The Optimization Of Porcine Recombinant Visfatin Protein And Its Role In Immune Inflammatory Response Of Weaned Piglets

Visfatin is a new type of fat cell factor,which is closely related to the body’s inflammation and immune response.Immune stress in weaned piglets is one of the important reasons that affect the performance of piglets.The main pathological change is the inflammatory response,but eventually the growth of piglets is hindered,causing huge economic losses to animal production.Previous studies in our laboratory have shown that in lipopolysaccharide(LPS)-induced weaned piglet immune stress model,the positive expression products of Visfatin in white and red pulp of spleen are significantly increased,speculating that Visfatin as an inflammatory mediator may be participated in the occurrence and development of immune stress and inflammation in weaned piglets.In order to obtain a large number of porcine recombinant lipoprotein with low endotoxin content and biological activity,and to clarify their role in the immune stress response of weaned piglets.In this study,firstly,prokaryotic expression,protein purification,biological activity verification,Western Blot,q RT-PCR and ELISA technology are used to prepare pig recombinant Visfatin.Then on the basis of successfully constructed weaned piglet immune stress model induced by LPS,the role of Visfatin in the immune and inflammatory response of weaned piglets was studied by q RT-PCR,ELISA,immunohistochemistry and TUNNEL.The specific research results are as follows:1 Prokaryotic expression of porcine recombinant Visfatin and optimization of purification conditionsUsing the total RNA of porcine liver tissue as a template,PCR technology was used to amplify the porcine Visfatin gene,which was transformed into the p EASY-T1 vector and then transformed into Trans5α.After extracting the plasmid,it was simultaneously digested with p ET-28 a and p ET-30 a by Bam HⅠ and XhoⅠ;The target gene fragments of Visfatin were ligated into p ET-28 a and p ET-30 a plasmids respectively,and transformed into DH5α to clone the expression plasmid containing the target gene.The recombinant expression plasmids were extracted and transformed into E.coli Trans BL21 and Transetta respectively.After induced by IPTG,and the protein was purified by the purification system AKTAprime10,and detected by SDS-PAGE and Western Blot technology.The results show that Visfatin-p ET28a-Transetta can stably express a large number of high-purity recombinant recombinant porcine Visfatin at 16 ℃ for 16 h,which is the optimal expression strain.2 Endotoxin removal and detection in porcine recombinant VisfatinVarious countries have strict requirements on the endotoxin content of biological products.In order to ensure the safety of the use of biological products,endotoxin must be removed.In this experiment,endotoxin removal reagents were used to remove endotoxins from porcine recombinant Visfatin,and the endotoxin detection limulus kit was used to detect the removal effect.The results show that the residual amount of endotoxin after removal is less than 1EU/m L,which is within the safe range,preventing its possible influence on the judgment of subsequent test results.3 Biological activity verification of porcine recombinant VisfatinThe endotoxin-removed porcine recombinant Visfatin acted on Raw264.7 cells.The results of CCK-8 showed that with the increase of Visfatin concentration,the cell survival rate increased first and then decreased,which were all higher than that of the negative control group(0ng/m L).When the concentration was 700 ng/m L,the cell survival rate was the highest,and the difference is extremely significant(p?0.0001),suggesting that endolipin can promote the survival of Raw264.7 cells.Then set up PBS group,Visfatin group and Visfatin+Poly.B group,and incubate with cells for 6h.The results of q RT-PCR and ELISA showed that compared with the PBS group,the m RNA and protein expression levels of the inflammatory factors IL-1β,TNF-α and MCP-1 in the Visfatin group were significantly increased.And there was no significant difference in the m RNA and protein expression levels of inflammatory factors in the Visfatin+Poly.B group compared with the Visfatin group.The above results indicate that the porcine recombinant Visfatin protein promotes the inflammatory activity of Raw264.7 cells,while the residual endotoxin does not work,proving that the porcine recombinant Visfatin protein has biological activity.4 Effect of porcine recombinant Visfatin on the expression of inflammatory factors in weaned pigletsLPS induction was used to construct an immune stress model for weaned piglets.The body weight and spleen weight of each pig were determined,and the spleen index(spleen weight(g)/ body weight(kg))was calculated.The results showed that the spleen weight and spleen index increased significantly in the LPS group compared with the PBS group(p?0.001),but not in the Visfatin group.While the spleen weight and spleen index in LPS Visfatin group were significantly decreased compared with LPS group,and the difference was extremely significant(p?0.01).Using q RT-PCR and ELISA techniques to detect the expression of inflammatory factors IL-1β,IL-6 and MCP-1 in the weaned piglets spleen tissues of each group.The results showed that compared with the PBS group,the m RNA and protein expression levels of IL-1β,IL-6 and MCP-1 in the Visfatin group were not significantly different,while those in the LPS group were significantly increased.The m RNA and protein expression levels of IL-1β,IL-6 and MCP-1 in the LPS+Visfatin group were significantly reduced compared with the LPS group.The above results indicate that LPS activates the immune system of the spleen and induces lymphocytes to synthesize a large number of inflammatory cytokines,resulting in hyperactive and enlarged spleen function,while Visfatin can reduce the inflammation of spleen tissue caused by LPS by down-regulating the expression of inflammatory factors damage.5 Effect of porcine recombinant Visfatin on antioxidant function of weaned pigletsThe expression of malondialdehyde(MDA),lysozyme(LZM),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in the peripheral serum of pigs in each group was detected by ELISA.The results showed that compared with the PBS group,there was no significant difference in the expression of MDA in the Visfatin group,but the expression of GSH-Px and SOD were significantly increased.While compared with the PBS group,the expression of MDA in the LPS group was significantly increased(p?0.01),the expression of GSH-Px did not change significantly,SOD and LZM both increased significantly.Compared with LPS group,but the expression of MDA in LPS+Visfatin group was significantly reduced,and the difference was significant(p?0.05).But the expression of GSH-Px,SOD and LZM in LPS+Visfatin group were significantly increased.The results showed that Visfatin reduces the oxidative damage to piglets caused by weaning stress by enhancing the body’s antioxidant function and after LPS stimulation,the body experienced severe oxidative damage.Exogenous injection of Visfatin reduced the content of MDA in the serum and increased the enzyme activity of SOD,GSH-Px and LZM.It is proved that Visfatin reduces the damage to the body caused by immune stress by improving the antioxidant function function of weaned piglets.6 Effect of porcine recombinant Visfatin on the proliferation and apoptosis of peripheral immune organ cells of weaned pigletsImmunohistochemistry,TUNEL technology and Leica Image Scope software were used to study and statistically analyze the proliferation and apoptosis of cells in different groups of spleen and mesenteric lymph node tissues.Immunohistochemical detection of proliferating cell nuclear antigen(PCNA)showed that: proliferating cell positive signal PCNA was mainly distributed in the germinal center of spleen and mesenteric lymph node tissue.Compared with PBS group,the positive rate of PCNA in spleen and mesenteric lymph nodes in Visfatin group was significantly increased,while the difference of mean difference in LPS group was significantly reduced.Compared with LPS group,the positive rate of PCNA in spleen and mesenteric lymph nodes in LPS+Visfatin group was significantly increased(p<0.01).TUNEL test results showed that: compared with the PBS group,the positive rate of apoptotic cells in the spleen and mesenteric lymph node tissues of the Visfatin group tended to decrease,while the differences of the LPS group were significantly increased.Compared with the LPS group,the positive rates of apoptotic cells in spleen and mesenteric lymph nodes in LPS+Visfatin group were significantly reduced.In addition,using q RT-PCR technology to detect apoptosis-related genes in spleen tissues showed that compared with the PBS group,the expression of FAS and caspase-3 in the LPS group were significantly up-regulated;However,caspase-3 in the Visfatin group tended to decrease.Compared with the LPS group,the expression of FAS and caspase-3 in the LPS+Visfatin group was significantly reduced.The above results indicate that Visfatin improves the immune function of weanling stress piglets by promoting the proliferation and inhibiting apoptosis of peripheral immune organs spleen and mesenteric lymph node tissue cells and LPS activates the mitochondrial apoptotic pathway,thereby inhibiting cell proliferation and promoting apoptosis of the spleen and mesenteric lymph nodes.The exogenous injection of Visfatin can inhibit the apoptosis of peripheral immune organ cells induced by LPS,promote the proliferation of peripheral immune organ cells,reduce the expression of FAS and caspase-3,and improve the immune status of the body when immune stress occurs.