Cloning And Expression Of Alpha-toxin Gene Of Clostridium Perfringens And Initial Research Of Indirect ELISA

Rabbit clostridial disease is an important disease that threat rabbit production. An acute rabbit infectious disease characteristic of dark diarrhea or gumlike blood feces and cecum bleeding and stomach mucosa bleeding occurrencd in a rabbit farm. A Clostridium perfringens strain was isolated through pathogen isolation,morphologic observation, biochemical reaction and half-nested PCR reaction. Sensive drug were selected by drug sensive experiment.This experiment result provide reference for the establishment of precaution measure of this disease.According to the published genome sequences of a-toxin gene of Clostridium perfringens on GenBank, the study analysised the secondary structure of amino acid sequence, hydrophobicity, antigenic determinant, transmembrane region and antigenic region of a-toxin gene of Clostridium perfringens. Therefore, the study choosed the region which was 837 bp in length between 130～966 nt of a-toxin gene and expressed compatibly in prokaryotic cells.Then the region was used for PCR amplification,cloning, sequencing and bioinformatics analysis. The sequenced results shows that:the sequence was 837 bp in length. According to the nucleic acid homology analysis, The nucleotide sequence homology with those a-toxin genes of Clostridium perfringens from different isolates on GeneBank were 99%. Bioinformatics analysis of this sequence shows that:the protein was hydrophilic and highly antigenic, having no obvius transmembrane region and possessing 11 antigenic determinants.Then the fragment was subcloned into EcoR I/HindⅢsite of prokaryotic expression vector PET-32a(+) and recombinant expression plasmid PET-32a(+)-αwas constructed successfully.The plasmid was transformed into Eschercha coil BL21 and 53KD recombinant protein was expression with IPTG inducer.The expression product were mainly non-soluble inclusion body.The expression conditions were determind by optimizing induction temperature,induction time and concentration of IPTG.An indirect ELISA method detecting antibody against alpha-toxin of Clostridium perfringens was estabished based on recombinant protein as coating antigen. It is positive when the OD450≥0.30,and it negative when OD450<0.30. The method can be applied in detection of antibody against alpha-toxin of Clostridium perfringens.