人多肽:N-乙酰氨基半乳糖转移酶2(ppGalNAc-T2)RNA干扰载体的构建及鉴定

Construction and identification of RNA interference expression plasmid of human UDP-galNAc: polypeptide N-acetylgalactosaminyltransferase 2

目的:构建稳定的转染人多肽:N-乙酰氨基半乳糖转移酶2(ppGalNAc-T2)基因的RNA i载体。方法:遵循RNA干扰目标序列的选取原则,对ppGalNAc-T2基因mRNA序列设计五条可能的小干扰RNA(siRNA),PCR方法扩增得到siRNA内源表达系统,转染至人胃癌细胞株SGC7901,运用RT-PCR方法检测筛选得到其中能高效引起ppGal-NAc-T2基因表达沉默的siRNA;化学合成带荧光标记的该段RNA O ligo,转染细胞并用荧光显微镜观察转染情况,进一步鉴定该siRNA对ppGalNAc-T2的抑制作用;构建ppGalNAc-T2的RNA干扰真核表达载体,酶切及测序鉴定后转染SGC7901细胞并经G418筛选得到稳定的ppGalNAc-T2基因敲减细胞株,RT-PCR方法检测该转染细胞株ppGalNAc-T2表达水平。结果:采用RNA i技术,将高表达ppGalNAc-T2的SGC7901细胞的T2表达水平明显降低。结论:成功构建稳定的ppGalNAc-T2基因敲减细胞株,为今后进一步深入研究ppGalNAc-T2的生物学功能奠定了基础。

Objective： To construct the stable RNA interference expression plasmid of ppGalNAc-T2. Methods： Following the principle of selecting RNA interference target sequence, designed five potential small interference RNAs （siRNA） of ppGalNAc-T2 mRNA. With PCR amplified siRNA endogeny express system, transfected them into SGC7901 cells. Used RT-PCR to detect and select the siRNA with the highest ppGalNAc-T2 inhibit efficacy. Chemical synthesized this fluorescently-labeled RNA Oligo, transfected into SGC7901 cells and observed with fluorescence microscope, and accredited the depressant effect of siRNA to ppGalNAc-T2. Subcloned this siRNA to the RNAi eukaryotic expression plasmid pSilenCircle. The siRNA expression vectors were constructed and confirmed after the enzyme digestion analysis and the DNA sequencing, transfected into cells, then was screened by G418. The transfected cell was detected by RT-PCR. Results： The siRNA expression vectors were constructed and confirmed by RNAi technology. Conclusion： The stable cells with ppGalNAc-T2 knockdown will play an important role in the advanced research of the biological function of ppGalNAc-T2.