Preparation Of PD-L1 ScFv,Screening Of Induction Conditions And Preliminary Identification Of Its Activity In Vitro

Background:With the development of medicine,the current treatment of tumors is not limited to surgery,radiotherapy and chemotherapy or targeted drugs.As a new type of cancer treatment,immunotherapy has ushered in great research progress after several years of development,especially the research results of PD-1(Programmed cell death protein 1)and PDL1(Programmed cell death 1 ligand 1)antibodies are particularly outstanding.Immunotherapeutic antibody drugs generally have high affinity and specificity,and have played outstanding effects in the clinical treatment of cancers such as melanoma,Hodgkin’s lymphoma,mesothelioma,and glioblastoma.However,antibody drugs have shortcomings such as unclear immune-related toxicity,pharmacokinetics,and insufficient penetration of the tumor microenvironment,which hinder the clinical application of antibody drugs.In this context,small-molecule antibody drugs have gradually developed.Because small-molecule antibodies have a small molecular weight and a clear structure,they can effectively solve the deficiencies of antibody drugs.Therefore,the development of more superior small molecule antibodies to make up for the shortcomings of antibody drugs is one of the hot research nowadays.Objective:This subject is to prepare a small molecule PD-L1 single-chain antibody protein so that it can be expressed in a prokaryotic system.At the same time,in order to increase the soluble expression of the target protein,focus on screening suitable protein induction conditions,and determine the biological activity of the extracted soluble protein.This topic provides a reliable basis for the development of small molecule immunotherapy drugs.Methods:In this experiment,the NCBI website analyzed and found the variable region of the light chain(PDB: 5X8L＿K)and the variable region of the heavy chain(PDB: 5X8L＿F)of the human Atezolizumab protein sequence that has achieved great commercial success.After they were translated into nucleotide sequences,the codon preference of E.coli was optimized.At the same time,the prokaryotic Omp A signal peptide sequence(Gen Bank: KF041825.1)was inserted into the sequence,and the PD-L1 scFv gene sequence was re-synthesized and cloned in the p ET-28a+plasmid containing the T7 promoter.We extracted the recombinant plasmid and transformed it into BL21 competent cells to induce expression.And screen the conditions that are conducive to the soluble expression of the target protein,and use the Ni column to purify the protein.The purified PD-L1 scFv protein was subjected to protein gel electrophoresis(SDS-PAGE),immunofluorescence experiment,and cell proliferation experiment for biological identification.Results:The PD-L1 scFv gene sequence was successfully constructed,and the sequence conformed to the codon preference of E.coli.After transforming the recombinant p ET-28a+ plasmid into BL21 competent cells,the optimal induction expression conditions were explored as follows:low temperature 16℃,optimal induction concentration of IPTG 0.2 m M,and optimal induction duration 7 h.Under this condition,PD-L1 scFv protein can be expressed soluble.The yield of PD-L1 scFv protein after purification and extraction by Ni column was about 2.4 mg,and the yield was about 70.1%.After the purified protein was subjected to dialysis and vacuum concentration treatment,the final concentration was 0.01 μM,0.05 μM,0.1 μM,and 0.3 μM to perform immunofluorescence experiments with human breast cancer cells MDA-MB-231.Under a confocal microscope,it can be observed that all concentrations of PD-L1 scFv protein can bind to the surface of human breast cancer cells.In addition,we also used CCK-8 to determine that the PD-L1 scFv protein has no obvious toxicity to normal human liver cells LO2.Conclusions:The nucleotide sequence of PD-L1 scFv synthesized in this experiment has been optimized for E.coli codon preference and can be soluble expression in a prokaryotic expression system.At the same time,it is proved that the purified PD-L1 scFv protein has the biological activity of combining with PD-L1 on the surface of breast cancer cells,and has no obvious toxicity to normal human liver cells LO2.This experiment provides a reliable basis for the development of small molecule immunotherapy drugs.