布鲁氏菌分泌蛋白BMCO基因缺失株的构建及生物学特性研究

Construction and Biological Characteristics of Brucella Secreting Protein BMCO Gene Deletion Strain

【目的】试验旨在构建牛种布鲁氏菌S2308的多铜氧化酶(BMCO)基因缺失株,探究缺失株的生长特性及在宿主细胞中的存活能力,并分析BMCO蛋白的结构。【方法】利用PCR扩增BMCO基因上、下游同源臂和Kan基因,通过融合PCR技术将3段基因进行融合。融合片段与pMD19-T载体连接,制作牛布鲁氏菌S2308感受态细胞,1800 V电压、400Ω电阻电转化至牛种布鲁氏菌S2308感受态细胞中,涂布于Kan抗性的布鲁氏菌固体培养基中,筛选挑取阳性菌落,连续培养10代,检测第10代阳性菌落的遗传稳定性和生长变化趋势,将pBBR1MCS-4-BMCO融合质粒电转至能够稳定遗传BMCO基因缺失的牛种布鲁氏菌中,培养筛选阳性菌落。以感染复数(MOI)100分别用亲本株、缺失株、回补株侵染小鼠巨噬细胞RAW264.7,通过平板计数法分别检测3种菌株在细胞内的生存能力。【结果】试验成功获得片段大小为522、539、1054 bp的BMCO基因上、下游同源臂和Kan基因;成功构建pMD19-T-BMCO-Kan融合片段重组载体;获得稳定遗传的BMCO基因缺失株,命名为S2308ΔBMCO;成功构建BMCO基因回补株,命名为ΔBMCO::BMCOS2308;缺失株和亲本株表现的生长曲线相同,均在12 h达到对数生长期,30 h进入平台期;侵染小鼠巨噬细胞RWA264.7后,与亲本株S2308相比,S2308ΔBMCO株在胞内的生存能力极显著降低(P<0.01)。生物信息学分析结果表明,BMCO属于疏水蛋白,定位于胞质且含有大量的无规则卷曲、α-螺旋和延伸链,预示该蛋白具有多个结合位点。【结论】本研究成功构建了布鲁氏菌BMCO基因的缺失株和回补株,BMCO基因的缺失不影响其生长性能,但其在宿主细胞内的存活能力显著性降低,初步分析了BMCO蛋白的结构,为后续布鲁氏菌分泌蛋白的功能研究奠定基础。

【Objective】The purpose of this study was to construct the multi copper oxidase(BMCO)gene deletion strain of Brucella bovis S2308,explore the growth characteristics of the deletion strain and its viability in host cells,and analyze the structure of BMCO protein.【Method】The upstream and downstream homologous arms of BMCO gene and Kan gene were amplified by PCR,and the three genes were fused by fusion PCR.The fusion fragment was connected with pMD19-T vector to make Brucella bovis S2308 competent cells.1800 V voltage and 400Ωresistance were transferred to Brucella bovis S2308 competent cells and coated in Kan-resistant Brucella solid medium.Positive colonies were selected and cultured continuously for 10 generations.The positive genetic stability and growth trend of the 10th generation positive colonies were detected.The pBBR1MCS-4-BMCO fusion plasmid was electrotransferred into Brucella bovis which could stably inherit BMCO gene deletion,and the positive colonies were cultured and screened.The mouse macrophage RAW264.7 was infected with parent strain,deletion strain and supplementary strain with plural infection with MOI 100.The viability of the three strains in the cells was detected by plate counting method.【Result】The results showed that the upstream and downstream homologous arms of BMCO gene and Kan gene with fragment size of 522,539 and 1054 bp were successfully obtained,the recombinant vector of pMD19-T-BMCO-Kan fusion fragment was successfully constructed,the stable genetic deletion strain of BMCO gene was obtained,named S2308ΔBMCO,and the complementary strain of BMCO gene was successfully constructed,namedΔBMCO::BMCOS2308.The growth curve of the deleted strain was the same as that of the parent strain,which reached logarithmic growth phase at 12 h and entered the plateau phase at 30 h.After infecting mouse macrophage RWA264.7,compared with the parent strain S2308,the viability of the S2308ΔBMCO was extremely significantly decreased(P<0.01).Bioinformatics analysis showed that BMCO was a hydrophobic protein,located in the cytoplasm and contained a large number of random coil,alpha helix and extended chain,indicating that the protein had multiple binding sites.【Conclusion】The deletion and replacement strains of BMCO gene of Brucella were successfully constructed.The deletion of BMCO gene did not affect its growth performance,but its viability in host cells decreased significantly.The structure of BMCO protein was preliminarily analyzed,which would lay a foundation for the further study of the function of protein secreted by Brucella.