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山东医药2023 年第 63 卷第 13 期

pcDNA3/HA-moesinT558A和pcDNA3/HA-moesinT558D

基因突变体构建与验证

刘红霞1，陈力2，王运林3，罗卓章1，黄巧冰41 广东省人民医院南海医院内分泌科，广东佛山528000；2 广东省人民医院南海医院肾内科；

3 佛山市第二人民医院内分泌科；4 南方医科大学基础医学院

摘要：目的　构建抑制型pcDNA3/HA-moesinT558A与激活型pcDNA3/HA-moesinT558D基因突变体，并在人脐静脉内皮细胞（HUVECs）中验证。方法　设计特定的moesinT558A、moesinT558D基因突变引物，以pcDNA3/HA-moesin为模板，采用逆向PCR技术进行体外定点突变，以丙氨酸、天冬氨酸取代苏氨酸，获得抑制型pcDNA3/HA-moesinT558A和激活型pcDNA3/HA-moesinT558D基因突变体并测序。体外培养HUVECs，随机分为对照1组、pcDNA3/HA空载体组、pcDNA3/HA-moesin组以及对照2组、pcDNA3/HA-moesinT558A组、pcDNA3/HA-moesinT558D组，pcDNA3/HA空载体组、pcDNA3/HA-moesin组、pcDNA3/HA-moesinT558A组、pcDNA3/HA-moesinT558D组分别转染pcDNA3/HA空载体、pcDNA3/HA-moesin、pcDNA3/HA-moesinT558A、pcDNA3/HA-moesinT558D，对照1组与对照2组不予转染。转染24 h，收集细胞，采用实时荧光定量PCR法检测moesin mRNA表达，采用Western blotting法和免疫荧光法检测moesin蛋白表达。结果　经测序，目的基因moesinT558A、moesinT558D序列完全正确，第558位苏氨酸分别突变为丙氨酸、天冬氨酸，抑制型pcDNA3/HA-moesinT558A和激活型pcDNA3/HA-moesinT558D基因突变体构建成功。pcDNA3/HA-moesin组moesin mRNA相对表达量显著高于对照1组和pcDNA3/HA空载体组（t分别为16.688、16.649，P均<0.05），而pcDNA3/HA空载体组与对照1组比较差异无统计学意义（t=1.925，P>0.05）。pcDNA3/HA-moesinT558D组、pcDNA3/HA-moesinT558A组moesin mRNA相对表达量均显著高于对照2组（t分别为13.809、9.260，P均<0.05），pcDNA3/HA-moesinT558D组与pcDNA3/HA-moesinT558A组比较差异无统计学意义（t=2.364，P>0.05）。Western blotting结果显示，pcDNA3/HA-moesinT558A组和pcDNA3/HA-moesinT558D组均能观察到HA-moesin蛋白表达，而对照1组未观察到HA-moesin蛋白表达。免疫荧光结果显示，pcDNA3/HA-moesin组、pcDNA3/HA-moesinT558A组和pcDNA3/HA-moesinT558D组均能观察到HA-moesin荧光，而对照2组未观察到HA-moesin荧光。结论　成功构建了抑制型pcDNA3/HA-moesinT558A和激活型pcDNA3/HA-moesinT558D基因突变体；经验证，这两种基因突变体均能促进moesin表达。关键词：膜突蛋白；pcDNA3/HA-moesinT558A；pcDNA3/HA-moesinT558D；人脐静脉内皮细胞doi：10.3969/j.issn.1002-266X.2023.13.002 中图分类号：R34 文献标志码：A 文章编号：1002-266X（2023）13-0006-05

Construction and validation of pcDNA3/HA-moesinT558A and pcDNA3/HA-moesinT558D gene mutants

LIU Hongxia1， CHEN Li， WANG Yunlin， LUO Zhuozhang， HUANG Qiaobing1 Department of Endocrinology， Nanhai Hospital， Guangdong Provincial People's Hospital， Foshan 528000， China

Abstract： Objective To construct suppressed pcDNA3/HA-moesinT558A and activated pcDNA3/HA-moesinT558D gene mutants and to verify their expression in human umbilical vein endothelial cells （HUVECs）. Methods Specific moesinT558A and moesinT558D gene mutation primers were designed， and pcDNA3/HA-moesin was used as the template for in vitro targeted mutagenesis by reverse PCR， and threonine was replaced with alanine and aspartate to obtain suppressed pcDNA3/HA-moesinT558A and activated pcDNA3/HA-moesinT558D gene mutants and then they were sequenced. HUVECs cells were cultured in vitro and randomly divided into the control 1 group， pcDNA3/HA empty vector group， pcDNA3/HA-moesin group， and control 2 group， pcDNA3/HA-moesinT558A group， and pcDNA3/HA-moesinT558D group； the pcDNA3/HA empty vector group， pcDNA3/HA-moesin group， pcDNA3/HA-moesinT558A group， and pcDNA3/HA-moesinT558D group were transfected with pcDNA3/HA empty vector， pcDNA3/HA-moesin， pcDNA3/HA-moesinT558A， and pcDNA3/HA-

基金项目：国家自然科学基金资助项目（81870210）。第一作者简介：刘红霞（1985-），女，硕士研究生，副主任医师，主要研究方向为内分泌代谢疾病诊治。E-mail： l125201848@163.com通信作者简介：黄巧冰（1963-），女，博士研究生，教授，主要研究方向为糖尿病微血管并发症诊治。E-mail： bing＠fimmu.

com开放科学（资源服务）标识码（OSID）6

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moesinT558D ， respectively， while the control 1 group and control 2 group were not transfected. The cells were transfected for 24 h. The moesin mRNA expression was detected by real-time fluorescence quantitative PCR， and the moesin protein expression was detected by Western blotting and immunofluorescence. Results After sequencing， the sequences of target genes moesinT558A and moesinT558D were completely correct， and the threonine at position 558 mutated into alanine and aspar⁃tate， respectively； the suppressed pcDNA3/HA-moesinT558A and activated pcDNA3/HA-moesinT558D mutants were success⁃fully constructed. The relative expression of moesin mRNA in the pcDNA3/HA-moesin group was significantly higher than that in the control 1 group and the pcDNA3/HA empty vector group （t=16.688， 16.649， both P<0.05）， while there was no statistically significant difference between the pcDNA3/HA empty vector group and the control 1 group （t=1.925， P>0.05）. The relative expression levels of moesin mRNA in the pcDNA3/HA-moesinT558D and pcDNA3/HA-moesinT558A groups were significantly higher than that in the control 2 group （t=3.809， 9.260， both P<0.05）， and the difference between the pcDNA3/HA-moesinT558D group and the pcDNA3/HA-moesinT558A group was not statistically significant （t=2.364， P>0.05）. Western blotting showed that HA-moesin protein expression was observed in both the pcDNA3/HA-moesinT558A and pcDNA3/HA-moesinT558D groups， while HA-moesin protein expression was not observed in the control 1 group. Immunofluorescence results showed that HA-moesin fluorescence was observed in the pcDNA3/HA-moesin group， pcDNA3/HA-moesinT558A group and pcDNA3/HA-moesinT558D group， while HA-moesin fluorescence was not observed in the control 2 group. Conclusion The suppressed pcDNA3/HA-moesinT558A and activated pcDNA3/HA-moesinT558D gene mutants were successfully constructed， and it was verified that both gene mutants could promote moesin expression.

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pcDNA3使用说明

pcDNA3使用说明

pcDNA3.1(+)

pcDNA3.1(-)

Catalog nos. V790-20 and V795-20, respectively

Version I

081401

28-0104

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ii Table of Contents

Table of Contents ......................................................................................................... iii

Important Information ................................................................................................... v

Purchaser Notification ................................................................................................. vi

Methods .........................................................................................................................1

Overview...................................................................................................................................................................1

弓形虫pcDNA3-ROP1 DNA疫苗免疫小鼠诱导体液免疫应答及保护性的初步观察

弓形虫pcDNA3-ROP1 DNA疫苗免疫小鼠诱导体液免疫应答及保护性的初步观察

第９卷第３期　２００８年６月　北华大学学报（自然科学版）　ＪＯＵＲＮＡＬ　ＯＦ　ＢＥＩＨＵＡ　ＵＮＩＶＥＲＳＩＴＹ（Ｎａｔｕｒａｌ　Ｓｃｉｅｎｃｅ）　Ｖｏ１．９　Ｎｏ．３　Ｊｕｎ．２００８　

文章编号：１００９－４８２２（２００８）０３－０２３９－０２　

弓形虫ｐｃＤＮＡ３一ＲＯＰ１　ＤＮＡ疫苗免疫小鼠诱导体液　

免疫应答及保护性的初步观察　

李薇，刘　波，丑莉莉　

（北华大学医学检验学院，吉林吉林１３２０１３）　

摘要：目的观察弓形虫ｐｃＤＮＡ３一ＲＯＰ１　ＤＮＡ疫苗免疫ＢＡＬＢ／Ｃ小鼠诱导其体内产生的体液免疫应答及抗虫感　染的免疫保护作用．方法重组质粒用生理盐水稀释后，肌注免疫ＢＡＬＢ／Ｃ小鼠．分别于免疫后５周和１０周用　ＥＬＩＳＡ法测定ＩｇＧ抗体滴度；免疫鼠腹腔注射弓形虫速殖子攻击感染．结果经两次检测，均可测到特异性ＩｇＧ　抗体，且抗体的滴度随免疫时间的延长而增高；弓形虫速殖子腹腔攻击感染免疫组小鼠的平均存活时间较对照　组延长，统计学有显著性差异（Ｐ＜０．０１）．结论弓形虫ｐｃＤＮＡ３一ＲＯＰ１质粒ＤＮＡ疫苗能诱导ＢＡＬＢ／Ｃ小鼠产生　特异性体液免疫应答及部分抗虫免疫保护作用．　关键词：弓形虫；￣ＤＮＡ３一ＲＯＰ１　ＤＮＡ疫苗；体液免疫　中图分类号：Ｒ３８２．５　文献标识码：Ａ　

Ｏｎ　ｔｈｅ　Ｈｕｍｏｒａｌ　ａｎｄ　Ｐｒｏｔｅｃｔｉｖｅ　Ｉｍｍｕｎｅ　Ｒｅｓｐｏｎｓｅｓ　Ｉｎｄｕｃｅｄ　

ｂｙ　ｐｃＤＮＡ３一ＲＯＰ１　ＤＮＡ　Ｖａｃｃｉｎｅ　ｏｆ　Ｔｏｘｏｐｌａｓｍａ　ｇｏｎｄｉｉ　ｉｎ　Ｍｉｃｅ　

ＬＩ　Ｗｅｉ，ＬＩＵ　Ｂｏ，ＣＨＯＵ　Ｌｉ—ｌｉ　

（Ｍｅｄｉｃａｌ　Ｉｎｓｐｅｃｔｉｏｎ　Ｃｏｌｌｅｇｅ　ｏｆＢｅｉｈｎａ　Ｕｎｉｖｅｒｓｉｔｙ，Ｊｉｌｉｎ　１３２０１３，Ｃｈｉｎａ）　

Ａｂｓｔｒａｃｔ：Ｏｂｊｅｃｔｉｖｅ　Ｔｏ　ｏｂｓｅｒｖｅ　ｔｈｅ　ｈｕｍｏｒａｌ　ａｎｄ　ｐｒｏｔｅｃｔｉｖｅ　ｉｍｍｕｎｅ　ｒｅｓｐｏｎｓｅｓ　ｉｎｄｕｃｅｄ　ｂｙ　ｔｈｅ　ｐｃＤＮＡ３一ＲＯＰ１　ＤＮＡ　ｖａｃｃｉｎｅ　ｏｆ　Ｔｏｘｏｐｌａｓｍａ　ｇｏｎｄｉｉ　ｉｎ　ｍｉｃｅ．Ｍｅｔｈｏｄｓ　Ｉ￣ｒｇｅ　ｓｃａｌｅ　ｐｒｅｐａｒａｔｉｏｎ　ｏｆ　ｔｈｅ　ｒｅｃｏｍｂｉｎａｎｔ　ｐｌａｓｍｉｄ　

真核表达质粒PcDNA3.1(+)-带有白蛋白信号肽的Vasostatin(120-180aa)的构建及其在体内外细胞中的表

真核表达质粒PcDNA3.1(+)-带有白蛋白信号肽的Vasostatin(120-180aa)的构建及其在体内外细胞中的表

中国实验诊断学２０１１年１月　第１５卷箜　塑　

文章编号：１００７—４２８７（２０１１）０１—００１１—０４　

真核表达质粒ＰｅＤＮＡ３．１（＋）一带有白蛋白信号肽　

的Ｖａｓｏｓｔａｔｉｎ（１２０—１８０ａａ）的构　建及其在体内外　

细胞中的表达　

刘建巨　，关继奎　，王琳　，赵秀梅　，郑建秋　，李丹　，闫庆慧　

（１．哈尔滨医科大学第一临床医学院眼科医院，黑龙江哈尔滨１５０００１；２．大庆油田总医院）　

摘要：目的构建真核表达质粒ＰｃＤＮＡ３．１（＋）一带有白蛋白信号肽的Ｖａｓｏｓｔａｔｉｎ（１２０—１８０ａａ）并了解其在体内外真核　

细胞中的表达。方法将带有白蛋白信号肽的Ｖａｓｏｓｔａｔｉｎ（１２０－１８０ａａ）的逆转录聚合酶链式反应（ＲＴ－ＰＣＲ）产物克隆至　

ｐｃＤＮＡ３．１（＋）真核表达载体上，经酶切鉴定及测序分析证明构建成功后，以脂质体介导法转染体外培养的真核２９３Ｔ　

细胞及注射人鼠骨骼肌细胞中，了解其在体内外真核细胞中的表达情况。结果　我们获取了包含带有白蛋白信号肽　

的Ｖａｓｏｓｔａｔｉｎ（１２０—１８０ａａ）成熟肽及ＨＡ抗原在内的一段ｃＤＮＡ片段，并成功地与真核表达载体ＰｃＤＮＡ３．１（＋）连接，经测　

序表明，其中含有的带有白蛋白信号肽的Ｖａｓｏｓｔａｆｉｎ（１２０—１８０ａａ）成熟肽的序列与Ｇｅｎｅｂａｎｋ上报道的序列完全一致。即　

我们成功构建了真核表达质粒ＰｃＤＮＡ３．１（＋）一带有白蛋白信号肽的Ｖａｓｏｓｔａｔｉｎ（１２０。１８０ａａ）。之后，在转染了该质粒的体　外真核２９３Ｔ细胞的上清液中及骨骼肌注射了该质粒的鼠血液中，利用Ｗｅｓｔｅｒｎ－ｂｌｏｔ法检测到了目的基因的蛋白质。结　

论带有白蛋白信号肽的真核表达质粒ＰｃＤＮＡ３．１（＋）一Ｖａｓｏｓｔａｔｉｎ（１２０—１８０ａａ）增强了蛋白分泌表达能力，这为进一步应　

用该质粒治疗全身新生血管类病奠定了一定基础．　

关键词：Ｖａｓｏｓｔａｆｉｎ（１２０－１８０ａａ）；２９３Ｔ细胞；基因克隆；真核表达　

真核表达质粒pcDNA3.1a-FGFR1构建及意义

真核表达质粒pcDNA3.1a-FGFR1构建及意义

真核表达质粒pcDNA3.1a-FGFR1构建及意义

王笑;严金川;龚杰

【摘 要】Objective: To construct a recombinant eukaryotic expression

plasmid pcDNA3. 1 a-FGFR1 .Methods: The fragnent of FGFR1 was

amplificated by PCR, after purification the PCR products of FGFR1 and

eukaryotic expression plasmid pcDNA3. 1 a were cut and FGFR1 were

inserted into pcDNA3. 1 a,and then transformed into DH5α strain and

positive clone was selected. The recombinant plasmid was confirmed by

restriction endonuclease analysed and sequencing test. Results: DNA

sequencing and restriction endonuclease digestion analysis indicated that

the eukaryotic expression plasmid pcDNA3. 1 a-FGFR1 was constructed

successfully. Conclusion : Construction of pcDNA3. 1 a-FGFR1

eukaryotic expression plasmid was successful. This study for the next