半滑舌鳎食欲素B的体外重组制备与生物活性分析

摘要

为了认识食欲素(orexin)编码的多肽orexinB对半滑舌鳎摄食的调控作用及机制,利用原核表达载体pET-32a成功构建了重组半滑舌鳎orexin B/pET-32a质粒,转化至大肠杆菌BL21后,经IPTG诱导获得了N端含6个组氨酸分子标签的orexinB重组蛋白.重组蛋白大小为21.14 ku,在温度37℃条件下以1.0 mmol/L的IPTG诱导6h的orexinB重组蛋白表达量最高,占菌体总蛋白的43.5％,并主要分泌于上清液中.Western blotting免疫印迹表明,获得的orexinB重组蛋白可被6×His抗体特异性识别.Ni2+-NTA亲和层析柱纯化可获得高纯度的半滑舌鳎orexinB重组蛋白.离体孵育实验表明,orexinB重组蛋白能促进下丘脑神经肽Y(NPY肽)的分泌和NPY,orexin mRNA的表达,表明获得的重组蛋白在激素和基因水平上调控下丘脑摄食相关神经肽的表达,具有明显的生物活性.研究结果可为半滑舌鳎orexinB蛋白的批量制备及高效促食生物制剂的研制提供技术支撑.%In 1998,the orexin was independently discovered by two different laboratories,and it was latterly proved to have wide functions in feeding promotion,energy balance regulation,growth and reproduction in vertebrates.It is secreted and released from hypothalamus and can encode two peptides,orexin A and orexin B.They exhibit high conservation in sequence during evolution,and the prior research verified that they both have important functions in feeding regulation and energy metabolism in mammals and fish.Cynoglossus semilaevis is an important economic farmed flatfish and a favorite table fish in China.Its farming industry is expanding,however,the endocrine mechanisms underlying growth and feeding actions remain limited.To explore the possible endocrine mechanisms for food intake regulation of C.semilaevis,the matured peptide fragment of orexin B was amplified and subcloned into the prokaryotic expression vector pET-32a to successfully construct orexin B/pET-32a recombinant plasmid.The recombinant plasmid was high-efficiently expressed in E.coli BL21 strains after being induced by IPTG with special fusion polypeptides containing His6 at their N-terminus.The molecular weight of obtained orexin B polypeptide is 21.14 ku and maximally accounted for 43.5％ of the whole bacterial protein post 6h induction with 1.0 mmol/L IPTG at 37 ℃.Western blotting analysis indicated that the recombinant polypeptides had the antigenicity to His6 antibody.The recombinant protein could be purified using Ni2+-NTA affinity chromatography.In vitro incubation of hypothalamus with recombinant expression orexin B of C.semilaevis indicated that it could promote the secretion and release of NYP peptide from hypothalamus,and upregulate the expression level of NPY mRNA.Meanwhile,the recombinant orexin B protein also could upregulate the expression level of orexin mRNA in hypothalamus at low concentration.All these verified that the obtained recombinant orexin B protein has biological activity in the present study.The present results would be helpful for better understanding the role of orexin B in feeding regulation and development of high-efficient feed additive for farming industry of C.semilaevis.