嵌合抗原受体G250-CD8-28BBZ的构建及表达

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.

Construction and expression of chimeric antigen receptor G250-CD8-28BBZ

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 IgKsignal 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 obser-vation, Western blot and flow eytometry. Results The results of enzyme digestion and sequencing showed that the cod-ing sequence of G250 - CD8 -28BBZ was right and was inserted into the vector correctly. The results of fluorescence ob-servation, Western blot and flow cytometry showed that T cells were infected successfully, G250-CD8-28BBZ was ex- pressed 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.