The Analysis Of ?2 Toxin Effect Harboring By CpA And The Whole-genome Sequence Of Strain JXJA17 From Piglets

Clostridium Perfringens(Cp)is widely used in soil,feed,food and human and animal intestines.Under the certain conditions,it can cause people and various animal diseases.The pathogenic effect of this bacterium is mainly due to the toxins it produces.Cp produces 17-18 or more toxins,which are classified into A,B,C,D and E according to the four toxins ?,?,?,and ?.CpA was considered to be a normal anaerobic bacterium in the intestinal microflora of piglets and its effects on the pathogenesis of NEC in piglets have been observed recently.The influence of piglet NEC on the modern pig industry has become increasingly significant in Jiangxi Province and even in China.With the limited use of antibiotics in feed,the impact of the disease on the aquaculture will be more seriously.The factors that cause piglet NEC are related to microbial infection,nutrition and environmental factors,which are very complicated.Therefore,in this experiment,Cp type A was isolated from diarrhea fecal of piglets by MPCR,and the model of mice was established for the toxicity.After the plasmid was extracted and the toxin was detected,the JXJA17 strain was decided for high-throughput sequencing analysis.The toxin effects of the beta2 were studied by yeast two-hybrid test.1.Isolation and animal testing of Clostridium perfringens type A carrying ?2 toxinIn the experiment,Cp was isolated from feces of diarrheal piglets in six regions of Jiangxi Province by using modified TSC culture medium.Biochemical and 16 SrDNA molecular identification of the isolated Cp strains was performed.The genotyping of Cp and the carrying rate of CpA ?2 toxin gene was determined by multiplex PCR.CPE toxin gene also was detected.And then select 15 strains to extract plasmid,detecting plasmid carring beta2 and CPE toxin.Furthemore,The CpA(?2+/?2-)isolate was subjected to a mouse toxicity test.The results showed that the Cp isolation rate was 100% in 65 samples of suckling piglets.Among them,the ?2 toxin carrying rate of the CpA strain was 98%.Only one CpA(?2-)strain was isolated,the isolated CpA strain usually carried 2~3 or more plasmids,and the ?2 toxin gene was located in the CpA plasmid that the CPE toxin gene wasn't found in;it was derived from diarrhea CpA(?2+),CpA(?2)-)and health(from the laboratory)CpA(?2 +)culture supernatant injected in mice,all can cause degree of death in mice.The experiment of rabbit small intestinal loops showed that the toxicity of CpA(beta2+/beta2-)from diarrhea was stronger than that from health(beta2+)source.It is indicated that the rate of CpA and beta2 are high.Beta2 toxicity test suggests that toxin effect may be associated with beta2 toxin gene expression and host intestinal environment,not for strain source health or diarrhea piglets and carring beta2 toxin.2.Sequencing analysis of isolated strain JXJA17 Cp(?2+)in jiangxi provinceThe selected strain JXJA 17 was analyzed by high-throughput microbial genome de novo sequencing.The results showed that the JXJA 17 genome was 3,324,072 bp in length and contained 9 intact plasmids encoding a total of 3396 genes.The genome encodes 3092 DNA sequences,30 rRNA genes(10 5S,10 16 S,and 10 23S),95 tRNA genes,and 0 non-coding RNAs.The nine plasmid sizes were: 58,796,38,284,62,027,40,125,36,275,12,326,12,133,3,550 and 11,801 bp,respectively.The comparison of toxin database shows that only the ? toxin and ?2 toxin are involved,and there are more than 20 kinds resistance genes.The pathogenic strain of JXJA17 through genomic circle mapping,eggNOG and KEGG functional annotation,and CRISPR prediction will help to understand epidemiological changes and evolutionary characteristics of bacteria.In this study,whole genome sequencing was performed on the pathogenic strain of JXJA17 to understand the characteristics of the CpA genome,especially high beta2 toxin genes and to provide a genetic reference for the study of molecular epidemiological characteristics of clostridial enteritis in China.3.?2 toxin yeast two-hybrid systemTo study the interaction of ?2 toxin effect between the bacterial and intestinal epithelial cells proteins,the ?2 toxin was amplified based on the JXJA17 sequencing results,the yeast two-hybrid assay was carried out.The results showed that the ?2 toxin transferred into the yeast did not inhibit the growth of the yeast.The pGBKT7-?2 toxin fusion protein was successfully expressed in yeast,intestine porcine epithelial cell is the protein that interacts with the beta 2 toxin,and the mechanism of interaction protein needs to be further study.In this experiment,the yeast two-hybrid system of beta 2 toxin was used to provide a test reference for further study on the toxicity of beta 2.