Establishment of Echinococcus granulosus AgB8/1 recombinant protein expression cystem

Objective To construct pET32a-AgB8/1 antigen prokaryotic expression recombinant plasmid and expression of its recombinant protein.Methods Total RNA was extracted from protoscoleces of Echinococcus granulosus and reverse transcribed into cDNA.The cDNA encoding mature form of EgAgB8/1 antigen was amplified by PCR using gene specific primers,after sequence confirmation,based on the gene fragments,a nucleotide sequence encoding EgAgB8/1 antigen was artificially synthetized.The synthetized nucleotide sequence encoding the EgAgB8/1 antigen was conformed by sequencing after cloning into pUCm-T vector,then the target sequence was directionally ligated into pET32a plasmid after duoble digestion with restriction enzymes for prokaryotic expression,and the constructed recombinant plasmid pET32a-AgB8/1 was transformed into E.coli BL21(DE3) LysS,and the recombinant protein expression was induced by isopropyl-1-thio-β-galactopyranoside(IPTG).The recombinant protein expression was analyzed by SDS-PAGE.Results Sequence analysis revealed that the nucleotide sequence encoding for AgB8/1 recombinant protein was directionally cloned into pET32a plasmid.SDS-PAGE analysis confirmed that the recombinant protein AgB8/1 fused with Trx was succesfully expressed in E.coli BL21,with its relative molecule mass of expressed product of about 28 kDa.Conclusion The recombinant plasmid pET32a-AgB8/1 is successfully constructed and the recombinant protein EgAgB8/1 is expressed.The results obtained in this study will provide a foundation for further study on its immune characteristics in the future.