程序性死亡蛋白1(PD-1)在转染的HEK293T细胞的表达和鉴定

PD-1 overexpression in HEK293T cells transfected with pMH3-PD1

目的优化HEK293T细胞转染条件,比较程序性死亡蛋白1(PD-1)在不同真核载体的表达水平以高效获取目标蛋白。方法设计L9(33)正交试验,对细胞转染条件进行优化;扩增人PD-1胞外段基因,构建PD-1重组蛋白表达载体,瞬时转染HEK293T细胞,通过ELISA、Western blot对pCMV3、pcDNA3. 1和pMH3三种真核表达载体的蛋白表达水平进行比较。结果PD-1胞外段蛋白编码序列成功构建到pc DNA3. 1和pMH3真核表达载体上,目的蛋白成功表达。在最佳转染条件下,PD-1重组蛋白在pMH3中的表达量最高,其次为pCMV3、pcDNA3. 1的表达量最低。结论人PD-1胞外段蛋白在pMH3-PD1真核载体转染的HEK293T细胞中表达量最高。

Objective To optimize the transfection conditions of HEK293T cells and compare the expression levels of human programmed death 1(hPD-1)in different eukaryotic vectors to obtain the target protein efficiently.Methods L9(33) orthogonal test was designed to optimize the conditions of cell transfection.The DNA sequence of hPD-1 extracellular domain gene was amplified by PCR and then cloned into different vectors:pcDNA3.1,pCMV3 and pMH3,PD-1 recombinant protein was expressed in HEK293T cells transiently and the expression levels was evaluated by ELISA and Western blot analysis. Results The gene of hPD-1 extracellular domain was successfully cloned into pcDNA3.1 and pMH3 eukaryotic expression vectors,and the target protein was successfully expressed.Under the optimal transfection conditions,the expression level of hPD-1 recombinant protein in pMH3 vector was the highest,followed by that of pCMV3 and pcDNA3.1 vectors.Conclusion The extracellular domain of hPD-1 is successfully expressed and the expression level of pMH3-PD1 was the highest among the three vectors tested.