靶向CD19嵌合抗原受体载体的构建及其稳转细胞体外活化效应的研究  

Construction of Targeting CD19 Chimeric Antigen Receptor Vector and Activation Effect of Stably Transfected Jurkat Cells In Vitro

作　　者：王振禹 姜英浩[1] 许承明[1] 阮班军 叶子晨[1] 翟东昇 卢兹凡[1] WANG Zhenyu JIANG Yinghao XU Chengming RUAN Banjun YE Zichen ZHAI Dongsheng LU Zifan(Department of Pharmacogenomics, Fourth Military Medical University, Xi＇ an, 710032, China)

机构地区：[1]第四军医大学药学系药物基因组学教研室,西安市710032

出　　处：《医学分子生物学杂志》2017年第1期1-5,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.31571215）

摘　　要：目的构建靶向CD19嵌合抗原受体载体并通过建立稳转细胞系检测其在体外的活化效应。方法合成获取CD19的CAR片段，转入pCMV6-Entry质粒，构建pCMV6-Entry—CAR载体。通过重组酶的作用将pCMV6一Entry—CAR载体和pLenti6．3／V5-DEST载体进行重组。形成pLenti6．3／V5．DEST．CAR载体，将其包装入慢病毒并感染Jurkat细胞建立稳转细胞系。将稳转的Jurkat细胞和Raji细胞共孵育后，利用ELISA检测上清IL-2的含量，判断Jurkat细胞的活化状况。结果成功构建了pLenti6．3／V5-DEST-CAR载体，感染筛选后的Jurkat细胞可稳定表达CD19的CAR，与Raji细胞共孵育后细胞上清中IL-2的浓度增加至22pg/ml。结论实验中构建的pLenti6．3／V5-DEST—CAR载体能够稳定转染并表达于Jurkat细胞系中，并能有效促进其激活效应。为下一步优化CART细胞提供基础。Objective To construct a recombinant vector harboring CD19 chimeric antigen receptor and detect its action effect in vitro by establishing a stable transfection cell line. Methods CAR fragments and pCMV6-Entry fragments were extracted and connected to construct the pCMV6- Entry-CAR vector. PCMV6-Entry-CAR vector and pLenti6.3/V5-DEST vector were reconstituted by recombinant enzyme to create the pLenti6. 3/V5-DEST-CAR vector, which was packaged into virus and infected Jurkat cells to establish a stable cell line. The level of IL- 2 was detected by ELISA after co-incubation of Jurkat cells and Raji cells, and the action effect of Jurkat cells was determined. Results The pLenti6.3/V5-DEST-CAR vector was constructed successfully. CAR of CD19 could be stably expressed in Jurkat cells after infection and screening. The content of IL-2 was increased to 22 pg/mL in the supernatants of cells co-incubated with Raji cells. Conclusion The recombinant vector pLenti6. 3/V5-DEST-CAR could be stably transfected into Jurkat cells, which were verified to have action effects. Our study provides a foundation for further optimizing CART cells.

关 键 词：CD19 嵌合抗原受体 载体构建 增殖 

分 类 号：Q789[生物学—分子生物学]