炭疽芽孢杆菌A16R株eag基因缺失突变株构建

【目的】构建炭疽芽孢杆菌A16R株eag基因缺失突变株, 为研究eag基因的功能奠定了基础。【方法】本研究以我国人用炭疽杆菌活疫苗A16R株中eag基因为目的缺失基因，根据炭疽芽孢杆菌Ames株基因组序列，利用软件设计了扩增上下游同源臂以及抗性基因引物，构建了重组质粒，将该重组质粒电击转入炭疽杆菌A16R感受态细胞中，利用同源重组原理筛选到炭疽杆菌A16R株eag基因缺失突变株。在分子水平及蛋白质组学方面对基因缺失突变株进行验证。【结果】成功构建了重组质粒，经同源重组后获得eag基因缺失突变株。PCR鉴定表明目的基因已经丢失；SDS PAGE表明野生株与突变株在93 KDa处有差异蛋白条带，经质谱鉴定分析该条带为目的基因所表达的EA1蛋白；双向电泳结果显示突变株与野生株比较明显缺失3个蛋白点，经质谱分析后确定这3个点都是EA1蛋白。【结论】成功获得炭疽芽孢杆菌A16R株eag基因缺失突变株，为深入研究eag基因的功能奠定了基础，同时也为炭疽芽孢杆菌重要基因功能的研究建立了一个良好的技术平台。

Construction of eag deletion mutant of Bacillus anthracis vaccine strain A16R

Objective] Construction of eag deletion mutant of Bacillus anthracis vaccine strain A16R. Methods] To study the function of the gene eag of Bacillus anthracis vaccine strain A16R, according to the sequence of Bacillus anthracis Ames strain, we designed primers and constructed a recombinant plasmid by the spectinomycin resistance cassette, upstream homologous fragment and downstream homologous fragment of eag cloned in tandem in pKSV7. We introduced the recombinant into A16R by electroporation and screened the mutant using the principle of homologous recombination. We checked the mutant using the PCR and proteomics. Results] We constructed the recombinant plasmid successfully and got the eag deletion mutant. PCR results showed the gene eag was deleted; SDS PAGE showed evident differences between prime strain and mutant strain. Two-dimensional gel electrophoresis results displayed three protein points identified as EA1 by mass chromatographic analysis presented in prime strain had absented from the mutant strain. Conclusion] We constructed eag deletion mutant of Bacillus anthracis vaccine strain A16R?. This research will be helpful to study the functions of eag gene and the functions of the important genes of Bacillus anthracis.