Isolation,Identification Of Pasteurellosis Multocida From Yak And Primary Study On Immunological Effect Of Recombinant OmpH、OmpA Of Pasteurellosis Multocida On Mice

Objective:Pasteurellosis is an animal acute infectious disease caused by a particular serotype of PasteurellaMultocida(Pasteurellosis multocida, Pm),such as cattle and buffalo hemorrhagic septicemia, lung disease, rabbits sepsis, wild and domestic birds avian cholera. It caused huge economic losses to the farmingaround the world. Because of its many serotypes, narrow vaccine immune spectrum, a shorter protection period and bacterial drug resistance, the disease has not been effectively controlled. In this study, Pasteurella multocida will be isolated and identified from clinical sample, the ompH and the ompA sequence will be analysed by software; The ompH and the ompA will be prokaryotic expressed and purified; OmpH and OmpA recombinant protein will be immunized mice and detected immune effect. The results will provide a theoretical basis for the effective control of pasteurellosis of epidemic and vaccine development.Method:1. Bacteria were isolated from the clinical infected Yak samples, and were identified by biochemical assays and PCR of KMT-1, Its capsular serotypes were determined by PCR. Drug resistance and virulence were evaluated by drug sensitivity test and artificial infected mice.2. The primers were designed according to ompH and ompA gene in GenBank. The ompH and ompA full legth segment were amplified from isolates, cloned and sequenced. Sequences were analysed with software such as DNAStar, DNAman and so on.3. The ompH and ompA segment without signal peptide sequence were cloned into T-vector, and constructed expression vector pET-28a-ompH,pET-28a-ompA respectively.The expression vector pET-28a-ompH,pET-28a-ompA transformed into E. coli BL21(DE3) competent cell. Recombinant expression E.coli was induced by IPTG. Recombinant protein were purified through Ni column, and detected by Western-blot method.4. The purified recombinant protein were prepared corresponding monovalent vaccine and bivalent vaccine. Recombinant protein vaccine and inactivated whole cell vaccine respectively immunized mice by subcutaneous injection.The indirect ELISA was used to evaluation antibody level.The immunized mice were challenged with2LD50Yak P.multocida at9days after third immunization and the immune protective efficacy was observed.Results:1. The identification results showed that these bacteria belonged to Pasteurella multocida, these isolates were susceptible to gentamycin, streptomycin, tetracycline, oxytetracycline, sulfa drugs. The capsular serotypes is type B, LD50is48.3CFU/mL for mouse.2. The ORF of ompH, ompA genes of these isolates were1002bp,1077bp, respectively encode334,359amino acids, the size is35.6743kDa,38.3432kDa.Compared with the representative strains corresponding genes, the homogeneity were82.3%-99.7%,85.7%-99.7%, it showed that both of them have high conservation. Further analysis revealed that ompH, ompA genes of these isolates shared shared99.7%homogeneity with P52strain isolated from cattle in India,which serotype is also B:2. 3. The prokaryotic expression vector pET-28a-ompH and pET-28a-ompA were highly expressed in E.coli. The molecular weight of fusion protein was about38.0kDa and40.4kDa, and existed in the form of inclusion bodies. The Western-blot results showed that recombinant proteins produce a single blot bands with serum of mouse which was infected Pasteurella multocida isolates.It showed recombinant proteins possesed antigenicity.The purity recombinant proteins were obtained, which provides a base for further study of its immunological characteristics.4. The antibody detection showed that antibody levels were increased in all groups except PBS control group. But antibody were significantly increased in inactivated whole cell group and rOmpA group. PBS group antibody levels are maintained at a low level. The protection rate were80%,40%respectively in inactivated whole cell group and rOmpA group, but the protection rate were only0%in other three groups. However, rOmpH, rOmpH+rOmpA group can postpone the time of death.Conclusion:The research showed that ompH and ompA gene of Pasteurella multocida strains from yak is highly conserved; recombinant protein OmpH and OmpA can stimulate the mice to produce specific antibodies; recombinant protein OmpA can provided protection against2LD50challenge of same type of Pasteurella multocida, but still lower than inactivated whole cell vaccine, however, the recombinant protein OmpH can not provided protection against2LD50challenge of same type of Pasteurella multocida. Therefore, Pasteurella multocida subunit vaccine still need further research.