Expression Of Plc And Netb Of Clostridium Perfringens And Characterization Of Monoclonal Antibodies Against Plc And Netb Proteins

Clostridium perfringens had long been recognized as a significant cause of gastrointestinal diseases in human and domestic animals, which can occasionally be found in the environment. The bacteria caused a wide variety of diseases such as necrotic enteritis and enterotoxemia in a variety of animal species, gas gangrene and food poisoning in human. A wide array of enteric toxins produced by C. perfringens played major roles in the pathogenesis of these diseases.The plc gene encoding C. perfringens alpha toxin and the NetB gene excluding signal peptides were amplified by PCR, which were cloned into prokaryotic expression vector pGEX-6P-1, constructing two recombinant expression plasmids pGEX-plc and pGEX-NetB. The pGEX-plc and pGEX-NetB were transformed into E. coli BL21(DE3). The highest level expressions of recombinant protein were induced by IPTG at1.0mmol/L for5hours, mainly in inclusion form with molecular weight of66kDa and62kDa as certified by SDS-PAGE. The purified expressed proteins were used to prepare the monoclonal antibodies (McAbs)The proteins were purified by cutting gels and used to immunize BALB/c mice. The third day after boost immunization, spleen cells from the immunized BALB/c mice were fused with myeloma cell line SP2/0. By the indirect ELISA methods, there were nine and five hybridoma cell lines secreting specific monoclonal antibodies against plc and NetB, which were obtained and named H8C11, F4D9, F4C3, H11H12, F4G12, H8F8, A10H9, A10E5, G7H8and D6H8, F9G3, E7G2, E11C11, E7B11, respectively. The subclasses of A10E5and A10H9were IgG2a, the subclass of D6H8was IgG2b, and other McAbs were IgG1subclass. Western blot showed that all McAbs could react specifically with plc protein and NetB protein separately. A10H9and A10E5could react with alpha toxin of the culture supernatant of C. perfringens. Ascites of H8C11, F4D9, F4C3, D6H8, F9G3and E7G2were purified by caprylic acid-saturate ammoniumsulphate method, and SDS-PAGE analysis indicated that there were H chain and L chain obviously without visible mixed proteins. The titers of McAbs of ascites were higher than that of culture supernatant of hybridoma cells by indirect ELISA methods. And the hybridoma cells could stably secret McAbs after passaging. The results of counting chromosome number showed that the chromosome average number of the nine hybridoma cells against plc was91.1, and that of five hybridoma cells against NetB was88.2, much more than that of SP2/0, cell. The immunofluorescence assay showed that seven McAbs against plc could bind the bacteria surface. Alpha toxin neutralization experiment results indicated that all obtained McAbs against plc had alpha toxin-neutralizing activity.