Establishment of Echinococcus granulosus AgB8/1-AgB8/2 chimeric recombinant protein expression system

In order to construct the pET32a-AgB8/1-AgB8/2 chimeric antigen prokaryotic expression recombinant plasmid and the expression of its recombinant protein,the total RNA was extracted from protoscoleces of Echinococcus granulosus,and reverse transcribed into cDNA,the cDNA encoding mature form of EgAgB8/1and EgAgB8/2 antigen were amplified by PCR using gene specific primers.Based on the both gene fragments,a nucleotide sequence encoding EgAgB8/1-EgAgB8/2 chimeric antigen were artificially synthesized after sequence confirmation.The synthesized nucleotide sequence encoding EgAgB8/1-EgAgB8/2 chimeric antigen were conformed by sequencing after cloning into pUCm-T vector,then the target sequence was directionally ligated into pET32a plasmid after double digestion with restriction enzymes for prokaryotic expression.The constructed recombinant plasmid pET32a-AgB8/1-AgB8/2 was transformed into E.coli BL21(DE3) LysS,and the recombinant chimeric protein expression was induced by IPTG.The recombinant protein expression was analyzed by SDS-PAGE.Sequence analysis revealed that the nucleotide sequence encoding for AgB8/1-AgB8/2 chimeric protein was directionally cloned into pET32a plasmid.SDS-PAGE analysis confirmed that the recombinant chimeric protein AgB8/1-AgB8/2 fused with Trx was successfully expressed in E.coli BL21,with its relative molecule mass of about 38 kD.In this study,the recombinant plasmid pET32a-AgB8/1-AgB8/2 is successfully constructed and the recombinant chimeric protein EgAgB8/1-EgAgB8/2 was expressed. The results obtained in this study will provide a foundation for further study on its immune characteristics in the future.