摘要
为了构建tTS真核表达载体,转染HepG2细胞,建立稳定转染的HepG2细胞系,采用PCR方法,以质粒pTet-tTS为模板扩增tTS基因的编码区序列,利用DNA重组技术将其定向插入到真核表达载体pApoE-rtTA-Neo,经酶切和测序鉴定后,用脂质体转染法转染HepG2细胞,通过G418筛选,建立稳定转染的HepG2细胞系,用RT-PCR、Western blot法检年测tTS的表达,成功构建了pApoE-rtTA-tTS-Neo真核表达载体,并建立了稳定转染的HepG2细胞系,成功地表达目的基因。真核表达载体成功构建和稳定转染HepG2细胞系的建立为进一步研究tTS的功能奠定良好的实验基础。
In order to construct eukaryotic expression vector of the tetracyclinecontrolled transcriptional silencer（tTS）,and transfect HepG2 cells to establish stable HepG2 cell line.the full length tTS DNA fragment was amplified by PCR from the pTet-tTS was inserted into eukaryotic expression vector pApoE-rtTA-Neo.After the identification by digestion and sequencing on the recombinant eukaryotic expression vector pApoE-rtTA-tTS-Neo,the recombinant was transfected into HepG2 cell by lipofectamine 2000.After screening culture by G418,stable transfected HepG2 cell line was established,and the transcription and expression of tTS were identified by RT-PCR,Western blotting and immunofluorescence.The eukaryotic expression vector pApoE-rtTA-tTS-Neo was constructed successfully.The stable transfected HepG2 cell line was established.The tTS protein was expressed successfully.Conclusion：The construction of the eukaryotic expression vector pApoE-rtTA-tTS-Neo and the establishment of stable transfected HepG2 cell line provided solid foundation for further experimental studies on the function of tTS.
出处
《山西农业大学学报（自然科学版）》
CAS
2010年第4期336-339,共4页
Journal of Shanxi Agricultural University(Natural Science Edition)
基金
广东省科技计划项目(2007A060305013)
