| 李梦苑,叶传涛,范 超,张沛欣,张 颖,贾战生.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[J].现代生物医学进展英文版,2018,(8):1441-1446. |
| 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 |
| Received:October 29, 2017 Revised:November 23, 2017 |
| DOI：10.13241/j.cnki.pmb.2018.08.008 |
| 中文关键词: 潜伏膜蛋白1型 慢病毒 嵌合抗原受体 |
| 英文关键词: Latent membrane protein 1 Lentivirus Chimeric antigen receptor |
| 基金项目:国家自然科学基金项目（81601749） |
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| 中文摘要: |
| 摘要 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GenBank中LMP-1单链抗体、CD28及CD3ξ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pCDH-anti-LMP-1-IgG-Fc-CD28-CD3ξ（CAR）及pCDH-CD28-CD3ξ（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。 |
| 英文摘要: |
| ABSTRACT Objective: To prepare the recombinant lentivirus over-expressing anti -latent membrane protein -1(anti-LMP-1) single chain variable fragment（scFv） antibody gene, then construct and identify anti-LMP-1 gene modified human peripheral blood mononuclear cells (PBMCs). Methods: The primers of anti-LMP-1 scFv、CD28 and CD3ξ were designed according to the GenBank. IgG1-Fc were obtained from existing pVitro-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-CD3ξ, hereafter refer to as CAR; control vector pCDH-CD28-CD3ξ, 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. |
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