肿瘤相关糖蛋白-72特异性T细胞活化融合基因的分子克隆

Molecular cloning of fuse gene to coactivate TAG-72 specific T lymphocytes

目的克隆正常人T细胞CD3ξ胞内区、跨膜区基因,将其与肿瘤相关抗原肿瘤相关糖蛋白-72(TAG-72)特异性单链抗体基因克隆入真核表达载体。方法 用逆转录-聚合酶链反应(RT-PCR)法从正常人外周血T细胞扩增出CD3ξ胞内区、跨膜区基因;应用PCR法扩增抗TAG-72特异性单链抗体(scFv)基因;将该单链抗体与CD3ξ基因顺序克隆构建重组真核表达载体scFv-CD3ξ-pcDNA3.0并测序。结果抗TAG-72 scFv cDNA片段为729 bp,与已知的序列相符;CD3ξ胞内区、跨膜区的cDNA片段为306 bp,与Gene Bank公布的序列一致。重组的表达载体经酶切琼脂糖电泳及测序加以证实。结论成功构建了含抗肿瘤相关抗原TAG-72scFv及CD3ξ胞内区、跨膜区序列的重组表达载体scFv-CD3ξ-pcDNA3.0,为制备消化道肿瘤靶向性嵌合锚定T细胞奠定了实验基础。

Objective To clone the constructed transmembrane and intracellular domains of the signal transducing chain of CD3ξ in combination with TAG-72-specific single chain variable fragment (scFv) into a eukaryotic expression vector. Methods The transmembrane and intracellular domains of CD3ξ cDNA was amplified from human T lymphocyte using RT-PCR to clone into a eukaryotic expression vector, and anti-TAG-72 scFv cDNA was inserted to upstream of CD3ξ. Results A 729-base pair of anti-TAG-72 scFv was in accordance with sequence concerned;a 306-base pair of cDNA of the transmembrane and intracellular domains of CD3ξ was confirmed as sequence concerning of Genebank. Conclusion We constructed a eukaryotic expression vector encoding fusing gene to activate tumor-associated antigen-specific T lymphocyte, for generation of modified T lymphocytes to gastrointestinal tumors.