Study On The Nucleic Acid Vaccine Of Newcastle Disease Virus And Avian Infectious Bronchitis Virus

Nucleic acid vaccine is a new-type vaccine which is developed in 1990s and can induce humoral immunity and cell-mediated immunity by muscles injected or gene-gun inoculability, and nucleic acid vaccine which has no any poison effect is different from other traditional vaccines, so it is named "the third-generation vaccine". Newcastle disease (ND) and Avian infectious bronchitis (IB) are both very important viral infectious diseases that endanger seriously the raising-chickens' industries in our country all along. Although attenuated vaccine has been developed successfully and been applied abroad, it is often interfered by the maternal antibody, so the immunity of vaccine often fails. To search for a new-type vaccine which can counteract the maternal antibody and shorten or reduce the blank period after immunity and inoculability on one-day chicklings, we have a study on the NDV and IBV nucleic acid vaccine.In order to study the immunity of NDV nucleic acid vaccine, the NDV F gene was amplified from cloning plasmid of NDVZF gene by polymerase chain reaction (PCR), and it was recombinated into eukaryotic expression vector pcDNA 3.1/V5-His-TOPO. 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 pcDNANDVZF. With the function of liposome, pcDNANDVZF was transfected into CEF cells and detected by indirect immunofluorescence assay (IFA) which proved that pcDNANDVZF was expressed as F protein in CEF cells well. At last, pcDNANDVZF (NDV nucleic acid vaccine) were injected separately into muscles of the little mice and one-day old SPF chicklings and commercial chicklings at a different dose, the result showed that NDV nucleic acid vaccine had the good immunogenicity.In order to assess the probability of IBV vaccine on controlling IB, IBV S1 gene was inserted into the eukaryotic expression vector pcDNA3.1/-V5-His-TOPO. A eukaryotic expression recombinant plasmid pcDNAIBV-ZS1, containing start codon and stop codon, was constructed. With the function of liposome, the recombinant plasmids were transfected into CEF cells and detected by IFA. Results demonstrated that SI protein could be expressed in a large amout in CEF cells. The pcDNAIBVZSl (nucleic acid vaccine) were injected separately into muscles of the little mice and one-day old SPF chicklings and commercial chicklings. After two weeks, the serums of immuned animals were collected to make the indirect ELIS A, the results showed that IBV nucleic acid vaccine could induce the specific antibodies in bodies and had the good immunogenicity.In recent years, thymosin has been an immuneopotiating agent, which is very concerned and used widely in medicine clinic by people, and which has an advance effect on prevention and cure in veterinary clinic. In order to enhance the immunity of NDV and IBV nucleic acid vaccine and assess the combination effect of nucleic acid vaccine and thymosin, three simple and suitable methods of extracting thymosin from the animal's thymus had been devised by analyzing all the methods reported nowadays in the experiment. The immune activity and content of thymosin were confirmed by E rosette test, T lymphocyte conversion and ultraviolet spectrophotometry, then we established that the method of freeze-melt and extracting with acid and alkali was a better way to extract thymosin by compareting the different ways. At last, we used two simple and suitable methods to extract the chickens' thymosin. By E rosette test, T lymphocyte conversion and ultraviolet spectrophotometry comparison, thymosin immune activity and content were determinated. Compared the results of two ways, we found that all the immune activity of thymosin extracted was very high and also had no species differentiation, so it established a better foundation on carrying on the research of the combination effect of nucleic acid vaccine and immuneopotiating agent.