pBR002-HLA-DRB1＊ 0803转基因载体构建、表达及其抗原递呈功能研究  

Construction of transgenic vector pBR002-HLA-DRB1* 0803 and its expression and antigen-presenting function

作　　者：张旭晖[1] 何韦韦 谭文婷[1] 但芸婕[1] 李辉文[1] 王勇[2] 邓国宏[1] 

机构地区：[1]第三军医大学,西南医院全军感染病研究所,感染病研究重庆市重点实验室,重庆400038 [2]第三军医大学基础医学部实验动物学教研室,重庆400038

出　　处：《第三军医大学学报》2016年第9期916-920,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金重点项目(81330038);重庆市前沿与应用基础研究计划项目(CSTC2014jcyj A10114)~~

摘　　要：目的构建可以表达人类HLA-DRB1*0803的转基因载体,并在细胞水平验证其表达及抗原递呈功能。方法提取人WATANABE细胞株(基因型为HLA-DRB1*0803纯合子)的总RNA,RTPCR扩增DRB1*0803基因片段,与小鼠Eαpromoter和兔β-globin基因内含子序列同时导入p BR002-lacz载体。pBR002-HLA-DRB1*0803转基因载体转染小鼠DCS细胞和B细胞系,用Western blot和免疫荧光的方法对HLA-DRB1蛋白进行检测。流式细胞术检测转染小鼠DCS细胞对HBc Ag特异性CD4+T细胞的刺激和极化效应。结果经双酶切和测序验证,成功构建了p BR002-HLA-DRB1*0803转基因载体;转染载体后的小鼠DCS细胞和B细胞系均表达出了大小约为29×10~3的HLA-DRB1蛋白;免疫荧光结果表明HLA-DRB1蛋白主要表达于细胞质内。转染后的小鼠DCS细胞可将负载的HBcAg递呈给HLA-DRB1*0803阳性基因型的人CD4^+T细胞,产生明显的CD4^+T细胞应答。结论成功构建了HLA-DRB1*0803转基因载体,该载体可以在细胞水平特异性表达HLA-DRB1蛋白,并具有HLADRB1*0803特异性抗原递呈功能。Objective To construct a transgenic vector of human HLA-DRB1＊ 0803 gene,and validate its expression and antigen-presenting function in vitro. Methods The c DNA encoding the HLADRB1＊ 0803 gene was reverse transcribed and amplified by RT-PCR from the DRB1＊ 0803 BLCL cell lines.The sequences of mouse Eα promoter and rabbit β-globin intron 1 were synthesized. The above 3 fragments were inserted into the plasmid p BR002-lacz in a particular order. Successfully constructed p BR002-HLADRB1＊ 0803 transgenic vector was used to transfect DCS cells and B cell line by Lipofectamine 2000,and the target proteins were detected by Western blotting and immunofluorescence assay. T cell proliferation assay were used to validate whether the transgenic DCS cells can present HBc Ag antigens to human CD4~＋T cells and polarize the CD4~＋T cells. Results A p BR002-HLA-DRB1＊ 0803 transgenic vector was successfully constructed,and verified by dual restriction enzyme and sanger sequencing. After transfection,the DCS cells and B cells expressed the target protein at a molecular weight of 29 × 103. Immunofluorescence assay results showed that the HLA-DRB1 proteins were expressed mainly in the cytoplasm of DCS cells after transfection. T cell proliferation assay showed that the transfected DCS cells presented the HBc Ag to the human T cells,resulting in a specific CD4~＋T cell response. Conclusion HLA-DRB1＊ 0803 transgenic vector is successfully constructed,which can express the HLA-DRB1＊ 0803 at the cellular level,present viral antigen and stimulate a HBc Ag-specific CD4~＋T cell response.

关 键 词：HLA-DR 转基因载体 乙型肝炎病毒 抗原递呈 

分 类 号：R392.33[医药卫生—免疫学] R392.11[医药卫生—基础医学]