Construction of recombinant retroviruses expressing anti-CD20 scFv/CD80 /CD28/zeta gene and expression in Jurkat cell line / 中国应用生理学杂志

ABSTRACT

<p><b>AIM</b>To construct recombinant retroviruses expressing anti-CD20 scFv/CD80/CD28/zeta gene and detect its expression in Jurkat cells.</p><p><b>METHOD</b>CD28-zetacDNA were amplified from plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/CD80 gene. The recombinant plasmids were transfected into PA 317 cells. Retroviruses were harvested from culture medium of PA 317 cells. Then NIH 3T3 were transfected with retroviruses. Objective gene expression was determined by PCR and FACS. Jurkat cells were transfected with high titer of retroviruses and resistant clones were obtained by G418 selection. Objective mRNA was determined by RT- PCR.</p><p><b>RESULTS</b>The recombinant eukaryotic vector was constructed successfully by PCR and enzyme digestion analysis and objective gene was amplified from NIH 3T3 cells transfected with retroviruses by PCR; FACS showed that objective protein could be expressed in NIH 3T3 cells. Objective gene was amplified from Jurkat cells transfected with retroviruses by RT-PCR.</p><p><b>CONCLUSION</b>Recombinant retrovirus expressing anti-CD20 scFv/CD80/CD28/zeta gene was successfully constructed and objective protein could be expressed in Jurkat cells.</p>