人源ADAM17基因干扰表达载体的构建与鉴定

Construction and identification of human ADAM17 gene RNA interference vector

目的构建靶向人去整合素-金属蛋白酶17(ADAM17)基因短发夹RNA(shRNA)的表达载体,并在人胰腺癌细胞株(PANC1)中鉴定其干扰效率。方法设计并合成ADAM17基因的shRNA片段,连接到经AgeⅠ和EcoRⅠ消化过的pLKO.1-puro载体中,构建干扰表达载体pLKO.1-puro ADAM17shRNA,测序鉴定;用干扰载体pLKO.1-puro ADAM17shRNA转染PANC1细胞,荧光定量聚合酶链式反应(q-PCR)和Western blot法检测干扰质粒的干扰效率;CCK-8法检测其对PANC1细胞增殖的影响。结果测序结果显示,成功构建了靶向人ADAM17基因shRNA的表达载体,且该干扰载体可将PANC1细胞中ADAM17的相对表达量下调65%以上,并能显著抑制PANC1细胞增殖。结论成功构建了ADAM17基因干扰质粒。

Objective To build human disintegrin domain and metalloproteinase17(ADAM17) short hairpin RNA (shRNA) gene expression vector,and to identify its interference efficiency in human pancreatic cancer cell line PANC1. Methods ADAM17 shRNA gene fragments was designed and compunded and connected to pLKO.1-puro vector which was double digested by AgeⅠand EcoRⅠ.Interference expression vector pLKO.1 Puro ADAM17 shRNA was constructed and sequenced.PANC1 cells were transfected with interference vector pLKO.1 Puro ADAM17 shRNA.The interference efficiency of interfering plasmids was detected by fluorescence quantitative PCR (q-PCR) and Western blot assay.CCK-8 assay was used to detect its effect on the proliferation of PANC1 cells. Results The sequencing results showed that shRNA expression vector targeting human ADAM17 gene was successfully constructed,and the interference vector could down-regulate the relative expression of ADAM17 in PANC1 cells by more than 65%,and significantly inhibit the proliferation of PANC1 cells. Conclusion ADAM17 gene interference plasmid vector was constructed successfully.