摘要
目的构建pEGFP—C3／Smad3真核表达质粒，并进行鉴定。为进一步研究Smad3在抑制乳腺癌生长机制中起到的作用提供实验基础。方法利用RT—PCR方法从MCF-7乳腺癌细胞中获得cDNA，应用PCR方法扩增、提取Smad3的基因片段，酶切后与pEGFP—C3真核表达载体连接，构建pEG—FP—C3／Smad3真核表达质粒，进行测序、酶切鉴定，表明质粒构建成功。将构建的质粒瞬时转染至MCF-7乳腺癌细胞中，通过荧光倒置显微镜观察、RT—PCR技术及Westenblot技术鉴定转染是否成功。结果本实验成功构建了pEGFP—C3／Smad3真核表达质粒。结论pEGFP—C3／Smad3真核表达质粒瞬时转染到MCF-7细胞中，可使Smad3在基因与蛋白水平的表达显著上调。
Objective The study was to construct and identify the eukaryotic expression plasmid pEGFP - C3/Smad3, and then transfect it into MCF - 7 breast cancer cells. To lay a foundation for further research on the biological function of Smad3 in the inhibition of breast cancer growth mechanism. Methods The cDNA was extracted from MCF -7 breast cancer ceils by RT - PCR, the Smad3 gene fragment was amplified and extracted by PCR, and the amplified products were inserted into pEGFP - C3 eukaryotic expression vector then sequenced and identified by restrictive endonuclease digestion. The constructed recombinant plasmid was transitorily trans- fected into the MCF- 7 breast cancer cells and the expression of Smad3 was observed by inverted fluorescence microscope, RT - PCR technology and westen blot. Results The experiment was successfully constructed the eu- karyotic expression plasmid pEGFP - C3/Smad3 and transiently transfected into MCF - 7 ceils. Conclusion The eukaryotic expression plasmid pEGFP - C3/Smad3 was transiently transfected into MCF - 7 cells. Smad3 gene and protein levels can be significantly raised.
出处
《实用肿瘤学杂志》
CAS
2013年第1期6-10,共5页
Practical Oncology Journal
基金
黑龙江省留学归国科学基金项目（LC201009）
黑龙江省杰出青年科学基金（JC201203）
