| Avian coccidiosis is one of most common disease affecting the intensive poultry industry. Coccidia infection results death accompanied by severe depression in body weight gain, reduced feed efficiency. The losses due to coccidiosis in chicken industry has been estimated8billion dollars every year. The annual consumption of anticoccidial drugs is about0.3to0.6billion dollars in our country. At present, the measure of disease prevention and control is transforming drug-treatment strategies into immune prevention. Gametophyte subunit vaccine is a new type of chicken coccidiosis vaccine developed abroad, which is composed of Eimeria maxima gametophyte antigen purified. Because the vaccine antigens are gametophyte proteins, which were separated and purified by affinity column from intestinal epithelial cells of infected chicken. So the process is complicated and the cost is highe. Therefore, the development of low cost and prokaryotic expression of subunit vaccines has a tempting prospect. Therefore, this current study was undertaken to express the full-length Emgam56gene of E. maxima in Escherichia coli. The effect of rEmGam56vaccination on resistance to experimental E.maxima infection was evaluated by animal experiment. The experiment may provide a basical theory in preparing recombinant vaccine to resist coccidiosis.1. Prokaryotic expression of Emgam56gene of E. maximaThe Emgam56gene of E.maxima NT strain without signal peptide encoding region was amplified by polymerase chain reaction from plasmid named pGEM-T-gam56which contained the cDNA of Emgam56gene with two primer sequences designed according to the sequence of Emgam56gene and enzyme cutting sites of plasmid pET-28a(+). Amplicons were cloned into the pET-28a(+) plasmid vector to build a recombinant expression plasmid, named pET-28-Emggm56. Then the recombinant plasmid were transformed into E. coli. After induced by IPTG, the56kDa His-Em Gam56fusion protein was expressed, which were confirmed by SDS-PAGE and Western-blot analysis using the positive serum of chicken infected with E. maxima and mouse a.nti-gam56polyclonal antibody, respectively. The fusion protein was expressed as soluble form, and showed to be immunogenic according to the results of Western-blot analysis using the positive serum of chicken infected with E. maxima.2. Preparation of rEmGam82-Y of E. maxima Plasmid pGEX-6P-1-gam82-Y which was successfully constructed and preserved in our laboratory was transformed into E. coli BL21. The fusion protein was successfully expressed in E. coli after induced by IPTG. Insoluble53KDa GST-Gam82-Y fusion protein was successfully expressed. Based on the original inclusion body purification technology, some improvements were made. The purification of inclusion body has a better effect. After being concentrated with PEG8000, the final protein concentration was3mg/mL3. Protective efficacy of rEmGAM56of E. maximaTo determine the protective effects of the recombinant protein rEmGAM56, the evaluation indicators of protective immunities were assessed by mortality rate, relative weight rate, reduction of lesion scores, oocyst reduction rate. Compared with the group immunized with recombinant protein rEmGam82-Y and oocysts. The results indicated that:the group immunized with300μg recombinant protein has the best immunization protection effect, but it was still lower than the group immunized with oocysts. The difference is significant (P<0.05). The average weight gain and relative weight gain rate of recombinant protein rEmGam56of high dose group (300μg) is higher than the middle dose group (200μg) and low dose group (100μg). It is similar to the recombinant protein rEmGam82-Y of high dose group (300μg), but it is less than the group immunized with oocysts. And the difference is significant (P<0.05). The intestinal lesion score of recombinant protein rEmGam56of high dose group (300μg) is lower than that of the middle dose group and low dose group. It is similar to the recombinant protein rEmGam82-Y of high dose group, but it is higher than that of the group immunized with oocysts. The intestinal lesion score reduction rate of recombinant protein rEmGam56of high dose group is higher than the middle dose group and low dose group, but it is lower than the group immunized with oocysts. The results showed that the recombinant protein rEmGam56for E. maxima infection has certain protection, but it is lower than vaccined with live oocysts.4. Combined immunization protective efficacy of rEmGAM56and rEmGAM82-Y of E. maximaTo determine the combined immunization protective effects of the recombinant protein rEmGAM56and rEmGam82-Y, the evaluation indicators of protective immunities were assessed by mortality rate, relative weight rate, reduction of lesion scores, oocyst reduction rate. Compared with the group immunized with recombinant protein rEmGam82-Y, rEmGam56and oocysts. The results indicated that the average weight gain and relative weight gain rate of the combined immunization group, rEmGam56(150μg) and rEmGam82-Y (150μg), is higher than the group immunized with one recombinant protein, but it is less than the group immunized with oocysts. And the difference is significant (P<0.05). The average intestinal lesion score of combined immunization group is lower than that of the group immunized with one recombinant protein, but it is higher than that of the group immunized with oocysts. And the difference is significant (P<0.05). The intestinal lesion score reduction rate of combined immunization group is higher than the group immunized with oocysts, but it is lower than the group immunized oocysts. The results showed that the combined immunization group has a better protection for E. maxima infection, but it is lower than vaccined with live oocysts.5. Effects of different adjuvants on the protective efficacy of rEmGam56of E. maximaTo determine effects of different adjuvants on the effect of recombinant protein rEmGam56of E. maxima, the experiment selected three adjuvant:interleukin-2(IL-2), cell transfer factor and astragalus poly saccharide (APS). The evaluation indicators of protective immunities were assessed by relative weight gain, oocyst output and oocyst reduction rate. Compared with Freund’s adjuvant, rEmGam56without adjuvant (recombinant protein alone) and the group immunized oocysts. The results indicated that:the average weight gain, relative growth rate and lesion scores reduction rate of IL-2, cell transfer factor, APS, Freund’s adjuvant, recombinant protein alone are in a decreasing manner. The average weight gain of the group immunized with recombinant protein group was significantly lower than the average weight of the group immunized with oocysts. The difference to the group immunized with oocysts is significant(P<0.05). The mean intestinal lesion score of IL-2, cell transfer factor, APS, Freund’s adjuvant, recombinant protein alone is in a increasing manner. The difference to the group immunized oocysts is significant(P<0.05). The results showed that the IL-2, cell transfer factor and APS can enhance the protection of recombinant protein rEmGam56in a certain extent. |
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