CAJ | 학술논문

目的：将组蛋白去乙酰化酶2( HDAC2)基因克隆入pEGFP-C2载体,探讨构建的质粒转染进非洲绿猴肾成纤维细胞COS-7株相关蛋白和mRNA的表达及蛋白分布部位的情况。方法从人肝癌细胞( HepG2)中获得组蛋白去乙酰化酶2(HDAC2)的cDNA,采用Xho I和 BamH I双酶切,用T4连接酶将该 cDNA 连接 pEGFP-C2质粒,将构建成功的pEGFP-C2-HDAC2质粒经PCR、限制性内切酶酶切和测序鉴定后转染非洲绿猴肾成纤维细胞( COS-7),荧光显微镜下观察绿色荧光蛋白表达, RT-PCR和Western blot鉴定HDAC2的表达。结果双酶切鉴定可见HDAC2质粒片段,转染重组质粒后可观察到绿色荧光蛋白的表达,PCR结果可检测到HDAC2 mRNA表达,同时 Western blot可检测 HDAC2蛋白和GFP 蛋白的表达。结论成功构建了重组 pEGFP-C2-HDAC2表达质粒,HDAC2蛋白可与绿色荧光蛋白在COS-7细胞中融合表达。
목적：장조단백거을선화매2( HDAC2)기인극륭입pEGFP-C2재체,탐토구건적질립전염진비주록후신성섬유세포COS-7주상관단백화mRNA적표체급단백분포부위적정황。방법종인간암세포( HepG2)중획득조단백거을선화매2(HDAC2)적cDNA,채용Xho I화 BamH I쌍매절,용T4련접매장해 cDNA 련접 pEGFP-C2질립,장구건성공적pEGFP-C2-HDAC2질립경PCR、한제성내절매매절화측서감정후전염비주록후신성섬유세포( COS-7),형광현미경하관찰록색형광단백표체, RT-PCR화Western blot감정HDAC2적표체。결과쌍매절감정가견HDAC2질립편단,전염중조질립후가관찰도록색형광단백적표체,PCR결과가검측도HDAC2 mRNA표체,동시 Western blot가검측 HDAC2단백화GFP 단백적표체。결론성공구건료중조 pEGFP-C2-HDAC2표체질립,HDAC2단백가여록색형광단백재COS-7세포중융합표체。
Aims HDAC2 gene was cloned into pEGFP-C2 vector to explore the efficiency of the plasmid trans-fection in renal fibroblasts COS-7 cells to identify the expression of both mRNA and protein levels and to ob-serve the distribution of the protein. Methods The HDAC2 cDNA was amlified by PCR and cut with the double enzyme Xho I and BamH I, then inserted into the eukaryotic expression vector pEGFP-C2 with T4 en-zyme. The recombinant vector was verified by PCR, restriction enzymes cut and sequencing identification. Then it was transfected into COS-7 cells and the ex-pression of pEGFP-C2-HDAC2 was monitored by fluo- <br> rescence microscope and PCR. Results Fragments of HDAC2 could be seen after dealt with double diges-tion, and GFP could also be detected in the transfected COS-7 cells. HDAC2 gene expression could be detec-ted by PCR and Western blot. The fusion expression of pEGFP-C2-HDAC2 could be detected by Western blot. Conclusion Eukaryotic expression vector of HDAC2 has been successfully constructed, the fusion expres-sion of HDAC2 and GFP protein can be detected in COS-7 cells.