H-Ras结构域的原核表达及纯化

Prokaryotic Expression and Purification of the Fragment of Human H-Ras with GST-Tagged

目的:克隆人H-Ras部分片段基因,获得其原核表达产物,并对融合蛋白进行纯化。方法:采用PCR技术从质粒Myc-H-Ras中扩增H-Ras(1-165)和H-Ras(165-189)结构域片段,将其克隆到p GEX-KG载体中,在大肠杆菌Rossate中表达后,利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化,以SDS-PAGE和Western印迹鉴定表达与纯化产物。结果:从Myc-H-Ras质粒中分别扩增获得约500和100 bp的DNA片段,并克隆至p GEX-KG载体,经测序与目的序列完全一致;在大肠杆菌Rossate中诱导表达出相对分子质量分别为46×103和29×103的目的蛋白,经纯化后获得了纯度较高的重组蛋白GST-H-Ras(1-165)和GST-H-Ras(165-189)。结论:获得了重组蛋白GST-H-Ras(1-165)、GST-H-Ras(165-189),为后续深入研究H-Ras影响肿瘤细胞生长的机制奠定了实验基础。

Objective： To construct the prokaryotic expression vector of the fragment of human H-Ras gene, ob- tain the purified GST-H-Ras（1-165） and GST-H-Ras（165-189） protein. Methods： Human H-Ras（1-165） and H-Ras（165-189） gene coding regions were amplified from the plasmid of Myc-H-Ras by the technique of PCR, and were inserted into prokaryotic expression vector pGEX-KG respectively. The recombinant plasmid pGEX-KG- H-Ras （1- 165） and pGEX-KG-H-Ras （165- 189） were transformed into E.coli Rossate. The expressed products were purified by GST-Sepharose 4B beads and identified by SDS-PAGE and Western blot analysis. Results： The DNA fragment of H-Ras（1-165） about 500 bp and H-Ras（165-189） about 100 bp, which were amplified by PCR, successfully cloned into pGEX-KG and identified by sequencing. The recombinant protein GST-H-Ras（1- 165） at 46 kD was successfully induced, purified well as well as GST-H-Ras（165-189） that its Mr was 29 kD. Conclusion： The recombinant protein of GST-H-Ras（1-165） and GST-H-Ras（165-189） were obtained successful- ly, which lay the foundation for further research between Ras gene and tumor.