Prokaryotic Expression Of Eimeria Tenella Gam22Gene And Its Immunoprotecive To Chickens

Avian coccidiosis is a diseases of the major economic importance to the poultry industry. It is caused by the apicomplexan protozoa Eimeria which include Eimeria acervulina, E. brunette, E. maxima, E. mitis, E. necatrix, E. praecox and E. tenella. Among the seven pathogens, E. tenella has the most pathogenicity. Prophylactic feeding of coccidiostat drugs is the major disease control method used in commercial settings. However the extensive use of anticoccidials over the years has led to the emergence of drug resistance in the field, and consumer concern over the presence of residual drugs in poultry meat has led to the phasing out of the use of these agents. Current control strategies is being from chemoprophylaxis to immunoprophylaxis. At present there are two types of vaccines, live vaccine and subunit vaccine. The live vaccine has strong immunity which can protect against coccidiosis in the birds, but there are some defects shch as diffusion of pathogen, antigenic variation, high toxicity, reduction of feed conversion ratio and the difficulty in controlling the inoculation dosage, and so on. All of above, recombinant vaccine will be the most advantageous idea in controlling coccidiosis.The E. tenella gametocyte gene(Etgam22) was a multiple copy gene which was expressed in the phage of gametocyte. It played an important role in oocyst wall formation. Although there were some reports of this gene, but there were no research about gene expression in vitro and its role in immunoprotection. In this paper, Elgam22gene was cloned and expressed in E.coli. The protective immunity of recombinant protein EtGAM22was dectected with animal experiment. The results may provide a new method of controlling coccidiosis in poultry industry.1. Clone and sequence analysis of Etgam22geneThe gam22gene of E. tenella YZ strain was amplified by RT-PCR from total RNA of purified gametocytes, and cloned into pGEM-T Easy vector. The recombinant plasmid was identified by PCR, restriction endonuclease analysis and sequencing after blue/white selection. The results indicated that Etgam22gene was consisted of749nucleotides and included an complete opening read frame with a length of597bp which encoding198amino acids. Compared with E. tenella VT-2, the homology of nucleotides acid sequence was99.6%. Its predicting antigenicity of gam22protein by computer softwares showed that there were four antigenic determinats and three of them were located in the peptides where are proline-histidine rich domains. 2. Expression of Etgam22gene in E.coliThe Etgam22gene expressing fragment with the length of558bp was amplifyed by PCR from plasmid pGEM-T-gam22, then subcloned into express vector pET-28a(+). The fusion protein was expressed of23.8kDa successfully in E.coli after induced by IPTG, and mainly existed in the precipitation. According to the results of western-blot analysis using the positive serum of chicken infected with E. tenella and mouse anti-gam22polyclonal antibody the fusion protein was immunogenic. The fusion protein could combine with sera of chiekens infected E.necatrix and E.maxima, which indicated it had a good cross-protection. As to the level of anti-gam22polyclonal antibody produced in the mouse, the recombinant protein had good immunogenicity.3. Immunofluorescence localization of expression product of Etgam22gene in E.tenellaSeven-week-old chickens were infected with E. tenella Yangzhou strain. The caecum tissues of the chickens was removed at120h,132h,144h,156h,168h and180h post-infection respectively. After these caecum tissues were fixed and sectioned, the sections were strained with hematoxylin-eosin or the antibody(anti-GAM22) followed by goat anti-mouse IgG which was conjugated to Rhodamine respectively. Observation using fluorescence microscope, it was found that the fluorescence labelling appeared in WFBs in macrogamecocytes and oocyst walls. It could prove that the expression product of Etgam22gene in E.tenella was nvolved in oocyst walls formation.4. Protective efficacy of recombinant GAM22protein of E. tenellaTo determining the protective effects of the recombinant protein EtGAM22, the evaluation indicator of protective immunities were assessed by relative weight gain, oocyst output, and oocyst reduction. The results indicated that:the level of antibody, CD4+and CD8+of all the immunized groups were higher than the negative control group, the recombinant protein EtGAM22had the ability to increase the weight of chicken, cut down the pathological changes and decrease the oocyst reduction. The group given at100μg recombinant antigen had the best immunization protection effect, but was still lower than the group immunized oocysts.