【摘要】 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。更多还原

【Abstract】 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 lipofectamineTM2000.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.RT-PCR 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.更多还原