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获得人成纤维细胞生长因子受体2Ⅲc(FGFR2Ⅲc)及其S252W突变型重组腺病毒,感染乳腺癌细胞MDA-MB-231,为下一步研究FGFR2Ⅲc基因的功能和作用机制奠定基础。以本实验室保存的含FGFR2Ⅲc基因的质粒为模板,PCR扩增得到FGFR2Ⅲc基因,重叠延伸法PCR获得FGFR2ⅢcS252W突变型基因;分别将上述野生型和突变型基因克隆至腺病毒穿梭质粒pAdTrack-CMV上,得到重组穿梭质粒pAdTrack-FGFR2Ⅲc和pAdTrack-FGFR2ⅢcS252W,DNA测序证实。Pme I酶切后分别与腺病毒骨架质粒pAdEasy-1共转化BJ-5183感受态细菌同源重组,得到的重组表达质粒Ad-FGFR2Ⅲc和Ad-FGFR2ⅢcS252W Pac I酶切线性化后转染HEK293A细胞进行重组腺病毒的包装和扩增,通过GFP报告基因观察病毒表达情况。收集重组病毒颗粒并测定滴度,进一步感染乳腺癌细胞MDA-MB-231,RT-PCR和Western blotting方法检测目的基因的表达,3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法和流式细胞术分析细胞增殖情况。结果表明,成功构建了人FGFR2Ⅲc及其S252W突变型基因的重组腺病毒表达载体,获得的重组腺病毒颗粒能高效感染MDA-MB-231细胞,并表达目的基因。MTT结果显示FGFR2Ⅲc和S252W均能抑制MDA-MB-231细胞增殖,S252W抑制效果更加明显。流式细胞术表明FGFR2Ⅲc和S252W均能使MDA-MB-231细胞周期停滞于G0/G1期,抑制细胞增殖。
Obtaining FGFR2Ⅲc and its S252W mutant recombinant adenovirus and infecting breast cancer cell MDA-MB-231, which will lay the foundation for the further study on the function and mechanism of FGFR2Ⅲc gene. The FGFR2Ⅲc gene was amplified by PCR using the plasmid containing FGFR2Ⅲc gene preserved in our laboratory as a template, and the FGFR2ⅢcS252W mutant gene was amplified by overlap extension PCR. The wild type and mutant genes were cloned into the adenovirus shuttle plasmid pAdTrack-CMV The recombinant shuttle plasmid pAdTrack-FGFR2Ⅲc and pAdTrack-FGFR2ⅢcS252W were confirmed by DNA sequencing. After digested with Pme I, the recombinant plasmids were co-transformed into BJ-5183 competent cells by adenoviral backbone plasmid pAdEasy-1 respectively. The recombinant plasmids Ad-FGFR2Ⅲc and Ad-FGFR2ⅢcS252W Pac I were linearized and transfected into HEK293A cells Recombinant adenovirus packaging and amplification, through the GFP reporter gene expression observation of the virus. The recombinant virus particles were collected and the titer was determined. The cells were further infected with MDA-MB-231. The expression of target gene was detected by RT-PCR and Western blotting. The expression of 3- (4,5-dimethylthiazol-2) 5-diphenyltetrazolium bromide (MTT) method and flow cytometry analysis of cell proliferation. The results showed that the recombinant adenovirus vector of human FGFR2Ⅲc and its S252W mutant gene was successfully constructed. The obtained recombinant adenovirus could efficiently infect MDA-MB-231 cells and express the target gene. MTT results showed that both FGFR2Ⅲc and S252W could inhibit the proliferation of MDA-MB-231 cells, and the inhibitory effect of S252W was more obvious. Flow cytometry showed that both FGFR2Ⅲc and S252W could arrest the cell cycle of G0 / G1 phase and inhibit cell proliferation in MDA-MB-231 cells.