Establishment Of Echinococcus Granulosus EgAgB8/1 Recombinant Protein And EgAgB8/1-EgAgB8/2 Chimeric Recombinant Protein Expression Cystem And Serological Evaluation

Objective:Construction of pET32a-EgAgB8/1 antigen prokaryotic expression recombinant plasmid and pET32a-EgAgB8/1-EgAgB8/2 chimeric antigen prokaryotic expression recombinant plasmid, express and serologically evaluate The EgAgB8/1 and EgAgB8/1-EgAgB8/2 recombinant protein of Echinococcus granlosus for the purpose of usage in serological diagnosis in cystic echinococcus(CE).Methods:Total RNA was extracted from protoscoleces of Echinococcus granulosus, and reverse transcribed into cDNA, The cDNA encoding mature form of EgAgB8/1 and EgAgB8/2 antigen were amplified by PCR using gene specific primers, after sequence confirmation, based on the gene fragments, a nucleotide sequence encoding EgAgB8/1 antigen was amplified and EgAgB8/1-EgAgB8/2 chimeric antigen were artificially synthetized. The synthetized nucleotide sequence encoding the EgAgB8/1 antigen and EgAgB8/1-EgAgB8/2 chimeric antigen were conformed by sequencing after cloning into pMD18-T vector, then the target sequence were directionally ligated into pET32a plasmid after double digestion with restriction enzymes for prokaryotic expression, and the constructed recombinant plasmid pET32a- EgAgB8/1 and pET32a- EgAgB8/1-EgAgB8/2 were transformed into E.coli BL21 (DE3) LysS with induction of IPTG for expression. These recombinant proteins obtained were purified by ultrasonication. Samples of sera taken from patients with hydatidosis and cysticercosis were used to evaluate the serological reaction of these recombinant proteins.Results:Sequence analysis revealed that the nucleotide sequence encoding for EgAgB8/1 recombinant protein and EgAgB8/1-EgAgB8/2 chimeric protein were directionally cloned into pET32a plasmid. SDS-PAGE analysis confirmed that the recombinant protein EgAgB8/1 and EgAgB8/1-EgAgB8/2 fused with Trx were succesfully expressed in E.coli BL21, It was found that the expressed pET32a- EgAgB8/1 and pET32a- EgAgB8/1-EgAgB8/2 recombinant protein were detected as a band of 28kDa and 38kDa. As demonstrated by Western blotting, these recombinant proteins could react specifically with serum samples of patients with hydatidosis. In the serological reaction of these recombinant proteins with 16 serum samples of patientes with hydatidosis,11 serum samples showed positive reaction with recombinant protein EgAgB8/1,13 serum samples showed positive reaction with recombinant protein EgAgB8/1-EgAgB8/2. Meanwhile, in the serological reaction of these recombinant proteins with 12 serum samples of patientes with cysticercosis,5 serum samples showed positive reaction with recombinant protein EgAgB8/1,3 serum samples showed positive reaction with recombinant protein EgAgB8/1-EgAgB8/2.Conclusion:The recombinant plasmid pET32a- EgAgB8/1 and pET32a- EgAgB8/1-EgAgB8/2 are successfully constructed, the recombinant protein EgAgB8/1 and the recombinant chimeric protein EgAgB8/1-EgAgB8/2 were expressed. As demonstrated by Western blotting, it is evident that EgAgB8/1-EgAgB8/2 recombinant protein may be considered as a better antigen in serological diagnosis in cystic echinococcus (CE) then EgAgB8/1 recombinant protein.