The Biological Activity Of Genetic Enginerring Swine Interferon β And Comparison Of Three JEV EDⅢ Vaccine DNA Adjuvant

Japanese encephalitis (Japanese encephalitis, JE) is an acute infectious disease of central nervous system caused by the Japanese encephalitis virus (Japanese Encephalitis Virus, JEV). The disease widespread in many countries, not only on the pig industry caused huge economic losses, but also a serious threat to human health. There is no effective treatment methods for JE, and treatment of this disease is primarily based on the way immunization vaccines for prevention. Now the world’s vaccine to prevent this disease is mainly used in some of the traditional inactivated vaccine and the attenuated vaccine. There are some drawbacks of these traditional vaccines, such as the inactivated vaccine effect is not very strong, attenuated vaccine there is a strong risk of toxicity to return and so on. New and effective JE vaccine is urgently needed. Meanwhile, the urgent need to develop a safe, efficient, new anti-virus and immune enhancer for enhancing the body’s resistance and improving vaccine protection rate. Interferon and heat shock protein 70 are biologically active proteins playing an important role in the immune response. In this thesis, the biological activity of swine interferonβ, and a comparative study of immune-enhancing effects among the two types of bacterial Hsp70 and swine interferonβas nucleic acid adjuvant have been done. The experimental thesis includes the following experiments:1 Construction of recombinant vector:To study the biological activity of swine interferonβ, we constructed a recombinant expression vector pPICZa-A-SwIFN-β, swine interferonβto achieve secretion expression in yeast system. Swine interferon P was prepared for the follow-up; To compare the immune-enhancing effects among the two types of bacterial Hsp70 and swine interferonβas JEV EDIII protein subunit vaccine nucleic acid adjuvant, we constructed three recombinant eukaryotic expression vector pCDNA3.0-SwIFN-β, pCDNA3.0-H.Ps-Hsp70 and pCDNA3.0-MT-Hsp70, prepared for the follow-up animal tests.2 The experiment of recombinant swine P-interferon as a biologically active protein for JE virus:Study of swine interferonβinhibited JEV replication on BHK-21 cells, while content of IFN is 1000 units, it can protect 50% BHK-21 cells from infection. The real-time quantitative PCR results show that when the content of interferon is 10U, it did not show significant reduction of viral copies, while the 1000U and 105U when the viral copies by nearly 10 times; We chose three doses of swine interferonβ 0.1ug, lug and lOug for pigs texts as JEV EDIII protein subunit vaccine adjuvant. After a series of tests we found that highest titers of anti-encephalitis virus antibody and immune antibodies appeared in the lug swIFN-β+ TRX-EDIII experimental group, while the groups immunized with O.lug swIFN-β+ TRX-EDIII and lOug swIFN-β+ TRX-EDIII and control groups did not show significant improvement. For the purposes of inducing cytokines, IFN-y in the lug swIFN-β+ TRX-EDIII experimental group was significantly higher than other groups groups. But recombinant swine interferonβas an adjuvant was not significantly increased IL-4 induction level. The study found that swine interferonβcan inhibited JEV replication on the BHK-21 cells, and recombinant swine interferonβcan be used as a powerful adjuvant to significantly enhance immune response for JEV EDIII protein subunit vaccine.3 In this study, three recombinant eukaryotic expression vector pCDNA3.0-SwIFN-β, pCDNA3.0-H.Ps-Hsp70 and pCDNA3.0-MT-Hsp70, respectively, were used as JEV EDIII protein subunit vaccine adjuvant on pathogen-free pigs, after some testing:We found that pCDNA3.0-SwIFN-β, pCDNA3.0-H.Ps-Hsp70 and pCDNA3.0-MT-Hsp70 can induce a strong immune response for JEV EDIII protein subunit vaccine. The results of antibody titers and neutralizing antibody showed H.Ps-Hsp70 and MT-Hsp70 can stimulate stronger immune responses than swine interferonβ; For cytokines detecting, H.Ps-Hsp70 and MT-Hsp70 can both stimulate IL-4 and IFN-y secretion, while the swine interferonβmainly stimulated the expression of IFN-y but IL-4 secretion less affected. There was no significant difference between H.Ps-Hsp70 and M.T-Hsp70. The experiments show that Hsp70 can induce stronger immune response than swine interferonβfor JEV EDIII through a more comprehensive pathway. Possibly, Hsp70 can be a good candidate as JEV EDIII subunit vaccine adjuvant.