靶向性肿瘤相关抗原TAG-72的scFv-CD28融合基因的真核表达载体的构建

Construction of A Mammalian Expression Vector to Activate Tumor-Associated Antigen-Specific T Lymphocytes

目的 构建含抗肿瘤相关抗原TAG 72单链抗体及CD2 8胞内区及跨膜区融合基因的哺乳细胞表达载体 ,为制备消化道肿瘤靶向性嵌合锚定T细胞 ,并探讨其对消化道肿瘤的杀伤活性。方法 应用PCR法 ,将抗TAG 72单链抗体 (singlechainvariablefragment ,scFv)克隆入哺乳细胞表达载体pcDNA3 .0 ,在 5′端及 3′端分别引入相应酶切位点 ;用RT PCR法从正常人外周血T细胞克隆CD2 8胞内区及跨膜区的cDNA ,然后将其克隆于scFv的下游 ,并在 5′端及 3′端引入相应的酶切位点。结果 抗TAG 72scFvcDNA片段为 72 9bp ,与已知的序列相符 ;CD2 8胞内区及跨膜区的cDNA片段为 2 40bp ,与Genebank公布的序列一致。重组的表达载体经酶切琼脂糖电泳测序加以证实。结论 完成了抗肿瘤相关抗原TAG 72scFv及CD2 8胞内区及跨膜区融合基因scFv CD2 8 pcDNA3 .0的构建 ,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 A mammalian expression vector encoding TAG 72 specific single chain variable region(scFv) fused to the transmembrane and intracellular domains of the signal transducing chain of CD28 was constructed, to generate for targeting of genetically modified T cells to gastrointestinal cancer, and to investigate the cytotoxicity against TAG 72 positive target cells.Methods The transmembrane and intracellular domains of CD28 cDNA was amplified from human T lymphocytes using RT PCR to clone into a mammalin expression vecter, and CD28 fragments were ligated downstream of the anti TAG 72 scFv cDNA and sequence verified.Results A 729 base pair of anti TAG 72 scFv was in accordance with sequence concerned; a 240 base pair of cDNA of the transmembrane and intracellular domains of CD28 was confirmed as sequence concerning of Genebank.Conclusion We constructed a mammalian expression vector encoding fusing gene to activate tumor associated antigen specific T lymphocyte, for generation of modified T lymphocytes to gastrointestinal tumors.