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

Abstract: 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 clonemethod， 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.

CSTR: 32200.14.cjcb.2010.05.0004