人vasorin蛋白的基因克隆、表达及纯化简

目的：克隆、表达人vasorin（VASN）蛋白。方法：利用PCR方法从HepG2细胞的cDNA中扩增获得目的基因，并插入带有6xHis标签的原核高效可溶性表达载体pET28a中，构建重组表达质粒pET28a-VASN，将重组表达质粒转化大肠杆菌BL21（DE3），经IPTG诱导后目的基因获得表达，对融合目的蛋白进行Ni^2＋金属螯合柱纯化。结果：内切酶鉴定及基因序列测定证实重组表达质粒构建成功；对目的蛋白进行了原核表达，SDS-PAGE显示相对分子质量为61x10^3的特异表达条带；Western印迹证实目的蛋白为VASN，且主要以包涵体形式存在；对经尿素变性的表达产物进行了亲和层析纯化，有利于以后的变性、复性过程。结论：获得了人VASN融合蛋白，为其进一步的生物学功能研究奠定了基础。

Gene Cloning,Prokaryotic Expression and Purification of Human Vasorin Protein

Objetive： To clone, express the human vasorin（VASN） protein in E.coli. Methods： The gene of in terest were amplified by PCR from cDNA of HepG2 cells and inserted into the prokaryotic expression vector pET28a with N terminal 6× His tag to build recombinant expression plasmid pET28a-VASN. The recombinant plas mid was transformed into E.coli BL21（DE3）, and the expression of fusion protein was induced by IPTG. Ni^2＋ met al chelating column was utilized for high purification of the target protein. Results： Sequencing and restriction analysis revealed that the recombinant plasmid was successfully constructed. The fusion protein was expressed in E. coli, and SDS-PAGE showed a clear protein band with a relative molecular weight of 61 kD. Western blot con firmed the VASN was induced expressed in E.coli mainly in the form of inclusion bodies. The expression products were purified by affinity chromatography from urea treated inclusion body of E.coli. Conclusion： The fusion pro tein of human vasorin was successfully expressed and purified, which makes the foundation of further study on its biological function.