101K被膜蛋白____

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

http://www.100md.com 《齐鲁医学杂志》 2006年第1期

101K被膜蛋白,,],疱疹病毒6型，人；101K被膜蛋白；基因表达,1材料和方法,2结果,3讨论,[参考文献]

     [摘要] 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6B 101K被膜蛋白的主要抗原决定簇区(666～834aa)编码基因片段(nt2 347～2 853)，并导入T载体进行测序，与GenBank中HHV6标准毒株序列比较以进一步鉴定。目的基因及表达载体pThioHis A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，SDS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31 900的融合蛋白条带。结论HHV6 101K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。

    [关键词] 疱疹病毒6型，人；101K被膜蛋白；基因表达

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

    YAN LIPING, WANG YUN, WANG XIAOFENG, et al

    (Department of Clinical Laboratory, The Second Affiliated Hospital of Qingdao University Medical College, Qingdao 266021, China)

    [ABSTRACT]ObjectiveTo construct prokaryotic expression clones of human herpes virus 6 (HHV6) 101K protein genes.MethodsA 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 prokaryotic expression plasmid was confirmed by PCR and restricted endonuclease analysis and the recombinant protein was confirmed by SDSPAGE. ResultsThe 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.ConclusionThe successful construction of recombinant prokaryotic expression plasmid and expression of the recombinant protein provides valuable information for the diagnosis of active HHV6 infection. ......

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