Cloning and Expression of Alpha-Toxin Gene from Clostridium perfringens Type C / 微生物学通报

ABSTRACT

Alpha-toxin gene was amplified from chromosomal DNA of Clostridium perfringens type C by polymerase chain reaction(PCR). PCR product was inserted into vector pGEM-T directively. The cloned recombinant plasmid pXCPA1 possesses positive nucleotide sequence of alpha-toxin. A 1.2 kb alpha-toxin gene fragment was cleaved with restriction endonucleases NcoI/EcoRI from plasmid pXCPA1, and then inserted into an expression vector pET-28c which cleaved with NcoI/EcoRI by blunt-end ligation. The recombinant expression plasmid pETXA1 was studied in detail by restriction endonucleases analysis and nucleotide sequencing. The results showed that the recombinant expression pETXA1 possessed a positive alpha-toxin gene sequence and reading frame. BL21(DE3) (pETXA1) could produce alpha-toxin and the expressed products were recognized by alpha-toxin monoclonal antibodies, and the expression level of the alpha-toxin proteins were about 16.28% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis.