Cloning,Expression And Polyclonal Antibody Preparation Of TcdC Gene From Clostridium Difficile

Clostridium difficile is a gram-positive anaerobic and one of the most important pathogenic bacteria in human intestinal infection.Using antibacterial agents can lead to C.diffcile hypertrophy,secreting a large amount of toxin A(enterotoxin)and toxin B(cytotoxictoxin)which lead to a series of Clostridium difficile infection symptoms.Recently,C.difficile in water environment and related environmental detection rate increased,and along with the present of high virulent strains,showed that the infection rates and incidence of C.difficile in aquatic organisms,especially the in aquaculture organisms is increasing.Therefore,it is very important to study the pathogenic mechanism of C.difficile in the prevention and control of aquatic diseases.The C.difficile infection has become a global health problem.TcdA and B toxins in C.difficile are two important factors leading to diseases in organism.The expression of tcdA and B genes is regulated by many factors located in the pathogenicity locus(PaLoc).So far,it has been still debatable whether tcdC gene is regarded as a negative regulator,and the mechanism of tcdC gene is still unclear.In this proposal,the C.difficile standard strain(ATCC43255)is chosen as the target.The codon optimized tcdC gene was synthesized in vitro using overlap extension PCR.The two epitopes from tcdC gene were cloned into prokaryotic expression vectors,and then the correct recombinant plasmids were transformed into E.coli.A polyclonal antibody was developed by immunizing rabbits with the purified TcdC protein.The expression of TcdC in C.difficile was analyzed with the polyclonal antibody.The main results were as follows:(1)Codon optimization of the tcdC was performed,followed by analysis of codon relative adaptiveness based on relative frequency of synonymous condon(RFSC)by E.coli.The full length of tcdC fragment was synthesized in vitro using overlap extension PCR.(2)By molecular cloning methods,we constructed prokaryotic expression plasmid pBVGST-S(×2)?pBVGST-L?pBVIL1-S(×2)and pBVIL1-L.In prokaryotic expression system,the plasmid was induced by 42? water,which can effectively expressed fusion protein.The results showed that the molecular mass of the targeted proteins were consistent with the expectations and mainly expressed as inclusion bodies.The purity of the fusion proteins was high after purified by ion exchange chromatography and affinity chromatography.(3)The purified recombinant proteins were used to immunize rabbits to obtain the rabbit against TcdC-specific antigens polyclonal antibodies,which titers were all above 1:64000.The SDS-PAGE of purified antibodies suggested that only 50 KD and 25 KD protein strips were appeared which was consistent with MW of IgG's H and L strand.Western blot showed there was strong reactivity and specificity between TcdC proteins and the polyclonal antibodies.The TcdC from CD ATCC43255 can be specifically recognized by TcdC-L polyclonal antibody.We demonstrate that TcdC is a membrane associated protein in C.difficile.We also found that the maximum levels of TcdC were found during the exponential phase of growth,and levels decreased as the culture approached stationary phase.In conclusion,TcdC proteins were expressed and the polyclonal antibody against TcdC was prepared successfully,which laid foundation for researching the function and mechanism of tcdC gene in pathogenicity locus(PaLoc).