Expression And Immune Efficacy Of The HA Gene Of H3N2 Subtupe SIV

Through investigating the SIV serum, the scientists found that H3N2 SIV had widely lived in the swine. In order to guard against and control the SIV epidemic effectively and prevent the avian influenza virus through swine infecting human, developing an effectively vaccine is a pressing task. At present, the in activated oil emulsion vaccine was mainly used to guard against the SIV But the vaccine against the AIV including the traditional inactivated oil emulsion vaccine and subunit vaccine and recombinant living vector vaccine and gene vaccine etc. So the development of vaccine against SIV lags behind the development of vaccine against AIV.This experiment does take the SIV HA gene as the object of study, mainly has done following researches:1. Construction and expression of swine influenza virus HA gene prokaryotic vector. According to the characters of the HA protein, The recombinant plasmid pMD18-HA was digested with SacI and Not I to generate HA gene, and sub cloned into the prokaryotic expression vector PET-32a(+). The recombinant plasmid was transformed into BL21. The recombinant bacteria were induced by IPTG. The expression of HA protein was optimized with proper inducing conditions of 1mmol/L IPTG, 4 hours and 30℃temperature. After the SDS-PAGE electrophoresis, has not seen the sense of purpose protein, extrapolated possibly is because the expression fragment is long, causes the vector PET-32a(+) metabolism burden to overload either in the HA gene the influential HA expression sequence or the part.2. Construction and expression of swine influenza virus HA gene eukaryotic vector. Hemagglutinin gene is an important gene of protective antigen of swine Influenza Virus (SIV).In order to study HA gene vaccine, the HA gene of SIV, amplified by PCR, was subcloned into eukaryotic expressing vectors pcDNA3.1, and did the transfection with the Lipofectin reagent. the indirect immunofluorescent experiment certificated the expression of HA gene in Vero cell.3. Immune efficacy of the recombination plasmids expressing HA gene of H3N2 subtybe SIV in mice. There are many authors indicate that inactivated vaccine provides protective immunity against swine influenza virus infection in mice. some investigation suggest that the protective efficacy of DNA vaccine is adequate against swine influenza virus infection. In this study, we examined the antibody response to DNA vaccine and inactivated virus and the protection against H3N2 SIV challenge using mouse model. Mice were immunized with different immune formation. First, mice immunized twice with protein at an interval of 3 weeks. second, mice immunized twice with DNA at an interval of 3 weeks. Third, mice immunized with DNA priming and protein boosting. Three weeks after the second immunization, mice were challenged with a high dose (10⁴EID₅₀) of H3N2 SIV. The ability of protection against virus challenge in mice was evaluated by induction of serum antibody response, the lung virus titers of mice. The results demonstrate the immune response induced by different immune formation in mouse model has been proved to be able to protect against the challenge with H3N2 SIV. Meanwhile, by the statistical analysis of the weight changes of mice, there were high significant differences between the immunization groups and the control(P< 0.01).