| Capripoxvirus causes a severe and highly contagious disease in sheep or goats, it is the most important in all animal pox virus. Capripox is the one of the 93 animal diseases that must be reported by Office International des Epizooties (OIE) and group first diseases in China.P32 protein of capripoxvirus is the main structural protein on the virus envelope surface and contains neutralized antigen epitope.The CD58 is the lymphocyte function antigen (LFA-3). CD58 and CD2(CD58/CD2) make the immune system Activated. This article aims is that make the Capripoxvirus P32 and CD58 gene construct in the same vector. Play the role of CD58 in the immune adjuvant. To enhance the immune effect of P32 protein, for the further development of effective DNA vaccine.The lymphocytes isolated from the blood of healthy sheep. Using RT-PCR, the CD58 is amplified in the sensitized lymphocytes. The CD58 gene was inserted into pGEX-4T-1 vector, the positive plasmid,named pGEX-CD58.The recombinant pGEX-CD58 were expressed in Escherichia coli induced by IPTG.The molecular weights of expressed protein were about 53ku(GST fusion). The results of Western-blotting showed that the CD58 protein could be detected by the Sheep CD58 monoclonal antibody.It indicated that the CD58 protein be of immunoreactivity.Then the two genes (CD58 and P32 ) were cloned into the eukaryotic plasmid pBudCE4.1,the positive plasmid,named pBudCE4.1/CD58/P32. After plasmid purification, the acquired recombinant plasmids were transfected into BHK-21 cell with liposome mediated method. the expression of P32 and CD58 were tested by direct immunofluorescence assay ( IFA) or indirect immunofluorescence assay. The results showed that P32 and CD58 were successfully expressed in BHK-21 cells. Using BALB/c mice as animal models,and inject in intramuscular of BALB/c mice, Regular tests were carried out by ELISA and MTT assays. The results showed that humoral and cellular immunoresponse were elicited by pBudCE4.1/CD58/P32. |
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