周期型马来丝虫3-磷酸甘油醛脱氢酶/半胱氨酸蛋白酶抑制剂复合基因真核重组质粒的构建与表达  

Construction of eukaryotic recombinant expression plasmids with glyceraldehydes-3-phosphate dehydrogenase and cysteine protease inhibitor gene of periodic Brugia malayi and its expression in HeLa cells

作　　者：刘晓骏[1] 郭晓峰[1] 张赛楠[1] 陆施娟[1] 方浩[1] 徐邦生[1] 方政[1] 

机构地区：[1]江苏省南通大学医学院寄生虫学教研室,226001

出　　处：《中国地方病学杂志》2011年第4期371-375,共5页Chinese Jouranl of Endemiology

基　　金：江苏省高等学校大学生文践创新训练计划项目（2010639）;南通市应用研究计划项目（K2009027）

摘　　要：目的 构建周期型马来丝虫3-磷酸甘油醛脱氰酶/半胱氨酸蛋白酶抑制剂（GAPDH/CPI）复合基因真核重组表达质粒,为研制复合多价疫苗奠定基础.方法 依据GenBank中BmGAPDH及BmCPI基因序列设计引物,以周期型马来丝虫总RNA为模板,RT-PCR扩增目的 编码基因.PCR产物经TA克降后,克隆至载体pGEM-T Easy中.取阳性重组质粒pGEM-T/BmGAPDH和pGEM-T/BmCPI以及真核重组表达载体分别双酶切,将酶切后的载体和目的 片段进行连接,构建真核重组表达质粒pcDNA3.1（＋）/BmGAPDH/BmCPI.将复合基因重组质粒瞬时转染人HeLa细胞后,进行RT-PCR验证,将表达产物用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行分析和鉴定.结果 分别克隆了pGEM-T/BmGAPDH和pGEM-T/BmCPI重组质粒,经酶切鉴定分别得到877、621 bp特异性片段,与预期值吻合.成功构建了复合基因重组真核表达载体pcDNA3.1（＋）/BmGAPDH/BmCPI,酶切鉴定所产生的片段大小与预期吻合.复合基因真核重组表达质粒转染HeLa细胞后得到高水平表达,SDS-PAGE分析显示重组蛋白相对分子质量（Mr）约为54×103.结论 成功构建了周期型马来丝虫GAPDH/CPI复合基因重组表达质粒,并在哺乳动物细胞内得到正确表达.Objective To construct the eukaryotic expression plasmid containing glyceraldehydes-3-phosphate dehydrogenase （GAPDH） and cysteine protease inhibitor （ CPI ） gene from periodic Brugia malayi （Bm） and to lay foundation for studying multivalent vaccines. Methods Total RNA was extracted from periodic Bin. The BmGAPDH and BmCPI genes were amplified by RT-PCR. The PCR product was cloned and then subeloned into eukaryotic recombinant plasmid vector pcDNA3.1 （＋）. pcDNA3.1 （＋）/BmGAPDH/BmCPI was constructed. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification, and were transformed into HeLa cell subsequently. The transient expression of BmGAPDH and BmCPI were examined by RT-PCR. The expressed protein was identified by sodium dodeeylsulphate-polyacrylamide gel electrophoresis（SDS-PAGE）. Results Two specific bands of around 877 bp of BmGAPDH and 621 bp of BmCPI were amplified, consistent with the expected value. The same bands were obtained by double restriction enzyme digestion of recombinant plasmids or PCR using recombinant plasmid as template. BmGAPDH and BmCPI mRNA were highly expressed in transfeeted HeLa cell. The relative molecular mass （Mr） of the recombinant protein was about 54 × 103. Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1 （＋）/BmGAPDH/BmCPI has been constructed successfully and the protein is expressed correctly in mammalian cell.

关 键 词：丝虫 马来 甘油醛-3-磷酸脱氢酶类 半胱氨酸蛋白酶抑制剂 重组 遗传 HELA细胞 

分 类 号：R686[医药卫生—骨科学]