表达anti-CD19-CAR协同沉默PD-1的慢病毒载体重构

Reconstruction of the lentivirus vector expressing anti-CD19-CAR and co-silencing PD-1

本研究通过PCR方法扩增含EF1a启动子的DNA片段,利用同源重组酶将该DNA片段整合到pGreenPuro-shRNA慢病毒载体CMV启动子下游,获得新的多启动子慢病毒载体pGreenPuro-Dual。同时,在改造的慢病毒载体的CMV启动子下游插入鼠源anti-CD19-CAR片段,并在H1启动子下游EcoRI和BamHI酶切位点之间插入PD-1shRNA片段,重组获得pGreenPuro-Dual/anti-CD19-CAR/shRNA PD-1慢病毒载体,重组质粒与2个辅助质粒共转染HEK293FT细胞包装重组慢病毒颗粒。利用包装病毒感染HEK293T细胞后,荧光观察病毒感染效率,并提取细胞总蛋白Western blotting检测antiCD19-CAR以及PD-1的表达。结果显示表达anti-CD19-CAR以及shRNA PD-1的重组慢病毒成功感染了HEK293T细胞,鼠源anti-CD19-CAR成功表达,同时PD-1蛋白被有效沉默。

In this study, DNA fragment containing EFla promoter was amplified by PCR＇and was integrated into downstream of CMV promoter of pGreenPuro-shRNA lentivector via homologous recombination to generate a novel lentivector termed as pGreenPuro-Dual. Then, murine anti-CD19-CAR fragment was inserted into downstream of the CMV promoter, while PD-1 shRNAs, respectively, were inserted at the downstream of H1 promoter through EcoRI and BamHI enzyme restriction sites to obtain the recombinant lentiviral plasmid termed as pGreenPuro-Dual/anti-CD19-CAR/shRNA PD-1. Next, the recombinant plasmids were co-transfected into HEK293FT cells with 2 packaging plasmids to produce pseudovirus. After infection of HEK293T cells with the recombinant virion, the copGFP protein expression was observed under fluorescence microscope, and the whole cell lysis was used to detect the expression of anti-CD19-CAR and PD-1 by Western blotting. The results showed that HEK293T cells were successfully infected by recombinant lentivirus expression with anti-CD19-CAR and PD-1 shRNA. Murine anti-CD19-CAR was positively expressed and PD-1 was effectively silenced in cells.