HSV-TK/CD20双自杀基因系统慢病毒载体的构建及应用

Construction of recombinant lentivirus vectors carrying HSV-TK/CD20 double suicide gene and its application

目的构建同时表达单纯疱疹病毒胸苷激酶(HSV-TK)和RQR8膜蛋白双自杀可控开关的Jurkat细胞,使之可通过更昔洛韦药物(GCV)或利妥昔单抗(RTX)实现可控性细胞消除。方法 RQR8膜蛋白小分子含利妥昔单抗识别的模拟表位和抗CD34单抗表位。合成编码RQR8及HSV-TK的基因,构建慢病毒共表达载体pLV-RQR8-T2A-TK;脂质体Lipofectamine3000转染Jurkat细胞,流式荧光抗CD34单抗检测RQR8的表达,通过磁珠筛选获得RQR8+TK+Jurkat细胞;分别加入GCV或RTX检测自杀可控开关功能:CCK8法检测加药后细胞增殖情况,Annexin-V/PI检测细胞凋亡情况。结果慢病毒载体pLV-RQR8-T2A-TK酶切片段大约1 670 bp,测序进一步证实外源片段插入正确。慢病毒感染Jurkat细胞,感染率为45.21%,磁珠纯化得到99.4%纯度的RQR8+TK+Jurkat。GCV对RQR8+TK+Jurkat具有细胞毒性作用,GCV浓度为40μmol/L时,其存活率为(52.11±7.04)%,IC50为20.66μmol/L。80μmol/L GCV作用RQR8+TK+Jurkat细胞72 h后凋亡率达(41.28±2.78)%。在血清(含补体)存在情况下,320μg/ml RTX可通过补体介导的细胞杀伤作用杀伤RQR8+TK+Jurkat,其存活率为(29.64±4.52)%。结论双自杀可控开关慢病毒载体pLV-RQR8-T2A-TK构建成功,加入GCV或RTX可通过2种不同路径成功杀伤细胞,达到细胞清除目的,提高了安全性,为临床治疗过继免疫不良反应提供多种选择。

This study aimed to modify the Jurkat cells with HSV-TK and RQR8 double suicide gene, which are readily eliminated upon exposure to the ganciclovir(GCV) or Rituximab(RTX) alternatively. RQR8 is a small transmembrane protein containing a CD20 epitope recognized by Rituximab and a CD34 epitope tag. The DNA fragment encoding HSV-TK, T2 A and RQR8 was synthesized and inserted into lentivirus vector pLV firstly. Then, the constructed pLV-RQR8-T2 A-TK was transfected into Jurkat cells with lipofectamine3000. The expression of RQR8 gene was detected by anti-CD34 antibody, while RQR8+TK+Jurkat cells were purified by CD34 beads. The self-cleaning ability was detected by CCK-8 and Annexin-V/PI when RQR8+TK+Jurkat cells were exposed to GCV or RTX. The enzymes digestion and DNA sequencing confirmed the correct construction of the vector. The infection efficiency reached to45.21% and the purity of RQR8+TK+Jurkat reached to 99.4% after purified by anti-CD34 antibody-coated beads.GCV has cytotoxicity to RQR8+TK+Jurkat cell. When the concentration of GCV reached to 40 μmol/L, the survival rate was(52.11±7.04)% and the IC50 was 20.66 μmol/L. The apoptosis rate of RQR8+TK+Jurkat was enhanced to(41.28±2.78)% when treated with 40 μmol/L GCV for 72 h. In presence of complement-containing serum, 320 μg/ml RTX could kill RQR8+TK+Jurkat cells in a complement-dependent cytotoxicity way, and the survival rate of RQR8+TK+Jurkat cells was(29.64±4.52)%. In conclusion, the pLV-RQR8-T2 A-TK lentivirus vector has been constructed successfully. Gene-modified cells can be eliminated in different ways after GCV or RTX treatment,which provides multiple options for clinical treatment of ACT adverse reactions.