Application of 2A peptide technology on the forth generation CAR cell

• 1、College of Biotechnology, Tianjin University of Science & Technology, Tianjin, 300457
• 2、 Tianjin Key Laboratory of Marine Resources and Chemistry, (Tianjin University of Science & 25 Technology), Tianjin, 300457

Abstract：2A peptide technology is a polygene expression vector technology developed rapidly in recent years, it has the advantages of balanced upstream and downstream gene expression and no effect on the activity of the expressed protein. In this paper, a novel Forth CAR was designed. ZEGFR:1907 served as the extracellular recognition region of chimeric antigen receptor as the affinity of EGFR, and CD28 andCD3ζ were intracellular signal regions of chimeric antigen receptor of EGFR, this gene sequence was co-expressed with the target gene of PD1 mutant by using 2A peptide technology. Upon completion oApplication of 2A peptide technology on the co-expression of PD-L1 specific binding polypeptide and Chimeric Antigen Receptor cellf the above work, Lentivirus transfection was used to transfer the target gene into the target HeLa cell line, in addition, CAR subcellular localization was identified byfluorescence microscopy, and CAR expression and PD-L1 specific binding peptide secretion were identified by Western-bloting. The results demonstrate the feasibility of the Forth CAR design based on 2A peptide technology.

Keywords： Tumor 2A peptide technology PD-L1 specific binding peptide Chimeric antigen receptor(CAR)