为获得毒力较弱且能够区分疫苗免疫与自然感染的羊种布鲁氏菌候选疫苗株,本研究构建羊种布鲁氏菌Rev.1疫苗株VirB12基因缺失突变株.分别扩增Rev.1疫苗株VirB12基因上下游同源臂序列以及卡那霉素抗性基因,采用融合PCR方法将3个基因片段连接构建突变盒,连接至pMD 19-T载体,电转化入布鲁氏菌Rev.1感受态细胞筛选阳性克隆,获得Rev.1-AVirB 12突变株.Rev.1-AVirB 12连续传代15代未发生回复突变.羊种布鲁氏菌疫苗株Rev.1-AVirB12的构建为羊种布鲁氏菌疫苗研制奠定了基础.%To obtain a candidate vaccine strain of Brucella which was less virulent and capable of distinguishing between vaccine immunity and natural infection,a VirB12 gene deletion mutant of Brucell amelitensis Rev.1 vaccine strain was constructed.The upstream and downstream homology arms of VirB12 were fused with Kanamycin resistance gene cloned from pUC-19K by fusion PCR.The pMD-kan-VirB12 constructed with T vector was introduced into Brucell amelitensis Rev.1 by electroporation,and Rev.1-AVirB 12 mutant was obtained.Loss of the gene encoding VirB 12 and replacement of the marker gene were confirmed within 15 serial passages.The research showed that Rev.1-AVirB 12 mutant strain will provide fundamental basis for developing a new vaccine of Brucella melitensis.
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