多肽:N-乙酰氨基半乳糖转移酶2真核表达载体的构建及在SHG-44细胞的表达

Construction of Polypetide N-acetylgalactosaminyltransferase 2 Eukaryotic Expression Vector and Expression in SHG44 Cells

目的 :为探讨O 糖基化在肿瘤发生发展及转移中的作用和功能 ,构建pcDNA3/ppGalNAc T2真核表达质粒并转染人神经胶质瘤细胞SHG 4 4。方法 :采用PCR技术从pDONR2 0 1 T2得到ppGalNAc T2全长编码序列 ,亚克隆至真核表达载体pcDNA 3 1,脂质体介导基因转染人神经胶质瘤细胞SHG 4 4细胞 ,采用RT PCR法检测重组质粒的表达。结果 :酶切图谱分析和基因测序证实pcDNA3 1 T2真核表达质粒构建成功。RT PCR显示转染细胞有T2的表达。结论 :成功构建了真核表达载体pcDNA3 1 T2 ,并成功转染入SHG 4 4细胞 ,为进一步研究ppGalNAcT2的功能奠定了基础。

Objective: To construct the eukaryotic expression plasmid pcDNA3/ppGalNAc-T2 and transfect it into SHG44 cells for exploring the effect and function of genesis,development and metastasis in cancer. Methods:With PCR the target DNA fragment enconding ppGalNAcT2 was amplified from cloning plasmid pDONR201-T2 and subcloned to the eukaryotic expression plasmid pcDNA3.1,then transfected the recombinant plasmid into SHG44 cells,and detected the transcription of ppGalNAcT2 in transfected cells by RT-PCR.Results: Certificated by restriction enzymes digestion and DNA sequencing,the eukaryotic expression plasmid pcDNA3.1-T2 was constructed successfully and the T2 mRNA was detected by RT-PCR in transfected cells.Conclusion:The eukaryotic expression plasmid pcDNA3.1-T2 was successfully constructed and the recombinant plasmid was successfully transfected into SHG44 cells,which will lay the foundation for the study of its function.