丙型肝炎病毒核心蛋白酵母双杂交诱饵载体的构建及人胎肝cDNA文库的扩增、纯化和鉴定

1、丙型肝炎病毒核心蛋白酵母双杂交诱饵载体的构建及人胎肝cDNA文库的扩增、纯化和鉴定 作者：刘敏，张伟，陈天艳，蔺淑梅，赵良，刘锦锋，赵英仁，张树林【关键词】 丙型肝炎病毒核心蛋白 摘要：目的 用含丙型肝炎病毒核心(HCV core)蛋白的cDNA片段构建酵母双杂交诱饵载体，并进行人胎肝cDNA文库的扩增、纯化和鉴定。方法 PCR扩增含HCV core蛋白不同大小的cDNA片段，分别克隆入pUC19质粒，经测序正确后，再亚克隆入酵母双杂交诱饵载体pGBKT7中。扩增人胎肝cDNA文库并纯化、鉴定。结果 获得含HCV core蛋白的cDNA片段，并成功克隆入pGBKT7中。待转化的人肝cDNA文库

2、滴度在5108左右，纯化后的质粒 DNA质量浓度约1g/L。用EcoR、Xho双酶切显示插入片段大小不一。结论 成功构建了核心蛋白的酵母双杂交诱饵载体；扩增、纯化的人胎肝cDNA文库的多样性很好，适合于筛选。为用酵母双杂交技术研究与HCV core蛋白相互作用的蛋白打下坚实基础。关键词：丙型肝炎病毒核心蛋白；酵母双杂交；cDNA文库ABSTRACT: Objective To construct the bait vector of HCV core region in yeast twohybrid system, and to amplify, purify and evaluate th

3、e cDNA gene bank of human fetal liver. Methods The cDNA fragments encoding HCV core region were amplified by PCR, and then were cloned into pUC19. After being verified by sequencing, they were subcloned into the bait vector pGBKT7 of yeast twohybrid system, subsequently amplified and purified. And t

4、hen the cDNA gene bank of human fetal liver was evaluated. Results The cDNA fragments of HCV core region were amplified successfully.The cDNA gene bank of human fetal fetal liver was about 5108; the purified DNA was about 1g/L; and the built in fragments were different in size after digested with Ec

5、oR and Xho. Conclusion The bait vector HCV core in yeast twohybrid system 3 is constructed successfully. The diversity of cDNA of human fetal liver after amplified and purified is suitable for screening. These lay a rigid basis for further study of the HCV core protein and may help us in understandi

6、ng how HCV works.KEY WORDS:HCV core protein; yeast twohybrid system; cDNA gene bank丙型肝炎病毒(HCV)感染是引起慢性肝炎、肝硬化和肝细胞癌的主要原因，全球超过1.7亿人口血清HCV抗体阳性。目前对于HCV感染尚无有效的预防疫苗，病毒蛋白对HCV感染细胞的功能影响还很不明确，利巴韦林和干扰素联合治疗仅对部分病例有效。这就迫切要求我们进一步了解病毒与细胞间的相互作用及免疫逃避机制。缺乏HCV感染和复制的组织培养系统、缺乏合适的HCV感染动物模型是我们进行HCV发病机制研究所面临的主要障碍1。分子生物学技术的发展为

7、我们的研究提供了新的思路。蛋白质与蛋白质的相互作用是很多生命现象的基础。酵母双杂交系统是近年来新发展起来的一种分析真核细胞中蛋白蛋白、蛋白DNA、蛋白RNA相互作用的一种有效的基因分析方法。酵母双杂交Gal4系统3是在系统1和系统2基础上的改进，具有转化效率高，假阳性率低等优点，我们利用它来克隆与HCV 核心(core)蛋白相互作用蛋白的基因，以期为HCV core蛋白功能的研究提供新依据。1 材料与方法1.1 材料 含HCV全长cDNA质粒pCDNA3.0HCV由第四军医大学微生物教研室尹文博士惠赠；表达载体pGBKT7酵母菌种(美国Clontech公司)；质粒pUC19菌种(本研究室保存)

8、；PCR试剂盒、DNA重组所用的各种限制性内切酶、T4 DNA 连接酶(Gibco公司)；人肝酵母双杂交cDNA文库购自Clontech公司。1.2 方法 1.2.1 PCR扩增 克隆编码核心蛋白的引物：引物P1：5GCCGAATTCATGAGCA CGAATCCTAAACCT3；引物P2：5GCGTCGACTTAGGCTGAAGC GGGCACAGT3。PCR反应条件为：94 30s, 55 60s，72 60s，30个循环 。1.2.2 PCR产物克隆及鉴定 纯化的PCR产物用EcoR和Sal双酶切，定向克隆入pUC19质粒，转化大肠杆菌DH5，进行蓝白筛选。酶切鉴定出含插入片段的阳性克隆

9、命名为pUC19core蛋白，进行序列分析。1.2.3 构建及鉴定诱饵载体 用EcoR、Sal酶切pUC19core蛋白和表达载体pGBKT7, 回收core蛋白及pGBKT7载体片段，载体片段与插入片段连接后转化大肠杆菌DH5。经限制性内切酶分析，含正确插入片段的克隆命名为pGBKT7core蛋白。1.2.4 人胎肝cDNA文库的扩增、纯化与鉴定 按Clontech公司的说明书对文库进行扩增，用QIAGEN公司的操作手册对文库进行纯化。取纯化的文库质粒1L转化E.coli DH5感受态细胞，随机挑取10个单克隆提取质粒，EcoR、Xho双酶切鉴定cDNA文库的多样性。2 结果2.1 Core

10、蛋白基因片段编码区的扩增 可见扩增出与预期大小一致的cDNA片段(图1)。Core蛋白大小为588bp。图1 核心蛋白基因片段编码区扩增产物的琼脂糖凝胶电泳（略）Fig.1 AGE analysis of PCR products encoding cDNA fragments of HCV core region geneM: DNA PCR marker; 1-6: HCV core2.2 重组pUC19core蛋白质粒的酶切鉴定及序列分析 鉴定正确的pUC19core蛋白重组质粒分别做正反向测序，序列分析表明所扩增的core蛋白基因序列与登录的HCV core蛋白基因序列完全一致(图2)

11、。图2 重组pUC19core蛋白质粒的EcoR、Sal双酶切鉴定（略）Fig.2 AGE analysis of recombinant plasmid pUC19core digested with EcoR, SalM: DNA PCR marker ; 1-2: pUC19core recombinant plasmid2.3 重组pGBKT7core蛋白质粒的酶切分析 重组质粒pGBKT7core蛋白的酶切鉴定结果见图3。图3 重组pGBKT7core蛋白质粒的EcoR、Sal双酶切鉴定（略）Fig.3 AGE analysis of recombinant plasmid pGBK

12、T7core digested with EcoR, SalM: DNA PCR marker; 13: pGBKT7 core recombinant plasmid2.4 人胎肝cDNA文库的扩增、纯化与鉴定 经测定，待转化的人肝cDNA文库滴度在5108左右，纯化后的质粒 DNA浓度约1g/L。用EcoR，Xho双酶切鉴定cDNA文库的多样性。结果显示插入片断大小不一(图4)。说明文库的多样性很好，适合于筛选。图4 经EcoR、Xho双酶切的cDNA文库的琼脂糖凝胶电泳结果（略）Fig.4 AGE analysis of cDNA gene bank digested with EcoR

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