周期型马来丝虫复合基因真核表达体系的建立  

Establishment on control of eukaryotic expression system with complex gene of periodic brugia malayi

作　　者：钱一言 徐怿琳 陆施娟[1] 王慧[1] 方浩[1] 徐邦生[1] 方政[1] 

机构地区：[1]南通大学医学院病原生物学教研室,江苏南通226001

出　　处：《现代预防医学》2013年第23期4389-4392,共4页Modern Preventive Medicine

基　　金：江苏省高等学校大学生实践创新训练计划项目(2012JSSPITP1571);南通市应用研究计划(BK2012017)

摘　　要：目的将含周期型马来丝虫3-磷酸甘油醛脱氢酶(BmGAPDH)和半胱氨酸蛋白酶抑制剂(BmCPI)复合基因的重组质粒pcDNA3.1(+)-BmCPI/BmGAPDH转染人宫颈癌细胞(Hela)细胞获得复合重组质粒稳定转染细胞株,并纯化所表达的重组蛋白,为研制新型的抗周期型马来丝虫重组蛋白疫苗提供理论及实验依据。方法将成功构建的pcDNA3.1(+)-BmCPI/BmGAPDH和空载体pcDNA3.1(+)分别转染Hela细胞,以G418筛选转染细胞,进而通过RT-PCR和SDS-PAGE鉴定G418筛选后的单克隆抗性细胞株。对阳性克隆进行无血清悬浮培养,收集细胞及其培养液。亲和层析纯化所表达的重组蛋白并通过Western-blotting对纯化的重组蛋白进行生物学鉴定。结果重组真核表达质粒pcDNA3.1(+)-BmCPI/BmGAPDH经转染Hela细胞,G418持续筛选14 d获得稳定表达。表达产物经Western-blotting法鉴定能够与相应的免疫鼠血清反应,相对分子质量Mr约为54×103。结论成功建立了周期型马来丝虫复合重组质粒pcDNA3.1(+)-BmCPI/BmGAPDH稳定转染细胞株并获得了相应的重组蛋白。OBJECTIVE To obtain stable transfected cell strain by plasmid pcDNA3.1 （＋）-BmCPI/BmGAPDH transfected Hela cell respectively. Affnity Chromatography was used to extract the recombinant purpose protein of pcDNA3.1-BmCPI/Bm- GAPDH with high purity. METHODS Plasmid pcDNA3.1 （＋） and recombinant plasmid pcDNA3.1 （＋）-BmCPI/BmGAPDH transfected Hela cell by lipofectin respectively, G418 was used for screening. The monoclonal resisting cell strain screened by G418 was confirmed with RT-PCR and SDS-PAGE. The monoclonal was cuhured in suspension cuhure with serum-flee medi- a, then Affnity Chromatography was used to extract the recombinant protein with high purity. In addition, Western bloting were performed to check it out. RESULTS Expressive recombinant plasmid had been transfected successfully into Hela cells and expressed steadily. The monoclonal cell strains with resistance to G418 were formed on 14th day after Hela cell is trans- fected with recombinant plasmid. The expression product could react with the immune mice serum by Western bloting and molecular weight was approximately 54x103. CONCLUSION The eukaryotic expression recombinant plasmid and recombinant protein of pcDNA3.1 （＋）-BmCPI/BmGAPDH are successfully constructed. Keywords： Periodical Brugia malayi; Glyceraldehydes-3-phosphate dehydrogenase ; Cystatin; Complex gene ; Recombi- nant protein

关 键 词：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 重组蛋白 

分 类 号：R459.5[医药卫生—治疗学]