人类8型疱疹病毒K8.1基因原核表达重组质粒的构建

Constructure prokaryotic expression plasmid of KSHV K8.1 gene

目的:构建人类8型疱疹病毒(HHV-8)K8.1基因原核表达重组质粒,获得HHV-8外壳蛋白重组融合蛋白,为制备检测抗原建立平台。方法:根据基因库K8.1全长基因序列和pET41a载体的多克隆位点设计引物,分别在引物两端添加特异酶切位点,以pGEM-Teasy/K8.1质粒为模板,PCR扩增K8.1基因全长序列,克隆入原核表达载体pET-41a,构建原核表达质粒pET41a-K8.1;转化大肠杆菌DH5α,经酶切、测序鉴定其插入序列的正确性。结果:酶切鉴定表明在约500bp处可见酶切片段,测序结果表明构建的pET41a-K8.1原核表达质粒连接正确,插入的K8.1基因片断为494bp。结论:成功构建了pET41a-K8.1原核表达质粒,为获得重组融合HHV-8外壳蛋白建立了平台。

Objective： To construct the prokaryotic expression plasmid for pET41a-K8.1. Methods： Using recombinant plasmid pGEM Teasy/K8.1 as the template, EcoR Ⅰ and Xho Ⅰ restriction digestion sites were amplified by PCR and subsequently inserted into prokaryotic vector, pET-41a. The recombinant plasmid was transformed into E. coli BL21 （DE3） and then identified by double enzyme digesting and sequencing. Results： The insert target DNA was 494 pb, EcoR Ⅰ and Xho Ⅰ restriction digestion identified that there was DNA band at about 500 pb position, sequencing showing the insert DNA was connected correctly.Conclusion： Recombinant plasmid pET41a-K8.1 is successfully constructed. Further study is needed to identify its immunity.