流产布氏杆菌rfbD基因缺失株的构建及其生物学特性

Construction and biological characterization of rfbD gene-deleted Brucella abortusmutant strain ΔrfbD

为了研究流产布氏杆菌rfbD基因缺失株的生物学特性和毒性变化,采用同源重组的方法构建该缺失株。以pDS132质粒为模板扩增sacB片段,通过NdeⅠ酶切位点,用T4连接酶将sacB片段与pUC19质粒连接,获得自杀质粒SacB-pUC19;用重组PCR方法扩增rfbD ORF内缺失668bp的缺失突变核,通过PstⅠ、SmaⅠ酶切位点,用T4连接酶将ΔrfbD缺失突变核与自杀质粒SacB-pUC19连接,获得突变载体SacB-pUC19-ΔrfbD;将SacB-pUC19-ΔrfbD电转化至流产布氏杆菌S2308感受态细胞中,运用sacB反向筛选构建流产布氏杆菌S2308 rfbD无痕突变株,并分析其在巨噬细胞RAW264.7和BALB/c小鼠体内的存活能力。结果显示,成功构建了流产布氏杆菌S2308 rfbD基因缺失株,该突变株在巨噬细胞和小鼠体内毒力减弱,存活能力下降,证明rfbD基因是流产布氏杆菌的毒力相关基因。

The objective of this study was to construct and characterize rfbD gene-deleted mutant Brucella abortus strain S2308. The sacB gene was amplified from plasmid pDS132 using polymerase chain reaction（PCR）. The gene was then inserted into the plasmid pUC19 at the Nde t digestion site to construct suicide plasmid SacB-pUC19. The rfbD gene with deletion of a 668 bp in the ORF was amplified by PCR and then inserted into the suicide plasmid SacB-pUC19 at the Pst I and Sma I digestion sites to construct SacB-pUC19-△rfbD,which was further electronically transformed into S2308 competent cells. A mutant strain was constructed and investigated for the survival ability in macrophage RAW264. 7 and BALB/c mice. The results showed that a rfbD gene-deleted mutant was successfully constructed,and the virulence and survival ability in macrophages in vitro and in mice in vivo were reduced. It was concluded tk the rfbD gene was associated with the Brucella virulence.