含anti-erbB2的重组表达载体的构建及其在T细胞株中的表达

Construction of a recombinant expression vector harboring anti-erbB-2 and its expression in T lymphoma cell line

目的:构建含融合基因anti-erbB2 scFv-Fc-CD28-CD3(ζ)的T细胞株,为erbB2过度表达肿瘤的靶向基因治疗奠定实验基础。方法:利用SWISS MODEL和PHYRE预测该融合蛋白的三级结构。利用PCR分别从重组质粒pPIC9k、pBullet和pLNCX上扩增出3段基因片段,即片段anti-erbB2 scFv、片段Fc-CD28-CD3(ζ)和信号肽序列signal。利用SOE-PCR将3段序列连接形成融合基因片段signal-anti-erbB2 scFv-Fc-CD28-CD3(ζ)。经TA克隆扩增及鉴定后,将融合基因片段与逆转录病毒表达载体pLNCX相连构建重组真核表达载体,脂质体方法转染人T淋巴细胞株Jurkat,G418筛选后用流式细胞术检测融合蛋白稳定表达情况。结果:经预测该融合蛋白在三级结构上可形成功能构象。经PCR、酶切及测序鉴定均证实成功构建重组真核表达载体pLNCX/signal-anti-erbB2 scFv-Fc-CD28-CD3(ζ)。经流式细胞术检测,在Jurkat细胞中表达量达23.68%。结论:应用分子克隆的方法成功地构建了重组真核表达载体pLNCX/anti-erbB2 scFv-Fc-CD28-CD3(ζ),融合基因能够在T淋巴细胞株中表达,为制备含该融合基因的原代T淋巴细胞,进行erbB2过度表达肿瘤的靶向基因治疗研究奠定了基础。

Objective：To construct fusion gene anti-erbB2-scFv-Fc-CD28-CD3（ζ） and to express it in the human T lymphoma cell line,to build a practical basis of targeted therapy against erbB2 over-expressing tumors.Methods：Using Swiss Model and PHYRE,the tertiary structure of the fusion protein was predicted.DNA fragments anti-erbB2 scFv,Fc-CD28-CD3（ζ） and the signal were amplified from recombinant pPIC9K,pBullet and pLNCX respectively by PCR.The fusion gene fragment signal-anti-erbB2 scFv-Fc-CD28-CD3（ζ） was obtained by SOE-PCR.After amplifying through TA cloning and confirming the recombinant gene fragment,a recombinant eukaryotic expression vector pLNCX/signal-anti-erbB2 scFv-Fc-CD28-CD3（ζ） was constructed and further transfected to Jurkat cell line using Lipofectin2000,with stable cells selected by G418 and validated for fusion gene expression by FAM.Results：The structure prediction showed that the fusion protein could form functional tertiary structure.As verified by the methods of PCR,restriction enzyme digestion and sequencing,the recombinant eukaryotic expression vector pLNCX/signal-anti-erbB2 scFv-Fc-CD28-CD3（ζ） was constructed successfully.As analyzed by FAM,the fusion protein anti-erbB2 scFv-Fc-CD28-CD3（ζ） could be expressed in Jurkat cells at a stable level of 23.68%.Conclusion：By molecular clone method,the recombinant eukaryotic expression vector pLNCX/anti-erbB2 scFv-Fc-CD28-CD3（ζ） is constructed successfully and the fusion gene can be expressed in T lymphoma cell line.These results may provide a way to establish primary T lymphocyte harboring this fusion gene,and in turn build a practical basis of targeted therapy against erbB2 over-expressing tumors.