Protective Evaluation Of Inactivated Bovine Campylobacter Jejuni And Its Recombinant HSP43 Protein Immunogenicity

Campylobacter jejuni (C.jejuni) can cause neonatal calf diarrhea and winter dysentery of adult cattle, which has brought about enormous economic loss for the cattle industry.One pair of primers was designed basing on the published sequences of PorA gene of C.jejuni in GeneBank by PCR from the total DNA, then cloned into pMD18-T vector. The positive plasmid pMD18-T-N was got by identification. Then the gene of PorA protein was subcloned into the downstream of the pQE-30a(+) vector named as pQE-30a-PorA. We confirmed that the target gene was inserted into the pQE-30a(+) vector by PCR and restriction enzyme digestion. It was proved that the recombinant plasmid pQE-30a-PorA contained the gene of PorA protein, which had correct sequence by sequence analysis. The positive plasmid was transformed into the optimization competent E. coli BL21(DE3) and induced by lmmol/L IPTG. SDS-PAGE result showed that the gene of PorA protein was expressed in prokaryotic expression system. Additionally, the western blot assay proved the recombinant PorA protein of C.jejuni-DQ strain having good reactionogenicity.Taking the purified recombinant HSP43 protein as antigen, We established indirect ELISA method for detecting C.jejuni antibody. Detection of serum antibodies in mice modified reaction condition of ELISA:the optimal coating buffer was carbonate buffer solution (0.05M pH9.6), and the optimal coating antigen for microplate was 7.5μg/mL. Five percent skim milk was added as the optimal blocking agent and incubated for 1 hour at 37℃. The best serum dilution buffer was 5% skim milk, and the optimal dilution of sera samples was 1:100. The working concentration of goat anti-mouse IgG-HRP was 1:5 000. Detection of serum antibodies in bovine modified reaction condition of ELISA:the optimal coating buffer was carbonate buffer solution (0.05M pH9.6), and the optimal coating antigen for microplate was 2.5μg/mL. Five percent skim milk was added as the optimal blocking agent and incubated for 1 hour at 37℃. The best serum dilution buffer was 5% skim milk, and the optimal dilution of sera samples was 1:200. The working concentration of goat anti-bovine IgG-HRP IgG was 1:5 000. There is no any cross reaction between the antigen and the positive serum of S.enteritidis, E.coli, S.aureus. Compared with the serum neutralization testing, the results showed that the specificity.Take the C.jejuni and the rHSP43 as the research object, preparing three different adjuvant C.jejuni formaldehyde immunogen and the rHSP43 immunogen, then immuning the 8 week old clean grade mice in three immunological way, the indirect ELISA method detected the antibody growth and decline condition and evaluated the protective effect of two immunogen with the challenge test.As the result, in the C.jejuni immunogen group, the immune effect of the 5×1010CFU/mL group was similar to the 5×109CFU/mL group, compared the 5×107CFU/mL group and the former group, their difference are significantly(P<0.01); the immune effect of the injected intraperitoneal group and the subcutaneos injection group is simmilar (P> 0.05),their immune effect significantly better than the gastric perfusion method group(P<0.05);The immune effect of adding adjuvant group is significantly better than the group without adjuvant(P<0.05); As the result, the immune effect is best of the C.jejuni immunogen in the condition of 5×109CFU/mL, white oil adjuvant,subcutaneos injection way. The rHSP43 group, uder the white oil adjuvant,subcutaneos injection or injected intraperitoneal method the immune effect is the best.Immunization in mice after 21d with 1×1010 doses of C.jejuni attack bacteria, whole bacterial immunogen able to provide complete protection and substituting white oil adjuvant emulsion of rHSP43 only 75% protection.The results also showed that higher special serum IgG antibodies titers were detected in the bovine vaccinated group, which was significantly different from the control group (P<0.01).Under the same condition, the C.jejuni immunogen group is better than the rHSP43 group in the resisting infection effection respects.