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Differentiation of Multiplex PCR to Clostridium Perfringens and Cloning and Sequence Analysis of the Enterotoxin Gene

Author: TangYuan
Tutor: WenMing；LiYongMing；ZhouBiJun；WangKaiGong
School: Guizhou University
Course: Preventive Veterinary Medicine
Keywords: Clostridium perfringens multiplex PCR enterotoxin gene screening and identification clone sequence analysis
CLC: S852.61
Type: Master's thesis
Year: 2009
Downloads: 52
Quote: 1
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Abstract


Clostridium perfringens is an endospore-forming, gram-positive bacterium that ranks among the most important of the anaerobic pathogens affecting humans and domestic animals. Four aspects have been researched on the Clostridium perfringens in this study, including:1. Differentiation of multiplex PCR to Clostridium perfringens isolates from goat in Guizhou province A multiplex PCR method was developed according to alpha, beta, epsilon and lota toxin genes of Clostridium perfringens and applied to differentiate the gene type of 34 isolates from goat in Guizhou province. The results showed that 32 isolates of them were identified as Clostridium perfringens type A, while the other two isolates as type C and type D respectively, which were identical with that of serological method in the earlier studies. These results above suggested that the multiplex PCR method can be used for differentiation of serum type of Clostridium perfringens isolates clinically.2. Screening and identification of the enterotoxin positive strain of Clostridium perfringens A specific pairs of primers were designed and synthesized according to the Clostridium perfringens enterotoxin genes (cpe) from reference report, 34 Clostridium perfringens isolates from Guizhou province were amplified by PCR using the primer. The PCR product was cloned into pMD18-T vector and transformed into competent cell DH5a. The recombinant plasmid was identified by PCR and restriction enzyme then sequenced. The results showed that one of the 34 isolates had the purpose fragment. The gene sequence consisted of 233 bp, which shared 99.6 %-100 % sequence identity and 98.7%-100% amino acid sequence identity with reference strains in GenBank. It was concluded that one of cpe~+ Clostridium perfringens type C was obtained from 34 Clostridium perfringens isolates of Guizhou province.3. Cloning and sequence analysis of the enterotoxin gene from Clostridium perfringens type C Clostridium perfringens enterotoxin (CPE) is an important virulence factor involved in Clostridium perfringens food poisoning and other gastrointestinal illnesses. In our experiment, the enterotoxin gene (cpe) of Clostridium perfringens type C strain isolated from Guizhou province was amplified by PCR. The PCR product was cloned into pMD18-T vectors and transformed into competent cell DH5a. The recombinant plasmid was identified by PCR and restriction enzyme then sequenced. The results showed that the cloned gene was 960 bp in length and coding 319 amino acids, which shared 99.4 %-99.8 % sequence identity and 99.1 %-99.7 % amino acid sequence identity with reference strains in GenBank. It laid the basis for further investigation of the mechanism of disease caused by C. perfringens enterotoxin.4. Constraction of prokaryotic expression vector of the enterotoxin gene of Clostridium perfringens type C A pair of specific primers were designed and synthesized acoding to the published Clostridium perfringens enterotoxin gene, and was amplified for the gene from vector pMD18-T-cpe containing the gene. The PCR products retrieved from 1.0 % agarase gel was digested by BamH I and EcoR I and then directly ligased to prokaryotic expression vector pET32a digested by the same enzymes. The recombinant plasmid was transferred into competent cells of Escherishia Coli DH5a. The recombinant plasmid was identified by PCR and restriction enzyme then sequenced. The results showed that the Clostridium perfringens enterotoxin gene was constructed into prokaryotic expression vector Pet32a (the recombinant plasmid designated as pET32a-cpe). Our experiment reported here is basic step in fuction research for Clostridium perfringens enterotoxin gene.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Pathogenic bacteria
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