Research On Cloning,eukaryotic Expression And Immunogenicity Of IL-2 Gene And TNF-? Gene From Sika Deer

Tuberculosis is a zoonotic disease caused by Mycobacterium tuberculosis,which has become a hidden danger affecting the development of sika deer industry.At present,vaccination and antibiotic injection are often used to treat sika deer infectious diseases.with the increase of antibiotic resistance and the variation of infectious pathogens,the use of traditional vaccines and antibiotics to control infectious diseases is facing more and more severe challenges.Cytokines are a kind of proteins or small molecular peptides that can transmit information between cells and have the function of immune regulation and effect.Studies have shown that as a new type of immune adjuvant,cytokines can not only avoid the adverse reactions of conventional adjuvants,but also enhance the immune effect of the vaccine,improve the uniformity of antibodies and prolong the duration of antibodies.Its good immune enhancement effect has been confirmed in some swine and chicken disease vaccines,but there are few studies on using cytokines as immune adjuvants of sika deer.Therefore,the cloning,eukaryotic expression and immunogenicity of IL-2 gene and TNF-?gene of sika deer were carried out in order to provide theoretical basis for the development of cytokine-related immune adjuvants of sika deer.The results are as follows:1.Cloning of interleukin(Interleukin2,IL-2)and tumor necrosis factor(Tumor necrosis factor,TNF-?from sika deer.According to the IL-2 gene(U14682.1)in NCBI(U14682.1)and the TNF-?gene(U14683.1)sequence design primers,the blood m RNA of the plum deer is a template,and RT-PCR amplification is performed,and it is connected to the p MD18T vector.In order to sequen,biological information analysis is performed.The IL-2sequence analysis showed that the plum deer IL-2 gene was composed of 489 bp,encoding 163 amuro acids,and the plum deer IL-2 gene was compared with the relegant IL-2 gene nucleotide sequence;system evolutionary tree analysis showed that The genetic distance of the plum deer and the white-taose deer is a group;the homology analysis of amino acid sequences shows that the homology of the plum deer and the bull,Linme,African buffalo,82.1%?80.6%,The IL-2 protein molecular mass was 40.19 k D,the equipotential point(PI)was 10.86,and the protein was analyzed by pro-hydrophobicity and transmembrane structure,and the plum deer IL-2was found to be a hydrophilic external membrane protein.The TNF-?sequence analysis showed that the plum deer TNF-?gene was composed of 690 bp,encoded230 amino acids,and the plum deer TNF-?gene was compared with the relegant TNF-?gene.Difference.System evolutionary tree analysis shows that the close relationship between the plum deer and the colors is recently,both with buffalo,white tail deer with high homology.The homology analysis of amino acids showed that the plum deer was similar to the colors and white-tailed deer with a similarity of 98.9%,98.3%.The molecular mass of the plum deer TNF-?protein was 57.97kd,and the equipotential point(PI)was 5.81,through pro/hydrophobicity.The protein was analyzed with the transmembrane structure and found that the plum deer TNF-?was a hydrophobic transmembrane protein.2.Construction and expression of eukaryotic expression vector pc DNA3.1/V5His A-IL-2 and pc DNA3.1/V5His A-TNF-?.The amplified products were ligated into eukaryotic expression vector pc DNA3.1/V5His A and identified by restriction endonuclease digestion and sequencing.The correct plasmids were named pc DNA3.1/V5His A-IL-2 and pc DNA3.1/V5His A-TNF-?.The recombinant plasmid was transfected into Vero cells mediated by liposome.48 hours after transfection,the expression of IL-2 gene and TNF-?gene was observed under laser confocal microscope.SDS-PAGE and Western blot methods were used to verify the expression of IL-2 and TNF-?protein.The results showed that fluorescence could be detected in Vero cells 48 hours after pc DNA3.1/V5His A-IL-2 and p DNA3.1-/V5His A-TNF-?transfection,SDS-PAGE results showed that compared with the control group,40k D and 55k D proteins were found,which were consistent with the expected protein size,Western Blot results showed that IL-2 and TNF-?proteins began to express at 24 hours,and the expression increased gradually with the increase of time.3.The effect of sika deer IL-2,TNF-?gene expression on the infection of Vero cells by Mycobacterium smegmatis mc~2155.Pc DNA3.1/V5His A-IL-2 and pc DNA3.1/V5His A-TNF-?plasmids were transfected into Vero cells and infected with Mycobacterium smegmatis mc~2155.ELISA assay was used to detect that IL-2 plasmid transfection increased the expression of anti-inflammatory cytokines IL-2 in Vero cells,increased cell viability and inhibited Vero cell apoptosis.Transfection of TNF-?plasmid increased the expression of pro-inflammatory cytokines(IFN-?,TNF-?)in cells,and recruited the release of innate immune factor IL-2 of Vero cells,which could inhibit the growth of Vero cells infected with mc~2155,improve the survival rate of uninfected cells,and increase the apoptosis rate of infected mc~2155 cells.By CCK-8 assay,it was found that compared with the Vero cell model group infected by mc~2155,the cell survival rate of transfected plasmid IL-2Vero cells increased significantly after infecting mc~2155 bacteria for 12 hours,while the cell viability of transfected plasmid TNF-?Vero cells infecting mc~2155 cells decreased significantly.Flow cytometry showed that the Vero cells transfected with IL-2 plasmid were 2.47%lower than those in the mc~2155 infected Vero group after being infected by mc~2155,but the apoptosis rate in the TNF-?plasmid group was 12.94%higher than that in the model group.The results of fluorescence quantitative pcr showed that the expression product of IL-2gene could effectively inhibit the secretion of apoptotic genes and improve the apoptosis of Vero cells.The expression products of TNF-?gene can promote the secretion of apoptosis genes in infected mc~2155 bacteria,activate death receptors and death mitochondrial pathways,and accelerate the apoptosis of infected cells.To sum up,the cloning,eukaryotic expression and immunogenicity of sika deer IL-2 gene and TNF-?gene were studied in order to lay a foundation for the preparation of sika deer specific immune enhancer.The cytotoxic effect of sika deer TNF-?gene on cells infected by Mycobacterium smegmatis mc~2155 was used to provide a new idea for the treatment of tuberculosis infected sika deer.