Expression Of Porcine Type ? Interferon Research On The Construction,Expression And Activity Of Recombinant Lactobacillus Plantarum LP18

Type ? interferon(IFN-lambda)is a newly discovered cytokine with biological activities of antiviral,antimicrobial and immunomodulatory.And it can not produce similar blood and neurological side effects when compared with type I interferon,and it is expected to become a new type interferon with better clinical efficacy and less side effects in the future.Pig type III interferon was first discovered and synthesized in 2017 in China,which mainly acts on mucosal epithelial cells and plays an important role in anti-virus and anti-bacterial infection of the intestinal tract and other mucosal tissues.Lactic acid bacteria(LAB)is 'generally recognized as safe',and the LAB probiotics were found in humans and animals with functions of natural anti-bacterial and immune-enhancing properties.Lactobacillus is the only non-pathogenic strain from LAB.Some screened probiotics pf lactobacillus planterum can be taken orally,which not only put up with the gastric acid and bile salt in the small intestine,but also can be well colonized in the intestinal tract.Therefore,Lactobacillus plantarum can be made into an orally viable organism carrier secreting and delivering target proteins on the cell surface,which can enhance the immune response and stimulate the immune response in the body,and carry out the targeted therapy for diseases.In order to obtain the gene sequence of porcine type III interferon lambda 1(pIFN-?1)and to verify its expression and biological activities,in this study we PCR amplified the gene sequence of pIFN-?1 from the total RNA of lung macrophage CRL2845 cells by RT-PCR method,and analyzed genetic evolution and predicted signal peptide.The gene sequence of pIFN-?1 obtained from amplification was cloned into the pMD-19T Simple vector for sequencing pIFN-?1 and the pcDNA3.1 eukaryotic expression vector for expression.The recombinant plasmids of p19T-pIFN-?1 and pcDNA-pIFN-?1 were obtained through transforming,screening,extracting,and digesting conventional plasmids.After transfecting CRL2845 cells with liposome for 48h,the expression and intracellular localization of pIFN-?1 were analyzed by quantitative real-time PCR(qRT-PCR),Western blot and indirect immunofluorescence(IFA).The expression of interferon-stimulated genes ISG15 and related gens including OAS1,PKR,and IFITM3 was analyzed with the method of qRT-PCR for the total RNA of the collected transfected cells.Using the recombinant poxvirus VTT-EGFP expressing GFP to infect CRL2845 cells that transiently express pIFN-?1,anti-poxvirus effect of pIFN-?1 was analyzed with the method of flow cytometry.The results showed that the pIFN-?1 gene can be successfully obtained from and expressed in the porcine alveolar macrophage CRL2845,and the pIFN--?1 gene can induce the expression of interferon-stimulated genes of OAS1 and ISG1 after transient expression,and inhibits poxvirus infection in CRL2845 with the inhibition rate of 38%.The results indicate that the transient expression of pIFN?1 can induce the expression of interferon-stimulated genes selectively and meanwhile have antiviral activity.In order to obtain a recombinant lactobacillus that can express pIFN-?1 protein in Lactobacillus plantarum LP18.Trizol total RNA extraction kit as used to in the study extract the genome of Lactobacillus plantarum LP18,and the gene sequence of Signal Peptide 1320 was amplified from the genome of Lactobacillus plantarum LP18 with the technique of PCR.Using pIFN-?1 obtained from CRL2845 cells in the porcine alveolar macrophage cell line as a template,the amplification product of 1320-?1-MH,which contains the targeting peptide M and the His-tag,was obtained by PCR and the fusion PCR.After the plasmid pSIP411 was digested with the method of double digestion and the vector fragment was recovered,the amplification product of 1320-?1-MH was ligated to the vector fragment through seamless cloning to obtain the recombinant plasmid of pSIP-1320-?1-MH.Using Lactococcus lactis NZ3900 as an intermediate host,the recombinant plasmid of pSIP-1320-?1-MH was then transferred into Lactobacillus plantarum LP18 with the electroporation technique,and after screening positive clones,the expression of pIFN-?1 protein was analyzed with the method of Western blot.In the meantime,the expression of pIFN-?1 protein was optimized in such four aspects as induced temperature,induced time,induced concentration,and the number of recombinant bacteria with the optimization strategy of prokaryotic expression system.The results showed that the gene sequence of Signal Peptide 1320 was successfully amplified from the genome of Lactobacillus plantarum LP18;the recombinant plasmid of pSIP-1320-X1-MH was successfully obtained with the techniques of the fusion PCR and seamless cloning;the recombinant Lactobacillus plantarum LP18-pIFN?1 capable of expressing pIFN-?1 protein smoothly was obtained;meanwhile the optimization conditions for the expression was obtained:the optimal expression of pIFN-?1 protein was obtained under the conditions of being cultured with 100 ng/mL inducer for 12 hours at the temperature 37?,which laid a foundation for in vivo activity experimentsIn order to study the biological activity of recombinant Lactobacillus plantarum LP18-pIFN?1 in animals,a mouse death model of oral administration of Salmonella enteritidis was constructed,in which 120 mice were divided into a prevention group and a treatment group,with each group further divided into 6 subgroups and 10 mice per subgroup.Through analyzing the mice's surface hair conditions,mental state,body weight,feed intake,bacterial distribution and mortality with the methods of Bioinformatics and flow cytometry,the in vivo activity of the recombinant Lactobacillus LP18-pIFN?1 was verified,and its prophylactic and therapeutic effects were evaluated.The results showed that,of the prevention group,the mortality of the prevention subgroup that had taken the recombinant Lactobacillus plantarum LP18-pIFN?1 was 70%,and the mortality of other subgroups was 100%;of the treatment group,the mortality of all subgroups was 100%,the possible reason is that the intestinal wall structure was damaged lead to the recombinant Lactobacillus plantarum LP18-pIFN?1 can not be colonized.The recombinant Lactobacillus plantarum LP18-pIFN?1 can increase significantly the numbers of double positive cells of CD3+CD4+and CD3+CD8+in the mice of the prevention group(*P<0.05),and these numbers were significantly higher than the empty vector LP18 group and PBS control group(*P<0.05).Therefore,the results indicate that the recombinant Lactobacillus plantarum LP18-pIFN?1 has the in vivo anti-Salmonella enteritidis effect and can stimulate a cellular immune response in miceThe recombinant Lactobacillus plantarum LP18-pIFN?1 capable of expressing pIFN-?1 protein was successfully constructed in this study,and its protein expression was optimized.Simultaneously,an in vivo experiment was carried out to conduct preliminary research into the in vivo biological activity of the recombinant Lactobacillus plantarum LP18-pIFN?1,which lays a foundation for subsequent in-depth research.