融合IL2的慢病毒实现对T细胞的高效基因转导  

Potent Gene Transfer in Human Primary T Lymphocytes Using Interleukin-2-displaying Lentiviral Vectors

作　　者：王依依 杜晓[2] 胡志嵩 刘欣禹 欧阳清 赵晶[2] 杨安钢[6] 阎博[2] WANG Yiyi;DU Xiao;HU Zhisong;LIU Xinyu;OUYANG Qing;ZHAO Jing;YANG Angang;YAN Bo(School of Laboratory Medicine,Xinxiang Medical University,Xinxiang,Henan,453004,China;State Key Laboratory of Cancer Biology,Department of Biochemistry and Molecular Biology,Fourth Military Medical University,Xi’an,710032,China;Internal Medicine Department,95140th Military Hospital,Huizhou,Guangdong,516008,China;School of Student Brigade,Fourth Military Medical University,Xi’an,710032,China;Department of Nephrology,PLA General Hospital,Beijing,100853,China;Department of Immunology,Fourth Military Medical University,Xi’an,710032,China)

机构地区：[1]新乡医学院医学检验学院河南省新乡市,453004 [2]第四军医大学生物化学与分子生物学教研室,肿瘤生物学国家重点实验室,西安市710032 [3]中国人民解放军第95140军区医院内科,广东省惠州市516008 [4]第四军医大学学生大队学院,西安市710032 [5]中国人民解放军总医院肾内科,北京市100853 [6]第四军医大学免疫学教研室,西安市710032

出　　处：《医学分子生物学杂志》2022年第1期1-9,共9页Journal of Medical Molecular Biology

基　　金：国家自然科学基金重点项目(No.81630069);国家自然科学基金面上项目(No.81972870)。

摘　　要：目的制备融合IL2的慢病毒以提高对人原代T淋巴细胞的转导效率。方法将白细胞介素-2(IL2)基因定向克隆至水疱性口炎病毒G蛋白(VSV-G)编码基因5′端,构建新型慢病毒包装辅助质粒pMD2.IL2-G。使用不同剂量pMD2.IL2-G进行慢病毒包装,应用免疫印迹和ELISA比较病毒滴度,流式细胞术检测病毒对T细胞的转导效率。结果单独使用pMD2.IL2-G显著降低病毒得率。与传统慢病毒相比,pMD2.IL2-G与原始辅助质粒pMD2.G以1∶4混合制备的慢病毒,能够显著提高EGFP基因导入T细胞的效率(89.2%vs.40.2%),且病毒得率相当。结论融合IL2的慢病毒能够对T细胞进行高效的基因转导,为基因修饰T细胞的临床应用提供有力支持。Objective To construct the interleukin 2(IL2)-displaying lentivirus to improve the transduction efficiency of human primary T lymphocytes.Methods The IL2 gene was fused to the N-terminus of vesicular stomatitis virus G protein to produce the novel lentivirus-packaging plas-mid pMD2.IL2-G.The yields of lentiviral particles pseudotyped with different doses of pMD2.IL2-G were compared using immunoblotting and ELISA.The infected capacity of IL2-displaying lentiviruses to T lymphocytes from healthy donors was tested using flow cytometry.Results The yield of lentivi-ral particles pseudotyped with pMD2.IL2-G was significantly less than that of wild-type lentiviruses packaged by pMD2.G.The mixture of pMD2.IL2-G and pMD2.G at a ratio of 1:4 could obtain the equivalent yield of lentiviruses that of the wild-type,and significantly improve the transduction effi-ciency of enhanced green fluorescent protein into the T lymphocytes compared to the wild-type lenti-viruses(89.2%vs.40.2%).Conclusion Our results demonstrate that IL2-displaying lentiviral particles have a greatly enhanced transduction efficiency into human T lymphocytes.This research is expected to provide strong support for the clinical application of genetically modified T cells.

关 键 词：T细胞 包膜蛋白 融合白介素-2的慢病毒载体 转导效率 过继免疫疗法 

分 类 号：Q782[生物学—分子生物学]