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本研究通过构建叶酸结合蛋白1(FOLR1)基因的慢病毒表达载体并进行表达鉴定。采用PCR技术扩增出基因全长,克隆至pWPI载体质粒,重组阳性克隆行PCR和测序鉴定,将重组载体质粒与辅助质粒共同转染人肾上皮(293T)细胞包装病毒,病毒颗粒感染人卵巢上皮癌(SKOV3)细胞,流式分选,RT-PCR和Western blot检测病毒感染后细胞中的FOLR1基因和蛋白表达。经鉴定结果显示重组载体质粒构建成功,293T细胞高效包装此慢病毒,携带FOLR1基因的慢病毒感染SKOV3细胞后可见大量绿色荧光细胞,流式分选后经两种方法分别检测出FOLR1基因和蛋白稳定表达。这为探讨FOLR1基因在卵巢恶性肿瘤中的生物学功能提供了实验基础。
In this study, lentiviral expression vector of folate binding protein 1 (FOLR1) gene was constructed and identified. The full length of the gene was amplified by PCR and cloned into the pWPI vector. The recombinant positive clones were identified by PCR and sequencing. The recombinant plasmid and the helper plasmid were co-transfected into human renal epithelial (293T) cell packaging virus. The virus particles infect human ovary The expression of FOLR1 gene and protein in the cells after the virus infection was detected by flow cytometry, RT-PCR and Western blot. The recombinant plasmids were successfully constructed and 293T cells efficiently packaged this lentivirus. A large number of green fluorescent cells were found after infection with SKOV3 cells with lentivirus carrying FOLR1 gene. FOLR1 gene and protein were detected by flow cytometry Stable expression. This provides an experimental basis for exploring the biological function of FOLR1 gene in ovarian cancer.