Molecular Ecological Analysis Of Clostridium Perfringens β2 Toxin From Animals From Some Regions Of Jiangxi Province And The Establishment Of Its Detection Method

Clostridium perfringens（Cp）is a widely occurring zoonotic pathogen in the environment and animal intestine,capable of producing more than 20 toxins.Cp can be classified into types A-G according to the six toxins produced.β2 toxin,a toxin produced by all Cp,plays a crucial role in enterotoxaemia in sheep and goats and in piglet diarrhea.Because of the unstable expression ofβ2 toxin and the low expression amount,the detection ofβ2 toxin poses some difficulties.In this experiment,two assays were developed for the detection of Clostridium perfringensβ2 toxin based on recombinant toxin protein and its antibodies.At the same time,multiple sequence alignment of cpb2 genes in different animal-derived isolates was carried out in combination with molecular ecology research methods.The similarities and homologies were analyzed and phylogenetic trees were constructed.The expression profile of cpb2-positive strains was explored using immunoblotting and the two methods established.1.The recombinant protein was expressed in large quantities and purified using the p ET-32a-β2 BL21 recombinant strain preserved in our group.The preserved hybridoma cell line was resuscitated,the ascites was prepared after resuscitation and the anti-32a-β2 monoclonal antibody was purified.Prepared Mo S₂-MWCNTs modified with gold nanoparticles and used as a sensing platform after attachment of antibodies on the surface.The electrochemical immunosensor was successfully developed for the detection ofβ2 toxin in the range of 0.1-100 ng/m L with good linearity between the antigen concentration and the current signal in the detection range.The method has good repeatability,reproducibility,stability and specificity.2.The ELISA was developed using rabbit and mouse multiple antibodies preserved in our group,with the rabbit multiple antibodies as the capture antibody and the mouse multiple antibodies as the detection antibody.The method was used to test 332 clinical pig serum samples from different regions of Jiangxi,and the overall positive rate was 77.41%.3.A total of 139 fecal samples from different animal sources were collected from different regions of Jiangxi.44 strains of Clostridium perfringens（31.56%,44/139）were isolated,purified and identified.There were four F-type strains carrying cpe,all isolated from pigs（9.09%,4/44）.The rest were type A,distributed among different animals（90.91%,40/44）.A total of 21 cpb2-positive strains were identified（47.73%,21/44）,of which 7 strains with the typical cpb2 gene were distributed in pigs and cats and 14 strains carrying the atypical cpb2 gene were distributed in different species.In addition,the expression of these strains was characterized and a total of three strains of porcine origin expressedβ2 toxin,all of them carrying the typical cpb2 gene.In summary,two methods were developed for the detection of Clostridium perfringensβ2 toxin.Forty-four Clostridium perfringens strains were isolated from 139 samples and analyzed for molecular ecology.,21 of which were cpb2 positive.Multiple sequence comparisons showed that canine-derived strains HB and DD,sheep-derived strain 5-2 and pig-derived strain 2-3 had base deletions that could affect protein expression.Homology was stronger between atypical cpb2 and weaker between typical cpb2.Expression testing of cpb2-positive strains using immunoblotting with the two established assays identified three pig-derived strains expressingβ2 toxin.The established assay was shown to be feasible.