Construction of eukaryotic expression vector carrying human TSLC1 gene and its expression in Hep

DC-SIGN真核表达载体的构建及其稳定转染HeLa细胞系的建立王欣;詹媛;王琰;周恩民;丁铲;谭磊;陆凤;徐乐乐;仇旭升;宋翠萍;孙英杰;廖瑛;茅翔【摘要】为研究新城疫病毒(Newcastle disease virus, NDV)与树突状细胞特异性粘附分子-3-结合非整合素分子(dendritic cell specifi c intercellular adhesion molecule-3-grabbing non-integrin,DC-SIGN)的相互作用对NDV感染树突状细胞(dendritic cell,DC)的影响,本研究拟构建能够稳定表达DC-SIGN蛋白的细胞系,为研究DC-SIGN蛋白与病毒蛋白互作提供基础.以小鼠DCs提取的cDNA为模板,通过PCR方法获得DC-SIGN基因,连入真核表达载体,并命名为pcDNA-DC-SIGN,利用LipofectamineTM2000转染HeLa细胞,G418进行药物压力筛选,经RT-PCR和Western blot鉴定,获得了一株可以高效表达DC-SIGN蛋白的HeLa 细胞,且经过多次传代后仍然可以稳定表达DC-SIGN,说明该细胞系构建成功.%To investigate the infl uence of the interaction between Newcastle diseases virus (NDV) protein with dendritic cells specifi c adhesion molecules - 3 - grabbing non-integrin (DC-SIGN) on NDV infection of dendritic cell (DC), we generated a Hela cell line stably expressing DC - SIGN protein for studying DC - SIGN protein and virus protein interactions. The cDNA was extracted from mouse DCs and used as template. The DC - SIGN gene was amplifi ed in polymerase chain reaction (PCR) for construction of eukaryotic expression vector pcDNA-DC-SIGN, which was then transfected into HeLa cells using LipofectamineTM 2000. A HeLa cell line effi ciently expressing DC - SIGN protein was identifi ed through RT-PCR and Western blot, and the specifi c Hela cell line was stable after many passages.【期刊名称】《中国动物传染病学报》【年(卷),期】2015(023)003【总页数】5页(P12-16)【关键词】树突状细胞特异性粘附分子-3-结合非整合素分子;新城疫病毒;树突状细胞;HeLa细胞【作者】王欣;詹媛;王琰;周恩民;丁铲;谭磊;陆凤;徐乐乐;仇旭升;宋翠萍;孙英杰;廖瑛;茅翔【作者单位】西北农林科技大学动物医学院,杨凌 712100;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海 200241;西北农林科技大学动物医学院,杨凌 712100;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海200241;中国农业科学院上海兽医研究所,上海 200241;西北农林科技大学动物医学院,杨凌 712100;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海 200241;中国农业科学院上海兽医研究所,上海200241【正文语种】中文【中图分类】S852.659.5树突状细胞特异性粘附分子-3-结合非整合素分子（dendritic cell specific intercellular adhesion molecule-3 grabbing non integrin，DC-SIGN），又称CD209，由美国科学家在研究人类免疫缺陷病毒（Humanimmunodeficiency virus，HIV）感染机制过程中发现。

基因组学与应用生物学，2020年，第39卷，第11期，第4947-4952页研究报告Research Report南极鱼基因在Z F4细胞中抗寒功能的分析胡瑞芹w李根芳w陈良标@1上海海洋大学，海洋生物科学国际联合研究中心，上海,201306; 2上海海洋大学，水产科学国家级实验教学示范中心，上海,201306; 3上海海 洋大学，水产种质资源发掘与利用教育部重点实验室，上海,201306* 通信作者，***************.cn摘要为探究南极鱼Co/motWi/i基因在低温适应下的作用与应用，本研究从南极鱼m aw;-的CDNA文库中克隆得到CaM基因，通过构建真核表达载体并转染到ZF4细胞中，在低温胁迫下检 测细胞的存活率。

结果表明，南极鱼CaM基因能够在ZF4细胞中表达，并分布在细胞质中；在低温胁迫下,与对照组相比，过表达南极鱼基因的细胞的LDH毒性明显低于对照组，并且细胞活性明显高于对照 组。

因此，低温胁迫下南极鱼CaM基因的过表达能够显著提高ZF4细胞的抗寒能力。

本研宄结果证明了 一种候选的抗寒基因，为鱼类的抗寒育种提供了理论依据。

关键词南极鱼基因，低温胁迫，ZF4细胞，LDH细胞毒性，细胞活性Analysis on Cold Resistance of Antarctic Fish Calmodulin Gene in ZF4 CellsHu Ruiqin1Z3Li Genfang1,2,3Chen Liangbiao旧*1International Research Center for Marine Biosciences at Shanghai Ocean University, Shanghai, 201306; 2 National Demonstration Center for Experi­mental Fisheries Science Education, Ministry of Science and Technology, Shanghai Ocean University, Shanghai, 201306; 3 Key Laboratory of Explo­ration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, 201306*Corresponsingauthor,***************.cnDOI: 10.13417/j.gab.039_004947Abstract To explore the role and application of Calmodulin gene in Antarctic fish under low temperature adap­tation,the CaM gene was cloned from the cDNA library of Antarctic fish Dissostichus mawsoni,and the eukaryotic expression vector was constructed and transfected into ZF4 cells to examine the survival rate of the cells under low temperature stress.The results showed that the Antarctic fish CaM gene could be expressed in ZF4 cells and distributed in the cytoplasm.Under low temperature stress,compared with the control group,the LDH toxicity of the cells overexpressing the Antarctic fish CaM gene was significantly lower than that of the control group,and the cell activity was significantly higher.Therefore,the overexpression of CaM gene in Antarctic fish under low temperature stress can significantly improve the cold resistance of ZF4 cells.The results of this study demonstrate a candidate cold-resistant gene that provides a theoretical basis for cold-resistant breeding of fish.Keywords Antarctic fish CaM gene,Low temperature stress,ZF4 cells,LDH cytotoxicity,Cell viability温度是限制物种分布、影响个体生长和决定生 殖周期的一个基本环境因子。

论著流感病毒受体结合表位真核载体的构建及表达罗俊1,王欢1,戴军1,左斌1,裴德翠1,丰峰1,李婉宜1,邝玉1,王保宁1,蒋忠华1,李虹2,李明远1*(1.四川大学华西基础医学与法医学院微生物学教研室,四川成都610041;2.四川大学华西第二医院港大联合研究中心) 摘要 目的 构建甲型流感病毒血凝素(H A )信号肽(SP)与HA 唾液酸受体结合部位(RB)所含T 、B 表位的基因片段(RBT 、R BB)真核表达质粒,研究其在真核细胞中的表达与免疫特性。

方法 通过重叠延伸PCR 法(o ver lap PCR)将HA 的SP 分别与R B 、RBT 、RBB 通过一段多肽接头Gly4Ser 融合为SP RB 、SP RBT 和SP R BT ,将其分别插入pcD N A 3.1(+)载体,构建质粒,鉴定正确后转染M DCK 细胞,RT PCR 、免疫荧光、M T T 检测该质粒的表达和分泌。

结果 融合基因真核表达质粒构建成功,在M DCK 细胞中成功表达,转染细胞培养上清能刺激淋巴细胞增殖。

结论 SP RB 、SP RBT 、SP RBB 真核表达载体成功构建,表达产物能刺激淋巴细胞增值,为流感病毒唾液酸受体结合部位的T 、B 细胞表位免苗研究提供初步依据,也为流感核酸疫苗的研制奠定了基础。

关键词 流感病毒;T 细胞表位;B 细胞表位;免疫荧光技术;淋巴细胞增殖试验中图分类号 R373.13 文献标识码 A 文章编号 1673 5234(2011)01 0008 4[J our nal of Pathogen B iology .2011Jan;6(1):8-11.]C onstruction of eukaryotic expression plasmid for receptor binding of influenza virus and their expression LU O Jun 1,WANG H uan 1,DAI Jun 1,ZU O Bin 1,PEI Dei cui 1,FENG Feng 1,LI Wan yi,KU ANG Yu 1,WANG Bao ning 1,JIANG Zhong hua 1,LI H ong 2,LI M ing yuan 1 (1.D ep ar tment of M icrobiology ,West China School of Pr eclinical and For ens ic M edicine,Sichuan Univ er sity ,Chengdu 610041,China;2.T he J oint R esearch Center of W es t China Seco nd Univ er s ity H os p ital of S ichuan Univ er sity and the Faculty of M edicine,Univer sity of H ong K ong )Abstract Objective T o const ruct eukary otic ex pression vector s fo r the signal pept ide (SP )of hemagg lutinin (HA )o f influenza A vir us and its acylneuraminate recepto r binding (RB)sites incor po rating gene segments o f T and B epitopes (RBT and RBB,respectively)and to observe the expressio n o f t arg et pr otein by and immunolog ical characterist ics o f eu kar yot ic cells. M ethods A polypept ide linker Gly4Ser w as used to splice SP w ith RB,RBT ,and RBB by ov erlap P CR to r espectively construct SP RB,SP RBT ,and SP RBT fusio n g enes.T hese g enes w ere inserted into eukar yo tic expres sio n plasmids pcD NA 3.1(+).A fter identification,pcDN A3.1(+)/SP R B,pcDN A3.1(+)/SP RBT ,and pcDN A 3.1(+)/SP R BB wer e tr ansfected into M D CK cells.T he ex pr essio n o f these g enes was analy zed w ith RT P CR,immunoflu o rescence assay ,and lym phocyt e pr oliferation test with M T T . Results pcDN A 3.1(+)ex pr essio n vector s incor po ra ting SP RB,SP RBT ,and SP RBT fusion genes wer e successfully constr ucted.T he fusion pro tein w as detect ed in M DCK transfected with pcDN A 3.1(+)/SP RB,pcDN A 3.1(+)/SP RBT ,or pcD NA 3.1(+)/SP RBB and st imulated lym phocyte pro liferatio n. C onclusion Eukar yo tic ex pr essio n plasmids fo r SP RB,SP RBT and SP RBT fusion genes wer e successfully constructed and t he ex pression pro ducts stimulated lympho cyte pro liferatio n,establishing a solid foundation for fur ther study of receptor binding,immuno lo gical study o f T and B cell epitopes o f influenza v ir us,and study of DN A vaccines.Key words Influenza virus;T cell epito pe;B cell epitope;immunofluo rescence assay ;lymphocyte pro liferatio n test流感病毒的高度变异性常导致流感的流行,因此如何获得高效及安全的流感疫苗是科学工作者关注的课题。

Bax A Bad基因真核表达质粒构建及转染人胚肾细胞后的翻译水平观察王孜恒一2，周敬伟一2，侯筱宇一2(1徐州医科大学，江苏徐州221000；2江苏省脑病生物信息重B实验室)摘要：目的构建Bcl-2相关X蛋白(Bax)、Bcl-2相关细胞死亡因子(Bad)基因真核表达质粒pcDNA3.1-Bax、pcDNA3.1-Bad,并观察两质粒转染至人胚肾293(HEK293)细胞后Bax,Bad的表达。

方法采用TRIzol试剂提取大鼠脑组织总RNA,利用反转录一聚合酶链反应(RT-PCR)扩增Bax和Bad的互补DNA(cDNA)序列，将cDNA分别克隆至pcDNA3.1载体上。

采用脂质体转染法将序列正确的pcDNA3.1-Bax和pcDNA3.1-Bad真核表达质粒分别转染至HEK293细胞，免疫印迹法鉴定Bax,Bad在细胞中的表达。

结果双酶切和DNA测序分析显示Bax和Bad基因片段成功插入pcDNA3.1载体中，并且与其cDNA(Genbank：NM_017059.2和AF031227.1)序列完全一致。

免疫印迹显示pcDNA3.1-Bax,pcDNA3.1-Bad真核表达质粒能够在HEK293细胞中高效表达。

结论pcD-NA3.1-Bax、pcDNA3.1-Bad真核表达质粒构建成功，Bax、Bad在HEK293细胞中成功过表达。

关键词:Bcl-2相关X蛋白；Bcl-2相关细胞死亡因子;真核表达质粒;人胚肾细胞doi：10.3969/j.issn.1002-266X.2019.30.006中图分类号:R743.31文献标志码:A文章编号:1002-266X(2019)30-0021-04Construction of eukaryotic expression plasmid of Bcl-2-assaciated X protein and Bcl-2-associated agonist of cell death and its expression in human embryonic kidney cellsWANG Ziheng1,ZHOU Jingwei,HOU Xiaoyu(1Xuzhou Medical University,Xuzhou221000,China)Abstract:Objective To construct the eukaryotic expression plasmids pcDNA3 .1-Bax and pcDNA3 .1-Bad for Bcl-2-assaciated X protein(Bax)and Bcl-2-associated agonist of cell death(Bad),and then to observe their expression in hu­man embryonic kidney293(HEK293)cells.Methods Total RNA of rat brain tissues was extracted by using trizol rea­gent,cDNA sequences of Bax and Bad were amplified by reverse transcription-polymerase chain reaction(RT-PCR),and then were cloned into pcDNA3.1vectors.The constructed eukaryotic expression plasmids pcDNA3.1-Bax and pcDNA3.1-Bad were transfected into HEK293cells by liposome transfection,respectively.The expression of pcDNA3.1-Bax and pcD-NA3 .1-Bad plasmids in cells was identified by Western blotting.Results Double enzyme digestion and DNA sequencing results showed that Bax and Bad gene fragments were successfully inserted into pcDNA3.1vector,and were completely consistent with its cDNA sequences(Genbank:NM_017059.2and AF031227.1).Western blotting showed that the eu­karyotic expression plasmids pcDNA3.1-Bax and pcDNA3.1-Bad were highly expressed in HEK293cells.Conclusion The eukaryotic expression plasmids pcDNA3.1-Bax and pcDNA3.1-Bad are successfully constructed,and Bax and Bad are highly expressed in HEK293cells.Key words:Bcl-2-associated X protein;Bcl-2-associated agonist of cell death;eukaryotic expression plasmids;hu­man embryonic kidney cells凋亡最早是由英国病理学家Kerr JF教授于1972年根据细胞形态学改变提出的概念，是指细胞在接受外界信号刺激后启动的主动的程序性细胞死亡过程［］。

Constructi on of Exogenous Expression Vector of Tri -choder ma reeseiZHANG X i ao-xuan 1,WANG Ao -xue2*1.Chengdong Co llege ,Nort heast Agricultural Univ ers ity ,Har b in 150030;2.Co llege of Lif e Sc iences ,Nort heas tAgricult ural Universit y ,Har -bin 150030Abstract [Objecti ve]The st udy w as t o construc ta ex ogenous expression v ector f or Tri chode r m a ree se i .[M et ho d]U sing CBH I pro moter and t er m inat or of T.ree se i strain 40359,we cons tructed an expr ession vec t or o f T.ree se i s train 40359f or expressing Hp t gene and got s ix strains capab l e o f grow ing on basic m ediu m c ontaining 175mg /L of hygro myc i n B ,f urt her c onduc t ed hygr omycin resis t ance tes.t [R es ults ]In co mpar-i son w ith t he ori g inal strain(w il d t ype),hygro myc in r esis t ance t he six engineer ed strains was increased by 75%;t he hygro myc i n resis t ance c oul d inherit st ab l y .[Co ncl usio n]Our results laid bas is f or bi o l o g i c al study on T.ree se i atm ol e cul a r and genetic a lly engineeri n g levels .Key wo r ds T ri cho de r m a ree se i ;Vec t or ;Genetic transf or mati o nReceived :February 18,2011 Accept ed :March 21,2011*C orresponding author .E -mai:l wangaoxue @yahoo .co mC BH I pro mot er fro m Tri choder m a reese i is a very strong pro m oter that is usually used for vector constructi o n f or gene-t i c m i provement of T.reese i ,i .e .,inserting t he gene of inter -est(GOI)int o t he space bet w een CBHI promot er and ter m-i nat or sequence .The reco mbined frag ment is transf or med into fil a ment ous fungi prot oplas,t and located and int egrat ed into chromoso m e by us i n g t he m ethod of t argeted gene replace -ment and int egrati o n .This makes the GO I dri v en by t he strong pro mot er CBH I and the required homologous or heter -ologous prot ein expressed secret ory under the induction of t he l e ader peptide of C BH I .This method has been successf ull y used f or construct engineered filament ous fungistai n s overex -pressing various t arget prot eins .For inst ance ,MU Jing -yu e t a l .[1]from Jilin Universit y succeeded i n overexpression of As-pe rg ill u s n i g er glucose ox i d ase gene under the control of CBH I pro m oter fro m T.reese i .I n vie w of t his ,we att empt ed to ex -press hygro mycin B phos photrans f erase gene i n T.reese i by us i n g CBH I promot er and ter m i n at or sequence ,and carri e d out hygromyc i n B resist ance t est on the y i e lded resistant strains and reco mbinants ,am i i n g at prov i d ing basis for bi o log -i c al study on T.reese i at molecular and genetically engineer -i n g l e vels .Mat eri a ls and MethodsExperi m entalmateri al sStrai ns and reagents Tri chode r m a reese i strain 40359was purchas ed from Chi n a Center of IndustrialCulture Collection ;plas m id pCAM1305.1harboring hygromyc i n B phos photrans -ferase gene was preserved by our research group ;vectors pMD18and pU C19were purchas ed fro m TaKaRa ;EF Taq DNA pol y merase ,plas m id DNA isolation k it and prm i ers were purchas ed from Beijing Langang B i o t ech Co .,Ltd .;T 4-DNA ligase ,DL2000Marker ,RT -PCR k i,t CTAB ,Tris #C,l B -mer -capt oet hano,l RNase and agarose were all purchas ed fro m TaKaRa ;all the restrictive endonulceas es used were pur -chased from MB;I agarose gel extrac tion k it was purchased fro m T iangen Biot ech (Beijing)Co .,Lt d .All other reagents were home made products at analyti c pure grade .M ed i a r eci pe(for 1L) Basalmediu m:20g glucose ,15gKH 2PO 4,5g(NH 4)2SO 4,0.6g CaC l 2#2H 2O,0.6g M g -SO 4#7H 2O,0.005g FeSO 4#7H 2O ,0.0016g M n SO 4#H 2O,0.0014g ZnSO 4#7H 2O ,0.002g Co C l 2#6H 2O,pH=5.5;aut o -cl a ving at 121e f or 30m in (addition of 2%of agar f or solid m ediu m and 1%agar for sem-i soli d medium ).Regenerati o n m edia :(1)l o wer layermediu m :additi o n of1mol/L of s orb-i t oland 2%of agar into basalm ediu m;(2)upper layermed-i u m :addition of 1mol/L of sorbit ol and 1%of agar into basal m ediu m.Experi m entalmethods Pri m er s and their sequences used for PCR a mplifi cati on Based on sequence inf or mation from GenBank ,soft ware Gene runnerwas e mployed to design prm i ers as f ollo w s :amplificati o n of CBH I promoter and signalpeptide(pCBHI ):P1:Sense :5c -GC GCATGC AATTCTGGAGACGGCTT -GTT -3c :S ph ÑP2:Ant -i s ens e :5c -GCGT CGACCTCCAAGT GTTGC -CAT CGTA -3c :S a lÑamplification of C BH I t er m inator(t CBHI ):T1:Sense :5c -GCGCGGATCCAGGTCACCTTCT CCAA -CATCA -3c :Bam H ÑT2:Ant -i sense :5c -GCGCGAATT CCACGAAGAGCG -GCGATTCTA -3c :Eco R Ñamplification of Hp t :H1:Sense :5c -GCGC GTCGAC ATGCCTGAACTCAC -CGCGAC -3c S a l ÑH2:Ant -i sens e :5c -GC GC GGAT CC CGGTCGGCATC -TACT CT ATT -3c Bam H Ñ.A mplifi cati on of target frag ment (1)PCR reacti o n volu me (50L l)f or amplifying t he upstrea m nucleoti d e fragment of T.reese i cbh 1gene was composed of 312.5L mol/L dNTPs ,2.5L mol/L of prm i er ,500ng of t emplate ,3.5-3.6mmol/L of MgC l 2,2.5U EF Taq DN A poly merase .These reacti o n co mponents were first react ed at 94e f or 7m in predenat ur -ati o n before t he addition of EF Taq DNA poly meras e ,then 30cyc l e s at 94e f or 1m in ,60e f or 1m in and 72e for 2.5m in ,finally at 72e f or 10m i n .(2)PCR reaction volume(50L l)f or a mplif y ing t he downstream nuc l e otide fragment of T.reese i cbh 1gene was composed of 200L mol/L of dNTPs ,1.5L mol/L of prm i er ,500ng of te mpl a t e ,1.5-2.5mmol/L ofAgri c ultural B i o technol o gyAgricult ural Sc i e nc e&Technology ,2011,12(2):308-312Copyright k 2011,I nf or mati o n Institute ofHAAS .A ll ri g ht s reserv ed .MgC l2,1.5U EF Taq DNA poly meras e.These reacti o n co mpo-nent s were first reac t ed at94e f or7m i n predenat urationbef ore the addition of EF Taq DNA poly meras e,t hen30cycl e s at94e f or1m in,55e f or1.5m in and72e f or1.5m in,fi n ally at72 e f or10m in.(3)PCR reac tion volu me(50L l)f or a mplifying the Hp t gene was co mposed of312.5L mol/L of dNTPs,1.0 L mol/L of prm i er,500ng of t emplate,2.5mmol/L ofMgC l2, 1.5U EF Taq DNA poly merase.These reaction co mponents were first react ed at94e f or7m i n predenaturati o n bef ore t he additi o n of EF Taq DNA poly merase,then30cycles at94e f or30s,60e f or1m in and72e f or2.5m in,finally at72e f or10m in.Constr uction of expr ession vector Geno m ic DNA of T.reese i[2-3]and P l a s m i d contai n ing Hygro m ycin B phospho-transferase gene was extracted as te mplat e f or PCR amplif-i cati o n using prm i ers P1and P2t o a mplif y T.reese iCBHI pro-mot er and CBHI signal pepti d e gene;the reacti o n products were ligat ed i n t o p MD18-T vect or;t he y i e lded vect orwas then double-digest ed by us i n g S phÑand S a lÑf or use.Likew ise,T. reese iCBH I promot erwas a mp lifi e d using prm i ers T1and T2 and ligated int o p MD18-T vector;the y i e lded vector was then double-digest ed by using Bam HÑand Eco RÑf or use.Esche-ri ch i a co l i hygromyc i n B(HygB)phosphotransferase gene was amplified using prm i ers H1and H2from pl a s m id DNA har-boring this gene,and li g at ed int o pMD18-T vec t or;t he yielded vec t or was t hen double-digest ed by us i n g S a lÑand Bam HÑf or use.The frag m ents yiel d ed above were li g at ed by usi n g T4 ligase at16e overni g h;t t he y i e lded productwas transfor med i n t o co mpet ent E.co li cells v i a heat shock method and coat ed on LB sel e cti o n pl a te cont aining Amp and HygB,the pl a tes were incubat ed at37e overni g h.t The recombi n antwas inoc-ulated i n t o LB li q ui d medium cont aini n g Amp and HygB and in-cubat ed at37e f or12-16h(200r/m in).The yiel d ed turbid bact erial solution was used to extract plas m i d DNA f or PCR and enzy mati c di g estion.The constructs consist s of CBH I promoter-CBH I signal peptide gene-Hyg gene-C BH I ter m i n at or-pUC19vector,desig-nat ed as Pcbh-HygB.Deter m i nation on the resi stance of T.reesei strain to HygB B.Se m-i solid basal medi a contai n ing25,50,75, 100,125,150,175and200mg/L of HygB were respectivel y prepared and m i x ed w ith appropriat e vol u me of T.reese i strain40359.Them ixtures were poured into the l o wer l a yerof plat e and cultured at28e for the observation of mycelial grow t h.Transfor mati on o f the pr o toplast of T.reesei strai n40359 Prot opl a st preparati o n and transfor mati o n was ref erred to the method of PenttiL¾M.et a l.[3].Appropriat e volu me of transfor mati o n reacti o n soluti o n was coated on the lower layer of regeneration mediu m and i n cubated at28e f or12-16h, then a l a yer of se m-i solid fil a ment ous f ungiMM mediu m cont a-i ning125mg/L of HygB was poured on the pl a t e used above and i n cubat ed at28e for3-4d.The Hyg B-resist ant transfor ma-nt s were sel e ct ed f or furt hermolecular identificati o n.PCR i dentifi cation of transgen i c str ai ns PCR a mplificati o n was perf or med by using genom ic DNA as t empl a t e and t he upstream and downstrea m prm i ers of Hp t as prm i ers,w ith ge-no m ic DNA of non-transf or med strain40359as negati v e con-trol and plas m id DNA as positive contro.lHpt-spec ific prm i ers:s ense:5c-ATGCCT GAACTCACCGCGAC-3c;ant-i sense:5c-CGGTCGGCATCTACT CTATT-3cFuncti onal i dentificati on o f transgenic strai ns Transgenic strains were inoculat ed on PDA media w it hout hygro mycin f or 6conti n uous generations.The yiel d ed spores were scraped int o ddH2O and coat ed on solid m edia containing diff erent concentrations of HygB;these medi a were incubat ed under dark f or12-24h,then appropriate vol u me of s em-i s olid me-dium cont aining s ame concentration of hygromycin was poured into thes e plates.Likew ise,non-transf or m ed strain 40359was used as negative control f or observing myceli a l grow t h.Results and AnalysisA mplifi cati on o f pCBH I,tCBHI and Hpt genesGenom ic DNA of T.reese i strai n YB40359was extract ed by using m i proved CTAB method(F i g.1).The y i e lded ge-nom ic DNAs were allw it h molecul a rweight hi g her t han20kb and hi g hly pure,whi c h could be used f or subsequent exper-i men.t PCR results of all the Hp t gene,t CBH I and pCBHI as-su med a singl e band,w ith Hp t gene of approxm i ately1000bp, t CBH I of approxm i ately600bp,pCBH I approxm i ately1600bp (F i g.2).M:Marker15000;lanes1-8:G enom i c DNA samples.Fi g.1E l e ctrophoresis patt ern of geno m ic DNA of T.reese i strain40359M:2000bp m arker;lanes1and2:Hyg;3-4:t C BHI;4-5:pC BH I.Fi g.2Elec tr ophores is patt ern of PCR a mp lific ati o n result sC l oning of pCBHI,t CBHI and Hpt genesThe a mplified results of pCBHI,t C BH I and Hp t genes were ligat ed into pMD18T-vect or and incubat ed at16e over-309ZHANG X i a o-xuan e t a l.Construc tion of Exogenous Ex pres s ion Vec t or o f Tri chode r m a reese inigh.t The li g at ed products were transfor med i n t o E.co li viaheat shock method and coated on LB pl a t e contai n ing Amp .Follow ing incubation for another 12-16h ,t wo white col o nies f or each gene were select ed f or PCR identificati o n .The re -sults showed that all six col o ni e s produced a band consistent w ith the size of t arget genes(Fig .3).The reco mbinants were identifi e d by using double diges -tion of Kpn I and S ph ,I the digestion produc t s were identifi e d w ith agarose gel electrophoresis(F i g .4).The double endonu -c l e ase di g esti o ns all produced t w o frag ments ,t he large one is the vector bond hi g her t han2.5kb ,the s mallone is the t arget genes :l a ne 1,about 600bp of t CBH I ;lane 2,about1000bp of Hp t ;lane 3,about 1600bp of pC BH I .This suggests that the t arget genes have been insert ed int o the c l o ning vec t or and successf ull y transf or med .The sequencing results (Shanghai Sangon Bioengineeri n g Co .,Lt d .)were blast ed and f ound an identity of 100%.M :2000bp marker ;1-2:Hyg ;3-4:t CB HI ;5-6:pC BH I .F i g .3 Bac t eriu m -based PCR a mplifi c ation on recombinantsM :2000bpmarker ;1:P l a s m id cont aini n g t CBH 1gene ;2:P l a s -m i d cont aini n g Hp t gene ;3:P l a sm i d cont ainin g pCBH 1gene .F i g .4 I dentifi c ation of reco mbined plas m id by double -di g esti o nI den tifi cati on expr essi on vector by usi ng PCR amp lifi ca -ti on and double endonucl ease digesti on As i n di c ated fro m Fig .5,PCR amplification results of all pCBHI ,t CBHI and Hp t genes accord w it h t hat us ed f or liga -tion ;l a ne 4is f or a mplificati o n of pC BH I p l u s Hp t gene ,w ith a size of approxm i atel y 2600bp ;lane 5is f or a mplificati o n of Hpt plus t CBH I gene ,w it h a size of approxm i at ely 1650bp .Endonucl e ase di g esti o n on recombinant s is using Eco R Ñpro -duced a s i z e of approxm i at ely 6000bp ,accordi n g w it h t he expect ed s iz e ;double endonulcease di g esti o n on recomb-inants is using S a l Ñand Bam H Ñto i d entif y Hp t gene ,whichproduced a larger band of approxm i at ely 4800bp(pU C19+pCBH I +t CBH I )and a s maller one of approxm i atel y 1000bp (Hp t gene);double endonul c ease digestion on reco mbinants is using S ph Ñand S a l Ñto identif y pCBHI ,which produced a lar -ger band of approxm i at ely 3300bp (pUC19+Hyg+t CBHI )and a s maller one of approxm i at ely 1600bp(pCBH I );double endonulcease di g esti o n on recombinant s is us i n g Bam H Ñand PstIto i d entif y t CBH I ,which produced a l a rger band of approx -m i at ely 4300bp(pUC19+Hyg+pCBHI )and a s maller one of approxm i at ely 600bp(t CBHI ).Not e :M:2000bp marker ;1:pC BHI ;2:t C BH I ;3:Hyg ;4:pC BH I and Hyg ;5:Hyg and t C BH I .Fi g.5 PCR a mplific ati o n on ex pres s ion vectorM1:15000bp mar ker ;M2:2000bp marker ;1:Sin g l e enzymedi g estion us i n g E co R Ñ;2:Dou b l e di g estion of S a l Ñand Bam H Ñt o i d entif y Hpt ;3:Doubl e di g estion of S ph Ñand S a l Ñto identif y pCB-H I ;4:Doubl e digesti o n of Bam H Ñand Pst Ñt o identif y t CBH I .Fi g.6 I dentifi c ation of ex press i o n by double -di g esti o nTransfor mati on of T.r eesei 40359and scr eeni ng of trans -for mantsRef erring t o PenttiL ¾M(1987),2-5L lof single endonu -clease di g est ed pUC19-Hyg was m ixed w it h the prot opl a st of T.reese i strain 40359(100s L l),using non -transfor med pro -t oplast as blank contro.l After coating on regenerationmedium containing HygB and incubat ed at 28e f or 3-4d ,six tran -f or m ants capabl e of grow ing on regeneration mediu m cont a-i ning 175mg/L of HygB .These six transf or m ants had st able and l a sting hygro mycin resi s tance aft er several conti n uous gen -erati o ns of culture .For the contro,l non -transf or med prot oplast of T.reesei strain 40359di d not grow onmediu m cont ai n ing 125mg/L of hygromycin ,indicati n g t hat there is no self resist ance m ut ation inw il d t ype strai n s .I dentificati on of tr ansgeni c strai ns PCR amp lifi cati on Geno m i c DNAs of hygro m ycin -resist ant strains all assumed a s i n gl e clear and orderly band(Fig .7).310Agricultural Sc i e nc e&Technology Vo.l 12,No .2,2011This indicat es that the DNA s amples have good qualit y and could be used for subsequent PCR amplifi c ati o n .Tab l e 1Gro w th sit uation of T.see se i strains on hy gro myc in Bp l a tesHy gro m y c in c oncentration M mg /LT.see se i Z40359T.see se i4035950++++75++++100+++125++-150++-175+-200--++repr es ent s we ll gro w th ;+r epres ents poor gro w th ;-repres ents no gro w th .F i g .7 E l e ctrophoresis patt ern of geno m ic DNA of genetic a llyengineered s tr a i nAs shown in Fig .8,PCR amplification on Hp t geneshowed t hat plas m id and six resist ant transf omants all produc -ed a target band of about 1000bp ,whil e non -transfor medstrain 40359di d no.t The results well demonstrat e that Hp t gene has transf or med and i n t egrated int o T.seese i strain 40359,designed as Z40359.M :2000bp marker ;1:Pos itive contro;l 2:Negati v e contro;l3-8:Transgenic s trains .Fi g.8 PCR det ection of pUC19-Hp t transgenic strainsResi stance and stab ility of tr ansgenic T.seesei strains tohygro myci n One of the T.seese i Z40359strains was cu-lt ured on solidm ediu m for s i x conti n uous generati o ns t o det ect its resistance t o hygro mycin(Fig .9).The results sho wed t hat T.seesei Z40359strains were res i s tant to175mg/L of hygro -m ycin ,and the resist ance l a st ed f or six generations .This confir m s that T.seeseiZ40359has a heredit ary stable resis-t ance t o hygromycin.1:Gene tically engineer ed T.ree se i s train Z40359on mediu m cont aining 100mg /L of hygro m yc in B ;2:P riginal strain 40359on med i u mcont aining 100mg/L hygro myc in B ;3:G eneti c all y engineered T.ree se i s train Z40359on mediu m cont aining 175mg/L of hygr omycin B ;4:O ri g i n a l s tr a i n 40359onm ediu m cont aining 175mg/L of hygro myc in B .Fi g.9 Toleranc e o fgeneticall y engineered T.reese i strain Z40359t o hygro myc i nD i s cussi o nThe re m arkable advantages of T.seese i inc lude its well capacit y of protein synt hesis and excretion and it s eukaryoti c excretory m echanis m ,as well as its protein modification per -for mance sm i ilar w ith ma mmal s yst e m ,such as hi g h -mannose type and N -gl y cosy l a tion .Thes e advant ages hugely pro mot e the geneti c modifi c ati o n of T.seese i strain ,and that construction of strong ext ernalgene expressi o n syst e m is t he precondition of itsmol e cul a r biol o gy st udy and genetic m i provement [4].The maj o r co mponent of T.reesei -excret ed extracellular prot ein i s cellul o se(CBH I ).So f ar ,t he cellulose cont ent of produced by T.reese i reached 40g/L ,several hundredstm i es over common bacteria [4].In the present st udy ,we e m -ployed PCR technique t o cl o ne the promot er and ter m i n at or of T.reese i strain YB40359,and f urt her est ablished strong ex -ternal gene expression geneti c transf or m ation syst em in T.reese i .Our results confir med the strong f unc tion of CBH I pro m oter and also lai d basis f or molecul a r st udy and geneti cm i provement of T.reese i .References[1]MU J Y(母敬郁),WANG Q(王峤),YAN G CZ (杨纯中),e ta l .Re -co mbinant Aspe r g ill u s n i ger gl u cose ox i d as e ex press ed in Tri -chode r m a reese i (瑞氏木霉表达黑曲霉葡萄糖氧化酶)[J].Chines eJ ournal of Biot echnol o gy (生物工程学报),2006,22(1):82-86.[2]JI A NG J(冮洁),DU LX (杜连祥),LU FP(路福平),e t a l .Themethod f or chro mos o me DNA pr eparation fro m reco mbinant Tri -chode r m a t ee se i (基因工程菌里氏木霉染色体DNA 的提取方法)[J].Bi o tec hn o l o gy(生物技术),2004,14(2):24-26.[3]PE NTT I L A M,NEVALAI NEN H ,R 'TT ;M ,e ta l .A v ersatile trans -f or mation syst e m f or t he cell u lol y tic fila ment ous fungus T ri chode r -rn a reese i [J].Gene ,1987,61(2):155-164.[4]WANG DH(汪大虹),WU ZH(吴志红).Construc ti o n of het er oge -n eous genes ex pression s yst e m of fila ment ous f ungus Tri chode r m ar e ese i (丝状真菌瑞氏木霉外源基因表达系统的构建)[J].Chines e J ournal of Bioc hem i s t ry and Molecul a r B i o logy (中国生物化学与分子生物学报),2003,19(6):736-742.Respons i bl e editor :DU AN Yong -boRespons i bl e proofreader :WU X i ao -yan311ZHANG X i a o -xuan e t a l .Construc tion of Exogenous Ex pres s ion Vec t or o f Tri chode r m a reese i里氏木霉外源表达载体的构建(摘要)张晓烜1,王傲雪2*(1.东北农业大学成栋学院,黑龙江哈尔滨150030;2.东北农业大学生命科学学院,黑龙江哈尔滨150030)[目的]构建里氏木霉外源表达载体。

重组蛋白真核表达系统构建流程1.从细菌中提取真核表达所需的重组蛋白的基因序列。

2. Extract the gene sequence of the recombinant protein required for eukaryotic expression from bacteria.3.将基因序列插入真核表达载体中。

4. Insert the gene sequence into the eukaryotic expression vector.5.确保基因插入的正确性，通过测序鉴定。

6. Ensure the accuracy of gene insertion through sequencing identification.7.将载体导入宿主真核细胞中。

8. Import the vector into the host eukaryotic cells.9.筛选出带有重组基因的真核细胞系。

10. Select eukaryotic cell lines with the recombinant gene.11.使用适当的诱导剂刺激真核细胞进行重组蛋白合成。

12. Stimulate eukaryotic cells with appropriate inducersto synthesize recombinant proteins.13.收集真核细胞培养上清液中的重组蛋白。

14. Collect recombinant proteins from the culture supernatant of eukaryotic cells.15.对纯化的蛋白进行鉴定和检测。

16. Identify and test the purified protein.17.通过离心等方法将蛋白精制并提纯。

18. Centrifuge and purify the protein by centrifugation and other methods.19.确定蛋白的浓度和纯度。

禽流感病毒HA-M2融合表位与EGFP蛋白在鸭胚成纤维细胞中的表达王善辉;王文秀;沈志强;池贤凤;高三阳【摘要】本研究选择禽流感病毒H9N2株的HA的两个保守T细胞表位基因和M2基因胞外保守序列(M2e),经人工合成并通过PCR扩增获得HA-M2融合表位基因,将其与含有增强型绿色荧光蛋白的载体pEGFP-N1连接,构建了重组真核表达质粒HA-M2-pEGFP-N1.采用脂质体法转染体外培养的鸭胚成纤维细胞(DEF)后,通过RT-PCR、直接荧光观察和间接免疫荧光检测,结果表明,成功构建了重组真核表达质粒HA-M2-pEGFP-N1,且以HA抗原表位和保守的M2e序列为基础构建的重组蛋白能够在鸭胚成纤维细胞中成功表达.本试验为研制新型的禽流感通用疫苗,进一步探索HA基因与靶细胞相互作用及AIV的感染机理等奠定了基础.%In this study, two T cell epitopes genes of hamagglutinin conserved domains of avian influenza virus H9N2 strain and Ml gene of extracellular domain were synthetized and joined as HA-M2 multiple-epitopes fusion gene of avian influenza virus by RT-PCR. The recombinant eukaryotic expression plasmid of HA-M2 fusion gene containing enhanced green fluorescent protein (EGFP) report gene was constructed, and transfected by lipofectin 10 prepared duck embryo fibroblasts (DEF). The fluorescence expression was directly detected with fluorescence microscope, and the expression of HA-M2 was tested by RT-PCR and indirect immunofluorescence assay (IFA) respectively. The results indicated the successful construction of the eukaryotic expression plasmid HA-M2-pEGFP-Nl, and showed that HA-M2 gene was expressed efficiently in transfected DEF. It provided a basis forthe study of new universal influenza vaccines and further research on interaction of HA gene and its target cells and as for infection mechanismof AIV.【期刊名称】《中国畜牧兽医》【年(卷),期】2012(039)011【总页数】4页(P27-30)【关键词】禽流感;融合表位基因;增强型绿色荧光蛋白;鸭胚成纤维细胞【作者】王善辉;王文秀;沈志强;池贤凤;高三阳【作者单位】山东省滨州畜牧兽医研究院,山东滨州 256600;山东省滨州畜牧兽医研究院,山东滨州 256600;山东省滨州畜牧兽医研究院,山东滨州 256600;山东绿都生物科技有限公司,山东滨州 256600;山东省滨州畜牧兽医研究院,山东滨州256600【正文语种】中文【中图分类】S858.3禽流感是由A型流感病毒引起的禽类急性呼吸道传染病。

·１４２０·ｐｃＤＮＡ３．０－ＣＢＰ真核表达质粒的构建及对肺腺癌ＳＰＣ—Ａ１细胞的影响周栋魏万胜苏云峰王志强王成苟云久【摘要】目的观察外源性ＣＢＰ基因稳定转染对人肺腺癌ＳＰＣ－Ａ１细胞体外牛长的影响。

方法构建ＣＢＰ真核表达质粒ｐｃＤＮＡ３．０．ＣＢＰ，应用ｐｃＤＮＡ３．０－ＣＢＰ和窄载体质粒ｐｃＤＮＡ３．Ｏ（一），脂质体转染法转染体外培养的人肺腺癌细胞株ＳＰＣ—Ａ１，Ｇ４１８（８００ｍｓ／Ｌ）筛选出抗性克隆。

Ｗｅｓｔｅｒｎｂｌｏｔ检测转染前后ＣＢＰ蛋白水平变化，噻唑蓝（Ｍ１Ｔｒ）比色法分析细胞生长抑制作用，Ｔｒａｎｓｗｅｌｌ体外侵袭实验和Ｗｏｕｎｄ．ｈｅａｌｉｎｇ实验对细胞进行侵袭和迁移能力研究。

结果转染ＣＢＰ基因的细胞株有目的基因整合和相应蛋白高表达。

ＭＴＹ检测ｐｃＤＮＡ３．０－ＣＢＰ转染组活细胞吸光度（Ｏ．２７８７±０．０７８６）低于未转染组（０．５０８９±０．１３０１）和ｐｅＤＮＡ３．Ｏ（一）空载体转染组（０．４８０４±０．１５４７）。

ｐｃＤＮＡ３．０－ＣＢＰ转染组与两对照组吸光度的差异有统计学意义（Ｐ＜０．０１）。

细胞侵袭实验表明ｐｃＤＮＡ３．０．ＣＢＰ转染组穿透滤膜数（３．５２±０．７７）明显少于未转染组（８．１７±０．８６）和空载体转染组（８．２２±１．３０），转染组与两对照组侵袭力的差异有统计学意义（Ｐ＜０．０１）。

细胞迁移实验转染组细胞迁移数（７１．３０±７．６８）明显少于未转染组（１１９．４０±８．３８）和空载体转染组（１１１．４１±１２．ｓ６），转染组与两对照组迁移力的差异有统计学意义（Ｐ＜０．０１）。

结论外源性ＣＢＰ基因稳定转染可抑制人肺腺癌ＳＰＣ．Ａ１细胞的恶性表型，町能通过对Ｓｒｃ家族激酶（ＳＦＫｓ）的负反馈调节抑制肿瘤细胞的增殖、侵袭。

【关键词】肺腺癌；基因转染；侵袭ＣｏｎｓｔｒｕｃｔｉｏｎｏｆｐｃＤＮＡ３．０．ＣＢＰｅｕｋａｒｙｏｔｉｃｅｘｐｒｅｓｓｉｏｎｐｌａｓｍｉｄａｎｄｉｔｓｅｆｆｅｃｔｓｏｎｌｕｎｇａｄｅｎｃａｒｃｉｎｏ－ｎｌａｅｅｌｌｌｉｎｅＳＰＣ．ＡｌｚＪＦ，Ｄ￡，Ｄｏｎｇ，ＷＥ＇／Ｗａｎ．ｓｈｅｎｇ，ＳＵＹｕｎ一膨，ｌｇ，ｅｔａ１．ＤｅｐａｒｔｍｅｎｔｏｆＣａｒｄｉｏｔｈｏｒａｃｉｃＳｕｒｇｅｒｙ．ＳｅｃｏｎｄＨｏｓｐｉｔａｌｏ厂ＬａｎｚｈｏｕＵｎｉｖｅｒｓｉｔｙ。

动物医学进展,2006,27(6):72274Progress in Veterinary Medicine传染性法氏囊病病毒V P2基因真核表达载体的构建及初步表达3鲁宏伟1,闫　强1,陈明勇13,王　宾2(1.中国农业大学动物医学院,北京100094;2中国农业大学农业生物技术国家重点实验室,北京100094)中图分类号:S852.659.4;Q289文献标识码:A文章编号:100725038(2006)0620072203摘　要:根据GenBank已登录的传染性法氏囊病病毒V P2基因序列,设计1对特异引物,应用反转录2聚合酶链反应技术从标准毒株B87中扩增了V P2基因,将其克隆到p roVA X载体上,构建了p roVA X2V P2真核表达载体,在脂质体介导下转染Hela细胞,用R T2PCR方法从转录水平证实V P2在Hela细胞中有特异性表达。

关键词:传染性法氏囊病病毒;V P2基因;真核表达传染性法氏囊病(Infectious bursal disease, IBD)是由传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)引起鸡和火鸡的一种急性、热性、高度接触性传染病。

现已确认IBDV的主要病毒结构蛋白有4种,分别为V P1、V P2、V P3和V P4[1],其中,V P2是IBDV的主要结构蛋白,是主要的宿主保护性抗原,含有能诱导中和抗体的抗原决定簇,其诱导机体产生的中和抗体能被动地保护宿主免受IBDV的感染[2]。

本试验通过R T2PCR方法扩增了IBDV B87株的V P2基因,进行了克隆和序列分析,构建了IBDV V P2基因的真核表达载体并在体外获得了初步表达,从而为下一步研制IBDV 核酸疫苗打下了基础。

1　材料与方法1.1　材料1.1.1　IBDV毒株、载体、细胞和菌种　IBDV B87毒株购自北京中海动物保健科技公司;p roVAX载体、Hela细胞和J M109菌种均由中国农业大学农业生物技术国家重点实验室构建和保存。

论 著（基础研究）重组铜绿假单胞菌外毒素Ａ和ｐｃｒＶ基因质粒的构建及真核表达姜明子，冯旰珠 ［摘要］　目的　外毒素Ａ（ｔｏｘＡ基因编码）是铜绿假单胞菌毒力最强的因子之一，ＰｃｒＶ（ｐｃｒＶ基因编码）是铜绿假单胞菌Ⅲ型分泌系统的关键调控因子之一。

文中旨在构建重组ｔｏｘＡ和ｐｃｒＶ基因的铜绿假单胞菌核酸疫苗，并在ＨＥＫ－２９３细胞中表达目的蛋白。

　方法　ＰＣＲ法从铜绿假单胞菌基因组基因中扩增出ｔｏｘＡ和ｐｃｒＶ基因，点突变法对ｔｏｘＡ基因进行减毒优化，随后将突变的ｔｏｘＡｍ基因和ｐｃｒＶ基因分别插入ｐＩＲＥＳ真核表达质粒的２个多克隆位点，构建真核双表达重组质粒ｐＩＲＥＳ－ｔｏｘＡｍ－ｐｃｒＶ。

脂质体法将ｐＩＲＥＳ－ｔｏｘＡｍ－ｐｃｒＶ瞬时转染入ＨＥＫ－２９３细胞，通过Ｗｅｓｔｅｒｎｂｌｏｔ检测ｔｏｘＡｍ及ｐｃｒＶ在真核细胞中的表达。

　结果　构建的真核表达质粒ｐＩＲＥＳ－ｔｏｘＡｍ－ｐｃｒＶ，经脂质体转染ＨＥＫ－２９３细胞后，在其细胞内检测到目的蛋白的表达。

　结论　成功构建ｐＩＲＥＳ－ｔｏｘＡｍ－ｐｃｒＶ表达载体并在转染的真核细胞中得以有效的表达，为研究铜绿假单胞菌预防性疫苗奠定了实验基础。

［关键词］　铜绿假单胞菌；外毒素Ａ；核酸疫苗；ｐｃｒＶ基因 ［中图分类号］　Ｒ５６３ ［文献标志码］　Ａ ［文章编号］　１００８－８１９９（２０１４）０７－０６９４－０４基金项目：江苏省卫生厅面上项目（Ｈ２００９１１）作者单位：２１００１１南京，南京医科大学第二附属医院呼吸内科［姜明子（医学硕士研究生）、冯旰珠］通讯作者：冯旰珠，Ｅ－ｍａｉｌ：ｆｅｎｇｇｚ＠ｎｊｍｕ．ｅｄｕ．ｃｎＣｏｎｓｔｒｕｃｔｉｏｎａｎｄｅｕｋａｒｙｏｔｉｃｅｘｐｒｅｓｓｉｏｎｏｆａｒｅｃｏｍｂｉｎａｎｔｐｌａｓｍｉｄｅｎｃｏｄｉｎｇＰｓｅｕｄｏｍｏｎａｓａｅｒｕｇｉｎｏｓａｔｏｘＡａｎｄｔｙｐｅⅢｓｅｃｒｅｔｉｏｎｓｙｓｔｅｍｐｃｒＶＪＩＡＮＧＭｉｎｇ－ｚｉ，ＦＥＮＧＧａｎ－ｚｈｕ（ＤｅｐａｒｔｍｅｎｔｏｆＲｅｓｐｉｒａｔｏｒｙ，ｔｈｅＳｅｃｏｎｄＡｆｆｉｌｉａｔｅｄＨｏｓｐｉｔａｌｏｆＮａｎｊｉｎｇＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｎａｎｊｉｎｇ２１００１１，Ｊｉａｎｇｓｕ，Ｃｈｉｎａ） ［Ａｂｓｔｒａｃｔ］　Ｏｂｊｅｃｔｉｖｅ　ＥｘｏｔｏｘｉｎＡ（ｅｎｃｏｄｅｄｂｙｇｅｎｅｔｏｘＡ），ｏｎｅｏｆｔｈｅｍｏｓｔｔｏｘｉｃｐｒｏｔｅｉｎｓｅｃｒｅｔｅｄｂｙｐｓｅｕｄｏｍｏｎａｓａｅｒｕｇｉ－ｎｏｓａ（Ｐ．ａ．），ａｎｄＰｃｒＶ（ｅｎｃｏｄｅｄｂｙｇｅｎｅｐｃｒＶ），ｋｅｙｃｏｍｐｏｎｅｎｔｔｏｔｙｐｅⅢｓｅｃｒｅｔｉｏｎｓｙｓｔｅｍｏｆＰ．ａ．，ｂｏｔｈｍａｔｔｅｒｓｉｇｎｉｆｉｃａｎｔｌｙｔｏｔｈｅｖｉｒｕｌｅｎｃｅｏｆＰ．ａ．ＴｈｅａｒｔｉｃｌｅｗａｓｔｏｃｏｎｓｔｒｕｃｔａｎｏｖｅｌＤＮＡｖａｃｃｉｎｅｅｎｃｏｄｉｎｇａｍｕｔａｔｅｄｔｏｘＡｇｅｎｅａｎｄｔｈｅｐｃｒＶｇｅｎｅｏｆＰ．ａ．ａｎｄｉ－ｄｅｎｔｉｆｙｇｅｎｅｅｘｐｒｅｓｓｉｏｎｓｉｎｅｕｋａｒｙｏｔｉｃｃｅｌｌｓ．　Ｍｅｔｈｏｄｓ　ＴｈｅｇｅｎｅｓｏｆｔｏｘＡａｎｄｐｃｒＶｗｅｒｅａｍｐｌｉｆｉｅｄｂｙＰＣＲ，ａｎｄｔｈｅｔｏｘＡｇｅｎｅｗａｓｍｕｔａｔｅｄｔｏｒｅｄｕｃｅｔｈｅｔｏｘｉｃｉｔｙｏｆＥｘｏｔｏｘｉｎＡ．ＴｈｅｎｇｅｎｅｆｒａｇｍｅｎｔｓｔｏｘＡｍａｎｄｐｃｒＶｗｅｒｅｉｎｓｅｒｔｅｄｉｎｔｏｅｕｋａｒｙｏｔｉｃｅｘｐｒｅｓｓｉｏｎｐｌａｓｍｉｄｐＩＲＥＳｓｉｍｕｌｔａｎｅｏｕｓｌｙｔｏｃｏｎｓｔｒｕｃｔａｒｅｃｏｍｂｉｎａｎｔＤＮＡｖａｃｃｉｎｅｐＩＲＥＳ－ｔｏｘＡｍ－ｐｃｒＶ．ＴｈｅｎｏｖｅｌｐｌａｓｍｉｄｗａｓｔｒａｎｓｆｅｃｔｅｄｉｎｔｏＨＥＫ－２９３ｃｅｌｌｓｂｙｌｉｐｏｆｅｃｔａｍｉｎｅ２０００．ＴｈｅｅｘｐｒｅｓｓｉｏｎｓｏｆｔｏｘＡｍａｎｄｐｃｒＶｗｅｒｅｄｅｔｅｃｔｅｄｂｙＷｅｓｔｅｒｎｂｌｏｔ．　Ｒｅｓｕｌｔｓ　Ｇｅｌｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓｄｅｍｏｎ－ｓｔｒａｔｅｄｔｈｅｔａｒｇｅｔｇｅｎｅｆｒａｇｍｅｎｔｓｅｎｃｏｄｉｎｇＥｘｏｔｏｘｉｎＡａｎｄＰｃｒＶ．ＷｅｓｔｅｒｎｂｌｏｔｅｘｈｉｂｉｔｅｄｐｒｏｔｅｉｎｓｅｎｃｏｄｅｄｔｏｘＡａｎｄｐｃｒＶｅｘｐｒｅｓｓｅｄｂｙＨＥＫ２９３ｃｅｌｌｓ．　Ｃｏｎｃｌｕｓｉｏｎ　ＴｈｅｒｅｃｏｍｂｉｎａｎｔｐｌａｓｍｉｄｐＩＲＥＳ－ｔｏｘＡｍ－ｐｃｒＶｗａｓｓｕｃｃｅｓｓｆｕｌｌｙｃｏｎｓｔｒｕｃｔｅｄ．Ｗｅｓｔｅｒｎｂｌｏｔａｎａｌｙｓｉｓｉｎｄｉ－ｃａｔｅｄｔｈｅｅｘｐｒｅｓｓｉｏｎｓｏｆｔｏｘＡｍａｎｄｐｃｒＶｉｎＨＥＫ－２９３ｃｅｌｌｓ．ＩｔｍａｙｂｅｕｓｅｄａｓａｐｏｔｅｎｔｉａｌｃａｎｄｉｄａｔｅｏｆｐｒｅｖｅｎｔｉｖｅｖａｃｃｉｎｅｏｆＰｓｅｕｄｏ－ｍｏｎａｓａｅｒｕｇｉｎｏｓａ． ［Ｋｅｙｗｏｒｄｓ］　Ｐｓｅｕｄｏｍｏｎａｓａｅｒｕｇｉｎｏｓａ；ＥｘｏｔｏｘｉｎＡ；ＤＮＡｖａｃｃｉｎｅ；ＰｃｒＶ０　引 言 铜绿假单胞菌（ｐｓｅｕｄｏｍｏｎａｓａｅｒｕｇｉｎｏｓａ，ＰＡ）是常见的条件致病菌，在各种医院感染中居于前位，也是慢性阻塞性肺疾病及气管扩张症反复感染者的常见的致病菌。

LXR α-shRNA 真核表达质粒的构建及抑制LXR α表达的有效序列筛选张琴彭俊1沈薇（重庆医科大学附属第二医院消化内科，重庆400010）〔摘要〕目的构建和筛选对肝X 受体α（LXR α）mRNA 和蛋白有抑制作用的LXR α质粒。

方法从NCBI 网站获得人的LXR αcDNA 序列，根据RNA 干扰设计原则，设计三条理论上最佳的siRNA 序列，相应的双链DNA 插入pGenesil-1.1载体中，经酶切和测序鉴定符合设计要求后，用转染试剂PolyJetTM 将三条质粒转染至HepG2.2.15细胞中并观察转染效果。

然后用RT-PCR 和Western 印迹方法分析各组LXR α的表达，筛选出干扰效率最佳的质粒。

结果三个质粒经测序分析与设计的序列完全一致，酶切凝胶电泳证实质粒构建成功。

三个质粒均能抑制LXR αmRNA 和蛋白的表达，与shRNA-LXR α2和shRNA-LXR α3质粒比较shRNA-LXR α1质粒表达更低。

结论成功构建了LXR αRNAi 表达载体，且三条质粒均能高效抑制转染细胞中LXR α的表达，其中第一条质粒shRNA-LXR α1干扰效率最好，从而为脂肪肝的治疗提供了新的方法和材料。

〔关键词〕肝X 受体α；RNA 干扰；质粒；构建〔中图分类号〕R394.3〔文献标识码〕A〔文章编号〕1005-9202（2012）08-1632-04；doi ：10.3969/j.issn.1005-9202.2012.08.038Construction of eukaryotic expression plasmid of LXR αand screening of effective sequences to inhibit LXR αexpressionZHANG Qin ，PENG Jun ，SHEN Wei.Department of Gastroenterology ，the Second Affiliated Hospital ，Chongqing Medical University ，Chongqing 400010，China 【Abstract 】Objective To construct and screen the plasmid of LXR αgene that can inhibit LXR αmRNA and protein expression.Methods Human LXR αcDNA sequence was obtained from NCBI website.Three small interfering RNA sequences were selected through online design software according to RNAi design principles.The corresponding double-stranded DNA was connected with pGenesil-1.1plas-mid vector ，namely shRNA-LXR α1，shRNA-LXR α2and shRNA-LXR α3.Digestion and DNA sequencing confirmed the cDNA sequence andthe orientation of the insert.Three plasmids were transfected into HepG2.2.15cells with PolyJetTM -reagent respectively to assess the trans-fection efficiency under fluorescence microscope and to determine the expression of LXR αgene ，with RT-PCR and Western blot.Results It was designed successfully of three plasmids demonstrated by digestion and DNA sequencing analysis.Through PolyJetTM reagent ，shRNA-LXR αplasmid was transfected into HepG2.2.15cells successfully.Target mRNA and protein was demonstrated by RT-PCR and Westernblot ，which displayed that three plasmids could effectively inhibit the expression of LXR αand the first shRNA-LXR αhad the most effect.Conclusions Eukaryotic expression recombinant shRNA-LXR αcould be constructed successfully.All of the plasmids could inhibit LXR αmRNA and protein expression ，especially the first one.It will be helpful for the therapy of fatty liver disease.【Key words 】LXR αgene ；RNA interference ；Plasmid ；Construction基金项目：重庆市自然科学基金计划项目（CSTC ，2008BB5404）1凉山州第一人民医院通讯作者：沈薇（1956-），女，教授，博士生导师，主要从事非酒精性脂肪肝性肝病研究。

中国热带医学2010年第10卷第9期CHINA TROPICAL MEDICINE Vol.10No.9September2010［论著］多嘧啶束结合蛋白相关剪切因子（Polypyrimidine tract-binding protein-associated splicing factor，PSF）是Patton等1993年首先鉴定出来的，由于PSF能与多嘧啶束结合蛋白（Polypyrimidine tract-binding protein，PTB）形成复合体，并参与前mRNA剪切，故被命名为PSF[1]。

PSF是一种能与DNA和RNA结合的多功能蛋白，参与多种核内事件，如参与前mRNA 的剪切、DNA的解螺旋、参与双链断裂DNA修复、稳定配对DNA末端、参与转录调节等过程[2]。

最近研究发现，PSF是新型核结构域“Paraspeckles”的组成成分[3]。

PSF蛋白在核内功能的多样性已吸引学者们对其生物功能进行研究。

为此，我们构建PSF基因真核表达载体并在小鼠肝癌细胞（Hepa1-6）内表达，观察PSF胞内定位及内毒素主要成分脂多糖（Lipopolysaccharide，LPS）对其定位的影响，为进一步研究PSF 在基因表达调控中作用及其生物功能提供重要的工具。

1材料和方法1.1材料Bio-Peofll凝胶图像分析仪（Vilber Lourmat）；PCR 仪（Eppendorf）；IM T-2倒置显微镜（Olympus）；荧光显微镜（Leica）；RNeasy M ini Kit总RNA制备试剂盒、PolyFect脂质体转染试剂（QIAGEN）；ReverTra Ace-α-TM kit、限制性核酸内切酶、Ligation High DNA连接酶、Kod plus DNA聚合酶（TOYOBO）；LPS、DAPI细胞核染料（Sigma）；鼠源抗血凝素（HA）标签抗体（Cell Signaling）；Alexa-Flour488标记羊抗鼠IgG抗体（M olecular Probes）；引物由上海英俊公司合成。

人源JAZF1基因真核表达载体的构建及其对胚胎癌细胞凋亡的影响陈倩，李超，杨诚诚，母彬，龚欢，黄超*（四川农业大学动物医学院，实验动物疾病模型研究室，成都611130）摘要:【目的】构建人源转录因子锌指并列基因1（JAZF1）真核表达载体，并探究过表达JAZF1对胚胎癌细胞存活的影响。

【方法】以胚胎癌细胞NCCIT 的cDNA 为模板扩增JAZF1基因片段，并将其与pRK5-myc 载体连接，构建重组质粒，转染至NCCIT 细胞；通过实时荧光定量PCR 和DAPI 染色法分别检测过表达JAZF1对NCCIT 细胞中炎性因子IL1-β、IL8、TGF-β转录水平的影响及对NCCIT 细胞存活的影响。

【结果】过表达JAZF1基因后，促炎因子IL1-β（P <0.05）、IL8（P <0.01）转录水平明显下降，而抑炎因子TGF-β的mRNA 表达水平无明显变化（P >0.05），同时NCCIT 细胞出现更明显的细胞凋亡（P <0.01）。

【结论】JAZF1可能通过抑制促炎因子IL1-β和IL8的表达来抑制炎症发展，进而抑制畸胎瘤进展。

关键词:锌指并列基因1；胚胎癌；炎性因子；细胞凋亡中图分类号:Q78文献标志码:Ａ文章编号:1000-2650（2021）01-0108-06Construction of Eukaryotic Expression Vector of Human Juxtaposedwith Another Zincfinger Gene 1and Its Influence on EmbryonalCarcinoma Cell ApoptosisCHEN Qian ，LI Chao ，YANG Chengcheng ，MU Bin ，GONG Huan ，HUANG Chao *（College of Veterinary Medicine ，Sichuan Agricultural University ，Chengdu 611130，China ）Abstract:【Objective 】The eukaryotic expression vector of human juxtaposed with another zincfinger gene1(JAZF1)was constructedto explore the effect of JAZF1on embryonal carcinoma cell survival.【Method 】The JAZF1gene fragment was amplified using the cDNA of embryonic cancer cell NCCIT as a template ，and wascloned into pRK5-myc vector to construct a recombinant plasmid.The recombinant plasmid wastransfected into NCCIT cells ，while the cell viability and the expression of transcription factorIL1-β，IL8and TGF-βwere following evaluated by DAPI staining and quantitative real-time PCR ，respectively.【Result 】Transfected with JAZF1，the transcriptional levels of proinflammatory factor IL1-βand IL8in NCCIT cells showed a significant suppression ，while enhanced expression of antiinflammatory factor T GF-茁was observed.Meanwhile ，we found increased cellularapoptosis in JAZF1-transfected NCCITcells.【Conclusion 】JAZF1may suppressinflammation by inhibiting the expression of IL1-βand IL8，and then inhibit the progressionofembryonic cancer.Keywords:human juxtaposed with another zinc finger gene 1(JZAF1)；embryonal carcinoma ；inflammat-oryfactor ；apoptosis第39卷第1期2021年2月收稿日期:2020-07-25基金项目:四川农业大学学科建设双支计划（2018-2019）。

醛缩酶B基因真核表达质粒的构建与鉴定发表时间：2011-06-09T16:16:17.700Z 来源：《中外健康文摘》2011年第12期供稿作者：赵洪波巴亚斯任建林[导读] 这一载体的成功构建为下一步研究ALDOB在肝细胞癌发生、发展中所起的作用提供了前提基础。

赵洪波巴亚斯任建林 (厦门大学附属中山医院消化内科 361004)【中图分类号】R446.1 【文献标识码】A 【文章编号】1672-5085 (2011)12-0053-03【摘要】目的构建含ALDOB基因的重组PCMV5质粒并进行鉴定。

方法以人HEPG2细胞RNA为模板，用RT-PCR方法扩增出ALDOB基因。

将PCR产物克隆进真核载体PCMV5内，构建含ALDOB基因的重组真核表达质粒。

重组质粒转染293T细胞，用免疫荧光和Western blot等方法检测ALDOB在293T细胞中的表达。

结果核酸序列分析的结果表明，克隆的ALDOB基因与GenBank中已登记ALDOB基因序列100%同源。

免疫荧光结果显示，ALDOB蛋白在细胞质表达；Western blot结果显示，在约40KD位置有目的条带，与预期的重组ALDOB蛋白大小一致。

结论成功构建了含ALDOB基因的真核表达质粒。

【关键词】 ALDOB 质粒真核表达Construction and verification of eukaryotic expression plasmid carrying human ALDOB gene【Abstract】 Objective To construct and certificate an eukaryotic expression plasmid carrying human ALDOB gene. Methods ALDOB gene was amplified by RT-PCR with the total RNA of human HepG2 cell as template.The PCR fragments were cloned into pCMV5 vector to construct recombinant eukaryotic expression plasmids. 293T cells were transfected with the recombinant plasmids. Immunofluorescence staining and Western blot were used to certificate the expressions of ALDOB-myc in 293T cells. Results The sequence of ALDOB was 100% homology with human ALDOB gene previously registered in GenBank. The result of Immunofluorescence staining manifest recombinate protein of ALDOB-myc protein expression in 293T cells; An interesting band about 40kD was visible in the resuult of Western blot, which was consistent with expected with expected size of recombinate protein of ALDOB-myc expressed in 293T cells. Conclusion pCMV5-ALDOB-myc has been constructrd successfully.【Key words】 ALDOB Plasmid Eukaryotic expression醛缩酶（ALD）与机体能量代谢密切相关。

论著文章编号:1007-8738(2004)05-0552-04抗CD3scFv 基因的真核表达及其生物学活性鉴定杨章民,胡劲松,来宝长,王一理*,司履生(西安交通大学生命科学与技术学院癌症研究所,陕西西安710061)收稿日期:2004-01-14; 修回日期:2004-03-25基金项目:国家卫生部科学研究基金资助项目(No.98 1 232);陕西省自然科学基金资助项目(No.2002C118)作者简介:杨章民(1966-),男,陕西丹凤人,副教授,博士.Tel:(029)82655429;Email:yz hangmin@yahoo.c om* Corres ponding author,Tel:(029)82655499Eukaryotic expression of anti CD3single chain Fv antibody gene and the charac terization of its bioactivitiesY ANG Zhang min,HU Jin song ,LAI Bao chang,WANG Yi li *,SI L shengInstitute for Cancer Research,School of Life Science &T echnology,Xi an Jiaotong University,Xi an 710061,ChinaAbstractAIM:To express anti CD 3scF v in H ela cells and investigate its biological activity.METHODS:DN A fragment encoding anti CD 3scFv was ins erted into eukaryotic express ion vector pD is play .The recom binant expression vector was sequenced and then transfected into H ela cells by electroporation m ethod.The expression of anti CD 3s cFv was identified by in situ hybridiza tion.In vitro T lym phocyte activation was then detected by 3H TdR incoporation method.Anti CD 3scFv gene transfected Hela cells were co cultured w ith and T cells cytotoxicity was m ea sured by MTT colori m etry.RESULTS:Anti CD 3s cFv gene was correctly inserted into pDisplay and expressed in Hela cells.The secreted anti CD 3scFv was able to activate T lymphocy tes in the presence of anti CD 28m Ab.Cytotoxicity could be observed when anti CD 3scFv gene transfected Hela cell s were m ixed and co cultured with T lym phocytes .CONC LUSION:Anti CD 3scFv expressed by Hela cells can activate T lymphocytes.Keywords:anti CD 3single chain Fv antibody;eukaryotic expression;biological activity摘要目的:真核表达抗CD3单链抗体(scFv ),并研究其生物学活性。

㊀山东农业科学㊀２０２４ꎬ５６(２):１４４~１５０ＳｈａｎｄｏｎｇＡｇｒｉｃｕｌｔｕｒａｌＳｃｉｅｎｃｅｓ㊀ＤＯＩ:１０.１４０８３/ｊ.ｉｓｓｎ.１００１－４９４２.２０２４.０２.０２０收稿日期:２０２３－０４－１４基金项目:国家自然科学基金项目(３２３７３０９４ꎬ３２１０２７１０)ꎻ山东省自然科学基金项目(ＺＲ２０２１ＺＤ０８ꎬＺＲ２０２３ＭＣ０７６ꎬＺＲ２０２１ＭＣ１３９)ꎻ国家兽用生物制品工程技术研究中心开放课题(ＧＴＫＦ(２３)００９)ꎻ国家重点研发计划项目(２０２１ＹＦＤ１８００３００)作者简介:苗勤(１９９８ )ꎬ女ꎬ硕士研究生ꎬ研究方向为临床兽医学ꎮＥ－ｍａｉｌ:ｍｉａｏＭＱＭ＠１６３.ｃｏｍ通信作者:王林(１９８０ )ꎬ男ꎬ教授ꎬ主要从事临床兽医学研究ꎮＥ－ｍａｉｌ:ｗａｎｇｌｉｎ２０１３＠ｓｄａｕ.ｅｄｕ.ｃｎ杜以军(１９８１ )ꎬ男ꎬ研究员ꎬ主要从事动物抗病毒感染与免疫学方面研究ꎮＥ－ｍａｉｌ:ｄｕｙｉｊｕｎ０９１６＠１６３.ｃｏｍＦＭＤＶ３Ｄ基因真核表达质粒的构建、表达及对Ⅰ型ＩＦＮ信号通路的作用苗勤１ꎬ２ꎬ３ꎬ吴香菊２ꎬ３ꎬ齐静２ꎬ３ꎬ丛晓燕２ꎬ３ꎬ李均同２ꎬ３ꎬ王林１ꎬ杜以军２ꎬ３(１.山东农业大学动物科技学院ꎬ山东泰安㊀２７１０１８ꎻ２.山东省农业科学院畜牧兽医研究所/山东省畜禽疫病防治与繁育重点实验室ꎬ山东济南㊀２５０１００ꎻ３.农业农村部畜禽生物组学重点实验室ꎬ山东济南㊀２５０１００)㊀㊀摘要:本研究旨在构建口蹄疫病毒(Ｆｏｏｔ－ａｎｄ－ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓꎬＦＭＤＶ)３Ｄ基因真核表达质粒ꎬ并探究其对Ⅰ型干扰素(ｉｎｔｅｒｆｅｒｏｎꎬＩＦＮ)信号通路的作用ꎮ根据ＧｅｎＢａｎｋ序列合成３Ｄ基因并将其插入真核表达载体ｐＸＪ４１构建真核表达质粒ｐＸＪ４１－Ｍｙｃ－３Ｄꎬ经ＰＣＲ㊁双酶切及测序鉴定正确后分别转染ＨＥＫ－２９３Ｔ细胞和ＰＫ－１５细胞ꎬＷｅｓｔｅｒｎｂｌｏｔｔｉｎｇ及间接免疫荧光试验(ｉｎｄｉｒｅｃｔｉｍｍｕｎｏｆｌｕｏｒｅｓｃｅｎｃｅａｓｓａｙꎬＩＦＡ)检测３Ｄ蛋白在细胞内的表达及定位ꎮ通过双荧光素酶报告基因(Ｌｕｃｉｆｅｒａｓｅ)㊁Ｒｅａｌ－ｔｉｍｅＰＣＲ㊁ＴＣＩＤ５０等试验检测ＨＥＫ－２９３Ｔ细胞中过表达３Ｄ蛋白对水疱性口炎病毒(ＶｅｒｓｉｃｕｌａｒｓｔｏｍａｔｉｔｉｓｖｉｒｕｓꎬＶＳＶ)诱导的Ⅰ型ＩＦＮ信号通路的影响ꎮ结果显示ꎬ真核表达质粒ｐＸＪ４１－Ｍｙｃ－３Ｄ构建成功ꎻ３Ｄ蛋白在ＨＥＫ－２９３Ｔ细胞中表达ꎬ大小约为５５ｋＤａꎬ主要定位在细胞核中ꎻ３Ｄ蛋白抑制了ＶＳＶ诱导的ＩＦＮ－β启动子活性和ＩＦＮ－βｍＲＮＡ水平ꎬ促进了ＶＳＶ的复制ꎮ本研究为深入探究３Ｄ蛋白抑制Ⅰ型ＩＦＮ信号通路的作用机制奠定了基础ꎮ关键词:口蹄疫病毒ꎻ３Ｄ蛋白ꎻ真核表达ꎻⅠ型ＩＦＮ信号通路中图分类号:Ｓ８５２.６５㊀㊀文献标识号:Ａ㊀㊀文章编号:１００１－４９４２(２０２４)０２－０１４４－０７ＣｏｎｓｔｒｕｃｔｉｏｎａｎｄＥｘｐｒｅｓｓｉｏｎｏｆＥｕｋａｒｙｏｔｉｃＥｘｐｒｅｓｓｉｏｎＰｌａｓｍｉｄｏｆＦＭＤＶ３ＤＧｅｎｅａｎｄＩｔｓＥｆｆｅｃｔｏｎＴｙｐｅＩＩＦＮＳｉｇｎａｌｉｎｇＰａｔｈｗａｙＭｉａｏＱｉｎ１ꎬ２ꎬ３ꎬＷｕＸｉａｎｇｊｕ２ꎬ３ꎬＱｉＪｉｎｇ２ꎬ３ꎬＣｏｎｇＸｉａｏｙａｎ２ꎬ３ꎬＬｉＪｕｎｔｏｎｇ２ꎬ３ꎬＷａｎｇＬｉｎ１ꎬＤｕＹｉｊｕｎ２ꎬ３(１.ＣｏｌｌｅｇｅｏｆＶｅｔｅｒｉｎａｒｙＭｅｄｉｃｉｎｅꎬＳｈａｎｄｏｎｇＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｖｅｒｓｉｔｙꎬＴａｉａｎ２７１０１８ꎬＣｈｉｎａꎻ２.ＩｎｓｔｉｔｕｔｅｏｆＡｎｉｍａｌＳｃｉｅｎｃｅａｎｄＶｅｔｅｒｉｎａｒｙＭｅｄｉｃｉｎｅꎬＳｈａｎｄｏｎｇＡｃａｄｅｍｙｏｆＡｇｒｉｃｕｌｔｕｒａｌＳｃｉｅｎｃｅｓ/ＳｈａｎｄｏｎｇＫｅｙＬａｂｏｒａｔｏｒｙｏｆＡｎｉｍａｌＤｉｓｅａｓｅＣｏｎｔｒｏｌａｎｄＢｒｅｅｄｉｎｇꎬＪｉｎａｎ２５０１００ꎬＣｈｉｎａꎻ３.ＫｅｙＬａｂｏｒａｔｏｒｙｏｆＬｉｖｅｓｔｏｃｋａｎｄＰｏｕｌｔｒｙＭｕｌｔｉ￣ｏｍｉｃｓꎬＭｉｎｉｓｔｒｙｏｆＡｇｒｉｃｕｌｔｕｒｅａｎｄＲｕｒａｌＡｆｆａｉｒｓꎬＪｉｎａｎ２５０１００ꎬＣｈｉｎａ)Ａｂｓｔｒａｃｔ㊀Ｔｈｉｓｓｔｕｄｙａｉｍｅｄｔｏｃｏｎｓｔｒｕｃｔｔｈｅｅｕｋａｒｙｏｔｉｃｅｘｐｒｅｓｓｉｏｎｐｌａｓｍｉｄｏｆ３Ｄｇｅｎｅｏｆｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓ(ＦＭＤＶ)ａｎｄｅｘｐｌｏｒｅｉｔｓｅｆｆｅｃｔｓｏｎｔｙｐｅⅠｉｎｔｅｒｆｅｒｏｎ(ＩＦＮ)ｓｉｇｎａｌｉｎｇｐａｔｈｗａｙ.ＴｈｅｐｌａｓｍｉｄｐＸＪ４１￣Ｍｙｃ￣３Ｄｗａｓｃｏｎｓｔｒｕｃｔｅｄａｆｔｅｒ３ＤｇｅｎｅｗａｓｓｙｎｔｈｅｓｉｚｅｄａｃｃｏｒｄｉｎｇｔｏｔｈｅＧｅｎＢａｎｋｓｅｑｕｅｎｃｅａｎｄｉｎｓｅｒｔｅｄｉｎｔｏｔｈｅｅｕｋａｒｙｏｔｉｃｅｘｐｒｅｓｓｉｏｎｖｅｃｔｏｒｐＸＪ４１ꎬｗｈｉｃｈｗａｓｔｒａｎｓｆｅｃｔｅｄｔｏＨＥＫ￣２９３ＴａｎｄＰＫ￣１５ｃｅｌｌｓａｆｔｅｒｉｄｅｎｔｉ￣ｆｉｅｄｂｙＰＣＲꎬｄｏｕｂｌｅｄｉｇｅｓｔｉｏｎａｎｄｎｕｃｌｅｏｔｉｄｅｓｅｑｕｅｎｃｉｎｇ.Ｔｈｅｅｘｐｒｅｓｓｉｏｎａｎｄｃｅｌｌｕｌａｒｌｏｃａｌｉｚａｔｉｏｎｏｆ３Ｄｐｒｏ￣ｔｅｉｎｗｅｒｅｄｅｔｅｃｔｅｄｂｙＷｅｓｔｅｒｎｂｌｏｔｔｉｎｇａｎｄｉｎｄｉｒｅｃｔｉｍｍｕｎｏｆｌｕｏｒｅｓｃｅｎｃｅａｓｓａｙ(ＩＦＡ).Ｔｈｅｅｆｆｅｃｔｓｏｆ３Ｄｏｖｅｒ￣ｅｘｐｒｅｓｓｉｏｎｉｎＨＥＫ￣２９３ＴｃｅｌｌｓｏｎｔｙｐｅⅠＩＦＮｓｉｇｎａｌｉｎｇｐａｔｈｗａｙｉｎｄｕｃｅｄｂｙｖｅｒｓｉｃｕｌａｒｓｔｏｍａｔｉｔｉｓｖｉｒｕｓ(ＶＳＶ)ｗｅｒｅｆｕｒｔｈｅｒａｓｓａｙｅｄｂｙｄｕａｌｌｕｃｉｆｅｒａｓｅｒｅｐｏｒｔｅｒａｓｓａｙｓꎬＲｅａｌ￣ｔｉｍｅＰＣＲａｎｄＴＣＩＤ５０.ＴｈｅｒｅｓｕｌｔｓａｂｏｖｅｓｈｏｗｅｄｔｈａｔｔｈｅｅｕｋａｒｙｏｔｉｃｅｘｐｒｅｓｓｉｏｎｐｌａｓｍｉｄｐＸＪ４１￣Ｍｙｃ￣３Ｄｗａｓｃｏｎｓｔｒｕｃｔｅｄｓｕｃｃｅｓｓｆｕｌｌｙꎬｔｈｅ３ＤｐｒｏｔｅｉｎｃｏｕｌｄｂｅｅｘｐｒｅｓｓｅｄｉｎＨＥＫ￣２９３Ｔｃｅｌｌｓｗｉｔｈｔｈｅｓｉｚｅａｂｏｕｔ５５ｋＤａａｎｄｍａｉｎｌｙｌｏｃａｌｉｚｅｄｉｎｔｈｅｎｕｃｌｅｕｓꎬ３ＤｐｒｏｔｅｉｎｃｏｕｌｄｉｎｈｉｂｉｔｔｈｅｐｒｏｍｏｔｅｒａｃｔｉｖｉｔｙａｎｄｍＲＮＡｌｅｖｅｌｏｆＩＦＮ￣βｉｎｄｕｃｅｄｂｙＶＳＶａｎｄｐｒｏｍｏｔｅｒｅｐｌｉｃａｔｉｏｎｓｏｆｔｈｅｖｉｒｕｓ.Ｔｈｉｓｓｔｕｄｙｌａｉｄａｆｏｕｎｄａｔｉｏｎｆｏｒｆｕｒｔｈｅｒｅｘｐｌｏｒｉｎｇｔｈｅｍｅｃｈａｎｉｓｍｏｆ３ＤｐｒｏｔｅｉｎｉｎｈｉｂｉｔｉｎｇｔｙｐｅⅠＩＦＮｓｉｇｎａｌｉｎｇｐａｔｈｗａｙ.Ｋｅｙｗｏｒｄｓ㊀Ｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓꎻ３ＤｐｒｏｔｅｉｎꎻＥｕｋａｒｙｏｔｉｃｅｘｐｒｅｓｓｉｏｎｐｌａｓｍｉｄꎻＴｙｐｅⅠＩＦＮ㊀㊀口蹄疫(ｆｏｏｔ－ａｎｄ－ｍｏｕｔｈｄｉｓｅａｓｅꎬＦＭＤ)是由口蹄疫病毒(Ｆｏｏｔ－ａｎｄ－ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓꎬＦＭ￣ＤＶ)引起的一种急性㊁高度接触性人畜共患传染病ꎬ主要感染牛㊁羊㊁猪等偶蹄动物ꎬ给畜牧业造成了严重的经济损失ꎬ被世界卫生组织列为Ａ类动物疫病之首ꎬ我国也将其列为一类传染病[１]ꎮ口蹄疫病毒属小ＲＮＡ病毒科ꎬ是单股正链ＲＮＡ病毒ꎬ呈二十面体对称结构ꎮＦＭＤＶ基因组全长约８５００ｂｐꎬ仅有一个开放阅读框(ｏｐｅｎｒｅａｄｉｎｇｆｒａｍｅꎬＯＲＦ)ꎬ约６.５ｋｂꎬ编码一个大的多聚蛋白[２]ꎬ在Ｌ㊁２Ａ和３Ｃ的作用下裂解产生了病毒的４种结构蛋白[ＶＰ４(１Ａ)㊁ＶＰ２(１Ｂ)㊁ＶＰ３(１Ｃ)和ＶＰｌ(１Ｄ)]和８个非结构蛋白(Ｌ㊁２Ａ㊁２Ｂ㊁２Ｃ㊁３Ａ㊁３Ｂ㊁３Ｃ㊁３Ｄ)ꎮＦＭＤＶ有７种血清型ꎬ分别为Ａ型㊁Ｏ型㊁Ｃ型㊁亚洲Ⅰ型㊁ＳＡＴ１型㊁ＳＡＴ２型和ＳＡＴ３型ꎬ且各血清型之间无交叉保护[３]ꎮⅠ型ＩＦＮ是机体抵抗病毒天然免疫的核心ꎬＦＭＤＶ感染细胞后ꎬ病毒复制产生的ＲＮＡ被宿主细胞的模式识别受体所识别ꎬ激活下游信号通路ꎬ刺激产生Ⅰ型ＩＦＮ和促炎细胞因子ꎬ从而启动抗病毒反应ꎮ已有研究表明ꎬＦＭＤＶ多种蛋白ꎬ如前导蛋白Ｌｐｒｏ㊁蛋白水解酶３Ｃｐｒｏ㊁非结构蛋白２Ｂ㊁２Ｃ等ꎬ参与调节先天性免疫信号通路ꎬ拮抗宿主天然免疫ꎬ促进病毒增殖[４]ꎮＦＭＤＶ３Ｄ蛋白是病毒的ＲＮＡ聚合酶ꎬ介导ＦＭＤＶ基因组的合成ꎬ且核苷酸和氨基酸序列高度保守[５]ꎮ此外ꎬ３Ｄ蛋白具有Ｔ细胞表位ꎬ是一种潜在的免疫增强剂ꎬ还可以作为免疫佐剂使用[６]ꎮ但３Ｄ蛋白在天然免疫中的作用尚不明确ꎬ本研究旨在构建ＦＭＤＶ３Ｄ基因的真核表达质粒ꎬ并探索３Ｄ蛋白对Ⅰ型ＩＦＮ信号通路的影响ꎬ为ＦＭＤ的防控提供新的靶向分子ꎮ１㊀材料与方法１.１㊀试验材料１.１.１㊀细胞与质粒㊀ＨＥＫ－２９３Ｔ细胞㊁ＰＫ－１５细胞㊁ｐＸＪ４１真核表达载体㊁荧光素酶报告基因质粒ｐＩＦＮ－β－Ｌｕｃ和内参报告质粒ｐＲＬ－ＴＫ均由山东省畜禽疫病防治与繁育重点实验室保存ꎮ１.１.２㊀主要试剂㊀ＰｒｉｍｅＳＴＡＲ®ＨＳＤＮＡＰｏｌｙｍｅｒ￣ａｓｅ㊁ＤＬ５０００ＤＮＡＭａｒｋｅｒ㊁ＤＬ２０００ＤＮＡＭａｒｋｅｒꎬｐＭＤ１８－Ｔ载体购自ＴａＫａＲａ宝生物工程(大连)有限公司ꎻＬｉｐｏｆｅｃｔａｍｉｎｅ®３０００Ｒｅａｇｅｎｔ㊁ＡｌｅｘａＦｌｕ￣ｏｒ４８８－羊抗鼠ＩｇＧ(Ｈ＋Ｌ)购自Ｉｎｖｉｔｒｏｇｅｎ公司ꎻＷｅｓｔｅｒｎ一抗稀释液购自碧云天生物技术有限公司ꎻＮＣ膜购自ＢＩＯ－ＲＡＤ公司ꎻ辣根过氧化物(ＨＲＰ)标记的羊抗兔抗体购自武汉博士德生物工程有限公司ꎻＳｐａｒｋｊａｄｅＥＣＬｓｕｐｅｒ(极超敏化学发光试剂盒)购自山东思科捷生物技术有限公司ꎻ感受态细胞ＤＨ５α㊁双荧光素酶报告基因试剂盒㊁ＲＮＡ－ｅａｓｙＩｓｏｌａｔｉｏｎＲｅａｇｅｎｔ试剂盒均购自南京诺唯赞生物科技有限公司ꎻ限制性内切酶ＨｉｎｄⅢ㊁ＫｐｎⅠ及Ｔ４ＤＮＡＬｉｇａｓｅ购自ＴｈｅｒｍｏＦｉｓｈｅｒＳｃｉｅｎｔｉｆｉｃ公司ꎻ质粒小提试剂盒㊁通用型ＤＮＡ纯化回收试剂盒购自北京天根生物工程有限公司ꎻＤＭＥＭ细胞培养基㊁Ｏｐｔｉ－ＭＥＭ培养基㊁胰蛋白酶－ＥＤＴＡ均购自Ｇｉｂｃｏ公司ꎻ抗荧光衰减封闭剂购自Ｓｏｌａｒｂｉｏ公司ꎻ鼠源㊁兔源Ｍｙｃ抗体购自Ｓｉｇｍａ－Ａｌｄｒｉｃｈ公司ꎻβ－ａｃｔｉｎ抗体购自上海泊湾生物科技有限公司ꎮ１.２㊀试验设计与方法１.２.１㊀ＦＭＤＶ３Ｄ基因真核表达质粒的构建与鉴５４１㊀第２期㊀㊀㊀㊀苗勤ꎬ等:ＦＭＤＶ３Ｄ基因真核表达质粒的构建㊁表达及对Ⅰ型ＩＦＮ信号通路的作用定㊀根据ＧｅｎＢａｎｋ公布的ＦＭＤＶＯ/ＢＹ/ＣＨＡ/２０１０(ＧｅｎＢａｎｋａｃｃｅｓｓｉｏｎｎｏ.ＪＮ９９８０８５)３Ｄ基因序列ꎬ由北京擎科生物技术有限公司合成３Ｄ基因并连接到ｐＭＤ１８－Ｔ载体上ꎮ结合真核表达载体ｐＸＪ４１上的酶切位点序列ꎬ加入保护性碱基及Ｍｙｃ标签序列ꎬ利用分子生物学软件ＰｒｉｍｅｒＰｒｅｍｉｅｒ５.０设计了一对扩增３Ｄ基因的特异性引物:Ｍｙｃ－３Ｄ－Ｆｗｄ(ＨｉｎｄⅢ)和Ｍｙｃ－３Ｄ－Ｒｅｖ(ＫｐｎⅠ)ꎬ引物由北京擎科生物技术有限公司合成ꎬ序列见表１ꎮ㊀㊀表１㊀ＰＣＲ引物及序列引物名称引物序列(５ －３ )Ｍｙｃ－３Ｄ－Ｆｗｄ(ＨｉｎｄⅢ)ＧＣＧＡＡＧＣＴＴＣＣＡＣＣＡＴＧＧＡＧＣＡＧＡＡＡＣＴＣＡＴＣＴＣＴＧＡＡＧＡＧＧＡＴＣＴＧＧＧＡＴＴＧＡＴＡＧＴＴＧＡＣＭｙｃ－３Ｄ－Ｒｅｖ(ＫｐｎＩ)ＴＣＴＧＧＴＡＣＣＴＧＣＧＴＣＡＣＣＧＣＡＣＡＣＧＧＣ㊀㊀以ｐＭＤ１８－Ｔ－３Ｄ克隆质粒为模板进行ＰＣＲ扩增ꎬＰＣＲ反应体系(总体系为２５μＬ)如下:上㊁下游引物(１０ｐｍｏｌ/μＬ)各１μＬꎬ模板(２００ｎｇ/μＬ)０.５μＬꎬｄＮＴＰ２μＬꎬ５ˑＰｒｉｍｅＳＴＡＲｂｕｆｆｅｒ５μＬꎬＰｒｉｍｅＳＴＡＲ®ＨＳＤＮＡＰｏｌｙｅｒａｓｅ(２.５Ｕ/μＬ)０.２５μＬꎬｄｄＨ２Ｏ补足至２５μＬꎮ反应条件如下:９８ħ预变性２ｍｉｎꎻ９８ħ变性１０ｓꎬ５８ħ退火３０ｓꎬ７２ħ延伸２ｍｉｎꎬ３０个循环ꎻ７２ħ延伸７ｍｉｎꎻ４ħ保存ꎮＰＣＲ产物进行琼脂糖凝胶电泳ꎬ用ＤＮＡ纯化回收试剂盒进行回收ꎮ将回收纯化的３Ｄ基因和ｐＸＪ４１载体用ＨｉｎｄⅢ/ＫｐｎⅠ双酶切ꎬ酶切产物胶回收纯化后用Ｔ４ＤＮＡＬｉｇａｓｅ进行１６ħ过夜连接ꎬ将连接产物转化至ＤＨ５α感受态细胞ꎬ３７ħ过夜培养１６ｈꎬ挑取单菌落接种于含有氨苄西林抗性的ＬＢ培养基中ꎬ３７ħ㊁２００ｒ/ｍｉｎ过夜培养后提取质粒ꎬ进行ＨｉｎｄⅢ/ＫｐｎⅠ双酶切鉴定ꎬ鉴定为阳性的质粒送北京擎科生物科技有限公司测序ꎬ命名为ｐＸＪ４１－Ｍｙｃ－３Ｄꎮ１.２.２㊀Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ检测ＦＭＤＶ３Ｄ蛋白的表达㊀将ＨＥＫ－２９３Ｔ细胞接种于６孔板中进行培养ꎬ待细胞密度达到７０％~９０％时ꎬ用Ｌｉｐｏｆｅｃｔａｍｉｎｅ®３０００转染试剂按说明书进行操作ꎬ转染质粒ｐＸＪ４１－Ｍｙｃ－３Ｄ和ｐＸＪ４１空载体各２μｇꎮ培养２４ｈ后ꎬ弃去上清ꎬ用等体积预冷的ＰＢＳ洗涤细胞２次ꎬ将６孔板置于冰上ꎬ加入细胞裂解液裂解细胞２０ｍｉｎ后ꎬ４ħ１２０００ｒ/ｍｉｎ离心１０ｍｉｎꎮ取上清液与上样缓冲液混合后煮沸１０ｍｉｎꎮ将处理好的样品进行１２％ＳＤＳ－ＰＡＧＥ凝胶电泳后ꎬ采用湿法(１００Ｖꎬ７０ｍｉｎ)将蛋白转印至ＮＣ膜ꎮ将ＮＣ膜转移至５％脱脂奶粉封闭液中ꎬ室温轻摇封闭１ｈꎬ用一抗稀释液稀释的兔抗Ｍｙｃ和兔抗β－ａｃｔｉｎ于４ħ过夜孵育ꎬ二抗为用５％脱脂奶粉稀释的辣根过氧化物(ＨＲＰ)标记的羊抗兔抗体ꎬ室温孵育１ｈꎬ洗膜后用ＳｐａｒｋｊａｄｅＥＣＬｓｕｐｅｒ(极超敏化学发光试剂盒)显色ꎬＢＩＯ－ＲＡＤ凝胶成像仪进行曝光ꎮ１.２.３㊀ＩＦＡ检测ＦＭＤＶ３Ｄ蛋白的细胞定位㊀用无菌镊子夹取细胞爬片置于２４孔板内ꎬ接种ＰＫ－１５细胞ꎬ待细胞密度达到４０％~５０％时ꎬ用Ｌｉｐｏ￣ｆｅｃｔａｍｉｎｅ®３０００转染试剂按说明书进行操作ꎬ各孔分别转染质粒ｐＸＪ４１－Ｍｙｃ－３Ｄ和ｐＸＪ４１空载体各０.５μｇꎮ培养２４ｈ后弃上清ꎬ用４％多聚甲醛固定４５ｍｉｎꎻＰＢＳ洗１０ｍｉｎˑ３次后ꎬ加入０.１％ＴｒｉｔｏｎＸ－１００室温通透３０ｍｉｎꎻＰＢＳ洗１０ｍｉｎˑ３次后ꎬ加入１ʒ６００稀释的鼠源Ｍｙｃ抗体室温孵育２ｈꎻＰＢＳ洗１０ｍｉｎˑ３次后ꎬ加入１ʒ１０００稀释的ＡｌｅｘａＦｌｕｏｒ４８８－羊抗鼠抗体ꎬ室温避光孵育１ｈꎻＰＢＳ洗１０ｍｉｎˑ３次后ꎬ加入１ʒ１００００稀释的ＤＡ￣ＰＩ室温孵育５ｍｉｎꎻＰＢＳ洗１０ｍｉｎˑ３次后ꎬ取出爬片置于滴有抗荧光衰减封闭剂的载玻片上ꎬ封片后于荧光显微镜下观察３Ｄ蛋白在细胞内的定位情况ꎮ１.２.４㊀Ｌｕｃｉｆｅｒａｓｅ检测ＦＭＤＶ３Ｄ蛋白对ＩＦＮ－β启动子的影响㊀将ＨＥＫ－２９３Ｔ细胞接种于１２孔板中进行培养ꎬ待细胞密度达到７０％~９０％时ꎬ用Ｌｉｐｏｆｅｃｔａｍｉｎｅ®３０００转染试剂按说明书进行操作ꎬ分别转染ｐＸＪ４１－Ｍｙｃ－３Ｄ和空载体ｐＸＪ４１各０.４μｇꎬ同时分别转染ＩＦＮ－β启动子报告基因质粒ｐＩＦＮ－β－Ｌｕｃ０.４μｇ和内参质粒ｐＲＬ－ＴＫ０.０４μｇꎮ２４ｈ后感染水疱性口炎病毒(Ｖｅｒｓｉｃｕｌａｒｓｔｏ￣ｍａｔｉｔｉｓｖｉｒｕｓꎬＶＳＶ)(ＭＯＩ＝０.１)ꎬ１０ｈ后ꎬ按双荧光素酶报告基因试剂盒说明书方法收取细胞进行检测ꎮ１.２.５㊀Ｒｅａｌ－ｔｉｍｅＰＣＲ检测ＦＭＤＶ３Ｄ蛋白对ＩＦＮ－βｍＲＮＡ的影响㊀将ＨＥＫ－２９３Ｔ细胞接种于２４６４１山东农业科学㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀第５６卷㊀孔板中进行培养ꎬ待细胞密度达到７０％~９０％时ꎬ用Ｌｉｐｏｆｅｃｔａｍｉｎｅ®３０００转染试剂按说明书进行操作ꎬ分别转染质粒ｐＸＪ４１－Ｍｙｃ－３Ｄ和空载体ｐＸＪ４１各０.５μｇꎬ２４ｈ后感染ＶＳＶ(ＭＯＩ＝０.１)ꎬ１０ｈ后ꎬ收集细胞使用ＲＮＡ－ｅａｓｙＩｓｏｌａｔｉｏｎＲｅａｇｅｎｔ试剂盒提取总ＲＮＡꎬ以其为模板反转录得到ｃＤ￣ＮＡꎬ以ＧＡＰＤＨ作为内参基因检测ＩＦＮ－βｍＲＮＡ水平ꎬＲｅａｌ－ｔｉｍｅＰＣＲ引物序列详见表２ꎮ㊀㊀表２㊀Ｒｅａｌ－ｔｉｍｅＰＣＲ引物信息引物名称引物序列(５ －３ )ｈＧＡＰＤＨ－ＦＡＴＧＧＣＣＴＴＣＣＧＴＧＴＣＣｈＧＡＰＤＨ－ＲＧＴＧＧＧＴＧＴＣＧＣＴＧＴＴＧＡｈＩＦＮ－β－ＦＧＣＡＣＴＧＧＣＴＧＧＡＡＴＧＡＧＡＣＴｈＩＦＮ－β－ＲＣＣＴＴＧＧＣＣＴＴＣＡＧＧＴＡＡＴＧ１.２.６㊀Ｒｅａｌ－ｔｉｍｅＰＣＲ检测病毒拷贝数㊀将ＨＥＫ－２９３Ｔ细胞接种于２４孔板中进行培养ꎬ待细胞密度达到７０％~９０％时ꎬ用Ｌｉｐｏｆｅｃｔａｍｉｎｅ®３０００转染试剂按说明书进行操作ꎬ分别转染质粒ｐＸＪ４１－Ｍｙｃ－３Ｄ和空载体ｐＸＪ４１各０.５μｇꎬ２４ｈ后感染ＶＳＶ(ＭＯＩ＝０.１)ꎬ１０ｈ后ꎬ细胞及其上清反复冻融后ꎬ一部分用于病毒滴度检测ꎻ一部分使用ＲＮＡ－ｅａｓｙＩｓｏｌａｔｉｏｎＲｅａｇｅｎｔ试剂盒提取总ＲＮＡꎬ以其为模板反转录得到ｃＤＮＡꎬ绝对荧光定量检测ＶＳＶ拷贝数ꎮ１.２.７㊀ＴＣＩＤ５０测定病毒滴度㊀将ＨＥＫ－２９３Ｔ细胞接种于９６孔板中进行培养ꎬ待细胞密度达到９０％时ꎬ将１.２.６中的样品１２０００ｒ/ｍｉｎ离心１０ｍｉｎ后取上清ꎬ用２％细胞维持液按１０倍梯度稀释ꎬ共１０个梯度ꎬ每个梯度４个重复ꎬ将稀释好的样品加入９６孔板中ꎬ每孔１００μＬꎬ并设置８个孔加入等体积细胞维持液做阴性对照ꎬ观察并记录细胞病变情况ꎬ按照Ｒｅｅｄ－Ｍｕｅｎｃｈ法计算ＴＣＩＤ５０ꎮ１.３㊀数据统计与分析使用ＧｒａｐｈＰａｄＰｒｉｓｍ５软件对试验数据进行统计学分析ꎮ２㊀结果与分析２.１㊀ｐＸＪ４１－Ｍｙｃ－３Ｄ真核表达质粒的构建及鉴定以ｐＭＤ１８－Ｔ－３Ｄ克隆质粒为模板ꎬＰＣＲ扩增获得３Ｄ基因ꎮ扩增产物经１％琼脂糖凝胶电泳可见大小约１５００ｂｐ的特异性目的片段ꎬ与预期大小相符(图１)ꎮ重组质粒ｐＸＪ４１－Ｍｙｃ－３Ｄ经ＨｉｎｄⅢ/ＫｐｎⅠ双酶切后得到预期大小的片段(图２)ꎬ测序结果表明ｐＸＪ４１－Ｍｙｃ－３Ｄ真核表达质粒构建成功ꎮＭ:ＤＬ２０００ＤＮＡＭａｒｋｅｒꎻ１㊁２:３Ｄ基因ＰＣＲ产物ꎮ图１㊀ＦＭＤＶ３Ｄ基因的ＰＣＲ扩增Ｍ:ＤＬ５０００ＤＮＡＭａｒｋｅｒꎻ１:ｐＸＪ４１－Ｍｙｃ－３Ｄ质粒的ＨｉｎｄⅢ/ＫｐｎⅠ双酶切产物ꎻ２:ｐＸＪ４１－Ｍｙｃ－３Ｄ质粒ꎮ图２㊀ｐＸＪ４１－Ｍｙｃ－３Ｄ质粒的双酶切鉴定２.２㊀Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ检测３Ｄ蛋白的表达经Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ检测到ｐＸＪ４１－Ｍｙｃ－３Ｄ在ＨＥＫ－２９３Ｔ细胞中成功表达ꎬ大小约５５ｋＤａ左右(图３)ꎮ图３㊀Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ鉴定ＨＥＫ－２９３Ｔ㊀细胞中３Ｄ蛋白的表达２.３㊀ＩＦＡ检测ＦＭＤＶ３Ｄ蛋白的细胞定位ＩＦＡ结果显示ꎬＦＭＤＶ３Ｄ蛋白在ＰＫ－１５细胞中的表达主要定位在细胞核内ꎬ细胞胞浆中也有少量表达(图４)ꎮ７４１㊀第２期㊀㊀㊀㊀苗勤ꎬ等:ＦＭＤＶ３Ｄ基因真核表达质粒的构建㊁表达及对Ⅰ型ＩＦＮ信号通路的作用图４㊀ＩＦＡ检测ＰＫ－１５细胞中３Ｄ蛋白的定位２.４㊀双荧光素酶报告基因检测ＦＭＤＶ３Ｄ蛋白对ＩＦＮ－β启动子的影响双荧光素酶报告基因检测结果显示ꎬＶＳＶ感染后ꎬｐＸＪ４１－Ｍｙｃ－３Ｄ转染组的ＩＦＮ－β启动子活性极显著低于空载体ｐＸＪ４１转染组ꎬ表明ＦＭＤＶ３Ｄ蛋白抑制了ＶＳＶ诱导的ＩＦＮ－β启动子活性(图５)ꎮ柱上∗∗表示不同转染组间差异极显著(Ｐ<０.０１)ꎬ下同ꎮ图５㊀ＦＭＤＶ３Ｄ蛋白对ＶＳＶ诱导的㊀㊀ＩＦＮ－β启动子活性影响２.５㊀Ｒｅａｌ－ｔｉｍｅＰＣＲ检测ＦＭＤＶ３Ｄ蛋白对ＩＦＮ－βｍＲＮＡ的影响Ｒｅａｌ－ｔｉｍｅＰＣＲ检测结果显示ꎬＶＳＶ刺激后ꎬｐＸＪ４１－Ｍｙｃ－３Ｄ转染组的ＩＦＮ－βｍＲＮＡ表达水平极显著低于空载体ｐＸＪ４１转染组ꎬ表明ＦＭＤＶ３Ｄ蛋白抑制了ＶＳＶ诱导的ＩＦＮ－βｍＲＮＡ表达水平(图６)ꎮ２.６㊀ＦＭＤＶ３Ｄ蛋白对ＶＳＶ增殖的影响Ｒｅａｌ－ｔｉｍｅＰＣＲ检测ＶＳＶ拷贝数结果显示ꎬｐＸＪ４１－Ｍｙｃ－３Ｄ转染组极显著高于空载体组(图７)ꎮＴＣＩＤ５０结果显示ꎬｐＸＪ４１－Ｍｙｃ－３Ｄ转染组ＶＳＶＴＣＩＤ５０明显高于空载体转染组(图８)ꎮ表明ＦＭＤＶ３Ｄ蛋白通过抑制Ⅰ型ＩＦＮ的产生促进了ＶＳＶ的复制ꎮ图６㊀ＦＭＤＶ３Ｄ蛋白对ＶＳＶ诱导的㊀㊀ＩＦＮ－βｍＲＮＡ水平的影响图７㊀ＦＭＤＶ３Ｄ蛋白对ＶＳＶ拷贝数的影响图８㊀ＦＭＤＶ３Ｄ蛋白对ＶＳＶ病毒滴度的影响３㊀讨论与结论口蹄疫是一种急性㊁高度接触性人畜共患传８４１山东农业科学㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀第５６卷㊀染病ꎬ我国主要以Ｏ型口蹄疫为主ꎬＡ型口蹄疫呈点状散发ꎬ加之境外疫情传入的影响ꎬ给我国的畜牧业带来严峻的挑战[７]ꎮＦＭＤＶ３Ｄ蛋白作为ＲＮＡ病毒依赖的ＲＮＡ聚合酶ꎬ催化病毒ＲＮＡ合成ꎬ在不同血清型中高度保守ꎬ且有研究表明３Ｄ在病毒复制早期产生[８]ꎬ感染后１.５ｈ首先检测到３Ｄ瞬时表达ꎬ在核仁中均匀分布[９]ꎮ本试验成功构建了真核表达质粒ｐＸＪ４１－Ｍｙｃ－３Ｄꎬ并在ＨＥＫ－２９３Ｔ细胞中进行了表达验证ꎮ通过ＩＦＡ检测到３Ｄ蛋白在ＰＫ－１５胞核和细胞胞浆中都有表达ꎬ且胞核内表达明显ꎬ显示３Ｄ主要定位于细胞核ꎬ与毕研丽等[１０]的研究结果一致ꎮ天然免疫又称先天性免疫或固有免疫ꎬ是机体对抗病毒感染的第一道防线[１１]ꎬ当病毒感染机体后模式识别受体(ｐａｔｔｅｒｎｒｅｃｏｇｎｉｔｉｏｎｒｅｃｅｐｔｏｒｓꎬＰＲＲｓ)识别病原相关模式分子(ｐａｔｈｏｇｅｎａｓｓｏｃｉａｔ￣ｅｄｍｏｌｅｃｕｌｅｐａｔｔｅｒｎｓꎬＰＡＭＰｓ)ꎬ激活下游信号通路ꎬ诱导产生Ⅰ型ＩＦＮ等细胞因子ꎬ进而启动免疫应答[１２]ꎮＰＲＲｓ家族包括Ｔｏｌｌ样受体(ＴＬＲｓ)㊁ＲＩＧ－Ｉ样受体(ＲＬＲｓ)㊁Ｎｏｄ样受体(ＮＬＲｓ)㊁Ｃ型凝集素受体(ＣＬＲｓ)[１３]ꎬ其中ＴＬＲｓ和ＲＬＲｓ主要识别病毒ＲＮＡ[１４]ꎮＦＭＤＶ在长期进化中形成了一系列策略逃逸宿主的天然免疫ꎬ从而促进自身复制ꎬ维持对宿主的持续感染[１５]ꎮ已有研究表明ꎬＦＭＤＶ编码的多种蛋白可参与调控先天性免疫信号通路[４]ꎬ例如Ｌｂｐｒｏ蛋白可以通过去除ＩＳＧ１５的泛素化阻断天然免疫的启动[１６]ꎬ裂解ＬＧＰ２抑制ＩＦＮ－β的表达[１７]ꎮ３Ｃｐｒｏ蛋白通过溶酶体路径降解蛋白激酶ＰＫＲ[１８]ꎬ也能通过抑制ＮＦ－κＢ信号通路促进病毒复制[１９]ꎮ３Ａ蛋白与ＲＩＧ－Ｉ㊁ＭＤＡ５和ＭＡＶＳ互作并抑制蛋白表达ꎬ抑制ＲＬＲ介导的ＩＦＮ－β产生[２０]ꎮ２Ｂ通过降解ＲＩＧ－Ｉ和ＭＤＡ５调节ＩＦＮ信号通路[２１]ꎮ２Ｂ和２Ｃ蛋白协同作用ꎬ影响ＭＨＣ－Ｉ复合物的形成ꎬ逃避机体的天然免疫[２２]ꎮ３Ｂ与ＲＩＧ－Ｉ互作ꎬ抑制ＲＩＧ－Ｉ介导的信号通路[２３]ꎮ本实验室前期发现ＦＭＤＶ３Ｃｐｒｏ通过阻断ＳＴＡＴ１/ＳＴＡＴ２核易位来拮抗干扰素信号通路[２４]ꎮＦＭＤＶＬｐｒｏ通过与ｓＲＮａｓｅＬ的Ｎ末端结构域相互作用ꎬ抑制细胞凋亡ꎬ拮抗ＯＡＳ/ＲＮａｓｅＬ通路ꎬ抑制抗病毒天然免疫[２５]ꎮ有研究表明ＦＭＤＶ非结构蛋白３Ｄ作为一种反式作用元件调控ＴＬＲ３信号通路的激活ꎬ促进ＴＬＲ３通路介导的Ⅰ型干扰素的产生[２６]ꎮ但天然免疫反应交错复杂ꎬ不同的ＰＡＭＰｓ可能激活多条信号通路ꎬ通过不同分子间相互作用共同完成天然免疫应答ꎮ本试验选用ＨＥＫ－２９３Ｔ作为研究细胞ꎬ由于该模式细胞中不含ＴＬＲ３受体ꎬ可排除ＴＬＲ３信号通路的影响ꎬ探究３Ｄ蛋白对Ⅰ型ＩＦＮ信号通路的作用ꎮ水疱性口炎病毒是一种负链ＲＮＡ病毒ꎬ属弹状病毒科水疱病毒属ꎬ是研究Ⅰ型ＩＦＮ信号通路常用的模式病毒ꎬ细胞感染ＶＳＶ后可激活Ⅰ型ＩＦＮ信号通路ꎮ本研究采用ＶＳＶ模拟病毒感染ꎬ通过双荧光素酶报告基因㊁Ｒｅａｌ－ｔｉｍｅＰＣＲ及ＴＣＩＤ５０试验ꎬ发现３Ｄ蛋白能够通过抑制Ⅰ型ＩＦＮ信号通路的活化抑制先天性免疫ꎬ进而促进ＶＳＶ的复制ꎮ综上ꎬＦＭＤＶ３Ｄ蛋白抑制了ＶＳＶ诱导的ＩＦＮ－β启动子的活性和ＩＦＮ－βｍＲＮＡ水平ꎬ促进了ＶＳＶ的复制ꎬ提示ＦＭＤＶ３Ｄ蛋白是病毒抵抗先天性免疫的重要因子ꎬ为其在Ⅰ型ＩＦＮ信号通路的作用及机制研究奠定了基础ꎮ参㊀考㊀文㊀献:[１]㊀高巧梅.口蹄疫的流行特点与防控措施[Ｊ].今日畜牧兽医ꎬ２０２２ꎬ３８(１２):３２－３４.[２]㊀ＭａｓｏｎＰＷꎬＧｒｕｂｍａｎＭＪꎬＢａｘｔＢ.Ｍｏｌｅｃｕｌａｒｂａｓｉｓｏｆｐａｔｈｏ￣ｇｅｎｅｓｉｓｏｆＦＭＤＶ[Ｊ].ＶｉｒｕｓＲｅｓｅａｒｃｈꎬ２００３ꎬ９１(１):９－３２. [３]㊀易梦杰ꎬ丁远学ꎬ马锐.口蹄疫病毒综述[Ｊ].湖北畜牧兽医ꎬ２０１５ꎬ３６(１１):１１－１３.[４]㊀张向乐ꎬ朱紫祥ꎬ郑海学.口蹄疫病毒抑制宿主天然免疫应答研究进展[Ｊ].中国动物传染病学报ꎬ２０１８ꎬ２６(４):１－５. [５]㊀ＧａｏＹꎬＳｕｎＳＱꎬＧｕｏＨＣ.ＢｉｏｌｏｇｉｃａｌｆｕｎｃｔｉｏｎｏｆＦｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓｎｏｎ￣ｓｔｒｕｃｔｕｒａｌｐｒｏｔｅｉｎｓａｎｄｎｏｎ￣ｃｏｄｉｎｇｅｌｅ￣ｍｅｎｔｓ[Ｊ].ＶｉｒｏｌｏｇｙＪｏｕｒｎａｌꎬ２０１６ꎬ１３(１):１０７. [６]㊀Ｇａｒｃíａ￣ＢｒｉｏｎｅｓＭＭꎬＢｌａｎｃｏＥꎬＣｈｉｖａＣꎬｅｔａｌ.Ｉｍｍｕｎｏｇｅｎｉｃｉ￣ｔｙａｎｄＴｃｅｌｌｒｅｃｏｇｎｉｔｉｏｎｉｎｓｗｉｎｅｏｆｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉ￣ｒｕｓｐｏｌｙｍｅｒａｓｅ３Ｄ[Ｊ].Ｖｉｒｏｌｏｇｙꎬ２００４ꎬ３２２(２):２６４－２７５. [７]㊀沈超ꎬ曾一歌.口蹄疫病毒持续感染的研究进展[Ｊ].华中师范大学学报(自然科学版)ꎬ２０２２ꎬ５６(４):６６２－６６９. [８]㊀孙静静ꎬ邵军军ꎬ常惠芸.口蹄疫病毒非结构蛋白３Ｄｐｏｌ的研究进展[Ｊ].中国人兽共患病学报ꎬ２０１１ꎬ２７(１２):１１３５－１１３８.９４１㊀第２期㊀㊀㊀㊀苗勤ꎬ等:ＦＭＤＶ３Ｄ基因真核表达质粒的构建㊁表达及对Ⅰ型ＩＦＮ信号通路的作用[９]㊀Ｇａｒｃíａ￣ＢｒｉｏｎｅｓＭꎬＲｏｓａｓＭＦꎬＧｏｎｚáｌｅｚ￣ＭａｇａｌｄｉＭꎬｅｔａｌ.Ｄｉｆｆｅｒｅｎｔｉａｌｄｉｓｔｒｉｂｕｔｉｏｎｏｆｎｏｎ￣ｓｔｒｕｃｔｕｒａｌｐｒｏｔｅｉｎｓｏｆｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓｉｎＢＨＫ￣２１ｃｅｌｌｓ[Ｊ].Ｖｉｒｏｌｏｇｙꎬ２００６ꎬ３４９(２):４０９－４２１.[１０]毕研丽ꎬ沈小燕ꎬ丛国正ꎬ等.口蹄疫病毒３Ｄ基因真核表达载体ｐＥＧＦＰ－３Ｄ的构建及其融合蛋白分子在ＢＨＫ细胞中的定位[Ｊ].华北农学报ꎬ２００８ꎬ２３(４):５－９.[１１]常兴妮ꎬ郑兴华ꎬ马凯凯ꎬ等.口蹄疫及天然免疫的研究进展[Ｊ].当代畜牧ꎬ２０１７(３):４３－４５.[１２]ＡｋｉｒａＳꎬＵｅｍａｔｓｕＳꎬＴａｋｅｕｃｈｉＯ.Ｐａｔｈｏｇｅｎｒｅｃｏｇｎｉｔｉｏｎａｎｄｉｎ￣ｎａｔｅｉｍｍｕｎｉｔｙ[Ｊ].Ｃｅｌｌꎬ２００６ꎬ１２４(４):７８３－８０１. [１３]ＴａｋｅｕｃｈｉＯꎬＡｋｉｒａＳ.Ｐａｔｔｅｒｎｒｅｃｏｇｎｉｔｉｏｎｒｅｃｅｐｔｏｒｓａｎｄｉｎｆｌａｍ￣ｍａｔｉｏｎ[Ｊ].Ｃｅｌｌꎬ２０１０ꎬ１４０(６):８０５－８２０.[１４]ＷｕＪＸꎬＣｈｅｎＺＪ.Ｉｎｎａｔｅｉｍｍｕｎｅｓｅｎｓｉｎｇａｎｄｓｉｇｎａｌｉｎｇｏｆｃｙ￣ｔｏｓｏｌｉｃｎｕｃｌｅｉｃａｃｉｄｓ[Ｊ].ＡｎｎｕａｌＲｅｖｉｅｗｏｆＩｍｍｕｎｏｌｏｇｙꎬ２０１４ꎬ３２(１):４６１－４８８.[１５]ＲｏｄｒíｇｕｅｚＰｕｌｉｄｏＭꎬＳáｉｚＭ.Ｍｏｌｅｃｕｌａｒｍｅｃｈａｎｉｓｍｓｏｆｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓｔａｒｇｅｔｉｎｇｔｈｅｈｏｓｔａｎｔｉｖｉｒａｌｒｅｓｐｏｎｓｅ[Ｊ].ＦｒｏｎｔｉｅｒｓｉｎＣｅｌｌｕｌａｒａｎｄＩｎｆｅｃｔｉｏｎＭｉｃｒｏｂｉｏｌｏｇｙꎬ２０１７ꎬ７:２５２.[１６]ＳｗａｔｅｋＫＮꎬＡｕｍａｙｒＭꎬＰｒｕｅｎｄａＪＮꎬｅｔａｌ.Ｉｒｒｅｖｅｒｓｉｂｌｅｉｎａｃ￣ｔｉｖａｔｉｏｎｏｆＩＳＧ１５ｂｙａｖｉｒａｌｌｅａｄｅｒｐｒｏｔｅａｓｅｅｎａｂｌｅｓａｌｔｅｒｎａｔｉｖｅｉｎｆｅｃｔｉｏｎｄｅｔｅｃｔｉｏｎｓｔｒａｔｅｇｉｅｓ[Ｊ].ＰｒｏｃｅｅｄｉｎｇｓｏｆｔｈｅＮａｔｉｏｎａｌＡｃａｄｅｍｙｏｆＳｃｉｅｎｃｅｓꎬ２０１８ꎬ１１５(１０):２３７１－２３７６. [１７]ＲｏｄｒíｇｕｅｚＰｕｌｉｄｏＭꎬＳáｎｃｈｅｚ￣ＡｐａｒｉｃｉｏＭＴꎬＭａｒｔíｎｅｚ￣ＳａｌａｓＥꎬｅｔａｌ.ＩｎｎａｔｅｉｍｍｕｎｅｓｅｎｓｏｒＬＧＰ２ｉｓｃｌｅａｖｅｄｂｙｔｈｅＬｅａｄｅｒｐｒｏｔｅａｓｅｏｆｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓ[Ｊ].ＰＬｏＳＰａｔｈｏｇｅｎｓꎬ２０１８ꎬ１４(６):ｅ１００７１３５.[１８]ＬｉＣＴꎬＺｈｕＺＸꎬＤｕＸＬꎬｅｔａｌ.Ｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓｉｎｄｕｃｅｓｌｙｓｏｓｏｍａｌｄｅｇｒａｄａｔｉｏｎｏｆｈｏｓｔｐｒｏｔｅｉｎｋｉｎａｓｅＰＫＲｂｙ３Ｃｐｒｏｔｅｉｎａｓｅｔｏｆａｃｉｌｉｔａｔｅｖｉｒｕｓｒｅｐｌｉｃａｔｉｏｎ[Ｊ].Ｖｉｒｏｌｏｇｙꎬ２０１７ꎬ５０９:２２２－２３１.[１９]ＦａｎＸＸꎬＨａｎＳＣꎬＹａｎＤꎬｅｔａｌ.Ｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉ￣ｒｕｓｉｎｆｅｃｔｉｏｎｓｕｐｐｒｅｓｓｅｓａｕｔｏｐｈａｇｙａｎｄＮＦ￣кＢａｎｔｉｖｉｒａｌｒｅｓｐｏｎ￣ｓｅｓｖｉａｄｅｇｒａｄａｔｉｏｎｏｆＡＴＧ５￣ＡＴＧ１２ｂｙ３Ｃｐｒｏ[Ｊ].ＣｅｌｌＤｅａｔｈ＆Ｄｉｓｅａｓｅꎬ２０１８ꎬ８(１):ｅ２５６１.[２０]ＬｉＤꎬＬｅｉＣＱꎬＸｕＺＳꎬｅｔａｌ.Ｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓｎｏｎ￣ｓｔｒｕｃｔｕｒａｌｐｒｏｔｅｉｎ３Ａｉｎｈｉｂｉｔｓｔｈｅｉｎｔｅｒｆｅｒｏｎ￣βｓｉｇｎａｌｉｎｇｐａｔｈｗａｙ[Ｊ].ＳｃｉｅｎｔｉｆｉｃＲｅｐｏｒｔｓꎬ２０１６ꎬ６(１):２１８８８. [２１]ＷｅｅｒａｗａｒｄｈａｎａＡꎬＵｄｄｉｎＭＢꎬＣｈｏｉＪＨꎬｅｔａｌ.Ｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓｎｏｎ￣ｓｔｒｕｃｔｕｒａｌｐｒｏｔｅｉｎ２ＢｄｏｗｎｒｅｇｕｌａｔｅｓｔｈｅＲＬＲｓｉｇｎａｌｉｎｇｐａｔｈｗａｙｖｉａｄｅｇｒａｄａｔｉｏｎｏｆＲＩＧ￣ＩａｎｄＭＤＡ５[Ｊ].ＦｒｏｎｔｉｅｒｓｉｎＩｍｍｕｎｏｌｏｇｙꎬ２０２２ꎬ１３:１０２０２６２. [２２]武艺ꎬ李璐ꎬ柴瑞兰ꎬ等.口蹄疫病毒蛋白颉颃宿主天然免疫的分子机制[Ｊ].黑龙江畜牧兽医ꎬ２０２２(１０):２３－２９ꎬ１３３.[２３]ＺｈａｎｇＸＬꎬＺｈｕＺＸꎬＷａｎｇＣＣꎬｅｔａｌ.Ｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓ￣ｅａｓｅｖｉｒｕｓ３ＢｐｒｏｔｅｉｎｉｎｔｅｒａｃｔｓｗｉｔｈｐａｔｔｅｒｎｒｅｃｏｇｎｉｔｉｏｎｒｅｃｅｐｔｏｒＲＩＧ￣ＩｔｏｂｌｏｃｋＲＩＧ￣Ｉ￣ｍｅｄｉａｔｅｄｉｍｍｕｎｅｓｉｇｎａｌｉｎｇａｎｄｉｎｈｉｂｉｔｈｏｓｔａｎｔｉｖｉｒａｌｒｅｓｐｏｎｓｅ[Ｊ].ＴｈｅＪｏｕｒｎａｌｏｆＩｍｍｕｎｏｌｏｇｙꎬ２０２０ꎬ２０５(８):２２０７－２２２１.[２４]ＤｕＹＪꎬＢｉＪＳꎬＬｉｕＪＹꎬｅｔａｌ.３Ｃｐｒｏｏｆｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓａｎｔａｇｏｎｉｚｅｓｔｈｅｉｎｔｅｒｆｅｒｏｎｓｉｇｎａｌｉｎｇｐａｔｈｗａｙｂｙｂｌｏｃｋｉｎｇＳＴＡＴ１/ＳＴＡＴ２ｎｕｃｌｅａｒｔｒａｎｓｌｏｃａｔｉｏｎ[Ｊ].ＪｏｕｒｎａｌｏｆＶｉｒｏｌｏｇｙꎬ２０１４ꎬ８８(９):４９０８－４９２０.[２５]ＳｕｉＣꎬＪｉａｎｇＤＤꎬＷｕＸＪꎬｅｔａｌ.Ｉｎｈｉｂｉｔｉｏｎｏｆａｎｔｉｖｉｒａｌｉｎｎａｔｅｉｍｍｕｎｉｔｙｂｙｆｏｏｔ￣ａｎｄ￣ｍｏｕｔｈｄｉｓｅａｓｅｖｉｒｕｓＬｐｒｏｔｈｒｏｕｇｈｉｎｔｅｒａｃ￣ｔｉｏｎｗｉｔｈｔｈｅＮ￣ｔｅｒｍｉｎａｌｄｏｍａｉｎｏｆａｗｉｎｅＲＮａｓｅＬ[Ｊ].ＪｏｕｒｎａｌｏｆＶｉｒｏｌｏｇｙꎬ２０２１ꎬ９５(１５):ｅ００３６１－２１.[２６]李露露ꎬ张敬ꎬ李丹ꎬ等.口蹄疫病毒３Ｄ蛋白促进ＴＬＲ３介导的Ⅰ型干扰素产生[Ｊ].畜牧兽医学报ꎬ２０２０ꎬ５１(８):１９２３－１９３１.０５１山东农业科学㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀第５６卷㊀。

EBNA2基因真核表达载体的构建及表达产物的功能(一)【关键词】EBNA2 ConstructionofeukaryoticexpressionvectorofEBNA2andfunctionaldetection ofitsproduct【Abstract】AIM:ToconstructtheeukaryoticexpressionvectorofEBNA2andtoexpressitine ukaryoticcellsanddetectthefunctionofitsproduct.METHODS:EBNA2genewa sobtainedbyPCRandDNArecombinationfromtheplasmidpSG5EBNA2.PCRpr oductofEBNA2bwastheninsertedintoplasmidpMD18Tandsequenced.Thefull lengthEBNA2genewascutandinsertedintoeukaryoticexpressionplasmidpCM ingLipofectamineTM2000,theplasmidpCMVEBNA2wastransfectedin toCos7andthendetectedbyWesternblotandimmunofluorescence.Theactivit yofEBNA2wasdetectedbyluciferasereporterassay.RESULTS:TheEBNA2gene wassuccessfullyclonedanditssequencewasidenticaltothatinGenBank.Afterp lasmidpCMVEBNA2wastransfectedintoCos7,theexpressedproductofEBNA2 wasdetectedbyWesternblotandimmunofluorescence.Thetranscriptionalacti vityofEBNA2wasdemonstratedbyreporterassay.CONCLUSION:Theeukaryoti cexpressionvectorpCMVEBNA2hasbeensuccessfullyconstructedandsuccess fullyexpressedineukaryoticcellline.TheEBNA2proteinhastranscriptionalactiv ation.【Keywords】EBNA2;clone;expression;geneticvector【摘要】目的：构建EB病毒核心抗原2（EBNA2）的真核表达载体，在真核细胞中表达,并检测其表达产物的功能.方法：应用DNA重组和PCR方法从质粒pSG5EBNA2中亚克隆EBNA2的编码基因，插入到真核表达载体pCMVHA中.应用脂质体的方法将重组质粒pCMVEBNA2瞬时转染Cos7细胞，通过Westernblot和免疫荧光方法检测EBNA2的表达.应用荧光素酶报告实验检测EBNA2蛋白的活性.结果：克隆EBNA2的编码基因，经序列分析证实与GenBank中的序列完全一致.以含有EBNA2编码基因的真核表达载体瞬时转染Cos7细胞，EBNA2基因的表达产物通过Westernblot和免疫荧光方法得到证实.荧光素酶报告实验显示所表达的蛋白具有转录激活活性.结论：成功构建EBNA2的真核表达载体，证实其在真核细胞Cos7可以表达，并具有转录激活功能.【关键词】EBNA2基因；克隆；表达；遗传载体0引言EB病毒核心抗原2（EBNA2）的编码产物是EB病毒体外感染细胞时最先表达的蛋白，作为一种转录因子，它可以调节病毒及细胞内多种基因表达，如LMP1，LMP2A〔1〕，CD21，CD23及cmyc等，同时还可以抑制细胞免疫球蛋白重链的转录.Notch信号途径是体内一个非常保守的信号途径，在体内分布十分广泛〔2〕.EBNA2可以与Notch信号途径中的重要分子RBPJκ相结合，激活下游靶基因，并通过模仿Notch信号途径对体内淋巴细胞的分化和发育方向起调控作用〔3〕.同时EBNA2还可以促进抑癌分子p53的磷酸化〔4〕，调节癌基因的表达.我们对EBNA2基因进行了克隆，并在真核细胞中表达，为进一步探讨其对小鼠免疫系统发育过程的影响奠定基础.1材料和方法1.1材料大肠杆菌XL10；质粒载体pCMVHA，pBluscript（-），pGA9816为本室保存.载体pMD18T购自TaKaRa公司.Cos7细胞为本室保存.脂质体LipofectamineTM2000及细胞培养试剂均购自Invitrogene公司.抗EBNA2抗体购自Dako公司，HRP标记抗小鼠IgG,FITC标记抗小鼠IgG 购自博士德公司.小量质粒提取试剂盒、DNA胶回收试剂盒均购自华瞬生物工程公司.PCR试剂盒、各种限制性内切酶购自TaKaRa公司.根据GenBank中EBNA2基因的序列，设计2条引物：引物2的5＇端加入XhoⅠ位点，引物1和引物2用于扩增EBNA2基因的3＇端部分（EBNA2b）.引物1：5＇CTCGGGGCCACCATGGTGGC3＇；引物2:5＇CGCCTCGAGTTACTGGATGGAGGGG3＇.1.2方法1.2.1EBNA2基因的克隆及真核表达载体的构建EcoRⅠ/BglⅡ/SalⅠ三酶切质粒pSG5EBNA2，胶回收 4.0kb大小片段，将回收片段插入经EcoRⅠ/BamHⅠ双酶切开的pBluscript（-）载体中，命名为pBSSKEBNA2.以pSG5EBNA2为模板，用引物1和引物2进行常规PCR反应，扩增EBNA2基因的3＇部分EBNA2b.PCR反应条件：于94℃变性5min后，按下列参数循环35次：即94℃变性30s，60℃退火30s，72℃延伸30s，最后于72℃延伸7min.PCR产物克隆到pMD18T载体中，NcoⅠ/XhoⅠ酶切鉴定阳性克隆，命名为pMDEBNA2b.用NcoⅠ/XhoⅠ从pMDEBNA2b 中切下EBNA2b片段插入用NcoⅠ/XhoⅠ切开的pBSSKEBNA2载体pBSSKEBNA2a中，NcoⅠ单酶切和EcoRⅠ/XhoⅠ双酶切鉴定，命名为pBSSKEBNA2（a+b）.pCMVEBNA2用EcoRⅠ/XhoⅠ双酶切质粒pBSSKEBNA2（a+b），回收1500bp片段，将其插入EcoRⅠ/XhoⅠ切开的pCMVHA载体中，酶切鉴定阳性克隆，命名为pCMVEBNA2.1.2.2EBNA2表达的检测①WesternblotCos7细胞于转染前1d接种于直径60mm中皿，接种4×105个细胞,接种16h后，应用LipofectamineTM200010μL转染质粒pCMVEBNA24μg.转染60h后裂解细胞收集蛋白上清，加入2×加样缓冲液，煮沸5min.然后进行120g/LSDSPAGE电泳，于180mA稳压状态下转膜3h.于室温用20g/L蛋白干粉封闭1h后，一抗antiEBNA2（1∶1000）4℃过夜.PBST洗涤3次，每次5min.二抗抗鼠IgGHRP（1∶4000）室温作用1h，同上洗涤后，进行放射性显影.②免疫荧光Cos7细胞于转染前1d接种于直径60mm中皿，于皿底事先加入圆形玻片，转染过程同上，对照组转染质粒pCMVHA.转染60h后取出爬有细胞的玻片，PBS洗涤3次，每次5min；10g/LBSA37℃封闭1h；一抗antiEBNA2（1∶200）37℃避光湿盒中染色1h；同上洗涤；二抗抗鼠IgGFITC（1∶500）37℃避光湿盒中染色1h；同上洗涤后，荧光显微镜观察.1.2.3共转染报告质粒检测荧光素酶强度细胞准备和转染过程同上，对照组转染：pGA98160.2μg，βgal0.2μg，pCMVHA1.6μg；检测组转染：pGA98160.2μg，βgal0.2μg，pCMVEBNA20.2μg，pCMVHA1.4μg.转染48h 后收细胞.反复液氮中冻溶裂解，充分震荡混匀，样品与反应底物1∶5比例混合后，照度仪中检测荧光素酶强度.。

第44卷第2期2021年3月河北农业大学学报JOURNAL OF HEBEI AGRICULTURAL UNIVERSITYVol.44 No.2Mar.2021绵羊CTSB基因过表达载体的构建及生物信息学分析韩红叶1，张丽萌2，刘爱菊1，马晓菲1，李悦欣1，高 旭1，王志刚1，田树军1,3(1. 河北农业大学 动物科技学院,河北 保定071001; 2. 郑州师范学院分子生物学实验室,郑州450044; 3.河北省牛羊胚胎技术创新中心, 河北 保定071001)摘要：为了构建组织蛋白酶B（Cathepsin B，CTSB）基因的真核表达载体，进一步研究其蛋白的结构和功能，本试验以绵羊卵巢组织的cDNA为模板，扩增绵羊卵巢CTSB基因的CDS编码区，将其插入真核表达载体中成功获得重组质粒pcDNA3.1-CTSB，利用生物学信息学分析软件对绵羊卵巢CTSB基因的结构和功能进行分析鉴定。

研究结果表明：成功构建了重组质粒pcDNA3.1-CTSB，发现CTSB基因主要在细胞外行使功能，CTSB蛋白具有翻译后磷酸化及糖基化修饰特性，CTSB与LGMN、NLRP3、CTSD蛋白之间存在一定互作关系，上述发现将为进一步探究绵羊CTSB基因的功能提供重要线索。

关键词：绵羊；CTSB；过表达载体；蛋白结构；蛋白功能中图分类号：S826开放科学（资源服务）标识码（OSID）：文献标志码：AConstruction of overexpression vector and bioinformaticsanalysis of sheep CTSB geneHAN Hongye1, ZHANG Limen2, LIU Aiju1, MA Xiaofei1, LI Yuexin1, GAO Xu1, WANG Zhigang1, TIAN Shujun1（1.College of Animal Science and Technology, Hebei Agricultural University, Baoding 071001, China; 2. Laboratoryof Molecular Biology, Zhengzhou Normal University, Zhengzhou 450044, China; 3. Hebei Technology InnovationCenter of Cattle and Sheep Embryo, Baoding 071001, China )Abstract: In order to construct the eukaryotic expression vector of cathepsin B (CTSB) gene and further study thestructure and function of CTSB protein, the CDS coding region of sheep CTSB gene was amplified by using thecDNA of sheep ovary tissue as a template, and it was inserted into the eukaryotic expression vector to successfullyobtain PCDNA3.1-CTSB. We analyzed the structure and function of the CTSB gene by bioinformatics software. Theresults showed that the sheep recombinant plasmid pcDNA3.1-CTSB was successfully constructed, and the CTSBgene was highly conserved during evolution and functions outside the cell. The CTSB protein had post-translationalphosphorylation and glycosylation modification, and there was a certain interaction between CTSB and LGMN,NLRP3, CTSD, which provided clues for further exploring the function of sheep CTSB gene.Keywords: sheep; CTSB ; overexpression vector; protein structure; protein function收稿日期：2020-12-25基金项目：河北农业产业技术体系（HBCT2018140202）；河北省重点研发计划（20326349D）.第一作者：韩红叶（1993- ），女，河北石家庄人，硕士研究生，从事动物繁殖研究.E-mail：******************** 通信作者：王志刚（1968- ），男，河北衡水人，硕士，研究员，从事动物繁殖调控技术研究.E-mail：**************.cn 田树军（1970- ），男，河北张家口人，博士，教授，从事动物胚胎工程及羊生产学工作研究.E-mail：***************本刊网址：http: // hauxb. hebau. edu. cn文章编号：1000-1573(2021)02-0093-04DOI：10.13320/ki.jauh.2021.003194第44卷河北农业大学学报组织蛋白酶B（CTSB）是半胱氨酸蛋白酶家族中的一员，在所有半胱氨酸组织蛋白酶中含量丰富，在生理和病理（如细胞凋亡、肿瘤的浸润转移等）中发挥作用［1］。

论著【收稿日期】2005-03-03;【修回日期】2005-05-25【作者简介】侍晓云(1966-),女,副主任医师,现为天津医科大学在读博士。

葡萄糖调控的胰岛素原真核表达载体的构建侍晓云1,畅继武2,邱明才1(11天津医科大学总医院内分泌科,天津300052;21天津医科大学第二医院、天津市泌尿外科研究所肿瘤免疫室,天津300211)摘　要:　【目的】构建胰岛素原真核表达载体,在肝细胞内实现葡萄糖调控下的成熟胰岛素的表达。

【方法】(1)RT 2PCR 方法从胎儿胰腺获取野生型人胰岛素原cDNA ;(2)重叠延伸PCR 法在人胰岛素原cDNA 上制造两个点突变,该胰岛素原cDNA 片段命名为胰岛素/furin (INS/furin );(3)将INS/furin 亚克隆至含葡萄糖反应元件(G lRE )的p (G lRE )3BP 21Luc 的多克隆位点上。

【结果】Hind Ⅲ和EcoRV 酶切及测序均证实载体p(G lRE )3BP 211×furin 构建成功。

【结论】含葡萄糖反应元件的点突变胰岛素原基因真核表达载体p (G lRE )3BP 211×furin 有望成为糖尿病基因治疗理想的候选载体之一。

关键词:葡萄糖反应元件;胰岛素/furin ;胰岛素原;表达载体【文章编号】　100825041(2005)0420252204　【中图分类号】　Q782　【文献标识码】　AConstruction of eukaryotic glucose 2regulated insulin expression vectorSHI Xiao 2yun ,CHANG Ji 2w u ,QU Ming 2cai (The G eneral H ospital of Tianjin Medical U niversity ,Tianjin 300052,China)Abstract :【Objective 】A eukaryotic expression vector was constructed in order to realize glucose 2regulated mature insulin gene express in hepatocytes.【Methods 】Native human promisulin cDNA was obtained from fetus pancreas by RT ing site 2directed mutagenesis (overlap extension PCR ),furin consensus cleavage sequence was introduced into proin 2sulin cDNA ,and the new sequence was named INS/furin ,which allowed for the recognition and processed of furin and efficient proteolytic maturation of proinsulin to insulin within most of the cells containing furin.Subquently ,INS/furin was subcloned into the multiple clone sites of plasmid p (G lRE )3BP 21Luc ,which containing glucose reaction element G lRE in its promotor.【R esults 】Double digestion with Hind Ⅲand EcoRV ,as well as sequencing verified the vector containing target gene in specific sites.【Conclusion 】The recombinant mammalian ex pression vector p (G lRE )3BP 211×furin ,containing glucose reaction element and site 2mutated proinsulin ,is a novel ideal candidate vector for diabetic gene therapy.K ey Words :G lucose reaction element ;Furin ;Proinsulin ;Ex press vector 糖尿病是以糖代谢异常为主的代谢性疾病,无论何种类型的糖尿病,为控制晚期并发症的长期的血糖控制均需使用胰岛素。