Preparation And Research Of Swine Influenza Virus Matrix Protein M2e Gene Fuse Recombinant Lactobacillus

Swine influenza(SI), which is found worldwide and causes one of the most prevalent infectious diseases, it is also more common in our country and caused significant economic loss for breeding industry. The research show that lactobacillus can express the exogenous protein, so it is a potential candidate for multifunctional probiotics. The lactobacillus is safety,inexpensive, and it is esay to cultivate, so it can be used as foreign protein expression vector.In this study, we select a foreign protein expression vector—Lactobacillus(Lactobacillus plantarum NC8) to deliver the gene of Swine influenza virus M2 e as major protective antigen protein integrates with the Fc fragment of IgG, combined with exogenous gene specific immune function and probiotic lactic acid bacteria developing recombinant lactobacillus oral vaccine of M2 e gene fusion the Fc fragment of IgG, and preliminary analysis of the immune function. The main contents and conclusions are as follows:(1) The preparation of recombinant lactobacillusMethods:By means of synthesis,digestion,recycle and other methods to compound four groups of recombinant plasmid, included plasmid pSIP409, plasmid pSIP409-3M2 e, plasmid pSIP409-3M2e-Fc(mutation), plasmid pSIP409-3M2e-Fc(no mutation),by means of enzymes slash to identify, the target recombinant plasmid electroporated into Lactobacillus plantarum, after analysising by SDS-PAGE and Western blotting to further detect exogenous gene, three fusion proteins of pSIP409-3M2 e,pSIP409-3M2e-Fc(mutation) and pSIP409-3M2e-Fc(no mutation) presented reactionogenicity as the polyclonal antibody of swine influenza virus, which obtained the target protein by SppIP peptide inducing expression in lactic acid bacteria, indicating that it has laid the foundation of constructing recombinant SIV lactobacillus oral preparation.Results:Through the methods that is introduce as above, four groups of recombinant plasmid are prepared successful, and the protein expressed by the recombinant lactobacillus presented reactionogenicity as the polyclonal antibody of swine influenza virus, it has laid the foundation of constructing recombinant SIV lactobacillus oral preparation.(2) Detecting the immune protective effect of the recombinant lactobacillus pSIP409-3M2e-FcMethods: Add SppIP in the recombinant lactic acid bacteria which can obtained the target protein by inducing expression.Use the method of gasteic perfusion to immune the mouse. And the first time we immune the mouse was the 1d,3d,5d,the second time we immune the mouse was the 26 d,28d,30 d.After the immunization we use the flow cytometry to detect the CD95+PNA+ change in value in mesenteric lymph nodes and paneth cell, and the B220+IgA+ change in value in mesenteric lymph nodes and the paneth cell. These index indicate the number of the B cell activation. Therefore we identify the immune protective effect of recombinant lactic acid bacteria easily. Then we use the ELISA kit to detect the secrete of the IL-4 and the IFN-γ in the blood of mouse. After the first immunization we gather the excrement of the mouse,and after the second immunization we gather the excrement of the mouse also,in order to detect the expression level of the SIgA. After the immunization we intraperitoneal anesthesia the mouse,and then conduct the toxicity attack test, the toxicity include the H9N2 and the H1N1, because we want to indicate that the recombinant vaccine can play the protective immunity in different subtypes of influenza viruses. Through the mental state and the change of the body weight, coupled with the result of the detection of the IL-4 and IFN-γ secreted in the blood. And analyse the relative expression of the IL-4 and IFN-γ before toxicity attack test and after toxicity attack test. Thereby we can verify the immune function of the recombinant lactobacillus.Results: Compared with the PBS group, after immuned by the recombinant lactobacillus pSIP409-3M2e-Fc(mutation), plasmid pSIP409-3M2e-Fc(no mutation) and pSIP409-3M2 e, the expression level of the CD95+PNA+B220+IgA+ in mesenteric lymph nodes and paneth cell, it has indicate that recombinant lactobacillus have improved significantly, and SIgA expression levels also increased significantly. Compared with the post-infection, the expression levels of IL-4 has decreased, while the expression of IFN-γ has increased. By monitoring these index, we can summarize that the recombinant lactobacillus pSIP409-3M2e-Fc(no mutation), pSIP409-3M2e-Fc(mutation) and pSIP409-3M2 e can provide immune protective immunity between different subtypes of influenza viruses,and the recombinant lactobacillus which was fused the Fc(no mutation) can be better than others.Conclusion:In general, the results of this study demonstrated that the protective immunity of pSIP409-3M2e-Fc(no mutation) was better than the pSIP409-3M2e-Fc(mutation) and the pSIP409-3M2 e, and their immune levels were higher than other control group. It has indicate that the Fc(no mutation) as a vaccine adjuvant can play the role of the immunological function, and it has also indicate that the recombinant gene fusion lactic acid bacteria have the ability to resist the SIV. The research provided dara for further study of enhanced immune SI genetically engineered Lactobacillus oral vaccine.