人纤溶酶原K5基因的结构改造及其在大肠杆菌中的表达、纯化与鉴定

Expression,purification and identification of a structure modified human plasminogen kringle 5 gene in Escherichia coli

目的改造人纤溶酶原Kringle 5(K5)基因,构建RGDRGD-liteK5融合表达质粒,并表达纯化所得RGDRGD-liteK5蛋白。方法以人纤溶酶原K5 cDNA为模板,通过PCR得到RGDRGD-liteK5基因片段,并克隆到质粒pGEX1-λT中,构建重组原核融合表达载体pGEX-RGDRGD-liteK5;在IPTG诱导下,观察融合蛋白在大肠杆菌Rosetta中的表达情况;利用亲合层析柱纯化表达产物,用Western blot分析鉴定表达产物。结果PCR扩增得到274 bp的片段,并成功插入pGEX1-λT质粒;含重组质粒的大肠杆菌在IPTG诱导下表达了特异性的融合蛋白,其分子量为36 kD;获得的融合蛋白经凝血酶酶切后,得到了分子量约为10 kD的RGDRGD-liteK5蛋白;Western blot证实了表达产物的正确性。结论成功地改造了人纤溶酶原K5基因,构建了重组融合表达质粒pGEX-RGDRGD-liteK5,并纯化了其表达产物。

OBJECTIVE To clone,express and purify a modified human plasminogen kringle 5 gene,which provides a basis for further studies on the function of RGDRGD - liteK5. METHODS The gene of RGDRGD - liteK5 was amplified from the K5 cDNA by PCR and inserted into pGEX1 - λT plasmid to construct the prokaryotic fusion expression vector. Expression of fusion protein in Escherichia coli Rosetta was detected after induction by IPTG. The expression product was collected by affinity chromatography with GSTrap FF column and analyzed by Western blot. RESULTS The acquired gene was 274 bp and it was inserted into pGEX1 - λT plasmid successfully. The Escherichia coli containing recombinant vector expressed fusion protein of 36 KD after induction by IPTG. Cleaved by thrombin, the protein of RGDRGD - liteK5 was obtained. Its molecular weight was about 10 KD. The fusion protein was confirmed by Western blot analysis. CONCLUSION The modification, expression and purification of human plasminogen kringle 5 gene are successful.