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目的构建副溶血性弧菌三种主要溶血毒素(TDH、TRH和TLH)的重组融合表达体系,给后续这三种溶血毒素的大量获取及其特异性抗体的制备、副溶血性弧菌的致病机理等研究提供基础材料。方法首先将副溶血性弧菌标准菌株tdh、trh与tlh基因分别接入克隆载体pGM-T,并转入E.coliTOP10,进行BamHI、HindIII双酶切鉴定和克隆测序。然后从重组克隆质粒中将tdh、trh与tlh基因目的片段分别双酶切出,将其连接到带His标签的表达载体pET30a(+)中后转入E.coliBL21(DE3)中,先通过BamHI、HindIII双酶切以鉴定所构建表达载体的准确性。接着重组表达菌株经IPTG诱导表达,利用SDS-PAGE电泳和Western-blotting来分析鉴定表达结果。结果表达产物的SDS-PAGE电泳结果显示TDH在29000、TRH在30000、TLH在52000附近处出现明显的表达蛋白条带。利用His标签的特异性抗体进行Western-bloting分析,实验结果显示29000、30000、52000附近处的表达蛋白与His抗体均有特异性反应。结论成功构建了TDH、TRH和TLH溶血毒素的原核表达体系,为进一步免疫原性和特异性抗体制备奠定基础。
Objective To construct a recombinant fusion expression system of three major hemolytic toxins (TDH, TRH and TLH) of Vibrio parahaemolyticus, and to obtain large amounts of subsequent three hemolytic toxins and preparation of their specific antibodies, Pathogenesis and other research to provide basic materials. Methods Firstly, the standard strains of Vibrio parahaemolyticus, tdh, trh and tlh, were cloned into the cloning vector pGM-T and transformed into E. coli TOP10 respectively for restriction enzyme digestion and cloning of BamHI and HindIII. Then, the target fragments of tdh, trh and tlh genes were double-digested respectively from the recombinant cloning plasmids and ligated to the His-tagged expression vector pET30a (+) and then transformed into E. coli BL21 (DE3) , HindIII double digestion to identify the construction of the expression vector accuracy. Then the recombinant expression strain was induced by IPTG, and the expression was confirmed by SDS-PAGE electrophoresis and Western-blotting. Results The results of SDS-PAGE showed that TDH was 29000, TRH was 30000 and TLH was obviously expressed in the vicinity of 52000. His-tag-specific antibodies were used for Western-bloting analysis. The results showed that the expressed protein at 29000, 30000 and 52000 reacted specifically with His antibody. Conclusion The prokaryotic expression system of TDH, TRH and TLH hemolytic toxin was successfully constructed, which laid the foundation for further immunogenicity and specific antibody preparation.