免疫绵羊利钠肽受体C胞外区对阿勒泰羊血液生理生化指标的影响

Effects of Immunization against Extracellular Domain of Sheep Natriuretic Peptide Receptor C on Blood Physiological and Biochemical Indexes of Altay Sheep

利钠肽(NP)具有促进机体脂肪分解的作用,但其半衰期受利钠肽受体C(NPRC)表达水平调控,通过免疫途径中和机体NPRC有望延长NP发挥作用的时间。本研究根据大肠杆菌对密码子的偏爱性对绵羊NPRC胞外区基因进行密码子优化,通过全基因合成技术合成优化后的NPRC胞外区(NPRCo)基因并构建大肠杆菌表达载体pET28a(+)-NPRCo。经IPTG诱导后NPRCo在E.coli BL21(DE3)中获得表达,经Ni-NTA纯化和Western Blotting鉴定获得重组NPRC胞外区(rNPRCo)。选取16只2月龄左右雌性阿勒泰羊,随机分为试验组和对照组,分别于颈部皮下免疫rNPRCo 200μg/只或生理盐水。2周后加强免疫1次(100μg/只)。分别于免疫前、免疫后第15天、第30天、第90天和第180天空腹静脉采集血液,分离血浆用于测定抗体和血液生理生化指标。结果表明,主动免疫rNPRCo后阿勒泰羊可持续产生高水平的抗体;主动免疫rNPRCo对阿勒泰羊血液中葡萄糖、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇浓度均无显著影响,但可显著升高免疫后第15天、第180天时甘油和第180天游离脂肪酸浓度,并显著降低免疫后第180天时血浆中甘油三酯浓度。由此可见,免疫重组绵羊利钠肽受体C胞外区蛋白质对雌性阿勒泰羊脂肪代谢具有一定的调节作用。

Natriuretic peptide(NP)plays an important role in promoting lipolysis,but its half-life is regulated by the expression level of NPRC(natriuretic peptide receptor C),so immunoneutralization of the NPRC may prolong the action time of NP plays.In this study,based on the codon bias of E.coli,codon optimization was performed on NPRC extracellular domain genes of sheep,and extracellular domain of ovine NPRC gene was heterologously expressed in E.coli by gene engineering technology to obtain an abundance of antigen.The optimized gene(named NPRCo)was synthesized by the gene synthesis technology and the expression vector pET28a(+)-NPRCo was constructed.After IPTG induction,NPRCo was expressed in E.coli BL21(DE3).The recombinant NPRCo protein(named rNPRCo)was purified by Ni-NTA and identified by Western blotting.Sixteen two-month old female Altay sheep were selected and divided into the immunization group and the control group.Animals in the immunization group were immunized against rNPRCo subcutaneously in the neck with 200μg/animal,and the control group injected with saline and adjuvant.Two weeks later,the immunization was boosted once with dose of 100μg/animal.Fasting blood samples were collected from veins before and 15th,30th,90th and 180th day after immunization,and plasma was separated for the determination of antibody and physiological and biochemical indexes.The results showed that in animal trial,Altay sheep could produce high level titer of antibody after immunization.Immunization with rNPRCo had no significant effect on the blood glucose,contents of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol,but it could significantly increase the content of glycerol and reduce the content of triglyceridein plasma,and increase the content of glycerol at 15th and 180th day and non-esterified fatty acids at 180th day,and significantly reduce the content of triglycerides in plasma at 180th day after immunization.In summary,active immunization against recombinant extracellular domain protein of sheep natriuretic peptide receptor C has certain regulation effect on lipid metabolism of female Altay sheep.