TAP1真核表达载体的构建及其对GES-1细胞HLA-I分子表达的影响  

Construction of human TAP1 expression vector and the effects of it on HLA-I expression in GES-1 cells in vitro

作　　者：邢凌霄[1] 邢欣[1] 万晓伟[1] 薛丽英[1] 张祥宏[1] 严霞[1] 王俊灵[1] 

机构地区：[1]河北医科大学病理学研究室,河北石家庄050017

出　　处：《细胞与分子免疫学杂志》2010年第4期329-332,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：河北省自然科学基金资助项目(C2007000819)

摘　　要：目的:构建抗原加工相关转运蛋白TAP1真核表达载体,并观察其对HLA-I分子表达的影响。方法:采用基因重组技术,构建含人TAP1基因全长的pcDNA3.1/V5-His-TAP1真核表达质粒,并采用细胞转染、RT-PCR、Western blot以及流式细胞术(FCM)观察TAP1转染对胃黏膜上皮(GES-1)细胞HLA-I分子表达的影响。结果:以人外周血单个核细胞总RNA为模板,经RT-PCR反应获得人全长TAP1基因,以pcDNA3.1/V5-HisB为载体,经酶切、连接、转化等基因重组技术,构建了含人TAP1基因全长的pcDNA3.1/V5-His-TAP1质粒,并经测序结果证实。以GES-1作为靶细胞进行转染,经RT-PCR和Westernblot证实,转染后TAP1基因表达明显增加,细胞适于所构建的TAP1质粒的转染。进一步检测了TAP1转染对GES-1细胞HLA-I类分子表达的影响。结果发现,TAP1转染组HLA-A、HLA-B、HLA-C(重链)在mR-NA水平表达明显增加,β2m(轻链)mRNA水平无明显影响。FCM及Western blot检测结果表明,TAP1转染可以上调HLA-I蛋白的表达。结论:成功构建了TAP1真核表达质粒,细胞转染后TAP1表达的增加,可引起细胞表面HLA-I分子表达的相应增加,从而证实了TAP1在HLA-I抗原表达以及抗原递呈途径中的重要作用。AIM：To construct the human TAP1 expression vector and to evaluate its effects on HLA-I expression in GES-1 cells in vitro. METHODS：The human TAP1 expression vector （pcDNA3.1/V5-His-TAP1） was constructed by gene recombination technology. The expression of HLA-I on human gastric epithelial cell line （GES-1 cells） after transfection was detected by RT-PCR,Western blot and flow cytometry （FCM）. RESULTS：Human full-length TAP1 gene was obtained from human peripheral blood mononuclear cells by RT-PCR reaction,then TAP1 gene was inserted into pcDNA3.1/V5-HisB vector to get the TAP1 expression vector （pcDNA3.1/V5-His-TAP1） by recombination technology including digestion with restriction enzymes,ligation and transformation. The vector was sequenced to ensure the sequence fidelity.To further evaluate the function of the TAP1 plasmid we constructed,GES-1 cells were selected as the target cell to be transfected. Firstly RT-PCR and Western blot results showed that the expression of TAP in GES-1 cells was increased after pcDNA3.1/V5-His-TAP1 transfection. Based on the high efficiency of transfection in GES-1 cell,we then decteded the expression of HLA-I. The results showed that the expressions of HLA-A,HLA-B and HLA-C at mRNA level were all increased by TAP1 transfection,but no change was found in β2m mRNA. HLA-I protein level was increased correspondingly with the TAP expression in cells by FCM and Western blot assay. CONCLUSION：The TAP1 expression vector was successfully constructed,and it can induce the expression of HLA-I on the GES-1 cells after TAP1 transfection. The results confirm that the TAP1 plays a cruical role in the HLA-I antigen expression and antigen presentation pathway.

关 键 词：HLA-I分子 TAP1 GES-1细胞 

分 类 号：R392.11[医药卫生—免疫学]