【摘要】 目的:制备及纯化针对增殖诱导配体基因(APRIL)的小干扰RNA(APRIL siRNA),为进一步研究APRIL基因在人胰腺癌中的作用奠定基础。方法:构建靶向胰腺癌CFPAC-1细胞株APRIL基因双链RNA(APRIL dsRNA)的表达质粒pET-22b-APRIL,在大肠杆菌中发酵表达APRIL dsRNA,并利用CF-11树脂亲和层析纯化;用大肠杆菌Ⅲ型RNA水解酶(RNaseⅢ)水解dsRNA,制备APRIL siRNA,应用DEAE树脂离子交换层析使RNaseⅢ与核苷酸分离,分子筛层析进一步分离纯化APRIL siRNA。将纯化后的APRIL siRNA转染CHO细胞,荧光显微镜下观察转染后CHO细胞APRIL蛋白的表达情况。结果:pET-22b-APRIL转染大肠杆菌后,经IPTG诱导能大量表达APRIL dsRNA,表达产物经CF-11树脂纯化能得到纯化的APRIL dsRNA;dsRNA经RNaseⅢ水解后,过DEAE树脂离子交换层析柱,经5%PAGE、12%SDS-PAGE电泳证实RNaseⅢ与核苷酸分离,进一步经分子筛层析分离纯化,得到纯化APRIL siRNA。荧光显微镜下观察结果表明,APRIL siR- NA转染CHO细胞能明显抑制APRIL蛋白的表达。结论:成功制备了靶向CFPAC-1细胞株的APRIL siRNA,为下一步敲减CFPAC-1细胞APRIL基因的研究奠定了基础。更多还原

【Abstract】 Objective:To prepare and purify siRNA targeting a proliferation-inducing ligand targeted(APRIL-siRNA),so as to provxde a basis for studying the role of APRIL in human pancreatic cancer.Methods:pET-22b-APRIL was constructed to express APRIL dsRNA of human pancreatic cancer cell line CFPAC-1 in E.coli and the product was purified by chromatography using CF-11 column.APRIL dsRNA was digested by RNaseⅢto prepare APRIL siRNA,then the reaction mixture was loaded onto a DEAE ion exchange chromatography to remove RNaseⅢfrom oligonucleotides,and size exclusion chromatography was used to purify 21 bp siRNA.The purified APRIL siRNA was used to transfect Chinese hamster ovary(CHO)cells and the expression of APRIL in CHO cells was observed under fluorescence microscope Results:APRIL dsRNA was successfully expressed in E.coli after IPTG induction and was purified by CF-11 column.dsRNA was hydrolyzed with RNaseⅢand was purified by DEAE ion exchange chromatography and size exclusion chromatography.15% nondenaturing PAGE and 12% SDS- PAGE confirmed that RNaseⅢwas removed from oligonucleotides and 21 bp siRNA was purified with size exclusion chromatography.It was also found that APRIL siRNA obviously depressed APRIL expression in CHO cells.Conclusion:We have successfully constructed APRIL siRNA targeting APRIL gene of CFPAC-1 cells with in vitro transcription,which provides a basis for knock-down of APRIL gene in CFPAC-1 cells.更多还原