o C up to kit expiry date.KStop Solution14 mLReady for useASSAY PROCEDUREAllow all reagents to stand at room temperature (20-25o C) for at least 30 minutes prior to use. Do notreconstitute AQP4-Biotin until step 2 below. AnEppendorf type repeating pipette is recommended forsteps 2, 5, 8, and 9.1. Pipette 50 μL(in duplicate) of patientsera, calibrators (B1-5) and controls (C1-2 and D) into respective wells. Leave onewell empty for blank.2. Reconstitute AQP4-Biotin and pipette25μL into each well (except blank).3. Cover the frame and shake the wells for2 hours at room temperature on an ELISAplate shaker (500 shakes per min).4. Use an ELISA plate washer to aspirateand wash the wells three times withdiluted wash solution (J). If a platewasher is not available, discard the wellcontents by briskly inverting the frame ofwells over a suitable receptacle, washthree times manually and tap the invertedwells gently on a clean dry absorbentsurface to remove excess wash.RESULT ANALYSISA calibration curve can be established by plotting calibrator concentration on the x-axis (log scale) against the absorbance of the calibrators on the y-axis (linear scale). The AQP4 Ab concentrations in patient sera can then be read off the calibration curve [plotted at RSR as a spline log/lin curve (smoothing factor = 0)]. Other data reduction systems can be used. The negative control can be assigned a value of 0.15 u/mL to assist in computer processing of assay results. Samples with AQP4 Ab concentrations above 80 u/mL can be diluted (e.g.10 x and/or 100 x) in AQP4 Ab negative serum. Some sera will not dilute in a linear way. TYPICAL RESULTS (Example only; not forAbsorbance readings at 405nm can be converted to 450nm absorbances by multiplying by the appropriate factor (3.4 in the case of equipment used at RSR).This cut off has been validated at RSR. However each laboratory should establish its own normal and pathological reference ranges for AQP4 Ab levels. Also it is recommended that each laboratory include its own panel of control samples in the assay. CLINICAL EVALUATION(The information below is derived from 450nm data) Clinical SpecificitySera from 358 individual healthy blood donors were tested in the AQP4 Ab ELISA Version 2 kit. 356 (99%) sera were identified as being negative for AQP4 Ab.Clinical SensitivityOf 62 sera from patients with NMO or NMO spectrum disorder (NMOSD) 48 (77%) were positive for AQP4 Ab.Lower Detection LimitThe negative control was assayed 20 times and the mean and standard deviation calculated. The lower detection limit at 2 standard deviations was0.17 u/mL.Clinical AccuracyAnalysis of 205 sera from patients with autoimmune diseases other than neuromyelitis optica spectrum disorders (NMOSD) indicated no interference from autoantibodies to the TSH receptor (n=110), glutamic acid decarboxylase (n=26), 21-hydroxylase (n=12), the acetylcholine receptor (n=10), thyroid peroxidase (n=15), thyroglobulin (n=10), IA-2 (n=7) or from rheumatoid factor (n=15) in the RSR AQP4 Ab ELISA Version 2. InterferenceNo interference was observed when samples were spiked with the following materials; bilirubin at 20 mg/dL or intralipid up to 3000 mg/dL. Interference was seen from haemoglobin at 500 mg/dL. SAFETY CONSIDERATIONSStreptavidin Peroxidase (SA-POD)Signal word: WarningHazard statement(s)H317: May cause an allergic skin reaction Precautionary statement(s)P280: Wear protective gloves/protective clothing/ eye protection/face protectionP302 + P352: IF ON SKIN: Wash with plenty of soap and waterP333 + P313: If skin irritation or rash occurs: Get medical advice/attentionP362 + P364: Take off contaminated clothing and wash it before reusePeroxidase Substrate (TMB)Signal word: DangerHazard statement(s)H360: May damage fertility or the unborn child Precautionary statement(s)P280: Wear protective gloves/protective clothing/eye protection/face protectionP308 + P313: IF exposed or concerned: Get medical advice/attentionThis kit is intended for use by professional persons only. Follow the instructions carefully. Observe expiry dates stated on the labels and the specified stability for reconstituted reagents. Refer to Safety Data Sheet for more detailed safety information. Avoid all actions likely to lead to ingestion. Avoid contact with skin and clothing. Wear protective clothing. Material of human origin used in the preparation of the kit has been tested and found non-reactive for HIV1 and 2 and HCV antibodies and HBsAg but should, none-the-less, be handled as potentially infectious. Wash hands thoroughly if contamination has occurred and before leaving the laboratory. Sterilise all potentially contaminated waste, including test specimens before disposal. Material of animal origin used in the preparation of the kit has been obtained from animals certified as healthy but these materials should be handled as potentially infectious. Some components contain small quantities of sodium azide as preservative. With all kit components, avoid ingestion, inhalation, injection or contact with skin, eyes or clothing. Avoid formation of heavy metal azides in the drainage system by flushing any kit component away with copious amounts of water.ASSAY PLANAllow all reagents and samples to reach room temperature (20-25 o C) before usePipette: 50 μL Calibrators, controls and patient seraPipette: 25 μL AQP4-Biotin (reconstituted) into each well (except blank)Incubate: 2 Hours at room temperature on an ELISA plate shaker at 500 shakes/min Aspirate/Decant: PlateWash: Plate three times and tap dry on absorbent material1Pipette: 100 μL SA-POD (diluted 1:20) into each well (except blank)Incubate: 20 Minutes at room temperature on a ELISA plate shaker at 500 shakes/min Aspirate/Decant: PlateWash: Plate three times and tap dry on absorbent material1, 2Pipette: 100 μL TMB into each well (including blank)Incubate: 20 Minutes at room temperature in the dark without shakingPipette: 100 μL Stop solution into each well (including blank) and shake for 5 seconds Read absorbance at 450nm and 405nm within 10 minutes of adding stop solution31It is not necessary to tap the plates dry after washing when an automatic plate washer is used2Use pure water for the final wash when washing manually3If it is possible to read at only one wavelength, 405nm may be used。

真核翻译延伸因子eEF1A 功能研究进展*高爽，查笑君，潘建伟【摘要】摘要:总结了近年来对于真核翻译延伸因子eEFlA 生物学功能和作用机制研究的一些新进展，具体包括:eEFlA 参与介导受损或错误折叠的蛋白质的降解;对微丝、微管骨架系统的组织与调控;参与调控细胞凋亡;参与细胞核输出氨酰-tRNA;与病毒基因组和RNA 依赖性RNA 聚合酶类(RNA-dependent RNA polymerase，RdRP)互作，参与病毒繁殖.阐述了对eEFlA 进行研究的意义和价值，并提供了新的见解和展望.【期刊名称】浙江师范大学学报（自然科学版）【年(卷),期】2013(036)004【总页数】6【关键词】关键词:真核生物;翻译延伸因子1A(eEF1A);功能;作用机制蛋白质翻译延伸因子(translation elongation factor，EF)最初从大肠杆菌(Escherichia coli)细胞中分离获得，具有三磷酸鸟苷(GTP)或鸟苷二磷酸(GDP)亲和性，参与肽链的延伸过程.在原核细胞中，有3 类延伸因子，分别被命名为EF-Tu(elongation factor thermo unstable)，EF-Ts(elongation factor thermo stable)和EF-G(elongation factor G);而在真核细胞中，相对应的分别为eEF1A(eukaryotic translation elongation factor 1A)，eEF1B(eukaryotic translation elongation factor 1B)和eEF2(eukaryotic translation elongation factor 2).蛋白质生物合成过程大致可分为3 个阶段:起始、延伸和终止.在肽链延伸阶段，EF1A 与GTP 结合产生EF1A·GTP 复合体，此复合体再与特异的氨酰-tRNA 结合并将其运送到核糖体A 位点，并伴随着GTP 的水解，最后EF1A·GDP 从核糖体释放出来.EF1A·GDP 经EF1B 催化又重新形成EF1A·GTP.在核糖体肽酰转移酶作用下，位于核糖体P 位点的多肽被转移到A 位点，与新进入的氨酰-tRNA 形成新的肽键，再由EF2 催化肽基-tRNA·mRNA 复合物从核糖体A 位点转移至P位点，空出的A 位点将接纳下一个新的氨酰-tRNA.重复此过程，蛋白多肽链将最终被合成［1-2］.eEF1A 胞内含量很高，仅次于肌动蛋白，其基因及表达调控十分保守.eEF1A 由一个多基因家族编码，不同的物种具有不同数量的eEF1A 同源基因，如:酵母中有2 个eEF1A 同源基因;拟南芥和水稻中分别有4 个eEF1A 同源基因;玉米中有10～15 个eEF1A 同源基因;人类有多于18 个eEF1A 同源基因.蛋白结构分析表明，eEF1A 具有3 个构象不同的功能结构域，结构域Ⅰ与GTP 结合，结构域Ⅱ与氨酰-tRNA 结合，结构域Ⅰ和Ⅱ还与e EF1Bα(组成eEF1B 的亚基之一)互作，结构域Ⅱ和Ⅲ共同参与和肌动蛋白的互作［3］.过去20 年的研究表明，eEF1A 除参与蛋白质翻译外，还具有多种生物学功能.本文主要就真核细胞eEF1A 在蛋白质降解、细胞骨架组织调控、细胞凋亡、核物质输出和病毒繁殖等过程中的生物学功能作一扼要综述.1 eEF1A 与蛋白降解泛素(ubiquitin)介导的蛋白降解是蛋白质代谢的主要机制之一，在动植物生长发育过程中具有重要的调控作用.最初发现eEF1A 作为一个必需因子参与泛素介导的N-α-蛋白降解过程［4］.eEF1A 的原核同源蛋白EF-Tu 被证实有类似分子伴侣的活性［5］.随后的研究表明，eEF1A 既能与合成中的新生肽链互作，也能与翻译后折叠错误的蛋白质结合［6］.进一步的证据表明:eEF1A能有效缓解参与蛋白降解的RAD23 和RPN10 功能缺失后所引起的细胞生长缓慢等表型［7］;eEF1A 能与蛋白酶体的19S 调节亚基RPT1 直接互作，RPT1 的功能缺失可降低eEF1A 与蛋白酶体的互作，同时胞内出现受损蛋白的代谢缺陷［7］;刀豆氨酸(canavanine)能诱导蛋白折叠错误而使后者进入泛素化降解途径，但当eEF1A 的GTP 结合域第156 位天冬氨酸(Asp)突变为天冬酰胺(Asn)后，细胞对刀豆氨酸表现出较高的抗性［7］.这些研究结果充分暗示，eEF1A 参与介导受损或错误折叠的蛋白从核糖体到蛋白酶体的过程.2 eEF1A 与细胞骨架细胞骨架是细胞内错综复杂的动态纤维状网络结构，除具有维持细胞形态和调控细胞增殖外，对蛋白质翻译的组织与调控也具有重要的生物学意义［8］.许多蛋白质翻译系统的组分，如氨酰-tRNA合成酶、真核起始因子eIF(eukaryotic initiation factor)和翻译延伸因子EF 直接或间接地与细胞骨架相连.来自哺乳动物细胞和酿酒酵母的证据表明，微丝骨架系统的任何缺陷均会影响肽链合成的正常进行［9］.尽管eEF1A 最初被鉴定为翻译系统的重要作用因子，但后续的研究表明eEF1A 是一类进化上保守的肌动蛋白结合蛋白(actin-binding protein)，具有调控肌动蛋白组装微丝的功能［10］.eEF1A 通过抑制微丝纤维末端肌动蛋白单体的加聚和解聚，从而调控微丝纤维的组装，最终影响与微丝相关的货物运输、定位及mRNA 的翻译［11］.eEF1A 与肌动蛋白纤维或氨酰-tRNA 的结合受胞内pH 调控:当pH 值逐渐增大时，促进eEF1A 与肌动蛋白的解离，利于eEF1A 与氨酰-tRNA 的结合和肽链合成;而pH 值逐渐减小时，促进eEF1A与肌动蛋白的结合.而且竞争性结合实验进一步证实，这两种结合是相互排斥的［12］.在海胆的受精过程中，胞内pH 值的增加作为信号刺激蛋白质合成［13］.eEF1A 能与微丝骨架调节蛋白Rho1p的下游靶蛋白Bni1p 互作［14］.这些研究结果表明，细胞通过pH 的变化调控eEF1A 介导的肽链延伸和微丝骨架组织之间的切换.为进一步提供eEF1A 在微丝骨架组织中的遗传学证据，超表达eEF1A 的酿酒酵母在没有显著影响蛋白质合成的前提下引起了微丝骨架的组织紊乱，细胞生长缓慢［15］.酿酒酵母eEF1A 遗传突变筛选获得2 类突变体:一类使蛋白质合成功能维持正常，但存在微丝骨架组织缺陷，其eEF1A与肌动蛋白结合的功能维持正常，但将肌动蛋白组装成束的功能下降［16］;另一类表现为更严重的微丝骨架组织紊乱，蛋白质翻译起始缺陷，细胞生长缓慢［9］.最近的体外实验发现，eEF1Bα 能抑制eEF1A 对肌动蛋白组装成束的生物学功能［17］.皮肤性人乳头瘤病毒HPV38 的E7 蛋白能与eEF1A结构域Ⅲ的C 末端区域结合，从而抑制后者对微丝骨架的组织功能［18］.eEF1A 除了参与微丝骨架的组织外，也参与微管蛋白的组装.在海胆卵中，首次发现eEF1A作为有丝分裂活动的重要组分［19］.体外实验表明，胡萝卜eEF1A 以一种Ca2+/钙调蛋白依赖的方式结合并促进微管组装成束，稳定微管骨架［20-21］.非洲爪蟾和哺乳动物eEF1A 均被鉴定具有切割微管的活性［22］.然而，eEF1A 参与微管组织的分子调控机制至今仍不清楚.3 eEF1A 与细胞凋亡早期的研究发现，体外培养的鼠成纤维细胞内eEF1A 表达水平与去除血清后诱导的细胞凋亡率呈正相关［23］.在过氧化氢诱导的细胞凋亡前，胞内eEF1A 表达水平迅速上升［24］.这些结果暗示eEF1A 能促进细胞凋亡.而另一研究中筛选细胞凋亡抑制因子时，分离得到了eEF1A［25］.这似乎与之前的研究结果互相矛盾，但后续的研究为其作出了解释.哺乳动物中存在功能差异的2种eEF1A 亚型，即eEF1A1 和eEF1A2，分别由不同的基因编码，氨基酸序列同源性约为92%［26］.尽管两者在多肽延伸过程中作用相似，但它们的表达模式却具有不同的时空特异性，eEF1A1 在各组织中广泛表达，而eEF1A2 似乎只在骨骼肌、心肌和脑细胞中表达［26-27］.在成肌细胞分化过程中，发现eEF1A1 具有促进细胞凋亡的作用，而eEF1A2 的作用则相反［28］.在研究脂毒性细胞凋亡机制时，也发现抑制eEF1A1 的表达可阻碍细胞凋亡［29］.这些研究结果表明，eEF1A1 和eEF1A2 表达水平的差异参与决定细胞的命运［27-28，30］.最近的研究表明，胁迫刺激如病毒感染等诱导的干扰素诱导蛋白IFIT1(interferon-induced protein with tetratricopeptide repeats-1)通过与eEF1A1 互作，从而促进细胞凋亡［31］.在人巨噬细胞内，艾滋病毒HIV-1 Nef 蛋白与eEF1A1 结合，通过eEF1A1 和tRNA 的核-质重定位从而抑制由内质网应激介导的细胞凋亡［32］.另有研究表明，eEF1A2 通过与抗氧化蛋白peroxiredoxinⅠ的互作，从而抑制由氧化胁迫诱导的细胞凋亡［33］.这些研究结果说明，eEF1A 通过与功能不同的靶蛋白互作，行使不同的功能.上述研究所涉及的都是依赖于半胱天冬酶的细胞凋亡.然而，越来越多的证据说明，细胞还有不依赖于半胱天冬酶的凋亡途径［34-35］.在四倍体细胞中发现了一种不依赖于半胱天冬酶的细胞凋亡，这种凋亡由eEF1A1 的表达下调引起，能帮助除去异常四倍体细胞和抑制肿瘤发生［36］.4 eEF1A 与核物质输出有实验证据表明eEF1A 参与细胞核物质的输出过程.在酿酒酵母中，表达突变的eEF1AE286K或E291K(tRNA 结合位点突变)的菌株表现为核输出氨酰-tRNA 障碍而累积于核中［37-38］.tRNA 氨酰化是eEF1A 与tRNA 有效结合的前提［39］，也是tRNA 被运输到核外的前提［37］.Exportin-5 属于细胞核质转运受体importinβ 家族.在哺乳动物中，eEF1A 通过氨酰-tRNA 与Exportin-5 结合形成输出复合物，随后eEF1A 与氨酰-tRNA 一同被输出到核外［40-41］.eEF1A 不仅参与核输出氨酰-tRNA，在哺乳动物细胞核输出蛋白质的过程中也起到重要的作用.转录依赖的核输出序列TD-NEM(transcription-dependent nuclear export motif)是一种新发现的核输出信号序列［42］，eEF1A 与TD-NEM 互作，参与介导了含有此信号序列的蛋白向核外输出的过程［43］.自从发现eEF1A 参与核物质输出以来，其在细胞核与质之间的穿梭成为了讨论的热点.由于正常条件下eEF1A 定位于核外，而且Exportin-5对eEF1A 的输出也确保了其在核外，所以人们推测其在核物质输出过程中所起的作用都是在核膜的胞质侧完成的.然而，在一个核物质输出受体Msn5 突变的酿酒酵母菌株的细胞核中能检测到eEF1A 的存在，暗示了其进入细胞核内参与核物质输出的可能性［44］.因此，eEF1A 参与核物质输出的具体作用位置仍有待进一步验证.5 eEF1A 与病毒繁殖eEF1A 作为细胞内最丰富的蛋白质之一，也参与了病毒生活史的循环.所报道的eEF1A 参与病毒复制大都来自于正链RNA 病毒，如登革热病毒(DV)［45］、黄萝卜花叶病病毒(TYMV)［46］、烟草花叶病毒(TMV)［47］、西尼罗河病毒(WNV)［48］和芜菁皱缩病毒(TCV)［49］;但也有报道表明其参与负链RNA病毒如水疱性口炎病毒(VSV)的复制［50］.eEF1A 能与这些病毒基因组3′非编码区的类tRNA 二级结构结合，并与它们各自编码的RNA 依赖性RNA 聚合酶类(RdRP)互作.eEF1A能刺激TCV 的RdRP 活性及负链RNA 的合成［49］.WNV 基因组的eEF1A 结合位点突变，引起eEF1A 结合障碍，表现为负链RNA 合成降低，病毒复制受阻［51］.然而，对于TYMV，eEF1A 对其基因组的结合似乎强烈抑制了负链RNA 的合成［46］.这可能是因为:病毒侵染早期，eEF1A 结合其基因组，阻碍了RdRP 以正链RNA 为模板的复制行为，但正链RNA 指导的翻译活动正常进行;当RdRP 等病毒蛋白质合成达到一定量时，RdRP与eEF1A 竞争并结合到基因组的3′端，合成负链RNA.在本氏烟中，eEF1A 表达下调抑制TMV 的复制和传播［52］.eEF1A 可能作为番茄丛矮病毒(TBSV)复制酶复合体的一个组分，通过提高复制辅助因子p33 的稳定性来促进病毒复制.从酿酒酵母中分离出的一个突变体eEF1AT22S，表现为p33 的半衰期缩减，病毒复制受阻［53］.以上研究结果表明，eEF1A 对病毒复制具有重要作用.病毒的复制由一系列程序化事件组成，eEF1A 可能帮助维持了这种程序.6 展望综上所述，eEF1A 是一类具有多种生物学功能的重要调控蛋白.对于eEF1A 生物学功能的研究具有重要的应用价值.由于eEF1A 参与调控细胞凋亡，为肿瘤疾病的防治提供了新的策略和靶点，经工程改良的eEF1A 很可能成为肿瘤治疗的重要药物.同时，eEF1A 也参与心血管系统的调节，为相关疾病的防治提出了新的思路［54］.eEF1A 作为细胞内含量第2 高的蛋白质，其基因的表达必定由强启动子启动，该启动子可用来提高外源基因的表达和一些蛋白质的工业生产［55］.另外，eEF1A 还可作为玉米、大麦和高粱胚乳赖氨酸含量的指示物，谷物籽粒的赖氨酸含量是判断其营养价值的重要指标［56］.目前，有关eEF1A 生物学功能的证据主要来自于酵母和哺乳动物，对于植物eEF1A 的生物学功能和作用机制知之甚少.因此，对于植物eEF1A的研究还有待进一步深入.参考文献:［1］Dever T E，Green R.The elongation，termination，and recycling phases of translation in eukaryotes［J］.Cold Spring Harb Perspect Biol，2012，4(7):a013706.［2］Kavaliauskas D，Nissen P，Knudsen C R.The busiest of all ribosomal assistants:elongation factor Tu［J］.Biochemistry，2012，51(13):2642 2651.［3］Sasikumar A N，Perez W B，Kinzy T G.The many roles of the eukaryotic elongation factor 1 complex［J］.Wiley Interdiscip Rev RNA，2012，3(4):543-555.［4］Gonen H，Smith C E，Siegel N R，et al.Protein synthesis elongation factor EF-1 alpha is essential for ubiquitin-dependent degradation of certain N alpha-acetylated proteins and may be substituted for by the bacterial elongation factor EF-Tu［J］.Proc Natl Acad Sci USA，1994，91(16):7648-7652.［5］Caldas T D，Yaagoubi A El，Richarme G.Chaperone properties of bacterial elongation factor EF-Tu［J］.J Biol Chem，1998，273(19):11478 11482.［6］Hotokezaka Y，Tobben U，Hotokezaka H，et al.Interaction of the eukaryotic elongation factor 1A 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Advances in the study of Alzheimer's disease Angue Nkoghe Francoise;Yunman Li【期刊名称】《生物医学研究杂志（英文版）》【年(卷),期】2005(019)006【摘要】Alzheimer's disease (AD) is the most common cause of dementia, and the only treatment currently available for the disease is acetylcholinesterase inhibitors. Recent progress in understanding the molecular and cellular pathophysiology of Alzheimer's disease has suggested possible pharmacological interventions, including acetylcholineseterase inhibitors; secretase inhibitors; cholesterol lowering drugs; metal chelators and amyloid immunization. The objective of this paper is to review the main drugs possibly used for AD and their future therapeutic effects.【总页数】5页(P279-283)【关键词】阿尔茨海默病;乙酰胆碱酯酶抑制剂;分泌酶抑制剂;降低药物;金属螯合剂;淀粉样蛋白免疫【作者】Angue Nkoghe Francoise;Yunman Li【作者单位】Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, P.R. China;Department of Pharmacology, China Pharmaceutical University, Nanjing 210009, P.R. China【正文语种】中文【中图分类】R1因版权原因，仅展示原文概要，查看原文内容请购买。

核盘菌通过类似整联蛋白（SSITL）抑制寄主的抗病反应目 录摘 要 (I)ABSTRACT (IV)缩略词表 (VIII)1. 前言综述 (1)1.1 核盘菌的危害及其防治 (1)1.1.1 核盘菌的危害及其生物学特性 (1)1.1.2 作物菌核病的防治研究 (1)1.2 植物病原菌与寄主植物的互作 (5)1.2.1 植物天然的的物理及生理生化防卫屏障 (5)1.2.2 植物的先天免疫系统 (6)1.2.3 植物的后天免疫系统 (10)1.2.4 植物的非寄主抗性 (13)1.2.5 不同类型植物病原菌的侵染策略以及互作方式 (14)1.2.6 核盘菌的侵染策略 (16)1.3基因功能研究的策略 (19)1.3.1丝状真菌的遗传转化的研究进展 (19)1.3.2基因的超标达、敲除和沉默 (20)1.3.3 蛋白质的定位 (24)1.4 Integrin以及Integrin–like基因的研究进展 (26)1.4.1 整联蛋白的结构 (27)1.4.2 整联蛋白的信号传导 (29)1.4.3整联蛋白在微生物中的生物学功能 (30)1.5 本项研究的目的和意义 (32)2. 材料与方法 (33)2.1 菌株及植物材料 (33)2.2 基因的生物信息学分析 (33)2.3 核酸的实验操作 (34)2.3.1 DNA的提取 (34)2.3.2 质粒的提取 (34)2.3.3 总RNA的提取 (35)2.3.4 RT和Real–Time PCR (35)2.3.5 Northern blot (36)2.4 蛋白质的实验操作 (37)2.4.1 SSITL的原核表达 (37)2.4.2 抗体血清的制备、效价（ELISA）以及特异性（Western blot）的检测 (37)2.4.3 SSITL的免疫胶体金亚细胞定位 (39)2.4.4 核盘菌侵染洋葱表皮过程中SSITL的免疫荧光定位 (40)2.5 相关载体的构建 (40)2.6 ATMT介导的真菌和植物转化 (41)2.7 生物学特性的实验研究 (43)2.7.1 生长速度、致病力、菌丝顶端分支以及菌落形态的观察 (43)华中农业大学2012届博士研究生学位论文2.7.2 菌核的培养、大小及重量的测定和菌核萌发的研究 (43)2.7.3 核盘菌产草酸能力的测定 (44)2.7.4 核盘菌侵染拟南芥叶片过程的观察 (45)2.8 SSITL与植物诱导抗性的关系 (45)2.8.1 核盘菌侵染拟南芥过程中SSITL基因的表达情况 (45)2.8.2 核盘菌侵侵染拟南芥过程中拟南芥局部抗性的动态变化 (45)2.8.3 核盘菌侵侵染拟南芥过程中拟南芥系统抗性的动态变化 (46)2.8.4 SSITL在植物中表达对植物的抗病性的影响 (46)3. 结果与分析 (47)3.1 SSITL的生物信息学分析 (47)3.1.1 SSITL的序列分析 (47)3.1.2 SSITL蛋白的同源比对分析及高级结构预测 (49)3.2 SSITL对核盘菌生物学特性的影响 (53)3.2.1 SSITL基因在核盘菌不同生长时期的表达 (53)3.2.2 SSITL基因沉默对核盘菌生物学特性的影响 (53)3.3 SSITL抗体的制备以及免疫胶体金亚细胞定位 (61)3.3.1 SSITL的原核诱导表达 (61)3.3.2 抗血清效价以及特异性测定 (63)3.3.3 SSITL蛋白的亚细胞定位 (63)3.4 SSITL基因在核盘菌与植物互作过程中的作用 (67)3.4.1 核盘菌侵染拟南芥时，SSITL基因的表达情况 (67)3.4.2 核盘菌SSITL对拟南芥局部防卫反应的影响 (68)3.4.3 核盘菌SSITL对拟南芥系统防卫反应的影响 (70)3.4.4 SSITL在寄主植物中瞬时表达对植物抗病性的影响 (74)3.4.5 SSITL在寄主植物中组成型表达对植物抗病性的影响 (79)3.4.6 SSITL的表达对烟草的影响 (81)4. 讨论 (83)4.1 SSITL基因生物学功能的深入探讨 (83)4.1.1 SSITL基因的序列分析 (83)4.1.2 SSITL基因的功能分析 (85)4.2 SSITL参与抑制植物诱导抗性 (87)4.2.1 SSITL基因在核盘菌侵染过程中被诱导表达 (88)4.2.2 SSITL参与抑制植物的局部抗性 (88)4.2.3 SSITL参与抑制植物的系统抗性 (89)4.2.4 SSITL基因在植物中表达后，植物的抗性受到抑制 (90)4.3 研究SSITL的互作蛋白以及作用机理 (90)4.4 结论与展望 (92)5. 参考文献 (94)附录： (116)博士期间发表的论文 (121)致 谢 (122)核盘菌通过类似整联蛋白（SSITL）抑制寄主的抗病反应摘 要核盘菌（Sclerotinia sclerotiorum）属于子囊菌门，是一种世界性分布的典型的死体营养型病原真菌。

NatureCellBiology2019第1期2019第1期导读Nature Cell Biology 20岁啦！1999年细胞生物学家Günter Blobel “因为发现蛋白质自身具有控制胞内运输和定位的信号”获得了诺贝尔生理学和医学奖。

1997年诞生的多莉羊在这一年重返大众视线，因为据报道，从生物学上讲，她出现早衰征兆。

人类研究的伦理问题引发激烈争论。

互联网已经渗透到我们日常生活中。

但即使全世界都对“千年虫”病毒感到焦虑，科学出版仍在继续向数字化存在迈进。

这就是Nature Cell Biology二十年前诞生的世界。

创刊伊始，我们的定位是“一个旨在促进细胞生物学各个领域之间交流的新的跨学科论坛”（“a new interdisciplinary forum designed to foster the exchange of ideas between all areas of cell biology”）。

当时我们认为“了解细胞如何发挥作用并相互交流是生物学的巨大挑战之一，而揭示这背后的分子机制是细胞生物学研究的核心”，由此也确定期刊的发表内容将涵盖细胞生物学的各个领域。

从左到右：99年创刊号，NCB-10周年，NCB-20周年二十年过去了，Nature Cell Biology的使命依旧不变，我们将继续发表和细胞生物学相关的科学发现。

为了庆祝创刊20年，我们推出题为“细胞生物20年”（‘20 years of cell biology’）的主题专刊。

专刊包括16篇特邀综述（Review）和评论（Perspective）。

尽管这16篇文章无法涵盖整个细胞生物学领域，但我们希望它们能体现出细胞生物领域的多样性和重要性。

同时，我们还把过去20年在Nature Cell Biology上发表的一部分研究论文做了一个合集。

因为篇幅限制，我们无法做到面面俱到，非常遗憾很多优秀的论文未能囊括进来。

西南医科大学学报2022年第45卷第1期Journal of Southwest Medical University Vol.45No.12022镓（Gallium ）是一种是灰蓝色或银白色的金属，以5~15mg/kg 的浓度存在于地壳中，常常从铝土矿、铅锌矿和铜铁矿中作为一种副产品获得[1-6]。

镓的熔点低，约为28.7646℃[7]，在室温状态下可接近液态。

对空气极为敏感，在空气中易氧化，生成氧化镓层，氧化镓在水中转化为氧化镓单氢氧化物（GaOOH ）的形式，其钝化程度比氧化镓要低得多[7]。

镓还具有能和卤素作用生成各种卤化物的特性，所以镓易于与大多数金属形成合金。

此外镓还是低毒性金属，镓的某些化学性质与铁（III ）相似，是一种应用非常广的金属材料。

更重要的是，越来越多的研究显示了其在生物医学应用领域中的潜力，本文对镓及其化合物在生物医学领域的应用现状作以下综述及展望。

1抗菌近些年来，抗菌类材料的应用越来越广泛，但是也随之出现了一些问题，抗生素的滥用导致了多种病原生物的抗生素耐药性，并成为全球健康问题，迫切需要新的抗菌策略。

抗菌剂主要分为天然抗菌剂、有机抗菌剂和无机抗菌剂三大类[8]。

无机抗菌剂具备稳定性高、耐药性好、对环境友好等特点，是未来绿色抗菌剂的重要方向[9]。

镓作为无机抗菌剂，对常见的细菌具有较好的抗菌活性，可作为体外抗菌剂单独使用，并对细菌生长及其生物膜的生成起到很好的抑制作用[10]，镓的化合物硝酸镓在与其他药物联用时能够明显抑制细菌生物膜的形成[11-15]。

有研究发现硝酸镓对金黄色葡萄球菌镓在医疗领域方面的应用现状及前景展望李铁1，刘人铭1，曹家桢1，旷苗2综述于波3审校1.长春中医药大学（长春130117）；2.中国生物技术发展中心（北京100039）；3.中国科学院微电子研究所（北京100029）【摘要】镓作为一种新的液体金属材料，不仅具有高导热性、高导电性和出色的机械性能等常规金属的基本属性，还具备典型液体性能。