Preparation Of Fusion Protein Of PD-L1 Single-chain Antibody And Verification Of Its Antitumor Activity

Objective:The nucleotide sequence of the single chain antibody recognizing the human PD-L1 protein was obtained by screening the human single chain antibody library by human PD-L1 protein using yeast surface presentation technique?We express this scFv-mFc protein by using pFuse eukaryotic expression vector to study its affinity and in vitro and in vivo inhibition of liver cancer cells(HepG2)?Methods: Recombinant plasmid pFuse-scFv was constructed by gene engineering?The recombinant plasmid pFuse-scFv was transfected into 293F(human embryonic kidney cells)and cultured in serum-free Pro 293a-CDM for 72 hours,then the fusion protein was collected,and use the Rapid Protein Liquid Phase Separation and Purification System to purify the scFv-mFc fusion protein?Then the fusion protein and the liver cancer cells were detected by immunohistochemistry;The affinity of fusion protein and liver cancer cells was analyzed by flow cytometry;ADCC was used to determine the proliferation of liver cancer cells;The nude mice inoculated with liver cancer cells,and use the fusion protein to verify its anti-tumor effect in the mice experiment ? Results:scFv-mFc fusion protein was secreted into serum-free culture medium by recombinant plasmid transfection into the 293 F cells;Immunohistochemistry and flow cytometry showed that the fusion protein and PD-L1 protein which expressed highly in the surface of human liver cancer cells have a strong binding capacity;ADCC showed that the fusion protein inhibited the proliferation of tumor cells in vitro;The results of tumor-bearing mice showed that the fusion protein inhibited the growth of the tumor?At the dose of 5mg / Kg,The tumor volume growth rate decreased from 14.90% to 3.72%,the two independent samples t test P <0.05,the difference was statistically significant? Conclusions:The fusion protein containing single chain antibody was successfully prepared,which had good binding ability to HepG2 cells and inhibited the proliferation of tumor cells in vitro and in vivo,and provided the laboratory basis for the development of targeted anti-tumor drugs?...