Synthesis of full length recombinant chimeric receptor anti-erbB2 scFv-CD28-ζ and construction of its eukaryotic expression vector

Objective To synthesize the full length gene of recombinants chimeric receptor anti-erbB2 scFv-CD28-ζ, and construct its eukaryotic expression vector in order to further study the anti-tumor immune response of NK cells. Methods Assisted by DNA2.0 and Gene2Oliga software, codon usage and secondary structure of RNA were optimized, and enzyme sites HindⅢ and EcoRI were induced into the gene. Single chain oligo with a length of 50-70bp was synthesized according to its gene sequencing, and ligated into the full length gene. The synthesized gene was cloned into plasmid PMD-18T and transferred into competent DH5a cells. The cloned DNA fragment was sequenced. The recombinant plasmid PMD-18T-anti-erbB2 scFv-CD28-ζ was digested with HindⅢ and EcoRI, the target gene was inserted into the corresponding restriction site on eukaryotic expression vector PCDNA3.1 (+) and transferred into COS-7 cells. Results The 1 803bp DNA fragment synthesized in this study was consistent with the target gene anti-erbB2 scFv-CD28-ζ both in size and in consequence. Conclusion After the recombinant expression plasmid is transfected into COS-7 cells, anti-erbB2 scFv-CD28-ζ is instantaneously expressed and transfected in vitro.