人疱疹病毒6型101K被膜蛋白主要抗原决定簇区原核表达克隆的构建和表达

CONSTRUCTION AND EXPRESSION OF PROKARYOTIC EXPRESSIVE CLONE OF HUMAN HERPES VIRUS 6 101K TEGUMENT PROTEIN MAJOR ANTIGEN EPITOPES

目的 构建人疱疹病毒6型（HHV-6）101K被膜蛋白的原核高效表达克隆，并进行原核表达。方法 PCR扩增HHV-6B101K被膜蛋白的主要抗原决定簇区（666～834aa）编码基因片段（nt2347～2853），并导入T载体进行测序，与GenBank中HHV6标准毒株序列比较以进一步鉴定。目的基因及表达载体pThioHisA分别经双酶切后连接，转化宿主菌E．coli BL21，应用PCR法及限制性核酸内切酶双重鉴定原核表达质粒，并经测序证实。IPTG诱导蛋白表达，SD＆PAGE电泳鉴定重组蛋白。结果 PCR获得的目的基因序列与GenBank中HHV-6B标准毒株Z29序列一致，重组质粒诱导菌表达产物出现相对分子质量为31900的融合蛋白条带。结论 HHV-6101K蛋白的原核表达克隆构建和表达成功，为进一步完善HHV-6活动性感染的血清学诊断提供了实验基础。

Objective To construct prokaryotie expression clones of human herpes virus 6 （HHV-6） 101K protein genes. Methods A fragment coding for HHV-6 101K major antigen epitopes （666-843 aa） was amplified by PCR technique. The PCR products were cloned into TA cloning vector for sequencing and subsequently compared with the sequence of HHV-6B Z29 strain in GeneBank. The target gene was inserted into the prokaryotic expression plasmid pThioHis A. After the transformation of E. coli BL21, the recombinant plasmid was induced with IPTG to express the 101K fusion proteins. Recombinant prokaryotie expression plasmid was confirmed by PCR and restricted endonuclease analysis and the recombinant protein was confirmed by SDS-PAGE. Results The sequence of the target gene was identical with that of HHV-6B Z29 strain in GenBank. The relative molecular weight 31 900 fusion proteins were expressed in E. coli BL21. Conclusion The successful construction of recombinant prokaryotic expression plasmid and expression of the recombinant protein provides valuable information for the diagnosis of active HHV-6 infection.