第39 卷 第4 期

    2003 年12 月

    青 岛 大 学 医 学 院 学 报 ACTA ACADEMIAE MEDICINAE QIN GDAO UNIVERSITATIS

    Vol . 39 , No. 4

    December 2003

    [收稿日期] 2003206213 ; [修订日期] 2003207220

    [作者简介] 朱元祺(19622) ,男,硕士,主管检验师。

    HIV21 SF2 株env基因 C1 与 C3 区的嵌合表达

    朱元祺1

    ,姜 岩2

    ,王 斌3

    ,钱冬萌3

    (1 青岛市第五人民医院检验科, 山东 青岛 266002 ; 2 青岛市胸科医院 检验科; 3 青岛大学医学院微生物学教研室)

    [摘要] ①目的 构建 HIV21 SF2 株 env 基因 C1 与 C3 区嵌合片段的克隆并在大肠杆菌中表达。②方法

    用 DNAstar 软件辅助设计引物 ,以含有 HIV21 SF2株前病毒基因组的 9B1R6 质粒为模板 ,PCR法分 别扩增出 env

    基因 C1和 C3段;两个片段经 KpnⅠ酶切、 T4连接酶连接和扩增后 ,再将经 EcoR Ⅰ、 Sal Ⅰ双酶切的嵌合片段插入

    表达载体p GEX24T22 中 ,构建重组表达质粒p GEX24T22/ C1 C3 ;重组质粒经酶切、测序鉴定后 ,以 TSS法转入大肠

    杆菌 DH5 α, IPTG诱导表达融合 蛋白和 SDS 2PAGE电泳分析表达蛋白图谱。③结果 经酶切和测序 分析 ,插入片

    段大小、方向和读码框架正确 ,无碱基错配 ,表明成功构建p GEX24T22/ C1C3 克隆;重组克隆在大肠杆菌 DH5 α中经

    IPTG诱导 ,高效表达出相对分子质量为 4. 3 万的 融合蛋白。④结论 重组质粒p GEX24T22/ C1C3 的构建和表达

    成功 ,为进一步研究该表达蛋白的活性和 HIV 疫苗的研制奠定了基础。

    [关键词] HIV21 ;env基因;基因表达;重组融合蛋白质类 ;质粒

    [中图分类号] R373. 9 [文献标识码] A [文章编号] 167224488 (2003) 0420421203

    CLONING AND EXPRESSIONOF THELINKED FRAGMENTOF C1 AND C3 CONSTANT REGIONS OF ENVELOPE GENE FROM

    HIV 21 S F2 STRAIN IN E. col i DH5 α ZHU Y uan2qi , JIA N G Yan , WA N G Bi n , et al (Department of Laboratory , The Fifth

    Peoplep s Hospital of Qingd ao , Qingdao 266002 , China)

    [ ABSTRACT] Objective To const ruct the clone of the link ed f ragment of C1 and C3 constant regions of envelope gene f rom

    HIV21 SF2 st rai n and express it in E. coli DH5 α. Methods The recombinant plasmid 9B1R6 containing HIV21 proviral genome of

    SF2 st rain was chosen as the template. Two f ragments of C1 and C3 cons tant region of envelope gene were amplified by PCR with several

    primer pairs wit h DNAstar Software. The two amplified f ragments digested by KpnⅠwere then ligat ed by T4 ligase. The linked f rag2

    ment modified by EcoRⅠ and Sal Ⅰwas inserted int o the multiple cloning site (MCS) of glutathione S2t ransferase ( GST) of plasmid

    pGEX24T22. The recombinant plasmid (pGEX24T22/ C1C3) was identified by rest ri ction endonuclease assay and its sequence was deter2

    mined by dideoxy2chain termi nation method. Finally , pGEX24T22/ C1C3 was t ransformed by TSS method into E. c oli DH5 αand induced

    with Isopropyl2 β 2D2thioglactoside ( IPIG) to expr ess GST2C1C3 fusion protein in it . The expressed proteins were analyzed with S DS2

    PAGE elect rophoresis. Results The recombinant pGEX24T22/ C1C3 was obtained. The sequen ce analysis showed that its reading

    f rame was correct and no mismatched bases ha ppened. And the fusion protein of GST2 C1C3 was efficiently expressed in E. coli DH5 α, the

    relative molecular weight being about 43 ×103

    . Conclusion The successful const ruction and expression of the reco mbinant plasmid

    pGEX24T22/ C1C3 lay a foundation for the further study of the activity of the protein and future research on viral vaccine against HIV21.

    [ KEY WORDS] HIV21 ; gene , envelope ; gene expression ; viral fusi on proteins ; plasmid

    HIV21 的基因组(RNA)约为 9. 7kb ,与大多数

    逆转录病毒一样 ,主要结构蛋白的基因为 gag、 pol

    和env ......