Production Of Anti EgAgB8/3 Recombinant Protein And Development Of Sandwich-ELISA Coproantigen Detection System For Diagnosis Of Dogs Infected With Echinococcus Spp

Objectiv:To clone a gene gene fragment encoding EgAgB8/3 antigen, Construction prokaryotic expression plasmid pET32a(+)-EgAgB8/3, expression of high purity EgAgB8/3 recombinant protein, to prepare anti recombinant EgAgB8/3 polyclonal antibodies and establishment of sandwich-ELISA coproantigen test for detection of EgAgB8/3 native antigen in feces of dogs infected with Echinococcus spp. Methods: Total RNA was isolated from fresh samples of E. granulosus adullt worms and reverse transcribed into cDNA, EgAgB8/3 gene fragment was amplified by PCR using cDNA as a template. pGEM-T/EgAgB8/3 recombinant plasmid was constructed and confirmed by enzyme digestion and sequencing. The target gene was diractionally cloned into pET32a(+) for prokaryotic express. The recombinant plasimid pET32a(+)-EgAgB8/3 was transformed into E.coli DH5a, and the positive clones screened by Ampicillin, identified by restriction endonuclease analysis and PCR amplification; and transformed into E.coli BL21(DE3)LysS. Recombinant protein expression was induced with IPTG. The target protein was purified using His-Binding-resin column, and immunized to animals, serum total IgGs from immunized animals were purified by ammonium sulfate precipitation method. A batch of purified IgGs were labeled using sodium iodide method, another batch of unlabeled IgGs were left as capture antibody. In Sandwich-ELISA system, the capture antibodies were used as coating antibosied, the crude protein extract from dog feces infected with Echinococcus spp were used as sandwich antigens, and the labeled IgGs were used as final conjugate. Results:The high purity EgAgB8/3 recombinant protein and the high titer mono-specific polyclonal antibodies were successfully prepared. The established Sandwich-ELISA system using labeled and unlabeled polyclonal antibodies revealed 85.0% sensitivity and 95.7% specificity to detect EgAgB8/3 native antigen in feces of dogs infected with Echinococcus spp. Conclusion:Successfully constructed the recombinant plasmid pET32a(+)-EgAgB8/3. The recombinant EgAgB8/3 protein has better immunogenic property, and the polyclonal antibodies obtained by animal immunization revealed high sensitivity and specificity. Successful development of The Sandwich-ELISA coproantigen detection system using EgAgB8/3 recombinant protein is providing scientific data for preparation of fast diagnostic Kit for dogs infected with Echinococcus spp. However, the stability and wide-range applicability of this method should be further evaluated using epidemiological mass data.