摘要:目的 构建携带炭疽芽孢杆菌保护性抗原(PA)的两种穿梭载体与上游强启动子。 方法 将解淀粉芽孢杆菌的α-淀粉酶启动子克隆到载体pbluescript-sk(+)上，构建载体pBLKSP;以A16R疫苗株(Tox+，Cap-，弱毒株)DNA为模板，设计合成内外侧引物巢式PCR扩增获得保护性抗原(PA)的全基因，先克隆到载体pBLKSP，再将其和质粒PUB110重组构建成两种穿梭载体。 结果 酶切鉴定显示所切下的片段大小均与预计相符。测序结果与文献报道序列及预计结果一致。 结论 成功构建了带有强启动子的两种穿梭载体。为在无毒炭疽疫苗株中的高效表达和炭疽芽孢杆菌的分子疫苗研究奠定了基础。

    关键词:炭疽芽孢杆菌;穿梭载体;重组;基因

    Construction of shuttle vector of anthrax toxin protective antigen with upstream strong promoter.

    LIU Rong-na，HUANG Han-ju，XU Jing-hua.

    (Department of Pathogenic Organism of Tongji Medical College Elementary School，Huazhong Science and Technology Unversity，Wuhan430030，Hubei，P.R.China)

    Abstract:Objective To construct two shuttle vector of anthrax toxin protective antigen with upstream strong promoter. Meth-ods The promoter region of theα-amylase gene of Bacillus amyloliquefacienswas cloned into pbluescript-sk(+)vector;the an-thrax toxin protective antigen whole gene was amplified by nested PCR from A16vaccine strain template.Firstly it was cloned into pBLKSP vector，then recombinated with PUB110vector to form two kinds of shuttle vectors. Results Restriction analysis and DNA sequencing confirmed that the cloned gene fragments encoded anthrax toxin protective antigen. Conclusion Two kinds of shuttle vectors have been successfully constructed.it has set foundation for further study on the highly effective expression in nontoxic an-thracis strain and molecule vaccine research of Bacillus anthracis. ......