FasL基因构建 蛋白表达及其对胃癌细胞株BGC823和MGC803生长的影响

Construction of Vector Expressing FasL and the Influences of FasL on the Growth of BGC823 and MGC803 Cells

目的:构建适于原核表达的重组蛋白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:获得人FasL cDNA全序列,将其分段设计引物,通过重叠PCR获得FasL基因。构建pGEX-5X-1/FasL表达载体。转化大肠杆菌BL21(DE3),IPTG诱导表达,GST柱纯化。采用MTT比色法、流式细胞法检测融合蛋白对胃癌细胞的作用。结果:通过重叠PCR获得了编码正确氨基酸序列的目的基因。表达的目的蛋白表达量占菌体总蛋白的30%以上。纯化后,蛋白纯度达95%以上。MTT比色法与流式细胞技术均表明纯化的融合蛋白能抑制胃癌细胞株BGC823和MGC803的生长,诱导其调亡增加。结论:重组蛋白FasL表达载体的成功构建、表达、纯化及活性分析,为进一步的抗肿瘤功能研究奠定了基础。

Objective： To construct a vector expressing FasL gene and to investigate the influences of FasL on the growth of BGC823 and MGC803 cells. Methods： The FasL gene was obtained through overlapping PCR and then was inserted into vector pGEX-5X-1 and expressed efficiently in E.coli BL21 （DE3）. The expressed products were purified through GST resin column. The effect of recombined-protein on gastric cancer cell lines BGC823 and MGC803 was detected by MTT colorimetric and flow cytometry. Results： The proteins were expressed mainly as secretion with the yield of more than 30% of total bacterial proteins. After purification, the purity of the proteins was all more than 95%. The MTT-F colorimetric and flow cytometry suggested that the purified protein could inhibit the growth of BGC823 and MGC803 cell lines through inducing tumor cell apoptosis. Conclusion： The construction of vector expressing FasL, the purification of FasL gene and analysis of its anti-tumor activities provide the foundation for further study.