Prokaryotic Coexpression Of ADF And 3-1E Genes Of Eimeria Acervulina And Its Immunprotective To Chickens

Avian coccidiosis is a serve problem for the poultry industry, caused by intracellular protozoa including several species of the coccidia. Although coccidiosis is mainly controlled by the use of chemotherapeutic agents, alternative control strategies are needed due to the increasing emergence of drug-resistant parasite strains in commercial settings. The reaserch constructed a recombinant vector to co-express ADF and 3-1E genes of Eimeria acervulina. It built solid foundation for the development of effective and safe vaccines of anticoccodial as well as new drugs.Based on the sequence alignment of the actin-depolymerizing factor(ADF) gene of E.acervulina,two primers were designed and synthesized. Using the total DNA of sporozoites of E.acervulina Baoding strain isolated from Hebei province of China as template, a partial segment of ADF gene was amplified by PCR.The gene fragment 729bp in length was cloned into pMD-18T vector, and the recombinant plasmid was identified by PCR,restriction enzyme analysis and sequencing. The homology analysis revealed that the nucleotide sequences similar of the ADF gene of the E.acervulina Baodong strain with reference sequence were 99.6%. The plasmid pMD-18T-ADF was cloned into pET32a(+) vector,constructed recombinant plasmid pet32a-ADF,then transformed into E.coil BL21 strain to Expression. The ADF fusion protein band of about 46kDa was identificated by SDS-PAGE. Western blot analysis indicated that the recombinant protein could react specifically with Eimeria acervulina polyclonal antibody. The gene could synthesis a fusion protein at high levels.The fusion gene of ADF and3-1E was cloned by SOE-PCR, inserting into pET-32a(+) vector, the recombinant vector of pET-32a-ADF-3-1E was constructed correctly. Then it was transformedinto E.coli BL21 (DE3) and induced with IPTG. The expressed protein was detected bySDS-PAGE, with the expected molecular sizes of 68 kDa. The expressed protein was consistent with the prediction.The immuoprotection induced by recombinant ADF-3-1E antigen in chicken.The chicken were inoculated with r ADF-3-1E at 7 days and 14 days, then were challenged with Eimeria acervulina oocysts. The results showed that the pET-32a-ADF-3-1E test chickens can produce stronger resistance effect: Insects attack the egg sac of chicken production fell 67.88%, lower intestinal lesion score 67.21%, and the relative weight of chicken rate of 88.36%, ACI value of 166.76.