羊型布氏菌WbkC基因真核表达质粒的构建及鉴定

Construction and Identification of Eukaryotic Expression Vector for WbkC Gene of Brucella melitensis

目的构建羊型布氏菌脂多糖O-侧链合成必需的甲酰基转移酶WbkC基因真核表达质粒,并进行鉴定。方法提取16M株羊型布氏菌基因组DNA,PCR扩增WbkC基因,克隆至psos载体上,构建诱饵重组质粒psos-WbkC,转化酵母菌株cdc25Hα,进行菌落PCR鉴定,并检测其在酵母双杂交系统中的自激活和定位情况。结果诱饵重组质粒psos-WbkC经PCR双酶切及测序证明构建正确;重组酵母菌经PCR鉴定,可扩增出约780 bp的目的基因;诱饵质粒在酵母双杂交系统中无自激活且定位正确。结论已构建了羊型布氏菌WbkC基因真核表达质粒psos-WbkC,可应用于酵母双杂交系统中,用于寻找巨噬细胞cDNA文库中与WbkC相互作用的蛋白质。

Objective To construct and identify the eukaryotic expression vector for WbkC gene necessary for synthesis of O-chain of lipopolysaccharide（LPS） of Brucella melitensis.Methods Genomic DNA of B.melitensis 16M strain was extracted,from which WbkC gene was amplified by PCR and cloned into vector psos.The constructed recombinant plasmid psos-WbkC as a bait was transformed to Saccharomyces cerevisiae cdc25Hα.The colonies were identified by PCR,and the auto-activation and location of recombinant plasmid psos-WbkC in yeast two-hybrid system were determined.Results PCR,restriction analysis and sequencing proved that recombinant plasmid psos-WbkC as a bait was constructed correctly.The target gene at a length of about 780 bp was amplified from recombinant S.cerevisiae by PCR.Recombinant plasmid psos-WbkC showed no auto-activation in yeast two-hybrid system,and was located correctly.Conclusion The eukaryotic expression vector psos-WbkC for WbkC gene of B.melitensis was constructed,which might be used for screening the proteins interacting with WbkC from macrophage cDNA library.