Construction And Application Of Eukaryotic Expression Plasmid Of NDV F And Thymopentin Gene

Nucleic acid vaccine is quite different in the chenmical structure from traditional vaccine. Many experiments show that the delivery of genetic material into an animal's cells can trigger some synthesiztion of the encoden proteins as well as of antibodies targeted against those proteins, so it is named"the third-generation vaccine". Newcastle disease (ND) is a very important viral infectious disease that endangers seriously the chickens industries in our country all along. Although some attenuationn vaccine has been developed successfully and been applied abroad, it is often interfered by the maternal antibody, so the immunity of vaccine often fails. Emergence of nucleic acid vaccine has brought immeasurable prospect to the gene therapy and immunological field. It can induce cellular immune response and resistance the interferences from Maternal Antibodies. But the immune response of nucleic acid vaccine has a low efficiency, therefore it hinders its practical application. As a good immun-modulating medicine, thymopentin has been extensively applied to medical clinic and has got a certain achievements in the experiments of veterinary control in recent years. In order to further enhance the immune effect of nucleic acid vaccine, this dissertation does the research of construction of eukaryotic expression plasmid of NDV F and thymopentin gene and detection of the immunogenicity.Depending on the F gene sequence of newcastle disease virus which were published by GeneBank, a pair of specific primers was designed, up primers including the gene of TP-5. The NDV F gene DNA fragment was obtained by polymerase chain reaction(PCR), using the recombination plasmid of NDVZ F gene as the template. The PCR products were recombined into pcDNA3.1/V5-His-TOPO expression vector and it was indentified successfully by the restriction enzyme analysis. Nucleotide sequences was compared with the original sequences, the result showed homologous rates were higher than 99%, there was no aberrance in startup codon and terminate codon, the recombinant plasmid was named pcDNATP-5NDVZF. With the function of liposome, pcDNATP-5NDVZF was transfected into CEF cells and detected by indirect immunofluorescence assay (IFA) which proved that pcDNATP-5NDVZF was expressed as F protein in CEF cells well.pcDNATP-5NDVZF and pcDNANDVZF were injected separately into muscles of the little mice and one-day old commercial chicklings, the result shew that the immunogenicity of pcDNATP-5NDVZF was more significant than pcDNANDVZF and nucleic acid vaccine can induce immune response and resistance the interferences from Maternal Antibodies. So this study indicated that the expression products of TP-5 gene could strengthen the immunogenicity of DNA vaccines.