文章摘要
半滑舌鳎两种leptin基因的体外重组表达和生物活性分析
Recombinant expression and bioactivity analysis of two leptin genes from Cynoglossus Semilaevis
投稿时间:2018-12-27  修订日期:2019-05-28
DOI:
中文关键词: 半滑舌鳎  leptin  原核表达  生物活性
英文关键词: Cynoglossus semilaevis  leptin  prokaryotic expression  bioactivity analysis
基金项目:国家重点研发计划政府间国际科技创新合作重点专项(2017YFE104400); 国家自然科学基金(31502145, 31602133, 31772829); 国家海水鱼产业技术体系(CARS-47); 农业部“一带一路”沿线热带国家水产养殖科技合作项目; 中国水产科学研究院黄海水产研究所基本科研业务费项目(20603022017016)
作者单位E-mail
张雅星 中国水产科学研究院黄海水产研究所 1500805828@163.com 
王滨 中国水产科学研究院黄海水产研究所  
柳学周 中国水产科学研究院黄海水产研究所  
徐永江 中国水产科学研究院黄海水产研究所 xuyj@ysfri.ac.cn 
史宝 中国水产科学研究院黄海水产研究所  
姜燕 中国水产科学研究院黄海水产研究所  
崔爱君 中国水产科学研究院黄海水产研究所  
张正荣 中国水产科学研究院黄海水产研究所  
孙冉冉 中国水产科学研究院黄海水产研究所  
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中文摘要:
      根据半滑舌鳎(Cynoglossus semilaevis) lepa和lepb编码氨基酸序列合成开放阅读框ORF肽段。利用原核表达载体pEQ30成功构建了半滑舌鳎lepa/pQE30和lepb/pQE30重组质粒,分别转化至大肠杆菌M15后经IPTG诱导获得了N端含6个组氨酸的半滑舌鳎LepA和LepB重组蛋白。获得的重组蛋白大小均为16 kDa,37℃下用0.5 mmol/L的IPTG诱导4 h后目的蛋白表达量最高,主要以包涵体形式存在。检测半滑舌鳎LepA和LepB重组蛋白浓度分别为0.3 mg/ml 和0.25 mg/ml。western-blotting免疫印迹和质谱分析表明,获得的LepA和LepB重组蛋白均具有免疫活性且序列正确。Ni2+-NTA亲和层析柱纯化可获得高纯度的半滑舌鳎LepA和LepB重组蛋白。离体孵育实验表明,获得的半滑舌鳎LepA和LepB重组蛋白能显著抑制半滑舌鳎下丘脑lepa、lepb和gnrh3 mRNA的表达水平,表明获得的重组蛋白具有明显的生物活性。研究结果可为探究leptin在半滑舌鳎生长发育中的调控作用机制提供重要载体和途径。
英文摘要:
      The peptides encoding ORF domains were synthesized based on the amino acid sequence of lepa and lepb from Cynoglossus semilaevis. The prokaryotic expression vector pEQ30 was used to construct the recombinant plasmids of lepa/pQE30 and lepb/pQE30, which were transformed into E.coli M15 strains and then induced by IPTG to obtain the recombinant proteins of LepA and LepB containing His 6 at the N-terminus. The obtained LepA and LepB polypeptides expressed in form of inclusion bodies with molecular weight were both 16 kDa, and the optimum condition for the highest expression of the target proteins were induction by 0.5 mmol/L IPTG at 37℃ for 4 hours. The concentrations of LepA and LepB recombinant proteins in tongue sole were 0.3 mg/mL and 0.25 mg/mL, respectively. The results of western-blotting and mass spectrometry analysis indicated that the obtained recombinant proteins of LepA and LepB had correct sequences and immunological activity. The proteins were purified by Ni2+-NTA affinity chromatography, and high-purity recombinant proteins of LepA and LepB were obtained. In vitro incubation of hypothalamus with recombinant LepA and LepB proteins from Cynoglossus semilaevis indicated that they could significantly inhibit the expression of endogenous lepa、lepb and gnrh3 mRNA, which verified that the obtained recombinant proteins have biological activities. The results could support exploration of the physiological role and regulation mechanism of leptin in growth and development of Cynoglossus semilaevis.
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