人多肽:N-乙酰氨基半乳糖转移酶2基因反义RNA表达质粒的构建及克隆鉴定

Construction of Human Polypeptide: N-Acetylgalactosaminyltransferase 2 Gene Antisense RNA Expression Plasmid and Its Functions

目的:研究反义封闭ppGalNAc-T2基因表达对胃癌细胞GC7901细胞增殖以及肿瘤细胞生物学行为的影响,构建反义表达载体并转染胃癌细胞GC7901。方法:在对几种肿瘤细胞的ppGalNAc-T2基因的表达水平分析后,以高表达ppGalNAc-T2的人胃癌细胞GC7901的总RNA为模板,利用RTPCR方法扩增两段不同长度ppGalNAc-T2基因片段,构建反义表达载体并转染胃癌细胞GC7901,通过G418筛选,建立一系列旨在封闭胃癌细胞GC7901细胞ppGalNAc-T2基因表达的亚细胞克隆。通过荧光显微镜、RT-PCR技术手段检测封闭反义ppGalNAc-T2基因RNA表达。结果:ppGalNAc-T2反义表达质粒载体pEGFP-FDT2和pEGFPFX-T2经限制性酶切及部分序列分析证明基因已正确插入载体中,荧光显微镜及RTPCR显示转染成功。结论:成功构建了ppGalNAc-T2反义表达质粒载体,反义封闭ppGalNAc-T2基因表达后,胃癌细胞GC7901ppGalNAc-T2的含量明显降低,为进一步研究ppGalNAc-T2奠定了基础。

Objective: To construct eukayotic antisense RNA expression vector ppGalNAc-T2 for exploring the effect of the cell proliferation on human stomachic cancer and the biological activity variation of cancer cells caused by antisense blocking of ppGalNAcT2 gene expression. Methods: After analyse expression level of ppGalNAcT2 in different cancer cells, ppGalNAc-T2 gene was amplified by RT-PCR using the total RNA extracted from GC7901 cell which expressed it highly,making use of antisense expressing vector of two different length ppGalNAcT2 gene segments and transfected into GC7901 cells. A series of subcellular clones aiming at the blocking of ppGalNAcT2 gene expression of GC7901 cells were established by means of G418 selection. Results: It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic antisense RNA expression vector pEGFP-FDT2 and pEGFP-FXT2 was correct. The antisense blocking of ppGalNAcT2 gene expression were tested by use of flow cytometer ,luorescent microscopy and RT-PCR. Conclusion: The eukayotic expression plasmid pEGFP-FDT2 and pEGFP-FXT2 was successfully constructed and the recombinant plasmid was successfully transfected into GC7901 cells.The ppGalNAcT2 level in the GC7901 cells was obviously redudced after antisense blocking. This will lay the foundation for the study of its function.