pcDNA3.1(+)-SAA真核表达载体构建、转染及其对人鼻咽癌细胞CNE-2增殖的影响  

Construction and tansfection of pcDNA3. 1( + )-SAA eukaryotic expression vector and its effect on CNE-2 cell proliferation

作　　者：江青山[1] 肖桃源[1] 邓文蓉[1] 沈宝茗[1] 

机构地区：[1]南华大学附属第一医院,湖南衡阳421001

出　　处：《山东医药》2013年第47期8-10,共3页Shandong Medical Journal

基　　金：湖南省卫生厅科研基金资助项目(B2010-047)

摘　　要：目的构建pcDNA3.1(+)-血清淀粉样蛋白A(SAA)真核表达载体,观察其在人鼻咽癌细胞CNE-2中的表达及对CNE-2增殖的影响。方法以CNE-2细胞的cDNA为模板,通过PCR扩增得到SAA片段,经酶切、纯化后与pcDNA3.1(+)连接;重组质粒经酶切分析及测序鉴定后,用脂质体转染法转染人鼻咽癌细胞CNE-2,用Western blot法检测转染前后该细胞中SAA蛋白的表达,并用MTT法检测转染重组质粒后的细胞增殖情况。结果 pcDNA3.1(+)-SAA经酶切、测序鉴定完全正确,真核表达载体构建成功;转染pcDNA3.1(+)-SAA真核表达载体后CNE-2细胞中SAA蛋白表达水平明显高增高;转染pcDNA3.1(+)-SAA后CNE-2细胞增殖速度加快。结论成功构建pcDNA3.1(+)-SAA真核表达载体,其能稳定表达于人鼻咽癌细胞CNE-2并促进细胞增殖;此为进一步研究SAA的生物学功能及鼻咽癌的新型治疗方法奠定了基础。Objective To construct an eukaryotic expression vector pcDNA3.1 （ ＋ ）-serum amyloid A （SAA） and to detect its expression in CNE-2 cells and the influence of its expression on CNE-2 cell proliferation. Methods Human SAA gene was amplified by PCR with CNE-2＇s cDNA as the template and the fragment was combined with plasmid pcDNA3.1 （ ＋ ）. The recombinant expression vector pcDNA3.1 （ ＋ ）-SAA was transfected into CNE-2 cell by using Lipofectamin. The expression level of SAA gene before and after transfection was detected by Western blotting. The influence of its ex pression on CNE-2 cell proliferation was further analyzed through MTI＂. Results After identification by restriction enzyme digestion and sequencing, the eukaryotic expression vector pcDNA3.1 （ ＋ ）-SAA was successfully constructed. The ex pression level of SAA in CNE-2 ceUs transfected by pcDNA3.1 （ ＋ ）-SAA was significantly higher. Meanwhile, MTF tes ting showed that CNE-2 cell proliferation was faster after being transfected with the recombinant plasmid. Conclusion The eukaryotic expression vector pcDNA3.1 （ ＋ ）-SAA is successfully constructed, stably expressed in CNE-2 cells and pro motes cell proliferation, which lays the foundation for the further study of the biological functions of SAA and new treatment method of nasopharyngeal carcinoma.

关 键 词：PCDNA3 1(+)-血清淀粉样蛋白A CNE-2细胞 转染 增殖 

分 类 号：R739.6[医药卫生—肿瘤]