人TSARG4真核表达载体的构建及稳定转染HeLa细胞系的建立

Construction of eukaryotic expression vector of human TSARG4 and establishment of its stable transfected HeLa cell line

目的:构建人TSARG4基因真核表达载体,转染HeLa细胞,建立稳定转染TSARG4的HeLa细胞系。方法:应用RT-PCR从人睾丸中扩增TSARG4的开放阅读框(ORF),并将PCR产物插入到pUCm-T载体中测序验证。随后,将TSARG4进一步克隆到pcDNA3.1(+)真核表达载体中。用脂质体将经过测序、验证的pcDNA3.1(+)/TSARG4质粒转染HeLa细胞,通过G418筛选建立TSARG4稳定转染的HeLa细胞系。RT-PCR和组织原位杂交技术检测TSARG4在稳定转染的HeLa细胞系中的表达。结果:成功构建了pcDNA3.1(+)/TSARG4表达质粒,建立了稳定转染的HeLa细胞系。RT-PCR和组织原位杂交检测结果表明,TSARG4基因在该细胞系中成功表达。结论:TSARG4真核表达载体成功构建和稳定转染HeLa细胞系的建立为进一步体外研究TSARG4的功能奠定了基础。

AIM： To construction of eukaryotic expression vector of human TSARG4 and establishment of its stable transfected Hela cell line. METHODS： The open reading frame （ORF） of TSARG4 was amplified from human testis by RT-PCR. The PCR products were cloned into pUCm-T vectors and sequenced. Then the cDNA fragment was subcloned into pcDNA3. 1 （ ＋ ）, a eukaryotic expression vector, The recombined plasmid pcDNA3. 1 （ ＋ ）/ TSARG4 was sequenced and transfected into Hela cell by lipofectamine^TM2000. After screening culture by G418, stable transfected HeLa cell line was established, and the expression of TSARG4 was identified by RT-PCR and in situ hybridization. RESULTS： The eukaryotic expression plasmid of pcDNA3. 1 （ ＋ ）/TSARG4 was successfully constructed and stable transfected HeLa cell line was established. RTPCR and in situ hybridization result revealed that TSARG4 was expressed successfully in HeLa cells. CONCLUSION： The recombinant eukaryotic expression vector of pcDNA3.1 （ ＋ ）/TSARG4 has been constructed successfully and Stably expressed in HeLa cell line, providing a foundation for further studies on the function of TSARG4 in vitro.