P6BMP2的克隆及其在大肠杆菌中的表达-东南大学学报医学版

P6BMP2的克隆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：R784, R785

出版年·卷·期（页码）：2007·26·第二期（124-127）

摘要：

目的:在大肠杆菌中克隆和表达骨形态发生蛋白2(BMP2)的变体P6BMP2并初步纯化.方法:根据P6肽的氨基酸序列,按照大肠杆菌偏爱密码子将P6肽的核酸序列设计在5′端引物,通过PCR方法合成P6BMP2基因,并将其克隆到大肠杆菌表达质粒pALEX中,经NcoⅠ、XhoⅠ双酶切和DNA序列测定验证.结果:通过双酶切和DNA序列分析,证实获得的基因片段与我们期望的相符合,重组质粒在大肠杆菌BL21(DE3)中,经IPTG诱导获得表达.表达产物经超声破碎和Heparin SepharoseTM 6 Fast Flow亲和层析纯化,获得重组蛋白.结论:构建了P6BMP2的表达质粒,获得P6BMP2的表达并建立了纯化方式,为P6BMP2的深入研究和应用奠定了坚实的基础.

Objective To clone and express P6BMP2 in E.coli and to purify it.Methods P6 gene was designed in 5′-primer with the codon usage preferential for E.coli.Using PCR method,P6BMP2 was synthesized,cloned into E.coli expression vector pALEX and verified by NcoⅠ and XhoⅠ digestion and DNA sequencing.Results The NcoⅠ and XhoⅠ digestion and DNA sequencing verified the obtained full length P6BMP2 gene and the cloning of P6BMP2 in expression vector pALEX.After IPTG induction,recombinant plasmid expressed P6BMP2 in E.coli BL21(DE3).After sonication,the recombinant P6BMP2 products were purified with SepharoseTM 6 Fast Flow affinity chromatography.Conclusion We have constructed the recombinant plasmid to express P6BMP2,obtained the expression of P6BMP2 after induction and developed a purification procedure.All those have laid solid foundation for the further research and potential application of P6BMP2.

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