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目的 采用反转录聚合酶链反应 (RT -PCR)法获得人α型叶酸受体 (folatereceptoralpha,FOLR - 1,FR - 1)基因序列 ,构建融合表达载体 pGEXFR - 1并进行表达。 方法将RT -PCR法扩增的FOLR - 1基因 ,克隆入融合表达载体 pGEX - 4T - 2中 ,使其受控于Ptac启动子 ,转化大肠杆菌DH5α。经异丙基 - β -D -硫代半乳糖苷 (IPTG)诱导 ,用SDS -PAGE检测表达产物。结果SDS -PAGE显示 ,FOLR - 1表达产物的相对分子质量 (Mr)约为 5 5 0 0 0 ,与预期的结果相符。Western -blot证实 ,在相应Mr处 ,有GST-FR1融合蛋白的显示条带。结论成功地构建了FOLR -1基因的融合表达载体并获得表达 ,为进一步探讨卵巢癌的免疫治疗提供了实验依据
OBJECTIVE: To construct the fusion expression vector pGEXFR - 1 and express the gene folatereceptoralpha (FOLR - 1, FR - 1) by reverse transcriptase polymerase chain reaction (RT - PCR) Methods The FOLR - 1 gene amplified by RT - PCR method was cloned into the fusion expression vector pGEX - 4T - 2, which was controlled by Ptac promoter and transformed into E. coli DH5α. The product was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the expression product was detected by SDS-PAGE. Results SDS-PAGE showed that the relative molecular mass (Mr) of FOLR - 1 expression product was about 5500, which was consistent with the expected results. Western-blot confirmed that at the corresponding Mr, a GST-FR1 fusion protein was displayed. Conclusion The fusion expression vector of FOLR -1 gene was successfully constructed and expressed, which provided an experimental basis for further exploring the immunotherapy of ovarian cancer