prk基因非融合表达载体的构建及其在大肠杆菌中的表达

The Construction of prk Gene Non-fusion Expression Vector and the Expression of Prk Protein in E. coli

目的:构建prk基因的非融合表达载体,并诱导表达。方法:PCR扩增并回收prk基因片段,将其与亚克隆载体pMD18-T重组,通过蓝/白斑筛选、酶谱分析和序列测定鉴定出重组质粒,然后从该重组质粒上切下prk基因片段,再克隆至非融合表达载体pBV220中,酶谱分析鉴定出正向重组质粒,诱导含有正向重组质粒的宿主菌表达蛋白,提取全菌总蛋白进行聚丙烯酰胺凝胶电泳(SDS-PAGE)分析。结果:成功构建并筛选出pBV220正向重组质粒,经热诱导表达,得到一可溶性的相对分子质量为65ku的重组蛋白。结论:pBV220-prk表达载体的成功构建和表达,说明扩增所得的基因具有正确的阅读框架,使获得天然Prk蛋白成为可能,为进一步研究prk基因的生物学功能奠定了基础。

Objective: To construct a recombinant plasmid and to induce the expression of the Prk protein. Methods: The prk gene was amplified by PCR and then it was recombined with pMD18-T vector. The recombinant plasmid was selected according to the color (white/blue) of the bacterial colony and the analysis of restriction enzyme. Then the prk gene was digested with EcoR I and Sal I and then inserted into the expression vector pBV220. The host bacteria containing the recombinant plasmid grew with heat induction, and the complete protein of the bacteria was extracted for SDS-PAGE. Results: Restriction endonucleases digestion of pBV220-prk revealed two correct bands. SDS-PAGE analysis showed that E. coli DH5 a containing the recombinant plasmid could produce a kind of soluble protein of 65 ku that amounted to 10. 9% of the total cellular soluble proteins. Conclusion: The construction of pBV220-prk and the expression of non-fusion protein were successful, which will benefit the study on the biological activity of prk gene.