副溶血性弧菌Vop T蛋白的表达及其抗体的毒力菌株特异性研究

Expression of Vibrio parahaemolyticus Vop T protein and the specificity of its antibody against virulent strain

目的研究副溶血性弧菌VopT蛋白的菌群特异性,为建立致病性副溶血性弧菌的快速检测方法以及探讨VopT的作用机制奠定基础。方法根据副溶血性弧菌VopT蛋白基因(VPA1327)序列设计合成引物,通过PCR从副溶血性弧菌致病株WX06152(血清型O3∶K6)中扩增出目的基因,构建原核表达载体pET-30a(+)-VPA1327并转化大肠杆菌BL21(DE3)。经IPTG诱导表达,重组蛋白应用MALDI-TOF-MS/MS鉴定和His镍柱纯化,并通过免疫BALB/c小鼠制备抗血清,建立副溶血性弧菌VopT蛋白的间接ELISA检测方法,并分析其敏感性和毒力菌株特异性。结果成功表达了大小为33kDa融合蛋白,肽指纹图谱与副溶血性弧菌VopT蛋白一致。重组VopT抗血清能够特异性检测副溶血性弧菌毒力株的全菌细胞及其裂解蛋白,与环境株及其它种属菌株无明显交叉反应,灵敏度达到105 CFU·mL-1。结论重组VopT的抗血清具有良好特异性,为探讨VopT致病机理和分泌机制研究提供了一定的参考依据。

The aim of this study is to determine group specificity of Vibrio parahaemolyticus Vop T protein and provide bases for establishing a rapid detection method and exploring the mechanism of VopT action.The gene VPA1327 which encode Vop T protein was amplified from pathogenic V.parahaemolyticus strain WX06152(serotype O3∶K6)by PCR using apair of synthesized primers.The resulted amplicon in the size of 711 bp was subcloned into vector pET-30a(+),and the resulted recombinant expression plasmid pET-VPA1327/DE3 was transformed into the E.coli BL21(DE3).After IPTG induction,a recombinant protein was identified using MALDI-TOF-MS/MS spectrum and purified by His affinity column.The antiserum against the VopT was successfully obtained by immunization of the purified recombinant protein to BALB/c mice,and an indirect ELISA for rapid detecting VopT protein of pathogenic V.parahaemolyticus was developed using the antiserum as the detective antibody.The result showed that the obtained fussion protein,in the size of 33 kDa,showed the same peptide fingerprint with the V.parahaemolyticus VopT protein.The developed indirect ELISA method can specifically detect bacterial cell and its lysate of V.parahaemolyticus with the lowest detective limit of 105 CFU mL-1.The result of crossing test showed that the VopT antiserum had good specificity against virulent strains of Vibrio parahaemolyticus,and no cross-reaction observed with environmental strains of V.parahaemolyticus and selected bacterial strains belonging to other species.The data present in this study provides a new way to understand VopT action mechanism of pathogenic V.parahaemolyticus strains.