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首页> 外文期刊>Biochemistry >Molecular Cloning,Characterization,and Expression Studies of Water Buffalo(Bubalus bubalis)Somatotropin
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Molecular Cloning,Characterization,and Expression Studies of Water Buffalo(Bubalus bubalis)Somatotropin

机译：水牛生长激素的分子克隆，表征及表达研究

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摘要

Cloning,high-level expression,and characterization of the somatotropin(ST)gene of an indigenous Nili-Ravi breed of water buffalo Bubalus bubalis(BbST)are described.Coding,non-coding,and promoter regions of BbST were amplified and sequenced.Sequence analysis revealed several silent and two interesting point mutations on comparison with STs of other vertebrate species.One interesting variation in the BbST sequence was the replacement of a conserved gluta-mine residue by arginine.A plasmid was also constructed for the production of BbST in Escherichia coli BL21(RIPL)CodonPlus,under the control of IPTG-inducible T7-lac promoter.High-level expression could be obtained by synthesizing a codon-optimized ST gene and expressing it in the form of inclusion bodies.The inclusion bodies represented over 20% of the E.coli cellular proteins.The biologically active conformation of purified BbST was confirmed by its efficient growth promoting activity in Nb2 cell proliferation assay.The expression system and purification strategy employed promise to be a useful approach to produce BbST for further use in structure-function studies and livestock industry.
机译：描述了水牛水牛（BbST）Nili-Ravi本地品种生长激素（ST）基因的克隆，高水平表达和鉴定。扩增并测序了BbST的编码区，非编码区和启动子区。序列分析显示，与其他脊椎动物物种的ST相比，有几个沉默和两个有趣的点突变.BbST序列的一个有趣的变化是用精氨酸替代了保守的谷氨酰胺残基，还构建了一个质粒用于生产BbST。大肠杆菌BL21（RIPL）CodonPlus在IPTG诱导的T7-lac启动子的控制下。通过合成密码子优化的ST基因并以包涵体的形式表达可得到高水平表达。 20％的大肠杆菌细胞蛋白。纯化的BbST在Nb2细胞增殖试验中具有有效的生长促进活性，从而证实了其生物活性构象。所采用的em和纯化策略有望成为生产BbST的有用方法，以进一步用于结构功能研究和畜牧业。

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