Screening And Expression Of Human-derived Oxidized Low-density Lipoprotein Antibodies

Objective:Atherosclerotic heart disease is one of the cardiovascular diseases that lead to increased morbidity and mortality in humans year by year.Oxidized low-density lipoproteins play a significant role in the formation of atherosclerotic plaques.With the breakthrough of immunotherapy in the field of oncology,more and more researchers are devoting their research to immunological approaches to explore an antibody that can specifically bind to atherosclerotic plaque components for the diagnosis and treatment of atherosclerotic diseases.The aim of this study is to screen for an antibody with high specificity for oxidized LDL and to initially explore the mechanism of action of the antibody,which is expected to play a role in the diagnosis and treatment of atherosclerotic diseases in the future.Methods:The synthesis of single-chain antibody fragments was performed using the pre-existing gene library of the group.The target protein was expressed on the phage shell by phage display technology,and the target gene with highly specific binding ability to oxidized low-density lipoprotein was screened out through layers of sieving and elution.Subsequently,the phage containing the target gene was introduced into the E.coli system for expression and purification of the target protein.The purified antibodies were tested by Western-blot for the binding ability of the target protein to oxidized LDL and plaque tissue.In addition,the effect of the antibody on the ability of macrophages to phagocytose oxidized LDL and the level of macrophage apoptosis was verified by further applying the antibody to mouse macrophages.Results:By using phage display technique,we screened the antibody with high specificity for oxidized LDL and named it ASH8.The immunoblotting results showed that the antibody could bind specifically to oxidized LDL.In cytological experiments,the level of oxidized LDL phagocytosis by RAW macrophages was significantly reduced in the ASH8 antibody-added group when compared with the control group;in addition,TUNEL results also showed that the level of apoptosis was significantly alleviated in the ASH8 antibody added group compared with the oxidized LDL group,and the ASH8 antibody had no significant toxicity on RAW macrophages.Conclusion:(1)ASH8 Antibody can specifically bind to and act on oxidized LDL;(2)ASH8 Antibody can inhibit the phagocytosis of ox-LDL by macrophages and reduce the occurrence of apoptosis.