人整合素β3亚基真核表达载体的构建及表达

The Construction and Expression of Eukaryotic Expression Vector Harboring Homo Sapiens Integrin beta 3 ORF Gene Segment

构建人整合素β3亚基全读码框(ORF)基因真核表达载体,为探讨整合素β3作为汉坦病毒(Hantavirus,HV)受体的特异性奠定基础。根据已公布的序列设计引物。用PCR方法从原核质粒中扩增出人整合素分子β3亚基ORF基因.应用TA克隆将其插入pcDNA3.1/V5-His-TOPO载体中,采用酶切和PCR鉴定,选取初筛正向插入的阳性克隆进行测序,序列分析表明与公布的人整合素β3亚基ORF核苷酸序列基本一致,并通过脂质体介导转染至CHO细胞中进行瞬时表达,经间接免疫荧光法检测证实pcDNA3.1-β3能在宿主细胞中高效表达。

To study the cellular entry of Hantavirus, we constructed dukaryotic expression vectors harboring Homo sapiens integrin β3 gene. The 2.3kb ORF region of human integrin β3 subunit cDNA was amplified from the plasmid containing cDNA of human integriri β3 by PCR. Then the PCR products with the blunt-ends were directly cloned into eukaryotic ecpression vector pcDNA 3.1/V5-His TOPO by TA Cloning. It shows that the integrin gene was inserted into the plasmid vector by restriction enzyme analysis and PCR, and the sequence is identical with the reported human integrin β3 ORF gene, then the recombinant expression plasmids were transfected into CHO cells in vitro. The transient expression products were analyzed by IFA. The results above suggested that the eukaryotic expression vectors, pcDNA3.1-β3, has been successfully constructed and the expressed protein could be detected distinctly and peculiarly. It lies a basis for the further study of hantavirus pathogenicity and biological characteristics.