遗传密码子扩充系统表达载体及其稳定细胞系的构建

Construction of Genetic Codon Expansion Expression Vector and Stably Transfected Cells

目的:构建异源表达遗传密码子扩充系统的HeLa细胞系。方法:用PCR方法扩增MmBocRS、PyltRNA基因,定向克隆到Lenti-EF1α-Puro载体中,构建Lenti-EF1α-BocRS、Lenti-PyltRNA表达质粒;在HEK293T细胞中进行病毒包装,用获得的慢病毒感染HeLa细胞,获得BocRS-t RNACUA稳定细胞株;分别应用实时定量PCR和Western印迹鉴定该稳定细胞系中MmBocRS和PyltRNA的表达;利用转染EGFP的琥珀突变体验证BocRS-t RNACUA稳定细胞株的功能。结果:酶切及测序表明Lenti-EF1α-BocRS和Lenti-PyltRNA质粒构建正确;实时定量PCR结果显示BocRSt RNACUA稳定细胞株中PyltRNA的表达量显著提高;Western印迹结果显示BocRS-t RNACUA稳定细胞株中MmBocRS基因的表达量明显高于空白对照;转染EGFP琥珀突变体结果表明,可以通过非天然氨基酸来控制EGFP的表达。结论:构建了能稳定表达MmBocRS和PyltRNA的BocRS-t RNACUA细胞株,并实现了通过非天然氨基酸控制蛋白表达,为研究定点插入非天然氨基酸提供了细胞模型。

Objective： To establish a HeLa cell line heterologous-expressing orthogonal aminoacyl-tRNA synthetase/transfer RNA（tRNA） pairs. Methods： MmBocRS and PyltRNA genes were amplified by PCR and cloned into Lenti-EF1α-Puro vector to construct Lenti-EF1α-BocRS and Lenti-PyltRNA expression plasmids. Virus packaging was carried out in HEK293T cells. HeLa cells were infected with the obtained lentivirus to obtain BocRS-tRNACUA stably transfected cells. The expression of MmBocRS and PyltRNA in the stably transfected cells were identified by real-time quantitative PCR and Western blot respectively. The function of BocRS-tRNACUA stably transfected cells was examined by transfected amber mutant of EGFP. Results： Lenti-EF1α-BocRS and Lenti-PyhRNA plasmids were constructed correctly. The results of RT-PCR showed that the expression of PyltRNA in BocRS-tRNACUA stably transfected cells was enhanced. Western blotting showed that the expression of MmBocRS gene in the stably transfected ceils was significantly higher than in the blank control group. Transfection of EGFP amber mutant revealed that EGFP expression was controlled by unnatural amino acids. Conclusion： The BocRS-tRNACUA cell line stably expressing MmBocRS and PyltRNA was constructed and the expression of the protein was controlled by unnatural amino acid. It provided a cellular model for studying the insertion of non-natural amino acids.