结合转基因修饰的手段,能有效提高细胞免疫治疗的疗效,但由于缺乏T细胞高活性表达系统,限制了该方法的应用。构建腺病毒介导的双荧光素酶启动子活性筛选系统,检测一系列常用强启动子(CMV、CMV(in)、CAG、EF1α及CASI)在T细胞株K562及Jurkat中的活性,发现EF1α启动子活性在T细胞株中的活性最高。在EF1α的基础上,增加mCMV增强子和hCMV增强子顺式作用元件,构成了2个新型嵌合型启动子CCEF及CCEF(in)。双荧光素酶检测系统检测结果表明,新构建的2个启动子在T细胞中的活性高于EF1α,且两者相比CCEF启动子活性更高(p<0.05)。利用CCEF启动子控制PD-1全长抗体基因,并将此表达框插入腺病毒基因组,构建重组腺病毒Ad-CCEF-anti-PD-1。通过Western blotting及ELISA方法检测病毒感染T细胞株后,抗体基因的表达效率。结果表明,Ad-CCEF-anti-PD-1感染T细胞后,PD-1抗体能在T细胞中正确表达,且表达量较高(K562中11.019μg/mL,Jurkat中9.078μg/mL),表明该腺病毒介导的T细胞高效的基因表达系统成功构建,具有良好的应用前景。 Combined with genetically modified means can effectively improve the efficacy of cellular immunotherapy, but due to the lack of T cell highly active expression system, limiting the application of the method. Adenovirus-mediated dual luciferase reporter activity screening system was constructed to detect the activity of a series of commonly used strong promoters (CMV, CMV (in), CAG, EF1α and CASI) in T cell lines K562 and Jurkat, and found that EF1α Promoter activity is highest in T cell lines. Based on EF1α, two novel chimeric promoters, CCEF and CCEF (in), were constructed by adding cis-acting elements of mCMV enhancer and hCMV enhancer. The results of dual luciferase assay showed that the two newly constructed promoters were more active in T cells than EF1α, and both of them were more active than CCEF promoter (p <0.05). The full length of PD-1 antibody was controlled by CCEF promoter. The expression vector was inserted into the adenovirus genome to construct recombinant adenovirus Ad-CCEF-anti-PD-1. Western blotting and ELISA were used to detect the expression efficiency of antibody gene after T-cell infection. The results showed that the PD-1 antibody could be correctly expressed in T cells after transfected with Ad-CCEF-anti-PD-1, and its expression was high (11.019μg / mL in K562 and 9.078μg / mL in Jurkat) The results showed that the adenovirus-mediated T cell highly efficient gene expression system was successfully constructed and had a good prospect of application.