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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
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摘要 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.
出处 《温州医学院学报》 CAS 2010年第5期431-435,共5页 Journal of Wenzhou Medical College
基金 浙江省自然科学基金资助项目(Y205171) 温州市科技局对外合作项目(H20080059)
关键词 ERBB2 基因融合 表达载体 T淋巴细胞 克隆 分子 erbB2 gene fusion expression vector T-lymphocytes cloning molecular
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