| Influenza A virus(IAV)is a respiratory virus of Orthomyxoviridae,which can infect different species across the interspecific barrier.Different genotypes and branches of IAV reassemble into Swine influenza virus(SIV)in human and bird hosts and spread in swine herds,resulting in Swine influenza(SI)epidemic.At present,vaccination is still the most effective protective measure to control SI.However,since the virus mainly infects the host through the respiratory mucosa,most of the vaccines on the market cannot produce good mucosal protective effects,and the virus itself is constantly evolving.E.coli Nissle 1917(Ec N)is a commercial probiotic used to treat gastrointestinal diseases.It has been found that Ec N,as a carrier bacteria,can be compatible with plasmids with different copy numbers,and then express the recombinant protein with the transformed exogenous DNA,thereby inducing the body to produce antigen-targeted mucosal immune response.Therefore,Ec N is widely used in genetic engineering techniques such as molecular biology.Based on this,this experiment took the receptor binding site(RBS)of Eurasian avian-like(EA)H1N1 SIV as the research object,constructed recombinant probiotics Ec N-RGP-RBS and Ec N-RGP-T-RBS,and evaluated the effect of recombinant probiotics through animal experiments.The experimental contents and results are as follows:(1)Construction and expression verification of recombinant probioticsFirstly,the HA1 gene of H1N1 strain was amplified,cloned and sequenced,and the sequencing results were compared on the NCBI website to clarify the subtype of the strain.Then,the primers were redesigned to amplify the RBS and trimer tag sequences of the strain,and the trimer tag and RBS were overlapped by PCR to obtain the target gene fragment of T-RBS.Secondly,the vector RGP on the RGP-1487 GFP plasmid was digested by double enzymes,and the target gene fragment was homologously recombined with the vector RGP to construct a prokaryotic expression vector capable of fusion expression of RBS and T-RBS.In addition,Ec N was used as a vector to express exogenous genes to construct recombinant probiotics Ec N-RGP-RBS and Ec N-RGP-T-RBS.Finally,Western Blot was used to verify the reactogenicity of the target gene fragment in Ec N.The results showed that the protein band expressed by recombinant probiotics Ec N-RGP-RBS was in line with the expected size.Ec N-RGP-T-RBS also expressed monomeric protein,but the protein content was higher than that of RBS group.(2)Evaluation of immune effect of recombinant probiotics on miceThe mice were orally immunized with recombinant probiotics Ec N-RGP-RBS and Ec N-RGP-T-RBS at a dose of 1.0×10~9CFU/200μl/mouse for 2 days,and 14 days after the first immunization,the booster immunization was carried out for 2 days.After 20 days of booster immunization,the activation level of DCs in PP nodes and the level of B220~+Ig A~+produced by B cells were detected by flow cytometry.The results showed that the recombinant probiotics Ec N-RGP-RBS and Ec N-RGP-T-RBS could promote the expression of CD40 and CD86 of DCs in PP of immunized mice.Moreover,the B cells in the PP node and spleen of the experimental group immunized mice could also produce a higher level of B220~+Ig A~+.Then,the number of B220~+Ig A~+cells in the ileum of mice was detected by immunofluorescence.The results showed that recombinant probiotics increased the number of B220~+Ig A~+double positive cells in the ileum of immunized mice.In addition,ELISA was used to detect the levels of specific antibodies in serum and feces of mice 14 days after primary immunization and 20 days after booster immunization.The results showed that recombinant probiotics promoted the secretion of specific antibodies in serum and feces of immunized mice.Finally,the expression levels of specific cytokines IFN-γand IL-4 in mouse spleen and mesenteric lymph(MLN)T cells were detected by flow cytometry.The results showed that the recombinant probiotics Ec N-RGP-RBS and Ec N-RGP-T-RBS could up-regulate the levels of specific cytokines in MLN and spleen T cells of immunized mice.The results showed that the recombinant probiotics had a good immune effect on mice.(3)Evaluation of the protective effect of recombinant probiotics on infected miceMice were challenged with influenza virus A/Puerto Rico/8/1934(H1N1)14 days after booster immunization.The body weight and mortality of mice were recorded for 14 days.The results showed that the recombinant probiotics in the experimental group could produce a 50%protection rate for infected mice.The probiotics in the GFP group could produce a 30%protection rate for infected mice.Then,HE staining was used to observe the pathological changes of the lungs of challenged mice and the number of influenza virus-infected cells in the lungs of mice was detected by indirect immunofluorescence.The results showed that the recombinant probiotics Ec N-RGP-RBS and Ec N-RGP-T-RBS had a certain protective effect on the lungs of infected mice,which could reduce the pathological damage of the lungs of mice and reduce the number of infected cells in the lungs of infected mice.The levels of MLN,mediastinal lymph nodes(Mdln)and germinal center activated B cells(B220~+CD95~+GL7~+)in the spleen were detected by flow cytometry 5 days after challenge.The results showed that recombinant probiotics could promote the activation of germinal center B cells in infected mice.Secondly,ELISA was used to detect the levels of Ig G antibody in serum and s Ig A in bronchoalveolar lavage fluid(BALF)of infected mice.The results showed that recombinant probiotics promoted the secretion of specific antibodies in infected mice.Finally,the levels of T cell-specific cytokines in MLN and Mdln were detected by flow cytometry.The results showed that recombinant probiotics up-regulated the levels of specific cytokines in T cells of infected mice.The results showed that the recombinant probiotics had a good protective effect on infected mice.In summary,this experiment takes the RBS of EA H1N1 as the research object.The two recombinant probiotics Ec N-RGP-RBS and Ec N-RGP-T-RBS can enhance the secretion of specific antibodies and T cell-specific cytokines in mice,and also have a certain protective effect on infected mice.Therefore,the two recombinant probiotics constructed in this experiment can induce specific immune responses in mice,providing ideas for the next development of new mucosal vaccines. |
