【摘要】 目的 构建嵌合抗原受体G250-CD8-28BBZ的慢病毒表达质粒,并研究该嵌合抗原受体在T细胞中的表达。方法 利用常规分子克隆技术将编码Igκ信号肽、抗G250单链抗体及信号结构CD8-28BBZ的编码序列拼接起来,并将得到的融合基因片段插入慢病毒表达载体pLVX-IRES-ZsGreen中,采用酶切及测序鉴定。慢病毒感染T细胞后,通过荧光显微镜、Western blot、流式细胞术检测G250-CD8-28BBZ在T细胞中的表达。结果 双酶切及测序结果显示重组质粒pLVX-G250-CD8-28BBZ构建正确;慢病毒感染后的T细胞能发出绿色荧光;T细胞表达的G250-CD8-28BBZ分子大小正确;慢病毒感染2周后,G250-CD8-28BBZ在T细胞中的阳性表达率为45.2%。结论 成功构建了嵌合抗原受体G250-CD8-28BBZ的慢病毒表达质粒,该嵌合抗原受体能够在T细胞内高效稳定表达。更多还原

【Abstract】 Objective To construct lentivirus expression plasmid of chimeric antigen receptor(CAR) G250-CD8-28BBZ and detect its expression in T cells.Methods The coding sequences of Igκsignal peptide,the scFv of G250,and signal domain CD8-28BBZ were spliced by normal molecule cloning techniques.Then,the fusion gene sequence was inserted into the Lentivirus vector pLVX-IRES-ZsGreen.The reconstructed plasmid was identified by restricted enzyme digestion and sequencing.The expression of G250-CD8-28BBZ in T cells was analyzed by fluorescence observation,Western blot and flow cytometry.Results The results of enzyme digestion and sequencing showed that the coding sequence of G250-CD8-28BBZ was right and was inserted into the vector correctly.The results of fluorescence observation,Western blot and flow cytometry showed that T cells were infected successfully,G250-CD8-28BBZ was expressed correctly and G250-CD8-28BBZ+T cells were 45.2%.Conclusion The plasmid PLVX-G250-CD8-28BBZ was constructed successfully.The chimeric antigen receptor G250-CD8-28BBZ could be expressed in T cells efficiently and stably.更多还原