The Epidemiological Investigation Of Clostridium Chauvoei Infection,and CctA Gene Eukaryotic Expression Vector Of Construction And Expression In Yanbian Area

Clostridium chauvoei infection, pathogen is clostridium chauvoei, it is an acute, febrile, septicemic infectious disease. The typical symptoms of the disease are the occurrence of inflammation and swelling of the air at the position of muscle fullness, when touching and pressing, there is a pronunciation of crepitation, it also has a certain local popularity and seasonality, and it usually occurs in the wet valley ranch, the weather is hot and rainy season. However in recent years, Yanbian area of Jilin province have been outbreaked clostridium chauvoei infection of bovine, which brought huge shocks and economic losses to the development of animal husbandry and breeding industry, at the same time, clostridium chauvoei also threated more to human security and production.According to the Cytotoxic A gene (CctA) sequence published of clostridium chauvoei on the NCBI, designed two pairs of specific primers. On the one hand, the study tested 165 cattle blood samples in Yanbian area by Blood smear staining microscopy and PCR, and used statistics for data analysis. On the other hand, separated the liver, spleen, heart and muscle tissues of suspected clostridium chauvoei infection of bovine, identified the bacteria by etiological examination、biochemical identification、 animal testing、molecular biology identification. The CctA gene by PCR was cloned into pMD19-T simple vector, analysed the correct CctA gene sequence, then subcloned into pVAX-1 of eukaryotic expression vector, and the constructed pVAXl-CctA expression plasmid was transfected into Vero cells for expression, finally used indirect immunofluorescence and Western blotting to test the expression.The results showed that the average positive rate of PCR assay for the detection of clostridium chauvoei infection was 15.2%, statistical analysis showed that bovine emphysema disease infection rate between the different regions and varieties were not significant (P>0.05); but the different ages and geographical conditions were significant (P<0.05); individual farmers rearing in the positive rate was significantly higher than the scale of farming methods (P<0.01), it illustrated that clostridium chauvoei infection in catties were affected more by the age groups, geographical conditions, breed and rearing methods. The size of amplification of clostridium chauvoei CctA gene was 501 bp, compared CctA gene nucleotide sequence with clostridium chauvoei ATCC 10092 strain sequence about homologous alignment analysis, the results showed that CctA gene of yanbian strain had three point mutations, and nucleotide sequence homology was 99.4%; and there were two point mutations in amino acid sequence, sequence homology was 98.8%. Transfection pVAXl-CctA group of cells had green fluorescence, the expression of protein molecular weight was about 19 kDa, it showed that pVAX1-CctA plasmid successfully expressed the CctA protein in Vero cells.The study could help to predict and analysis the information of isolate in this region, and provided the reference about prevention and control of clostridium chauvoei infection, the research laid a solid foundation for clostridium chauvoei infection of nucleic acid vaccine.