融合基因anti-erbB2 scFv-Fc-CD28-CD3(ζ)的构建及真核表达

Construction of Fusion Gene anti-erbB2-scFv-Fc-CD28-CD3(ζ) and Expression in Eukaryotic Cell

本研究利用SWISS-MODEL预测该融合蛋白的三级结构。利用PCR的方法分别从重组pPIC9k、重组pBullet和pSecTag2B上扩增出3段基因片段,即片段anti-erbB2 scFv(简称A)、片段Fc-CD28-CD3(ζ)(简称B)和信号肽序列(简称S)。利用SOE-PCR将3段序列连接形成融合基因片段S-A-B。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,电转染人淋巴瘤T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。经预测在anti-erbB2 scFv与Fc基因片段之间不加连接肽的融合蛋白,在三级结构上可形成更佳的功能构象。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/S-A-B(在A与B基因片段之间不加linker)。经流式细胞术检测,在转染的Jurkat细胞中融合蛋白表达率约为56.17%。本研究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),融合基因能够在淋巴瘤T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过表达肿瘤的靶向基因治疗研究奠定了实验基础。

SWISS-MODEL was used to predict the tertiary structure of the fusion protein.PCR was used to amplify DNA fragment anti-erbB2-scFv（abbreviated to A） from recombinant plasmid pPIC9K,signal（abbreviated to S） from plasmid pSecTag2B and Fc-CD28-CD3（ζ）（abbreviated to B） from recombinant plasmid pBullet respectively.SOE-PCR was used to construct fusion gene fragment S-A-B.After amplifying and confirming the recombinant gene fragment through TA cloning,a recombinant eukaryotic expression vector pLNCX/S-A-B was constructed and transfected to Jurkat cell line by electroporation,with stable cells selected by G418 and validated for fusion gene expression by FAM.The structure prediction showed that the fusion protein with no linker between gene fragments anti-erbB2-scFv and Fc could form better functional tertiary structure.Using the methods of PCR, restriction digest and sequencing,the recombinant eukaryotic expression vector pLNCX/S-A-B（without linker between gene fragments A and B） 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 56.17%.By using molecular clone method,the recombinant eukaryotic expression vector pLNCX/ anti-erbB2 scFv-Fc-CD28-CD3（ζ） was 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 of erbB2 over-expressing tumors.