牛生长抑素基因的构建及其在大肠杆菌中的表达-东南大学学报医学版

牛生长抑素基因的构建及其在大肠杆菌中的表达

单位：南京大学医药生物技术国家重点实验室,江苏南京,210093

分类号：Q786

出版年·卷·期（页码）：2003·22·第六期（376-379）

摘要：

目的:在大肠杆菌中克隆表达牛生长抑素基因并初步纯化.方法:根据牛生长抑素基因序列,按照大肠杆菌偏爱密码子设计合成2个DNA片段,经退火、Klenow酶补平及连接反应,获得牛生长抑素基因片段;经PCR扩增、EcoRⅠ和BamHⅠ酶切,牛生长抑素基因被克隆在表达质粒pALEX中.结果:将重组质粒转化BL21(DE3),经IPTG诱导获得表达.表达产物经超声破碎和GST-Sepharose 4B亲和层析纯化获得重组蛋白.结论:牛生长抑素基因得到了高水平表达并初步纯化,为表达产物的大量制备、进一步的结构功能关系及在畜牧业生产上的应用研究奠定了基础.

Objective To clone and express bovine somatostatin in Escherichia coli(E.coli) and to purify it.Methods Bovine somatostatin gene was designed with the codon usage preferential for E.coli. Two oligonucleotide fragments were annealed, filled in with Klenow fragment of DNA polymerase Ⅰ and then ligated with T4 ligase. After further PCR amplification, the full length gene encoding somatostatin was obtained. After EcoRⅠand BamHⅠdigestion, the gene was inserted into plasmid pALEX.Result The recombinant plasmid was transformed into E.coli BL21(DE3), and expressed upon IPTG induction. The expression level of recombinant GST-somatostatin was about 6% of total cellular proteins. After sonication, the recombinant GST-SS products were purified by GST-Sepharose 4B affinity chromatography.Conclusion Overexpression of bovine somatostatin gene offers a basis for large scale production and further research regarding the relationship between its structure and function.

参考文献：

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