Recombinant anti-CD20 scFv-CD80-CD28-ζ non-replicating retroviral Construction.doc

 PAGE \* MERGEFORMAT 13 Recombinant anti-CD20 scFv/CD80/CD28/ζ non-replicating retroviral Construction [Abstract] Objective: To construct pLNCX/anti-CD20 scFv/CD80/CD28/ζ recombinant eukaryotic expression vector transfected cell line PA 317, packaging and preparation of recombinant non-replicating retrovirus. Methods: DNA recombinant technology to pBULLET on the CD28-ζ cDNA inserted into the anti-CD20 scFv/CD80 already included in the eukaryotic retroviral vector pLNCX plasmid transfected PA 317 cell lines by G418 selection, the supernatant was harvested Non-replicating retrovirus, the virus infected NIH 3T3 cell line, using PCR, flow cytometry of target genes in NIH 3T3 cells, determine the virus titer. Results: After digestion and sequencing confirmed the successful construction of pLNCX/anti-CD20scFv/CD80/CD28/ζ ; using RT-PCR from virus-infected NIH 3T3 cells was amplified by the size of a target gene with the same DNA fragment; flow cytometry showed that the gene can be expressed in NIH 3T3 cells, the target protein. Conclusion: The successfully constructed and expressed a high titer of anti-CD20 scFv/CD80/CD28/ζ non-replicating retrovirus, and NIH 3T3 cells in expression of the target protein. [Keywords:] Single-chain antibody PA 317 retrovirus gene expression in NIH 3T3 cells, cells Abstract: Objective: To construct recombinant eukaryotic expression vectors of pLNCX/anti- CD20scFv/CD80/CD28/ζ , transfect into PA 317 cells and package recombinant retroviruses. Methods: CD28-ζ cDNA were amplified from plasmids pBULLET and inserted into pLNCX vectors that contained anti-CD20 scFv/CD80 gene. The recombinant plasmids were transfected into PA 317 cells and resistant clones were obtained by G418 selection. Retroviruses were harvested from culture medium of PA 317 cells. Then NIH 3T3 were transfected with retroviruses. After G418 selection, objective gene expression was determined with PCR and FACS. Results: The recombinant eukaryotic vector was constructed