Construction Of A Human Phage Display Library And Screening Anti-LOX-1 ScFv

Lectin-like oxidized low-density lipoprotein receptor-1(LOX-1),a 50 kDa type ? membrane protein,is the major receptor for oxidized low-density lipoprotein(ox-LDL)that belongs to C-type lectin family.LOX-1 is a novel therapeutic target in atherosclerosis and atherosclerosis-related disease.LOX-1 expression on the cell surface can undergo proteolysis at its membrane-proximal extracellular domain and be released as sLOX-1(soluble LOX-1,sLOX-1)into blood.Hence,the anti-LOX-1 antibody has widespread research prospect in the diagnosis and therapy of relative disease.Especially,the human antibodies were more effective and safer in clinical diagnosis and therapy,which is the popular issure.However,the LOX-1 antibodies from reports were of general animal source,and the research on human source antibody was rare.The phage diplay technology was the main method to obtain human antibody avoiding the animal immunization.The crucial advantage of phage display was the physical link between the phenotypes of antibody fragments displayed on the coat of the phage particle and the genetype inside the phage.Compared to the intact immunoglobulin molecule,scFv(single chain fragment variable,scFv)is composed of VH and VL joined together with a short flexible peptide linker.scFv is characterized by small molecule weight,low immunogenicity and strong penetration,which has advantages in the imaging and treatment of atherosclerotic plaque.Phage display was one of the most important methods to prepare human antibody.In this study,a human scFv phage display library composed of 6.2×108 individual clones was constructed.The KD of scFv-39 binding LOX-1 ECD screened from the library was 5.8×10-6M.The research supplied the basis of the development of antibody medicines targeting LOX-1.Firstly,the LOX-1 ECD was solubly expressed in E.coli and purified.The host cells,soluble protein tags and induction conditions were systematically optimized.The largest amount of soluble expression of LOX-1 ECD which was fused with MBP was observed in Rosetta gami2(DE3).The optimized induction conditions were at 30? with the IPTG concentration of 200?M.The fusion protein MBP-LOX-1 ECD was purified with MBP Trap and size exclusion chromatography.The MBP tag was removed using 2.5 U/mg of thrombin.The purified LOX-1 ECD with high binding activity with oxLDL was obtained with MBP Trap and size exclusion chromatography,and the purity was 95%.Secondly,a human scFv phage display library of 6.2 ×108 individual clones was constructed by molecule biology methods.The total RNA of PMBC of 20 patients with cardiovascular disease was extraccted and transcripted into cDNA.The heavy chain variable regions and the light chain variable regions were amplified through semi-nested PCR.The scFv gene was assembled through overlapping PCR.The scFv was liagased to the phagemid vector,and the recombinat vector was transformed into TG1.The library colnes were identified with PCR and the diversity was also evaluated.The capacity of the library was 6.2×108.The anti-LOX-1 ECD positive recombinant phages were screened from the human phage display.Three rounds biopanning was applied to the recombinant phages with immunotubes.The input and output phages were counted,meanwhile,the phages from each round were applied to identify the binding activity to LOX-1 ECD with polyclonal phage ELISA.The results showed that the recombinant clones were effectively enriched.40 individual clones from the enriched recombinant phages were randomly picked for monoclonal ELISA.10 of 40 clones were approved to be positive.The sequence of scFv-39 was analyzed by information methods.Meanwhile,the scFv-39 was cloned,solubly expressed and determined the activity of binding LOX-1 ECD.The CDRs regions of VH and VL genes were identified through IMGT tool.The structure characterization,physical and chemical properties were predicted using SOPMA tool and Genetyx soft.ScFv-39 was composed of 735 nucleic acids which translated into 245 amino acids.The average molecue weight was 26209.7 Da,and the isoelectric point was 8.98.ScFv-39 was solubly expressed using Brevibacillus choshinensis secretory expression system.ScFv-39 protein with 6×His tag at C-terminal was purified through His trap and size exclusion chromatography.The ELISA results showed that the KD of scFv-39 was 5.8×10-6M.