人巨细胞病毒包膜糖蛋白gB/AD-1片段的基因克隆和原核表达

目的克隆人巨细胞病毒包膜糖蛋白gB/AD-1片段并构建其原核表达系统。方法用PCR法从人巨细胞病毒AD169株基因组DNA中扩增gB/AD-1片段并克隆至pGEM-T载体,酶切后,亚克隆至表达载体pET-15b,构建重组表达质粒pET-15b/gB/AD-1,并转化大肠杆菌,IPTG诱导表达。表达产物经金属螯合层析一步纯化,并用Westernblot鉴定。结果gB/AD-1蛋白表达量达到35%。表达产物经纯化后,纯度大于95%。经Westernblot检测,表达产物与CMV阳性人血清发生特异性反应。结论已成功构建重组表达载体pET-15b/gB/AD-1,并在大肠杆菌中高水平表达gB/AD-1。

Gene Cloning and Prokaryotic Expression of Human Cytomegalovirus Glycoprotein gB/AD-1

Objective To clone the gene fragment gB/AD-1 encoding the glycoprotein of human cytomegalovirus(HCMV) and construct its prokaryotic expression system.Methods Amplify gB/AD-1 gene fragment from the genomic DNA of HCMV AD169 strain and clone into vector pGEM-T,then,after identification by sequencing,subclone to expression vector pET-15b. Transform the constructed recombinant plasmid pET-15b/gB/AD-1 to E.coli for expression of gB/AD-1 under induction of IPTG.Purify the expressed product by one-step immobilized metal ion affinity chromatography(IMAC) and identify by Western blot.Results The expressed gB/AD-1 contained 35% of total somatic protein.The purity of expressed product after purification was more than 95%.Western blot showed specific reaction of the expressed product with CMV antibody-positive human serum.Conclusion The recombinant plasmid pET-15b/gB/AD-1 was successfully constructed,and gB/AD-1 was highly expressed in E.coli.