整合素β3亚基真核表达载体的构建及αvβ3的表达

Construction of human integrin β3 eukaryotic expression vector and expression of αvβ3 efficient surface

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αvβ3在CHO细胞表面有效表达 .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αv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亚基细胞膜表达较共转染组弱 ;而 pcDNA3 αv单独转染组则未见有效的细胞膜表达 .结论 :人整合素αvβ3在CHO细胞表面的有效表达需要

AIM: To study efficient surface expression of homo sapiens integrin αvβ3 in β3 integrin deficient and HV insusceptible China hamster ovary (CHO) cells and to lay a basis for further study of cellular entry of hantavirus mediated by β3 integrins. METHODS: Eukaryotic expression vector, pcDNA3.1 β3, harboring (open reading frame) ORF region of human integrinβ3 subunit cDNA was constructed and then pcDNA3.1 β3 and pcDNA3 αv which harbored human integrin αv subunit cDNA were transfected into CHO cells simultaneously or separately. The exogenous gene expression was analyzed by indirect immunofluorescence assay (IFA). RESULTS: The eukaryotic expression vector, pcDNA3.1 β3, was constructed successfully. Effective expression of integrin αvβ3 on the surface of CHO cells was observed by IFA, while weak surface expression was detected in the transfection with pcDNA3.1 β3 alone. Effective surface expression was not seen in the transfection with pcDNA3 αv alone. CONCLUSION: Efficient surface expression of integrin αvβ3 in CHO cells depends on the presence of both αv and β3 subunits.