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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 recombinant expression vector RGD-FasL for prokaryotic expression and to analyze the expression and purification of recombinant protein. Methods RGD-FasL gene was inserted into the N terminus of FasL gene by overlapping PCR. The pGEX-5X-1 / RGD-FasL expression vector was constructed. The E.coli BL21 (DE3) was transformed into E.coli BL21 (DE3), induced by IPTG and purified by GST column. In vitro adhesion assay, MTT colorimetric assay, flow cytometry assay of fusion protein function. As a result, the target gene encoding the correct amino acid sequence was obtained by overlap PCR. The target protein is expressed in a secreted form, and the expression amount accounts for more than 30% of the total bacterial protein. After purification, the protein purity of more than 95%. In vitro adhesion experiments showed that the purified fusion protein could specifically bind to cervical cancer Hela cells. Both MTT assay and flow cytometry demonstrated that the purified fusion protein can specifically induce tumor cell apoptosis. Conclusion The successful construction, expression, purification and activity analysis of recombinant RGD-FasL expression vector lays the foundation for further functional studies.