| 摘要: |
| 根据半滑舌鳎(Cynoglossus semilaevis Günther)生长激素(GH)基因的cDNA全长序列设计引物克隆得到其全长552个碱基成熟肽序列。利用RT-PCR方法将扩增片段克隆到原核表达载体pET- 28a上, 实现了GH成熟肽在大肠杆菌BL21(DE3)中的融合表达。融合蛋白分子量为26 kDa, 在IPTG诱导4h时目的蛋白表达量最高, 占细菌总蛋白的41.5%, 主要以包涵体形式存在。Western-blotting分析表明GH融合蛋白可特异性地被6×His抗体识别。诱导表达后的菌液沉淀经纯化和复性后, 获得大小为26 kDa的纯化GH融合蛋白。以ELISA方法检测纯化后的GH融合蛋白显示其具有免疫学活性。本研究为认识半滑舌鳎生长轴的调控机制提供了基础资料。 |
| 关键词: 半滑舌鳎 GH 原核表达 活性分析 |
| DOI：10.11693/hyhz20131100167 |
| 分类号: |
| 基金项目:国家鲆鲽类现代产业技术体系项目, CARS-50 号; 国家863 计划项目, 2012AA10A413 号; 中央级公益性事业单位基本科研业务费项目, 20603022012022 号; 山东省自然科学基金项目, ZR2012CQ025 号 |
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| IN VITRO PROKARYOTIC RECOMBINANT EXPRESSION AND BIOACTIVITY ASSAY OF GROWTH HORMONE FROM CYNOGLOSSUS SEMILAEVIS GüNTHER |
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Liu Xuezhou,Liu Zhiliang,Xu Yongjiang and Wang Yanyan
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Yellow Sea Fisheries Research Institute, CAFS,College of Fisheries and Life Science, Shanghai Ocean University,Yellow Sea Fisheries Research Institute, CAFS,Yellow Sea Fisheries Research Institute, CAFS
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| Abstract: |
| We isolated a mature peptide domain of growth hormone (GH) gene from pituitary of Cynoglossus semilaevis Günther. The matured peptide is 552base-pair long and composed of 183 amino acids residues. This matured peptide fragment was subcloned into a prokaryotic expression vector pET-28a and was successfully expressed in Escherichia coli BL21 (DE3) plysS. SDS-PAGE analysis indicated that the obtained GH fusion protein expressed in the form of inclusion bodies in molecular weight of 26 kDa and maximally amounted to 41.5% of the whole protein in E. coli cell in 4 hours after being induced with IPTG. Western-blotting analysis indicated the fusion protein had the antigenicity to 6?His antibody. The IPTG-induced bacterial precipitation was denaturalized, purified, and annealed, and then the purified fusion protein in molecular weight of 26 kDa was obtained. ELISA detection of recombinant GH protein showed that the in vitro recombinant GH fusion protein has ideal antigen activity. The proliferation experiment showed that high-concen?tration recombinant GH protein could inhibit the proliferation of human breast cancer cells MDA231. Results from this study could be helpful for understanding the growth mechanisms of C. semilaevis Günther. |
| Key words: Cynoglossus semilaevis Günther growth hormone (GH) prokaryotic expression bioactivity assay |
