Construction And Immun Characteristics Study Of Eukaryotic Expression Vector Of Hypodermins Gene

Hypodermosis is a parasitic disease caused by the Hypoderma larvae yurn into the subcutaneous tissue.The harm is vary serious,has a very large impact on the cattle industry and leather industry and prevent the public health caused by zoonoses. There is a growing emphasis on disease prevention and control research.In the present study, the DNA fragments of HA-HC were amplified by PCR and spliced by overlapping extension from the genome DNA of recombinant cloning vectors pTHA and pTHC, and the fragment was ligated into pMD19-T simple vector to contruct recombinant cloning pTHAC. HA、HC、HACgenes were subcloned into pVAXl vector to construct recombinant plasmids pVHA、pVHC and pVHAC.The three plasmids were transfected into the mice embryonic fibroblasts (MEF) and the bovine embryonic fibroblasts in vitro to detect the expression of target genes.KunMing mise were intranmuscularly vaccinated with the foure plamids(pVHA、pVHC、pVHAC、 pVHA+pVHC) and the control vector pVAXl and PBS respectively.The animals in each group were immunized3times with2week intervals.The serum antibody titers were determined using indirect enzyme-linked immunosobent assay (iELISA) coated.Two weeks after the third immunization,splenic lymphocytes proliferation assay were detected by MTT method and secreted IFN-y of spleen were tested.The results of indirect ELISA showed the lewels of antibodies in all recombinant plasmids groups were significantly higher than the two control groups(P<0.01). Splenic lymphocytes proliferation experiments indicated the SI value in all recombinant plasmids groups were significantly higher than the two control groups(P<0.01), the groups of fusion gene was higher than the single gene pVHC group and lower than the single gene pVHA group(P<0.01). The IFN-y experiments showed the level of induced the value in all recombinant plasmids groups were significantly higher than the two control groups(P<0.01).So our experiment may be useful for the development of Hypodermosis vaccine.