人整合素αvβ3在CHO细胞表达的研究

Efficient surface expression of Homo sapiens integrin αvβ3 requires the presen ce of both subunits

目的 使人整合素αvβ3在CHO细胞表面有效表达 ,为从分子水平了解汉坦病毒 (han tavirus ,HV)的感染及致病机制。方法 构建含人整合素 β3ORF区基因的真核表达载体pcDNA3.1 β3;将其与含人整合素αv全长cDNA的质粒pcDNA3 αv分别及共转染至CHO细胞中进行表达 ;采用间接免疫荧光法、流式细胞仪、免疫沉淀及免疫印迹实验对外源基因的表达进行定性、定位及定量检测 ,并确证表达产物的免疫反应性。结果 人整合素αv、β3基因在共转染组细胞中有高效表达 ,目的蛋白主要定位于细胞膜上 ;β3基因在pcDNA3.1 β3单独转染组细胞中的表达明显弱于共转染组 ;而pcDNA3 αv单独转染组则未见有效的表达。结论 人整合素αvβ3在CHO细胞表面的有效表达需要 2个亚基共同参与。

Objective To study efficient surface expression of Homo sapiens integrin αvβ3 in β3-integrin-deficient and HV-insusceptibl e China hamster ovary (CHO) cells, and to offer a technical support for the furt her study of cell entry of hantavirus mediated by β3 integrins. Meth ods The 2.3kb ORF region of Homo sapiens integrin β3 subunit cDNA w as amplified from the plasmid containing cDNA of human integrin β3 by PCR,the products with the blunt-ends were directly cloned into eukaryotic expression ve ctor pcDNA 3.1/V5-His TOPO by TA cloning. The integrin gene was then inserted i nto the plasmid vector by restriction enzyme and PCR and the sequence is identic al with the reported Homo sapiens integrin β3 ORF gene. After expression in euk aryotic vector, pcDNA3.1-β3, harboring ORF region of Homo sapiens integrin β3 subunit cDNA was constructed, and then transfected into CHO cells with eukaryot ic expression vector pcDNA3-αv harboring Homo sapiens integrin αv subunit c DNA individually or collectively. The exogenous gene expression was analyzed b y indirect immunofluorescence assay (IFA) or flow cytometer (FCM). Biosynthetic labeling experiments and Immunoprecipitation were performed. Results The eukaryotic expression vector, pcDNA3.1-β3, was successfully c onstructed. Effective surface expression of integrin αvβ3 was demonstrated by IFA and FCM in cotransfection, while weak surface expression was detected on tra nsfection with pcDNA3.1-β3 alone. In contrast, effective surface expression wa s not seen when pcDNA3-αv was transfected alone. The immunoprecipitation of ly sates from αvβ3 transfectants resulted in coprecipitation of β3 subunit with pro-αv distinctly and peculiarly. A post-translational cleavage of pro-αv t o yield αv heavy chain was also observed. Conclusion Eff icient surface expression of integrin αvβ3 depends on the presence of both sub units.