Isolation And Identification Of Clostridium Perrfingens In Sheep, And Prokaryotic Expression And Immunogenicity Of Its Alpha Toxin

Objective:Clostridium perfringens..which widely distributed in water,soil,nature and animal intestinal, can lead variety of important diseases of many kinds of livestock,such as,the popularity gangrene,food poisoning, sudden death syndrome of cattle and sheep, gut toxemia diarrhea lamb, necrotic enteritis of poultry.Due to Clostridium perfringens infected to show acute onset,short duration,high mortality, it is often caused serious economic losses to the livestock and poultry industry. Therefore, to understand and master the popular toxin types and resistant strains status of Clostridium perfringens from sheep of Xinjiang.Carry prokaryotic expression and immunogenicity study of the main virulence genes will provide reference materials bacteria infection prevention and control.Methods:According the microscopy,biochemical tests and PCR method for separation and identification Clostridium perfringens from the submission dead sheep sample in sheep farm of Xinjiang from2010to2012.16S rRNA sequence was amplified by PCR and then sequencing and homology compared.Determine the toxinotyping of identified Clostridium perfringens strains,and analyzing the popular toxinotyping of the Xinjiang region.According to the methods that is anaerobes published by the People’s Republic of China,selected Penicillin,Clindamycin hydrochloride, metronidazole, ceftriaxone,ticarcillin to do sensitive test of separated strains and learn the drug resistance of Clostridium perfringens from Xinjiang.According to the GenBank (the Registry Number X17300.1) has published Clostridium perfringens alpha toxin gene sequences, to design primers for the mature peptide sequence of the alpha-toxin, and amplified by PCR, inserted into plasmid pET-28b to construct a recombinant expression vector pET-28b-CPA. Transformed the recombinant vector pET-28b-CPA which identified by PCR, double enzyme digestion and sequencing to the BL21(DE3)plysS.With IPTG inducible expression of recombinant bacteria. Expression products were detected the forms of expression and reactogenicity by SDS-PAGE and Western blot and optimize expression conditions to determine the optimal induction conditions. Adoption Ni-NTA batch purified alpha toxin then use the Bradford protein concentration assay kit determine the purified protein concentration, the volume of the purified protein with Freund’s adjuvant was prepared by combining subunit vaccine immunized mice,and then evaluation its immune effect by antibody detection and conteracting toxic substances experiment. Results:we isolated49Clostridium perfringens from the submission sample by staining, biochemical identification,16S rRNA detection analysis. Toxinotyping results show that type A accounted for61.2%(30/49), type B for2.04%(1/49),type D for36.7%(18/49),that illustrate the popular toxinotyping is A-type and D-type strain of Xinjiang region.The results of the susceptibility testing is that metronidazole79.6%, clindamycin49.0%, penicillin38.8%, ceftriaxone12.2%and ticarcillin10.2%of experimental isolated strains.The above studies have shown that the resistance situation is seriously of Clostridium perfringens from sheep.The experiment was successfully amplified alpha toxin mature peptide sequence, and its molecular weight size of1110bp; And successful construction recombinant expression vector pET-28b-CPA then transform to recipient strain,through the expression of target gene induction,test results showed that recombinant alpha-toxin protein molecular weight42.1kDa, with the expected size consistent and reactogenicity.Further optimization of expression conditions found the target gene in25℃, IPTG1.0mmol/L, induced4h best expression of target protein accounted for34.6%of the bacterial soluble protein, soluble expression. Preparation subunit vaccine with recombinant protein immunized mice, the results showed that the recombinant alpha toxin can stimulate antibody production immune response of anti-alpha-toxin,antibody reached its highest level in28days after immunization (1:6400), and provide some immune protection of immunized mice.Conclusion:This paper analyzes the prevalence of Clostridium perfringens from parts of sheep farm in Xinjiang, And do susceptibility test analysis with strains which isolated from clinical sample,in-depth discussion of the pathogenic mechanism of infection. And cloning, expression and purification its main toxin-a toxin,to obtained immune sera by immunization of mice,and laid the foundation for the preparation of Clostridium perfringens alpha toxin genetically engineered proteins.