The Expression And Immunogenic Resear Ch On P26-P40and P26-HSP70C Fusion Gene Of Mycoplasma Ovipneumonia Y98

Mycoplasma ovipneumoniae(MO) was the pathogen caused mycoplasmal pneumonia insheep and goats, especially the lambs of1-3months old. Up to now, MO had spread in theworld wide, which had brought heavy losses on sheep industry.In this study, gene P26、P40and HSP70C were amplified by PCR, and the template wasthe genome of MO Y98. The homologies of MO P26and P40to those of Mhp wererespectively79%and74%; and the sequence of HSP70C was the same as that published inGenebank. TGAs, which encoded tryptophan in the gene P26and P40were mutated to TGGby overlap extension PCR. Constructed the prokaryotic expression vectors, and those vectorswere respectively named pET-28a-P26, pET-28a-P40and pET-28a-HSP70C. Those vectorswere transformed into Ecoli. BL21(DE3) to construct the positive recombination strainsrespectively named BL21(DE3)(pET-28a-P26)、 BL21(DE3)(pET-28a-P40) andBL21(DE3)(pET-28a-HSP70C). Those strains were induced with IPTG. The products weredetected by SDS-PAGE, and specific bands about26、40and30KD (Ku) were found in linewith expectations. The results of Western blot showed that those3recombination proteins hadgood immunogenicity.Extracted the plasmids from DH5α（pET28a-P26）and DH5α（pET28a-P40）. Afterdigested by BamH I/Hind III, the product of pET28a-P26and pET28a-P40were linked totransform into E.coli BL21(DE3) to get the positive recombination strainsBL21(pET28a-P26-P40). In the same way, the positive recombination strain of P26-HSP70Cwas built. SDS-PAGE and Western blot showed that BL21(pET28a-P26-P40) andBL21(pET28a-P26-HSP70C) could respectively express about66kD and56kD protein in linewith expectations after induction. Moreover, the expressed P26-P40and P26-HSP70Cproteins existed both in form of solubility and inclusion body. Prepared the geneticengineering vaccines of P26、P40、P26-P40and P26-HSP70C to conduct animal experiments.Indirect ELISA showed that all the proteins had good immunogenicity and the antibody titersof serum from mice immunized by P26、P40、P26-P40and P26-HSP70C were respectively1:8000,1:32000,1:128000and1:32000. In addition, under the same diluted multiples, the titer of serum immunized by P26-P40protein was higher than that of serum immunized byP26and P40protein, which stated that the immunogenicity of fusion protein was better thanthat of single protein. And the titer of serum immunized by P26-HSP70C protein was higherthan that of serum immunized by P26protein only, which declared that HSP70C had playedthe role as immunologic adjuvant. All those results were in accord with the protective rates.The lamb immunity test further confirmed: P26-P40fusion protein also had goodimmunogenicity. In conclusion, the immune effect of fusion protein vaccine of two antigenswas better than that of single protein vaccine. The role of HSP70C as immunologic adjuvantmade the immunogenicity of P26-HSP70C fusion protein better than that of single protein.