| Swine flu is an acute, febrile, highly contagious respiratory infectious diseasecaused by type A influenza virus in swine, clinical symptoms have sudden high fever,cough, rhinorrhea, difficult breathing, failure or death. Currently, H1N1and H3N2subtype influenza viruses were mainly circulating in pigs, H1N1subtype that wasfound since1918, so far, had popularly existed and spread in pigs in the world, whichcaused huge economic losses to the pig industry and a serious threat to human health.Therefore, the prevention and control against swine H1N1subtype influenza not onlyplay an important role on veterinary fields, but also has important significance onpublic health fields.Vaccination is an effective way to prevent the infection of swine flu and toalleviate its symptoms. Because of no commercial swine H1N1influenza vaccine isproduced currently in China, this study attempts to establish the recombinant lentiviralexpression system expressing HA gene from H1N1subtype of swine influenza virusfor packaging recombinant lentivirus expressing HA with trimeric activity. Treatingthe recombinant lentivirus as a vector vaccine for immunizing animals, the HA proteinwith trimeric activity can be expressed constantly in vivo so as to induce humoral andcellular immunity in body and protective immunity against the swine influenza virus,further evaluating the overall effect of the recombinant lentivirus as the vaccine toprovide theoretical and experimental basis for prevention and control in swine flu inthe future.The structure of leucine zipper in vivo is beneficial to the formation of the stabledimer, the main representative is the yeast transcriptional activator factor GCN4. Ifleucine becomes isoleucine to form iosluecine zipper, this structure can moreeffectively prompt the protein to form trimeric structure, and to improve its biologicalactivity. Therefore, the subject is to help HA to form to trimeric structure by theisoleucine zipper (abbreviated as GCN4) to improve its antigen activity. Firstly, weamplified and identified the HA gene, and linked the HA gene with GCN4gene theninsert them into the lentiviral vector. Using lentiviral protein expression system therecombinant lentivirus was packaged by co-tranfecting293T cells using expressionplasmid, packaging plasmid and envelope plasmid, and then the recombinantlentivirus was identified by the method of transduction of293T cells and RT-PCRidentification, and the expression of target protein in293T cells was analyzed bySDS-PAGE and Western blot, and finally the HA protein purified by Nickel column was treated using BS3cross-linker and identified by Western blot. The results showedthat the amplified HA gene sequences were correct; the lentiviral expression vectorswith HA gene were correct by restriction enzyme digestion; these green fluorescentcells were observed under the fluorescence microscope after the recombinantlentiviruses transduced cells, the identification results of RT-PCR showed that themRNA of HA gene can carry out transcription in infected cells, which furtherdemonstrated the recombinant lentiviruses expressing HA and GCN4fusion genewere packaged successfully, and viruses titer in each group were rLV-HA-GCN4group1.1×10~6TU/mL, rLV-HA group2×10~6TU/mL, LV group1.3×10~8TU/mL; theresult of SDS-PAGE was correct, which showed that the packaged recombinantlentiviruses can stably express HA-GCN4(67KDa, approximately) after transducingcells. Western blot showed that the HA-GCN4can effectively combine with theircorresponding antibodies and showed the expressed HA-GCN4protein had goodreactogenicity; HA protein contained three forms of structure: monomer, dimer andtrimer, their molecular weight were approximately57kDa,114kDa and171kDarespectively, HA-GCN4protein only contained trimeric form of structure and itsmolecular weight was approximately181KDa, which showed GCN4can effectivelykeep the trimeric activity of HA.BALB/c mice were randomly divided into four groups: recombinant lentivirusrLV-HA-GCN4group, recombinant lentivirus rLV-HA group, LV empty vectorcontrol group and PBS control group. The immunogenicity immunized byrecombinant lentivirus in BALB/c and immune protection after challenge wereevaluated by monitoring changes of body weight, humoral and cellular immunitylevels. Changes of body weight in mice were monitored daily; the spleen lung indexwere calculated after sacrificing; HA-specific IgG antibody titer in mice serum wasdetected by ELISA; the ability of lymphocyte proliferation, the sorting of T cellsubtype and cytokine expression in mice were detected by FACS. The results showedthat the body weight of mice from rLV-HA-GCN4group was not significant beforechallenge compared to PBS group, contrarily it had a significant difference afterchallenge compared with PBS group (P<0.05), indicating that the challenge had noeffect on body weight of mice in the group; the spleen lung index from mice inrLV-HA-GCN4group had a significant difference after challenge compared with PBSgroup (P<0.05); the HA antibody titer reached to1:8000before challenge,approximately1:7000after challenge; recombinant lentivirus activated spleenlymphocytes (TV=0.3±0.11); recombinant lentiviruses can cause T lymphocyteresponse mainly based on CD4~+T cells in mice and the percentage content of CD4+Tcells was38.27±3.32compared with PBS group (P<0.01), CD8+T cell percentageincreased to20.9±0.98after challenge compared with PBS group (P<0.05); the expression content of cytokines IL-12, IFN-γ, TNF-α, IL-10and IL-4caused byrLV-HA-GCN4were4.37±0.52,5.83±0.46,5.67±0.23,1.8±0.15,2.87±0.31respectively. IFN-γ, TNF-α and IL-4may be H1N1virus-associated cytokines, andaccording to the expression of cytokines, indicated that the Th1-type cellular immuneresponse was primary.These results indicated that recombinant lentivirus expressing HA-GCN4caneffectively express in infected293T cells, and the GCN4sequence effectivelyenhanced trimeric activity of the HA gene; recombinant lentivirus induced BALB/cmice to produce good humoral immunity and cellular immune response effectivelyagainst swine H1N1influenza virus. Therefore, this study provided theoretical basisand experimental data for next the homologous animal experiments and theprevention and control of swine flu in the future. |
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