Construction Of Clostridium Perfringens Antigenic Protein-sheep Pox Vaccine Polyvalent Recombinant Virus

Clostridium perfringens is an acute infectious disease caused by a variety of bacteria in the genus Clostridium,which can cause diseases such as:sheep sudden gangrene,sheep fastidious disease,sheep enterotoxemia,lamb dysentery and sheep black disease.The symptoms of these five diseases are very similar,and their common characteristics are rapid onset and death.Clostridium perfringens is capable of producing a variety of virulence-producing proteins and is classified from type A to type E based on the virulence-producing capacity of the toxins（α,β,ε,ι,θand enterotoxin）.Live attenuated vaccines provide long-lasting immunity and are therefore considered to be the best choice for vaccination.The sheep pox virus gene is huge,so we used the sheep pox virus vaccine strain as a vector and combined it with the immunity gene of the Clostridium perfringens antigenic protein,thus constructing a multivalent recombinant virus,and this construct was used in a combined sheep pox vaccine to protect sheep from both sheep pox and Clostridium perfringens,achieving the clinical effect of one vaccine for multiple uses.The aim of this study was to construct three Clostridium perfringens toxin vectors expressed and purified for safety evaluation,and later to insert these three toxin genes into sheep pox virus vaccine expression vectors to better utilize sheep pox virus vaccine strains to construct multivalent recombinant viruses and to pave the way for further exploration of Clostridium perfringens antigenic protein-sheep pox vaccine multivalent recombinant virus development.In this study,the antigenicity of the protein was firstly predicted by the online software IEDB for Clostridium perfringensα,βandεtoxin genes,and then the appropriate gene sequences were intercepted after comparative analysis of gene sequences by DNAstar.The p ET-42b-α-β₂-ε-β₁plasmid was then used as a template to amplify theαtoxin gene,βtoxin gene andεtoxin gene by PCR,and then the three toxin genes were ligated to the p ET-42b plasmid by Nde I and Xho I digestion,respectively,to construct the p ET-42b-α,p ET-42b-βand p ET-42b-εrecombinant plasmids,after which the identified The successful plasmids were expressed and purified,and then the purified proteins were detected by immunoblotting,and then the safety of the purified proteins was evaluated by immunizing mice,and after three weeks of observation,the internal organs of mice were dissected for pathological sections.Next,p ET-42b-α-β₂-ε-β₁plasmid was used as the template,and PCR was performed to obtain the fragment required for this test:Clostridium perfringens toxinα-β₂-ε-β₁fragment,after which theα-β₂-ε-β₁gene fragment was ligated to p GM-TK13-P11-gpt-GFP plasmid by Fse I and Asc I digestion,and finally the recombinant transfer plasmid p GM-The plasmids were extracted with endotoxin-free kit and transfected with Lipofectamine 2000 in testicular cells of lambs infected with the weakly virulent vaccine strain of goat pox virus GTPV AV41,and observed by fluorescence microscopy for 12 h-48 h.The fluorescent spots were picked by low melting point agar fixation in DMED and repeatedly stored at-20℃～37℃.After repeated freeze-thawing for 3times at-20℃～37℃,the lamb testis cells were inoculated by fluorescence microscopy and then the fluorescent spots were picked,diluted and inoculated,and repeated 4～6 times until all cells were fluorescent,and then the genome was extracted for PCR identification.The results showed that the antigenicity ofα,βandεtoxin gene proteins was predicted by using the online biological software IEDB,and the antigenicity of the genes in the middle segment was found to be higher,and then the intercepted intervals with higher antigenicity were from 124 to 405 amino acids forαtoxin gene,34 to 343 amino acids forβtoxin gene,and 50 to 317 amino acids forεtoxin gene.Three expression recombinant plasmids,p ET-42b-α,p ET-42b-βand p ET-42b-ε,were successfully constructed,and then expressed and purified to obtain purified proteins of 32 KDa,35 KDa and 30 KDa size,and none of them died after three weeks after intraperitoneal injection into mice,indicating that the purified proteins were less toxic and the immunogenicity of the truncated toxin proteins was better.The pathological section results showed that the liver,large intestine and small intestine tissues were clear and intact,and no obvious pathological changes occurred in the experimental and control groups,indicating that the toxicity of the purified toxin protein had been reduced and would not cause lesion death.Later,the successfully constructed recombinant transfer vector p GM-TK13-p11-α-β₂-ε-β₁-gpt-GFP was transfected with testicular cells of lambs inoculated with goat pox virus GTPV AV41 vaccine strain to produce a large number of cells with green fluorescence,and then the fluorescent cells were picked and the virus suspension of the offspring was passed six times to identify the purified recombinant virus by PCR.