RGD-FasL基因的构建、表达、纯化及其活性分析

Construction,expression,and purification of RGD-FasL and analysis of its activities

目的构建适于原核表达的重组蛋白RGD-FasL表达载体,并进行重组蛋白的表达纯化及抗肿瘤活性分析。方法通过重叠PCR将RGD序列插入到FasL基因的N端,获得RGD-FasL基因,构建pGEX-5X-1/RGD-FasL表达载体。转化大肠杆菌BL21(DE3),IPTG诱导表达,GST柱纯化。采用体外黏附实验、MTT比色法、流式细胞法检测融合蛋白的功能。结果通过重叠PCR获得了编码正确氨基酸序列的目的基因。目的蛋白以分泌的形式表达,表达量占菌体总蛋白的30%以上。纯化后,蛋白纯度达95%以上。体外黏附实验表明所纯化的融合蛋白可与宫颈癌Hela细胞发生特异结合。MTT比色法与流式细胞技术均表明纯化的融合蛋白能特异性地诱导肿瘤细胞发生凋亡。结论重组蛋白RGD-FasL表达载体的成功构建、表达、纯化及活性分析,为进一步的功能研究奠定了基础。

Objective To construct, express, and purify RGD-FasL gene and analysis its anti-tumor activities. Methods The RGD- FasL gene was constructed by overlapping PCR through inserting the sequence of RGD at the N-terminate of FasL gene, and then was inserted into vector pGEX-5X-1 and expressed efficiently in E. coli BL21 （DE3） under optimization condition. The expressed products were purified by GST resin column. The function of recombined protein was detected by adhesion in vitro analysis, MTT colorimetric, and flow cytometry. Results The proteins were expressed mainly as secretion with a yield of more than 30% of total bacterial proteins. After purification, the purity of the proteins was more than 95 %. The results of adhesion in vitro analysis showed that the purified protein could eombine with Hela cells specifically. And the MTT colorimetric and flow cytometry suggested that the purified protein could induce tumor eell apoptosis. Conclusion The construction, expression, purification of RGD-FasL gene and analysis its anti-tumor activities provides a foundation for further studying.