| Natriuretic peptide receptor C(NPR-C),as a third member of the natriuretic peptide(Natriuretic peptides,NPs)family receptor,a non-guanosine cyclase-coupled receptor,is capable of highly binding to various natriuretic peptides and polypeptide compounds with homologous structures,regulating their biological functions,and reducing the half-life of the atrial natriuretic peptide(ANP).This purposes of this research were to evaluation the effect of active immunization with recombinant extracellular domain peptide of sheep NPR-C on blood physiological and biochemical indexes and gene expression in tail adipose tissue of Altay sheep,and to provide a reference for finding a new way to regulate fat deposition in domestic animals.Firstly,the coding region of sheep NPR-C extracellular domain was optimized based on the codon bias of E.coli.and the optimized gene was synthesized by whole-genome synthesis technique.The genetic engineering technique was used to construct E.coli expression vector pET28a(+)-NPR-C.After being induced by IPTG,extracellular domain of NPR-C was expressed in E.coli BL21(DE3).Then,the expressed recombinant extracellular domain of NPR-C was purified by Ni-NTA for antigen preparation.Secondly,a total of 16 2-month-old female Altay lambs with body weight of 13.95±2.38 kg were selected and randomly divided into the test group and the control group.The test group(n=8)was subcutaneously immunized with 200 μg(in a total volume of 900 μLwith adjuvant)/each of the purified protein in neck,and the control group administration of 900μL/each of the mixture of saline and adjuvant.Booster immunization once after 2 weeks(half dose of the first immunization).A volume of 5～8 mL fasting blood was collected before and after immunization on the day of 15,30,90 and 180 from jugular vein and plasma was isolated for the determination of antibody and blood physiological and biochemical indexes.Meanwhile,adipose tissue in tail was sampled by surgery and used for transcriptome sequencing.By differential expression gene screening,GO,KEGG pathway and protein network analysis to evaluate the effects of active immunizaion recombinant extracellular domain of NPR-C on gene expression in tail adipose tissue of Altay sheep.The results showed that the molecular weight of the recombinant extracellular domain of NPR-C was about 50.55 KDa,and the recombinant protein accounted for 24.58%of the total bacteria protein by IPTG induction for 4 h.After being purified,the concentration of the recombinant protein reached to 0.46 mg/mL.A high level of specific antibody was detected after 15 d of the recombinant extracellular domain of NPR-C immunization.After booster,the antibody level is further improved and maintained until the end of the test There was no significant effect on the intake,body weight,blood glucose,LDL-C,HDL-C and FFA of Altay sheep(P>0.05)by immunization.But it significantly increased the content of glycerin and reduced the content of triglyceride in plasma of Altay sheep(P<0.01).Through the analysis of hormone levels,it was found that immunization of extracellular domain of NPR-C significantly increased the levels of adiponectin(P<0.01),cGMP(P<0.01)and insulin(P<0.05),and ANP had an increasing trend(P=0.068),but had no significant effects on the activity of fatty acid synthetase and the level of leptin(P>0.05).RNA-seq showed that there were 1809,599,309 and 423 genes up-regulated and 2083,505,438 and 456 genes down-regulated on the 15th,30th,90th and 180th day after immunization with recombinant extracellular domain of NPR-C.The difference genes related to lipid metabolism were screened with the threshold set at |log2(foldchange)|≥0.58 and pvalue<0.05,it was found that PDE3,PDE5,RGS5,ADVY7,STK4,ABCA1,and PDE3B8 genes were significantly up-regulated,AMPK and ATF4 genes were significantly down-regulated on the 15 d after active immunization;On the 30 d,GSTK1,INSIG1 and MOGAT2 genes were significantly up-regulated.There was no difference gene related to lipid metabolism on the 90 and 180 d.On the 15 d after immunization,KEGG enrichment analysis showed that cGMP-PKG signaling pathway,regulation of lipolysis in adipocytes,and insulin signaling pathway were significantly up-regulated,non-alcoholic fatty liver disease and oxidative phosphorylation were down-regulated GO term enrichment analysis showed that regulation of GTPase activity,positive regulation of fat cell differentiation and GTPase activator activity were significantly up-regulated,while release of cytochrome c from mitochondria,NADH dehydrogenase(ubiquinone)activity,and electron carrier activity were significantly down-regulated On the 30th d after immunization,the pathways of non-alcoholic fatty liver disease,ftty acid metabolism,glycerolipid metabolism and oxidative phosphorylation were significantly up-regulated.On the 90th d after immunization,the cAMP signaling pathway was down-regulated and the fatty acid β-oxidation was up-regulated.On the 180th d after immunization,the terms of triglyceride metabolic process and lipid transporter activity were up-regulatedIn conclusion,Immunization with recombinant extracellular domain of ovine natriuretic peptide receptor C can promote lipolysis in tail adipose tissue of Altay sheep through regulating cGMP,cAMP,insulin signaling pathway and oxidative phosphorylation. |
