Construction and expression of CPA-LTB fusion gene

Alpha-toxin gene from chromosomal DNA of Clostridium perfringens type A and heat-labile toxin B unit gene from plasmid pEWD299 containing heat-labile toxin of Escherichia coli were amplified by PCR.PCR products were cleaved with restriction endonucleases and recovered.The recombinant plasmid pCPA-LTB containing CPA-LTB fusion gene was constructed by recombinant technique and then transformed into Escherichia coli BL21(DE3).The recombinant expression plasmid pCPA-LTB was studied in detail by restriction endonucleases analysis and nucleotide sequencing.The results showed that the recombinant expression pCPA-LTB possessed the positive alpha-toxin and heat-labile toxin B unit gene sequences and their reading frames.The CPA-LTB fusion proteins were expressed in recombinant strain BL21(DE3)(pCPA-LTB) with the expression level of the CPA-LTB fusion proteins to be about 12.5% of the total cellular protein by SDS-PAGE and thin-layer gel scanning analysis.