The Protective Immunity Of Recombinant Proteins Of Eimeria Against Four Chicken Coccidia

Chicken coccidiosis, caused by intestinal infection with Eimeria spp. invading intraepithelial cells, is spread worldwide and economically the most important parasitic disease of the poultry industry. At present, the controls of Eimeria infections are still mainly based on the anti-coccidial drugs. But, with the increasing number of drug-resistant strains and the more and more serious issues of drug residues. It makes animal-derived food safety has gradually become a global problem. Recombinant subunit vaccine is utilizing the DNA recombination technology, by means of transferring the gene that coding protective antigen into recipient bacterium or cell, to make the gene high expressed in the recipient bacterium and secrete the peptide chains of protective antigen. Then adding the recombinant protein into adjuvant to creat recombinant subunit vaccine.Lots of genes of protective antigens had been respectively cloned from sporulated oocysts of E.tenella、E.necatrix and E.acervulina at the laboratory of parasitology at NAU, and the prokaryotic expression recombinant plasmids had been constructed successfully. And genes of protective antigens eight fragments of EmTFP250had been respectively cloned from sporulated oocysts of E. maxima at the laboratory. All of these achievements are basis of following study.1Prokaryotic Expression of E.maxima Em6and Em8Genes and Purification of Recombinant ProteinsBy the means of DNA recombination technique, Em6and Em8genes were cloned into prokaryotic expression vector pET32a(+) and transformed into BL21(DE3). The recombinant proteins were expressed by inducing with IPTG. As the results of SDS-PAGE indicated, the Em6and Em8recombinant proteins were successfully expressed in E.coli with the relative molecular weight of about68and71kDa, respectively. The results of SDS-PAGE of the pellet and supernatant after ultrasonication showed that most of the recombinant proteins existed in the supernatant. As the results of western blot indicated, both of this Recombinant Protein were recognized strongly by serum from naturally infected chicken.2The Protection of the Different Recombinant Proteins against Eimeria spp.The protection of recombinant proteins pET28b(+)-TA4, pET32a(+)-SO7, pET28a(+)-NA4, pET32a(+)-NPmz19, pET28a(+)-LDH, pET28a(+)-3-1E, pET28a(+)-MIF, pET32a(+)-Em6and pET32a(+)-Em8were analysised. With200ug of the recombinant proteins, single or mixed,14-day-old chickens were intramuscularly immunized. A booster immunization was given by the same method as the first immunization7days later. The control groups were injected with PBS buffer. At28days of age, chickens were challenged with sporulated oocysts of the corresponding Eimeria spp. except the unchallenged control groups. Seven days post challenge, all the chickens were slaughtered. Efficacy of immunization was evaluated on the basis of oocyst decrease ratio, body-weight gain, lesion score, and anti-coccidial index (ACI). The results indicated that all of the recombinant proteins could provide protection against the challenge with the homologous Eimeria spp., and the effect of admixture recombinant proteins are better. Recombinant proteins TA4, NA4, LDH and Em8are the best for E.tenella, E.necatrix, E.acervulina and E. maxima, respectively.3The Cross-protection of the Recombinant Proteins against Heterogenous Eimeria spp.This study describes cross-protection experiments with recombinant proteins pET28b(+)-TA4, pET28a(+)-NA4, pET28a(+)-LDH and pET32a(+)-Em8to determine its efficacy against heterogenous Eimeria spp. With200μg of the corresponding recombinant protein,14-day-old chickens were intramuscularly immunized. A booster immunization was given by the same method as the first immunization7days later. The control groups were injected with PBS buffer. At28days of age, chickens were challenged with sporulated oocysts of the corresponding Eimeria spp. except the unchallenged control groups. Seven days post challenge, all the chickens were slaughtered. Efficacy of immunization was evaluated on the basis of oocyst decrease ratio, body-weight gain, lesion score, and anti-coccidial index (ACI). The results indicated that:the TA4recombinant protein could provide partial cross-protection against the challenge with E.necatrix, but little effect against E.acervulina and E.maxima; the NA4recombinant protein could provide partial cross-protection against the challenge with E.tenella and E.acervulina, but little effect against E.maxima; the LDH recombinant protein could provide partial cross-protection against the challenge with E.tenella and E.necatrix, but little effect against E.maxima; the Em8recombinant protein could provide partial cross-protection against the challenge with E.tenella and E.acervulina, but little effect against E.necatrix.4The Protection of the Admixture Recombinant Proteins against Eimeria spp.The protection of admixture recombinant proteins pET28b(+)-TA4, pET28a(+)-NA4, pET28a(+)-LDH and pET32a(+)-Em8were analysised. With200μg of the admixture recombinant proteins,14-day-old chickens were intramuscularly immunized. A booster immunization was given by the same method as the first immunization7days later. The control groups were injected with PBS buffer. At28days of age, chickens were challenged with sporulated oocysts of the corresponding Eimeria spp. except the unchallenged control groups. Seven days post challenge, all the chickens were slaughtered. Efficacy of immunization was evaluated on the basis of oocyst decrease ratio, body-weight gain, lesion score, and anti-coccidial index (ACI). The results indicated that admixture recombinant proteins could provide partial protection against the challenge with E.tenella, E.necatrix, E.acervulina and E.maxima.