Expression Of Emgam56 Gene In Pichia Pastoris And Its Immunoprotection Analysis

Avian coccidiosis is an intestinal infection caused by intracellular protozoan parasites belonging to Eimeria. It not only causes the death of chickens, but seriously impairs the growth and feed utilization. Currently the control strategy of coccidiosis mainly employs the use of anti-coccidial drugs and live oocysts vaccine. However, the use of anti-coccidial drugs has been greatly restricted with the continual emergence of drug-resistance and the public concern of safety in animal foodstuff. Meanwhile, the use of live oocyst vaccine has some drawbacks including the high production expenses, diffusion of pathogen and atavistic possibility of coccidia. Therefore, subunit vaccine is the most advantageous idea in controlling coccidiosis. As a type of subunit vaccine, CoxAbic is composed of three Eimeria maxima gametophyte antigen: EmGam56, EmGam82 and EmGam230. The gametophyte antigens are separated and purified by affinity column from intestinal epithelial cells of infected chicken. So the manufacturing of CoxAbic(?) is time-consuming and expensive. Using gene engineering technique to develope genetic engineering vaccine may overcome the problem.As one of the eukaryotic expression systems, pichia pastoris expression system not only has a simple genetic manipulation, fast growth, low fermentation cost advantages, but also has higher eukaryotic protein expression systems various post-translational modification. Therefore, the study was undertaken to express the Emgam56 gene of E. maxima gametophyte antigen in P. pastoris. The antigenicity and immunogenicity of the recombinant protein rEmGam56 were investigated. The protective immunity of P.pastoris and prokaryotic recombinant protein rEmGam56 were dectected with animal experiment. The results may provide a basis to develope sub-unit vaccine of recombinant gametophyte antigen.1. Expression of Emgam56 gene in P. pastoris and preliminary identificationThe Em,gam56 gene of E. maxima NT strain without signal peptide encoding region was amplified by PCR from the plasmid pGEM-T-gam56, and then inserted into the eukaryotic express vector pPICzaA to construct the recombinant plasmid which was named pPICzaA-Emgam56. After the identification by PCR, restriction endonuclease analysis and sequencing, the correct recombinant plasmid was linearized and transformed into P. pastoris strain GS115 by electroporation. Multi-copy recombinant strains were screened by Zeocin and cultured in BMMY flask for the expression. The size of recombinant protein was about 11.6 kDa and could be recognized by 6×HIS tag.2. Immunogenicity of recombinant protein rEmGam56 expressed in P. pastorisAccording to the results of western-blot analysis, the fusion protein had the same immunogenicity as the positive serum of chicken infected with E. maxima by using the mouse anti P. pastoris rEmGam56 polyclonal antibody and mouse anti prokaryotic rEmGam56 polyclonal antibody as primary antibody.And indirect-ELISA analysis showed the P. pastoris recombinant protein had good immunogenicity according to the level of anti-Emgam56 polyclonal antibody produced in the mouse and chicken.3. Protective efficacy of recombinant protein rEmGAM56 against E. maximaThe protective efficacy of P. pastoris recombinant protein rEmGam56 was assessed by mortality rate, relative weight gain, reduction of lesion scores and relative oocyst protuction. Compared with the groups immunized with prokaryotic rEmGam56 and live oocysts, the results indicated that:The groups immuned with 60μg of rEmGam56 expressed in P. pastoris and 300 μg of rEmGam56 expressed in E. coli had a better immunoprotection, the oocyst reduction rate were up to 34.22% and 50.20% respectively, but were still lower than the group of immuned with live oocysts. Compared with the group immuned with 60μg of rEmGam56 expressed in P. pastoris, the group immuned with 300μg of rEmGam56 expressed in E. coli had a better immunoprotection, but the level of antibody and cytokines (CD4+ and CD8+) of the two immunized groups were higher than the negative control group and adjuvant group.