副溶血性弧菌是广泛存在于水产品中的革兰阴性致病菌。鞭毛蛋白FlaA是副溶血性弧菌的一种特异性的蛋白质,以该蛋白质作为抗原所制备到的抗体可以作为检测副溶血性弧菌的探针。该研究通过分子生物学方法构建了重组FlaA的表达载体pET28a-flaA,并采用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导实现FlaA的大量表达。该重组蛋白以可溶性形式存在,经Ni-NTA亲和柱纯化后对家兔进行免疫,得到浓度为24.32 mg/mL、效价为1∶125,000的抗FlaA抗体。通过间接ELISA实验,测得该抗体与副溶血性弧菌反应的灵敏度为1.0×104CFU/mL,且该抗体与5种其他常见致病菌无明显交叉反应。

The Gram-negative bacterium Vibrio parahaemolyticus is a common pathogenic bacteria in aquatic products. Flagellin A,which is a specific protein in this strain,plays an important role during infection and induction of the host immune response. The polyclonal antibody against FlaA protein can be used as a probe in V. parahaemolyticus fast detection. In this study,the flaA gene from V. parahaemolyticus was amplified and the recombinant plasmid pET28a-flaA was constructed for production of FlaA. The FlaA was then expressed recombinantly with induction by isopropyl β-D-1-thiogalactopyranoside( IPTG). The soluble FlaA protein was purified by Ni-NTA affinity chromatography and the polyclonal antibody against FlaA was prepared by immunning rabbit. The final concentration of FlaA antibody was 24. 32 mg / mL with the titer of 1∶ 125,000. The ELISA estimation result showed that the FlaA polyclonal antibody had a sensibility of 1. 0 × 104CFU / mL. The FlaA polyclonal antibody showed no cross-reaction with 5 other kinds of pathogenic bacteria.