【摘要】 目的:分离、克隆人类疱疹病毒8型(HHV-8)包膜糖蛋白K8.1编码基因,并置于大肠杆菌中作融合表达。方法:设计一对PCR引物,在引物的5’端分别引入BamHⅠ、XhoⅠ酶切位点,以佛波酯(TPA)刺激的原发性渗出性淋巴瘤(PEL)细胞系BCBL-1细胞总DNA为模板,PCR扩增出K8.1编码基因,克隆至原核表达载体pGEX-6p-1中,构建含K8.1基因重组质粒。重组质粒转化大肠杆菌(E.coli)BL21感受态细胞,以异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白。结果:分离、克隆的411bp序列与基因数据库中所登记的HHV-8K8.1序列呈现100%同源性。IPTG诱导后的菌体,经SDS-聚丙烯酰胺凝胶电泳(PAGE),显示有一个41ku的融合表达蛋白产生。结论:HHV-8K8.1编码基因在E.coli中初步获得正确表达。更多还原

【Abstract】 Objective: To isolate and clone the envelope glycoprotein K8.1 co di ng gene of human herpesvirus 8(HHV-8)and evaluate the expression of K 8.1 in E.coli. Methods: A pair PCR primers for K8.1 gene was designed, which was engine ered the cut sites of BamHⅠand XhoⅠon the 5′ends to facilitate directional cl oning. K8.1 gene was amplified by PCR taking the total DNA of BCBL-1 cells, whi ch was obtained from primary effusion lymphoma(PEL)after stimulation of 12-O -tetradecanoylphorbol-13-acetate(TPA), as template. Then, PCR product was i nserted into plasmid pGEX-6p-1 to construct the recombinant expression plasmid named pGEX-6p-1+K8.1. The recombinant plasmid was transformed to E.coli comp etent cells BL21(DE3)and the expression of fusion protein was induced by isopr opyl-β-D-thiogalactoside(IPTG). Results: The result has demonstrated that a 411-base-pair(bp)sequence isolated in this study was 100%homology with K8 .1 gene of HHV-8,which was previously registered in GenBank. And a 41 ku of fu sion protein was exhibited on a 12%of sodium dodecyl sulfate-polyacrylamide ge l electrophoresis(SDS-PAGE)when the recombinant bacteria was induced by IPTG. Conclusion: The envelope glycoprotein K8.1 coding gene of HHV-8 was correctly expressed in E.coli BL21 cells. [更多还原