| Swine flu is widespread in the world and has brought great threat and economic losses. How to develop a safe and effective swine flu oral formulations(vaccine) has become a problem for researchers. As an intestinal probiotics, lactobacillus has been used to synthesize foreign proteins except the immunomodulatory function. However, the inner cell production of exogenous proteins somehow limits their function. Pgs A is a component of the poly-glutamate synthetase system PGA in Bacillus subtilis, which can stably anchors the exogenous proteins on cell surface and promote their roles extracellular. In this study, pgs A was used to anchor the NS1 protein of swine influenza virus(SIV) on lactobacillus, yielding a new functional lactobacillus, and its immunological effects were evaluated in mice model. The research contents and results are as follows:(1)Construction and expression of new functional lactobacillus pSIP409-pgsA-NS1 The recombinant plasmid was constructed by genetic engineering method and electroporated into lactobacillus. After the induction by induced peptide, the protein size, density and reactivity were determined by SDS-PAGE, BCA, and western blotting approaches. The anchoring of NS1 protein on lactobacillus was evaluated by FCM and IF. Results: The new functional lactobacillus p SIP409-pgs A-NS1 was successfully constructed and the protein size was verified by SDS-PAGE. The protein concentration was 0.11146 mg/m L shown in BCA test and western blotting verified the immunogenicity. Strong fluorescence signals were observed in FCM and IF assaies to prove that pgs A successfully anchored NS1 protein on the cell surface. The results layed the foundation for the next animal immunization and virus challenged experiment.(2)Immunological evaluation of new functional lactobacillus pSIP409-pgsA-NS1 Mice were immunized with new functional lactobacillus according to immunization protocol. The proliferation of spleen lymphocytes were then determined by MTS assay and the NS1 specific Ig G and SIg A were measured by ELISA. In addition, the activation of T and B cells in related immune organs were measured by flow cytometry. The mice were monitored after challenge to calculate body weight loss and survival rates and their lungs were removed and weighed to determine the lung index and lung index inhibitory rates. The cytokines production in serum were also determined by ELISA before and after challenge. Results: new functional lactobacillus effectively stimulate the proliferation of spleen lymphocytes, raise the levels of NS1 specific Ig G and SIg A in vivo and promote the activation of T lymphocytes and B lymphocytes. Mice in new functional lactobacillus group lost less weight and recovered faster,in addition, their survival rates were significantly higher than the other groups after challenge. Lung index, lung index inhibitory rate and lung biopsy showed that new functional lactobacillus could alleviate inflammation of the lungs in mice, reduce pathological damage in the lungs, and effectively prevent swine influenza virus infection.Conclusions: The p SIP409-pgs A-NS1 new functional lactobacillus was successfully constructed and expressed. As an anchor gene, pgs A could stably anchor NS1 protein on the cell surface of lactobacillus to play a role in the extracellular spaces. The new functional lactobacillus could stimulate the proliferation of mouse spleen lymphocytes, and production of NS1 specific Ig G and SIg A, enhance B lymphocytes and CD8 + T lymphocyte activation, which significantly improved the levels of humoral and cellular immunity. This study provides a basis for the development of anti-swine flu oral probiotics. |
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