人整合素β3亚基真核表达载体的构建及αIIbβ3在细胞表面的表达

Construction of human integrin β3 subunit eukaryotic expression vector and efficient surface expression of integrin αIIbβ3

目的 :构建人整合素 β3亚基真核表达载体 ,并探讨如何使人整合素αIIbβ3在CHO细胞表面有效表达 ,为进一步研究整合素β3亚基作为汉坦病毒 (hantavirus,HV)受体的特异性奠定基础。方法 :构建编码人整合素 β3真核表达载体 pcDNA3.1 β3。将其与编码人整合素αIIb亚基真核表达载体 ,分别及共转染至中国仓鼠卵巢 (Chinahamsterovary ,CHO)细胞中进行表达。采用间接免疫荧光法 (IFA)检测外源基因的表达。结果 :共转染组目的蛋白呈高效的细胞膜表达。pcDNA3.1 β3单独转染组 β3亚基在细胞膜的表达较共转染组弱 ;而pBJ1 αIIb单独转染组则未见有效的细胞膜表达。结论 :人整合素αIIbβ3在CHO细胞表面的有效表达需要两个亚基共同参与。

AIM: To establish efficient surface expression of Homo sapiens integrin αIIbβ3 in β3 integrin deficient and HV insusceptible CHO cells, so as to lay the foundation for the further study of cellular entry of hantavirus mediated by β3 integrins. METHODS: Eukaryotic expression vector pcDNA3.1 β3 harboring ORF region of human integrin β3 subunit cDNA was constructed, then pcDNA3.1 β3 and eukaryotic expression vector pBJ1 αIIb containing human integrin αIIb subunit cDNA were transfected into CHO cells alone or together. The expression of exogenous genes were analyzed by indirect immunofluorescence assay (IFA). RESULTS: The eukaryotic expression vector pcDNA3.1 β3 was constructed successfully. IFA examination showed that surface expression of integrin αIIb β3 was highly effective in cotransfection group, while surface expression was weak on CHO cells transfected with pcDNA3.1 β3 alone, and no effective surface expression was found in pBJ1 αIIb transfected group. CONCLUSION: Efficient surface expression of integrin αIIb β3 requires expression of both subunits.