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目的克隆正常人T细胞CD3ζ的胞内区、跨膜区基因,将其与二硫键稳定的人源化抗肝癌的单链抗体scFv的基因克隆入真核表达载体,为制备肝癌靶向性嵌合锚定T细胞奠定基础,并探讨其对肝癌的杀伤活性。方法应用PCR法将二硫键稳定的人源化的单链抗体scFv的基因克隆入真核细胞表达载体pcDNA3,在5’端及3’端分别引入相应酶切位点;用RT-PCR法从正常外周血人T细胞扩增出CD3ζ的胞内区、跨膜区基因,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。结果二硫键稳定的人源化抗肝癌的单链抗体scFv的cDNA片段为735bp,与已知的序列相符;CD3ζ的胞内区、跨膜区的cDNA片段为294bp,与GeneBank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。结论完成了二硫键稳定的人源化抗肝癌的单链抗体scFv及CD3ζ的胞内区、跨膜区融合基因真核表达载体pcDNA3-scFv-CD3ζ的构建,为制备肝癌靶向性嵌合锚定T细胞奠定了实验基础。
Objective To clone the intracellular and transmembrane genes of CD3ζ in normal human T cells and cloned them into the eukaryotic expression vector with the scFv of disulfide stabilized humanized anti-liver cancer scFv. Chimeric anchored T cells lay the foundation for the study of its killing activity on liver cancer. Methods The scFv gene of disulfide stabilized humanized scFv was cloned into the eukaryotic expression vector pcDNA3 by PCR and introduced into the corresponding restriction sites at the 5 ’ The CD3ζ intracellular domain and transmembrane region genes were amplified from normal peripheral blood human T cells and then cloned downstream of the scFv. The corresponding restriction sites were introduced at the 5 ’end and the 3’ end. Results The disulfide-stabilized humanized anti-HCC scFv cDNA fragment was 735bp, which was consistent with the known sequence. The cDNA fragment of intracellular domain and transmembrane region of CD3ζ was 294bp, which was consistent with the sequence published by GeneBank . Recombinant expression vector was confirmed by restriction enzyme digestion agarose gel electrophoresis and sequencing. Conclusion The construction of the eukaryotic expression vector pcDNA3-scFv-CD3ζ of disulfide-stabilized humanized anti-HCC scFv and CD3ζ intracellular domain and transmembrane fusion gene, Anchor T cells laid the foundation for the experiment.