新型基因佐剂SPreS2真核表达载体的构建及其瞬时表达  

Construction and verification of a recombinant plasmid encoding HBV surface genes S and PreS2 as a genetic adjuvant

作　　者：赵群力[1] 闵娟[1] 周怀瑜[1] 古钦民[1] 丛华[1] 李瑛[1] 

机构地区：[1]山东大学医学院寄生虫学教研室,济南250012

出　　处：《山东大学学报（医学版）》2010年第9期29-33,39,共6页Journal of Shandong University:Health Sciences

基　　金：山东省科学技术发展计划基金资助项目(No.2006GG3202045);山东省医药卫生科技发展计划基金资助项目(No.2005HZ028)

摘　　要：目的构建重组真核表达载体pVAX1-SPreS2作为基因佐剂,用以增强或调节弓形虫DNA疫苗的体液及细胞免疫效果。方法采用重叠延伸PCR技术,扩增乙肝病毒表面抗原S和PreS2基因,引入编码GSGSGS柔性多肽序列作为接头拼接S和PreS2基因,构建重组克隆载体pMD18T-SPreS2和重组真核表达载体pVAX1-SPreS2,分别进行PCR、酶切和测序鉴定;脂质体法将重组载体pVAX1-SPreS2转染人成纤维细胞,建立体外真核细胞瞬时表达系统,SDS-PAGE和Western blot检测重组载体转染HFF细胞瞬时表达状况及其表达产物的免疫活性。结果 PCR、酶切、测序结果表明,重组克隆载体pMD18T-SPreS2和重组真核表达载体pVAX1-SPreS2正确构建,融合基因SPreS2在真核细胞中可以瞬时表达,其表达产物具有一定的免疫活性。结论成功构建重组真核表达载体pVAX1-SPreS2,为增强或调节弓形虫DNA疫苗免疫效果提供一种新型基因佐剂。Objective To construct and verify a recombinant eukaryotic expression plasmid pVAX1-SPreS2 for the further development of a new genetic adjuvant to enhance or adjust humoral and cellular immune responses to a Toxoplasma gondii DNA vaccine.Methods The S and PreS2 genes from HBV were fused with a polypeptide linker （GSGSGS） by overlap-ping extension PCR,constructing the cloning plasmid pMD18T-SPreS2 and recombinant eukaryotic expression plasmid pVAX1-SPreS2.Then the construction was verified by PCR,restriction enzyme digestion and sequencing.The specific ex-pression protein SPreS2 was detected by SDS-PAGE and Western blot after transient transfection of pVAX1-SPreS2 into HFF cells,which were treated with lipofectin to induce the establishment of an in vitro eukaryotic cell transient expression system.Results PCR,restriction enzyme digestion and sequencing results showed the correct construction of the recom-binant cloning plasmid pMD18T-SPreS2 and eukaryotic expression plasmid pVAX1-SPreS2.It was found that the fusion gene SPreS2 was transiently expressed in eukaryotic cells and its product had,to some extent,an immunological activity.Conclusion The recombinant eukaryotic expression plasmid pVAX1-SPreS2 has been successfully constructed and it may function as a new genetic adjuvant for a T.gondii DNA vaccine.

关 键 词：基因佐剂 基因 SPreS2 真核表达载体 瞬时表达 

分 类 号：R382.5[医药卫生—医学寄生虫学]