Immunogenicity Analysis Of Prokaryotic Expression Product Of Rabbit Clostridium Perfringens(Type A) Alpha Toxin

Rabbit Clostridium perfringens disease caused by Clostridium perfringens (type A) is one kind of enteral infections. The disease can spread to all breeds and ages rabbits, and the incidence of the disease is higher in immature groups and the mortality can reach 100%, which influences the development of rabbits' breeding deeply. The study on alpha toxin - the main pathogenic factor is important.Alpha toxin gene from the Clostridium perfringens(type A) NJ strain was amplified by PCR and cloned into the vector pMD-18T, then the recombinant plasmid pMD18Tc a was constructed. Sequence analysis showed that the alpha toxin gene from the NJ strain was 1194bp and had a complete open reading frame which encoded 398 amino acids, forward 28 amino acids for signal peptide and rearward 370 amino acids for mature alpha toxin. Nucleotide sequence comparison indicated that alpha toxin gene of NJ strain shared 99. 27% identities with reference strain NCIB19681 (type B) NCIB10662 (type C) and L9(type D). Alpha toxin gene was subcloned into pPRO-EX HTb, and the research on the expression of recombinant plasmid pPROHTbC a in E. coli BL21 (DE3) was done.Subunit gene of alpha toxin was subcloned into expression vector pPRO-EX HTa and pET-30b, and the recombinant plasmids were transformed into BL21(DE3). SDS-PAGE analysis showed that the recombinant plasmid pPROHTaP could express 36KD fusion protein, as well as the recombinant plasmid pET30bP expressed 38KD fusion protein which was 30% of total bacterial protein of BL21 (DE3). The fusion protein form recombinant strain BL21(DE3) pET30bP a was purified by metal chelation affinity chromatography. All induced products from BL21(DE3) pPROHTaP a and BL2l(DE3) pET30bP had a positive reaction with alpha toxin antiserum by western-blot analysis.Fusion protein a toxinA and a toxinB became soluble protein after denaturation by urea, renaturation and concentration, which originally existed in inclusion body extracted from recombinant strain, a toxinB was purified by gel-filtration chromatographyImmunodiffusion results showed that fusion protein a toxinA and a toxinB had a immunoreaction with alpha toxin antiserum , and that anti-a toxinA serum and anti- a toxinB serum had immunoreaction with alpha toxin extractedClostridium perfringens(type A) .This study provided the theoretical basis and material for rabbit Clostridium perfringens disease epidemiology investigation, research on diagnostic reagent and genetic engineering vaccine.Candidate: Liu JiasenSpeciality: Preventive Veterinary ScienceSupervisor: Prof. Li YijingResearch Fellow Chen Hongyan...