嵌合抗原受体表皮生长因子受体Ⅲ型突变体单链抗体-ICOS-CD3ζ表达载体的构建与鉴定

Construction and identification of chimeric antigen receptor epidermal growth factor receptor variant Ⅲ scFv-ICOS-CD3ζ expression vector

目的 构建嵌合抗原受体表皮生长因子受体Ⅲ型突变体单链抗体（EGFRvⅢscFv）-ICOS-CD3ζ表达载体.方法 将靶向EGFRvⅢscFv（726 bp）与第2代嵌合抗原受体的铰链区、跨膜区、胞内信号区（Hinge-TM-ICOS-CD,648bp）通过重叠延伸聚合酶链反应（PCR）连接,克隆入表达载体pCDH-CMV-MCS-EF1-copGFP的EcoR Ⅰ和BamH Ⅰ位点,测序确定DNA序列正确后脂质体法转染293T细胞,Western blot检测目的基因表达.结果 EcoR Ⅰ和BamH Ⅰ双酶切和DNA测序显示嵌合抗原受体EGFRvⅢscFv-ICOS-CD3ζ各基因片段连接及序列正确,转染293T细胞48h后提取蛋白质,鼠抗人CDζ单抗免疫印迹显示目的基因为57×103的蛋白条带,与预算相对分子质量相符.结论 成功构建嵌合抗原受体EGFRvⅢscFv-ICOS-CD3ζ表达载体,为表达EGFRvⅢ的肿瘤靶向免疫治疗提供了研究基础.

Objective To construct and identify chimeric antigen receptor epidermal growth factor receptor variant Ⅲ （EGFRv Ⅲ） scFv-ICOS-CD3ζ expression vector.Methods Single chain variable fragment of EGFRv Ⅲ monoclonal antibody was connected with hinge,transmembrane and intracellular signal domain （Hinge-TM-ICOS-CDζ,648 bp） of second generation chimeric antigen receptor by splicing-by-overlap extension polymerase chain reaction （PCR）.The resulting EGFRv Ⅲ scFv-ICOS-CD3ζ fragment was cloned into EcoRⅠ and BamHⅠ sites of the vector pCDH-CMV-MCS-EF-copGFP.After confirmation by sequencing analysis,the expression vector was transfected by lipofectamine into 293T cells and the protein expression was tested by Western blotting.Results The gene fragments in expression cassette were digested by double restriction enzyme digestion using EcoR Ⅰ and BamH Ⅰ and the whole sequence was confirmed by DNA sequencing analysis.The expression of EGFRvⅢ scFv-ICOS-CD3ζ was verified by immunoblotting （mouse anti-human CD3ζ as first antibody） as a band of about 57 × 103.Conclusion The chimeric antigen receptor EGFRv Ⅲ scFv-ICOS-CD3ζ expression vector was constructed successfully,which established a basis for further research on the chimeric antigen receptor-based adoptive cancer immunotherapy.