LMP-1单链抗体基因修饰人外周血单个核细胞的构建及鉴定

Construction and Identification of Anti-EBV LMP-1 Single Chain Variable Fragment Antibody Modified Peripheral Blood Mononuclear Cells

目的:构建携带抗-1型潜伏膜蛋白(anti-LMP-1)的单链抗体序列的过表达载体并包装慢病毒,构建及鉴定LMP-1单链抗体基因修饰的人外周血单个核细胞(PBMCs)。方法:根据Gen Bank中LMP-1单链抗体、CD28及CD3ζ序列设计引物并合成,从已有pVitro-IgG-1载体钓取IgG-1-Fc段,并分别插入慢病毒表达载体,获得p CDH-anti-LMP-1-IgG-Fc-CD28-CD3ζ(CAR)及pCDH-CD28-CD3ζ(对照VEC)并测序鉴定,测序成功后采用三质粒包装系统(核心质粒pCDH、包装质粒pSPAX2和pMD2.G)分别包装慢病毒CAR及VEC;浓缩慢病毒并离心感染PBMCs,经荧光显微镜观察绿色荧光蛋白(GFP)的表达,并利用流式细胞术检测GFP+比率以鉴定目的蛋白的表达。结果:构建了过表达LMP-1单链抗体基因的质粒并成功包装慢病毒,浓缩慢病毒感染PBMCs后GFP+细胞数可达42.5%(VEC-PBMCs)及51.0%(CAR-PBMCs)。结论:为进一步研究anti-LMP-1修饰的PBMCs对LMP-1阳性肿瘤细胞的靶向杀伤奠定了实验基础。

Objective： To prepare the recombinant lentivirus over-expressing anti-latent membrane protein-1（anti-LMP-1） single chain variable fragment（sc Fv） antibody gene, then construct and identify anti-LMP-1 gene modified human peripheral blood mononuclear cells（PBMCs）. Methods： The primers of anti-LMP-1 sc Fv、CD28 and CD3ζ were designed according to the Gen Bank.IgG1-Fc were obtained from existing p Vitro-IgG-1 vector. All these sequences mentioned above were amplified and inserted into the lentiviral expression plasmid pCDH to construct protein expression vectors（pCDH-anti-LMP-1-IgG-Fc-CD28-CD3ζ, hereafter refer to as CAR; control vector pCDH-CD28-CD3ζ, hereafter refer to as VEC）. CAR and VEC lentiviruses were prepared through three-plasmid packaging system（shuttle plasmid pCDH, packaging plasmids pSPAX2 and pMD2.G）. The PBMCs were isolated from healthy donors and infected with concentrated pCDH-VEC and pCDH-CAR lentivirus separately. The expression of green fluorescent protein（GFP）, as a surrogate reporter of target protein, was detected by fluorescence microscope and flow cytometry. Results： Over-expression vectors,VEC and CAR, were successfully constructed. The concentrated recombinant lentiviruses containing VEC or CAR could infect PBMCs and GFP as a reporter protein could be observed through fluorescence microscope and flow cytometry. The percentage of GFP＋ cells among VEC or CAR lentivirus transfected PBMCs were 42.5% and 51%. Conclusion： The anti-LMP-1 gene modified PBMCs were successfully established, which lays an experimental foundation for further research on its cytotoxic effect on LMP-1 positive tumor cells.