The Typing Method Of Clostridium Perfingens By Multiplex PCR And The Cloning And Expression Of Its Alpha,Beta And Epsilon Toxin Genes And Preparation Of Antisera

Three aspects have been researched on the Clostridium perfringens in this study, including:Firstly, the multiplex PCR for the typing of Clostridium perfringens were established. Secondly, the Alpha,Beta and Epsilon toxins' gene of Clostridium perfringens were cloned and expressed. Thirdly, the specific antisera of the Alpha,Beta and Epsilon toxin were preparated with the recombinant proteins.Four pairs of specific primers forα,β,εandιtoxins' gene were designed in terms of the toxin's gene sequences published in GenBank. The multiplex PCR have been establishecd for the typing of the Clostridium perfringens, the amplifing segments is 202bp,403bp,585bp and 291bp respectively . 140 strains of Clostridium perfringens which are conserved in CVCC(China Veterinary Culture Collection Center) were typed with the multiplex PCR,at the same time, the typing results with the multiplex PCR were validated by the Toxin-Antitoxin Neutralization. As a result,the typing result with the two ways is accordant completely,it proved that multiplex PCR is exact and specific. The typing for Clostridium perfringens with the multiplex PCR is more quikly,simple and economy than the Toxin-Antitoxin Neutralization.Specific primers for genes coding complete mature peptides from Clostridium perfringens were designed in terms of the gene sequences published in GenBank. The segments of alpha, beta and epsilon toxins were proved with PCR ,which respectively is 1125bp,942bp and 918bp.With the technique of the gene's cloning and recombining, expression vectors ofα,βandεtoxin genes were constructed successfully, namedα-pET28a,β-pET32a andε-pET28a respectively. It prove that the insert position ,size of the segments and the reading frame are all correct with the PCR,the restriction digestion and the sequence analysis.The three expression vectors were transformed into the E.coli , which were namedα-pET28a (plys),β-pET32a (DE3) andε-pET28a (DE3) respectively, were induced with the IPTG. It has been proved that the aim gene was expressed in the E.coli and the expressed proteins had the reactinogenicity by the SDS and Western-blotting. The molecular weight of the recombinantα,βandεtoxins were 46.1kDa ,54.6kDa and 36.6kD.Three recombinant proteins all expressed in the Cytoplasm,Periplasm and the culture supernatant of recombinats, the alpha and beta recombinant protein expressed mostly as inclusion body's and have no biological activity. Recombinant epsilon protein expressed mostly as soluble protein's way in the Periplasm and had toxicity . The expressing quantity ofα-pET28a (plys),β-pET32a (DE3) andε-pET28a (DE3) account for 38.3% ,24.6% and 29.3% in the whole bacterium's protein respectively. The recombinant epsilon protein has high biological activity , the supernatant of lysate's culture has 15000 mice MLD per ml after activated with trypsin . the virulence is similar to the toxins produced by the virulent Clostridium perfringen of type D(C60-2).Using the recombinant alpha and beta toxin's inclusion body protein and epsilon toxoid protein, the monofator antitoxins of the alpha,beta and epsilon toxin were prepared. Their neutralization titer is : alpha antitoxin can neutralize 100 mice MLD of type A toxin per ml; beta antitoxin can neutralize 500 mice MLD of type C toxin per ml, epsilon antitoxin can neutralize 30000 mice MLD of type D toxin per ml.