摘要
目的 : 在大肠杆菌中表达重组单纯疱疹病毒 2型 (HSV - 2 )抗原。方法 : 用PCR方法从单纯疱疹病毒中扩增HSV - 2DNA片段 ,约 5 0 0bp ,克隆到pMD18T载体并对克隆的DNA片段进行序列分析。用限制酶EcoRI和BamHI消化pMD18T HSV - 2重组质粒 ,分离HSV - 2片段 ,并插入质粒表达载体pBV2 2 0的相应限制酶位点 ,酶谱分析鉴定重组表达载体pBV2 2 0 HSV - 2。转化菌株经 4 2℃诱导 ,用SDS-PAGE和Westernblot鉴定表达的重组蛋白。结果 : PCR扩增的DNA片段与HSV - 2的目的片段大小一致。重组质粒pMD18T HSV - 2的DNA序列分析显示克隆的DNA序列与文献报道的HSV - 2DNA序列一致。SDS -PAGE表明重组蛋白相对分子量为 180 0 0 ,表达量达菌体总蛋白的 2 0 %左右 ,Westernblot分析显示重组蛋白能特异地与抗HSV - 2抗体结合。结论 : 已成功构建了表达具有功能的重组HSV -
Objective:To clone and express recombinant HSV-2 antigen in E.coli.Methods: A HSV-2 DNA fragment with a length of about 500bp was amplified from the DNA of HSV strain by PCR and was cloned to plasmid pMD18T. And then the cloned DNA fragment was sequenced. The recombinant plasmid pMD18T/HSV-2 was digested with EcoRI and BamHI. HSV-2 fragment was isolated and inserted to the corresponding restriction site on prokaryotic expression vector pBV220. The recombinant plasmid pBV220/HSV-2 was identified by enzymogram and transformed to E.coli, and then expressed by induction at 42 ℃. The expressed product was identified by SDS-PAGE and Western blot.Results: The length of DNA fragment amplified by PCR was consistent with that of HSV-2 DNA. DNA sequencing of pMD18T/HSV-2 revealed that the cloned DNA sequence was identical to that of reported results. SDS-PAGE proved that the expressed product, with a relative molecular weight of 18000, and contained about 20% of total somatic protein. Western blot showed that the recombinant protein could specifically bind to anti-HSV-2 antibody. Conclusion:A recombinant bacterial strain for expressing HSV-2 was successfully constructed.
出处
《中国麻风皮肤病杂志》
北大核心
2004年第4期320-322,共3页
China Journal of Leprosy and Skin Diseases
