| Chicken coccidiosis is caused by chicken Eimeria parasites protozoiasis parasitizing in the intestinal epithelial cells, causing huge economic losses to the poultry industry. It's estimated that the annual direct and indirect economic losses in the world is about $1-$3 billion, and about three billion RMB in Chinese. Drug prevention is the main measures of prevention and control of chicken coccidiosis at present. With the resistance and legislative limitation of anticoccidial drugs, people pay more attention to the immune prevention. The vaccine in the market with a variety of live oocysts of Eimeria species has good immune protective effect. Due to the live vaccine easily outbreak coccidial infection with improper use and exists other security issues or higher production costs, the novel molecular vaccine such as sub unit vaccine become hot research in recent years.The antigen with good immunogenicity is the key to construct a new subunit molecular vaccine.Through studying the life history of coccidiosis, finding out the microneme locating in front of the sporozoite and merozoite is an important part of the apical complex, playing an important role in the invasion of host cells.At present, many kinds of microneme proteins have been proved to have good immunogenicity.The apical membrane antigen and microneme proteins of Eimeria tenella are important microneme protein, playing an important role in invasion of host cells, transitional and the intracellular survival. However, the complexity of the life history of Eimeria and an antigen protein appeared only in a part of the life cycle, may lead the subunit vaccines based on single antigen in anticoccidial only has some protective immunity.Therefore, it may be a fast and effective method to improve the immunogenicity of antigen.Coccidiosis is intracellular parasitic protozoa, causing cell specific immunity response. Cellular immunity mainly includes CD8+T lymphocytes(cytotoxic T cells) and CD4+T lymphocytes(helper T cells). CD4 + T cells and CD8 + T cells play a different role in different kinds of coccidial infection and Eimeria antigen in T cell epitopes are crucial to the induction of cellular immunity.In order to improve the immunogenicity of antigen,select different antigen protein expressed in different stages of coccidia according to the life cycle of Eimeria and antigen expression, and analysis theamino acid sequence of the protein. It is a feasible plan to improve the immunogenicity of the antigen by selecting the effective antigen epitopes.Adjuvant is a non-specific immune enhancer, which can improve the immunogenicity of the antigen,change the type of immune response, and prolong the immune period to enhance the body's immune response ability. The application of different adjuvants on immune produced different effects, choose the suitable adjuvant can enhance the body produce nonspecific and specific immune response, and can improve the effect of vaccine. Therefore, the adjuvant is one of the focus about enhancing the immunogenicity of antigens.This study selected the several important microneme protein genes of microneme protein 2(etmic2), microneme protein 3(Et MIC3) and apical membrane antigen 1(Et AMA1) expressed by the sporozoites and merozoites of coccidiosis in the invasion stage to analyse the protein amino acid sequences by biological software SYFPEITHI and DNAStar. T cell epitopes were selected as candidate antigen genes of subunit vaccine, the genes were cloned and sequenced, and then transformed into Escherichia coli. The expression of recombinant protein Et MIC2-Et AMA1-Et MIC3 was induced by IPTG. By SDS-PAGE and Western-blot identification, the results showed that recombinant Escherichia coli was successfully expressed recombinant protein Et MIC2-Et AMA1-Et MIC3. The recombinant protein Et MIC2-Et AMA1-Et MIC3 was emulsified with PBS, Freund adjuvant,pine pollen polysaccharide and Propolis Adjuvant to make the subunit vaccines. At 7 and 14 days old, chicks were immuned twice at neck subcutaneous. And seven days after the second immunization, except the group without immune and infection with coccidia(I group),all groups were inoculated 30000 fresh oocysts of Eimeria tenella. Results showed that the group immunized with recombinant protein has significant weight gain,and the lesion score and fecal egg counts were significantly decreased(P < 0.05) compared with the group of not immune but coccidian infection(II group). And emulsion adjuvant group(IV, V and VI group) compared with alone immunized with recombination protein group(Group III) showed the weight gain increased, the lesion score and the number of grams of fecal oocyst decreased. The ACI results showed that the recombinant protein can provide good protective immunity against coccidia infection and the propolis adjuvant enhancing the immune protective effect of the recombinant protein in resistance to Eimeria tenella invasion is the most ideal. The effect of lymphocyte proliferation and the ratio of CD4+ and CD8+ in peripheral blood lymphocytes showed that the recombinant protein could induce the cellular immunity, and the adjuvant could enhance the effect of the recombinant protein. In the aspect of specific serum antibody and mucosal immune antibody s Ig A, recombinant protein can induce effective humoral and mucosal antibody and the Propolis Adjuvant was better than the other two adjuvants in the induction of serum antibody and mucosal immune antibody s Ig A.Test results showed that the recombinant protein M2-A1-M3 can against Eimeria tenella infection to provide effective immune protection, and adjuvant can improve the immune protective effect of recombinant protein in different degree. Compared with the adjuvant of pine pollen and Freund adjuvant,the Propolis Adjuvant was more ideal in enhancing the immune effection. |
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