Constructions Of Expression Vectors Of Gene Gam82 From Eimeria Maxima Nantong Strain And Its Immunoprotecive 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. A novel vaccine named CoxAbic that, unlike the currently available vaccine is based on the concept of"transmission blocking immunity", immunisation of breeding hens prior to the start of lay, give rise to a protective IgG response, which is transferred to the developing embryo, providing protection to their offspring broiler chickens. The production of CoxAbic is expensive, time-consuming and laborious, because it relies on the affinity purification of the native gametocyte antigens from parasites. Thus, genetic engineering vaccine, based on the recombinant forms of gam82 protein(one of the components of CoxAbic) would be advantageous. In order to attempt a new method of controlling coccidiosis in poultry industry, the studies had been conducted as the following.1. Clone and sequence analysis of gene gam82 of E. maximaThe gam82 gene of Eimeria maxima NT strain was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of purified gametocytes. The products of RT-PCR were cloned into pGEM-T Easy vector. Positive clones were identificated with blue/white selection, restriction enzyme digestion and sequence analysis, respectively. Results indicated that gam82 gene of E. maxima NT strain included an opening read frame consisted of 1 791 bp, which encoded for a polypeptide of 596 amino acids. Homology of the ORF sequences of gam82 gene between E. maxima NT strains and E. maxima Houghton strains was 100 percent. The secondary structure and its antigenicity of gam82 protein were predicted by computer softwares, which showed that there were nineteen antigenic determinats and the eight of them were located in the peptides of 282 to 503 amino acids where included two tyrosine rich domains and was named gam82-Y in this research.2. Expression of gene gam82-Y of E. maxima in E.coliThe gam82 gene fragment of E. maxima NT strain, which was named gam82-Y, was amplified by polymerase chain reaction (PCR) from plasmid pGEM-T-gam82, and subcloned into express vector pGEX-6P-1. The positive plasmid named pGEX-6P1-gam82-Y were identified by restriction enzyme digestion and sequencing, respectively. After induced by IPTG, the 53 Ku GST-gam82-Y fusion protein was expressed, which were confirmed by SDS-PAGE and Western-blot analysis, respectively. The fusion protein was expressed in insoluble form, and showed to be immunogenic according to the result of western-blot analysis using the positive serum of chicken infected with E. maxima.3. Construction of the vaccine pcDNA3.1-gam82 of E. maximaUsing a pair of specific primes with the sites recognized by EcoRI and Hind III, the gam82 gene of E. maxima NT strain was amplified by polymerase chain reaction (PCR) from plasmid pGEM-T-gam82. The products of PCR were cloned into expression vector pcDNA3.1 to construct the vaccine pcDNA3.1-gam82. After positive clones were selected with ampicillin, the recombineant plasmid was confirmed by restriction enzyme digestion and sequence analysis, respectively. To evaluate the immune efficacy of the vaccine, lymphocyte proliferation assay and antibody determination was carried out. The results showed that chickens immunized with the vaccine produced significantly higher lymphocyte proliferation and antibody responses than the control birds (P<0.05).4. Protective efficacy of recombinant gam82-Y protein and the vaccine pcDNA3.1-gam82 of E. maximaTo determine the protective effects of recombinant gam82-Y protein and the vaccine pcDNA3.1-gam82, two animal experiments were performed. The protective immunities were assessed by relative weight gain, oocyst output, and oocyst reduction. The results of the first experiments showed that the protective efficacis in the group given at 0.5 mg recombinant gam82-Y antigen mixed with FCA and in the group given at 0.5 mg gam82-Y antigen only were higher than in other groups (P<0.05) expect the group immunized 500 oocysts and negative control group. The results of the second experiments showed that protective effects in the group immunized with pcDNA3.1-gam82 was the same as the group immunized with recombinant gam82-Y antigen, but were higher than in the non-immunized control group and the group immunized with pcDNA3.1 (P<0.05), and was still lower than in the group immunized 500 oocysts (P<0.05).