关键词: 副溶血弧菌, ompK, 多克隆抗体, 免疫磁珠

Abstract: It was to construct recombinant gene cloning and express the membrane surface protein outer the membrane protein K(ompK)of Vibrio parahaemolyticus, developing a method to detect Vibrio parahaemolyticus via immunomagnetic separation combined with color plate method. Using Primer 5 to design primers of ompK gene, the ompK gene was amplified by PCR subsequently and cloned into pET28a(+)prokaryotic expression vector, optimally transferred into E. coli BL21 to express. The expression products were purified by Nickel column and immunized mouse to prepare polyclonal antibody. Western blotting analysis was applied to identify the recombinant protein and ELISA was used to analyze its immunogenicity. The titer and cross reactivity of the polyclonal antibody were determined using indirect ELISA, then the polyclonal antibodies we prepared were coupled with protein G immunomagnetic beads to develop a chromogenic plating detection of Vibrio parahaemolyticus. The sensitivity of immunomagnetic beads reached to 10⁴ CFU/mL. We managed to clone and express Vibrio parahaemolyticus outer membrane protein ompK, in addition, we prepared mouse anti-Vibrio parahaemolyticus polyclonal antibody to create a new detection method using immunomagnetic beads, this method which combined with color plate method could save 72 hours comparing with traditional enrichment method, all process only need one-third of the time of traditional methods. When compared to the common immune detection methods, it was two magnitudes more sensitive.

Key words: Vibrio parahaemolyticus, ompK, Polyclonal antibody, Immunomagnetic beads