Preparation Of Anti-SraP_L-Lectin Antibody And Its Function In Anti-Infection Of Staphylococcus Aureus

Staphylococcus aureus serine-rich repeat protein（SRRP）also known as Serine rich adhesin for binding to platelets（SraP）,is a kind of cell wall anchor protein.The ligand-binding region L-Lectin of SraP can recognize and bind to Neu5Ac of the receptors on the cell surface.This kind of receptors are expressed in platelets,lungs,gastrointestinal tract and other cells of mammalian and studies have shown that SraP can mediate the binding between S.aureus and platelets and then promote the development of infective endocarditis.In this study,rSraP_L-Lectin was expressed by genetic engineering technology,followed by immunization of mice,anti-SraP_L-Lectin-Lectin antibody was prepared by cell fusion technology.The specificity of anti-SraP_L-Lectin-Lectin antibody was detected by Western blot and ELISA,in vitro and in vivo assays were carried out to explore the function of this antibody in anti-infection of S.aureus.Objective:Recombinant protein antigen SraP_L-Lectin-Lectin was expressed by genetic engineering technology and anti-SraP_L-Lectin antibody was prepared by cell fusion technology.In vivo and in vitro assays were carried out to determine the function of anti-SraP_L-Lectin-Lectin antibody in anti-infection of S.aureus for laying a foundation for the development of effective targeting for the treatment of S.aureus infection.Methods:1.Expression of rSraP_L-Lectin:S.aureus srap_L-Lectin-Lectin was amplified by PCR and specific amplification products were inserted into pET28a vector and then transferred into DH5αcompetent cells.The rpET28a-srap _L-Lectin plasmid was extracted and the sequence of srap_L-Lectin-Lectin geen was compared with database,the recombinant plasmid was transferred into BL21 competent cells.After being induced by IPTG,SDS-PAGE and Western blot were carried out to detect the expression of rSraP_L-Lectin.2.Preparation of anti-SraP_L-Lectin antibody:Mice were immunized with rSraP_L-Lectin-Lectin antigen,and the mouse spleen-SP2/0 hybridoma cells were prepared by cell fusion technique.Anti-SraP_L-Lectitn positive monoclonal cells were screened and then were injected into the abdominal cavity of mice for collecting ascites.The antibody was purified by ATKA purification system.SDS-PAGE and Western blot were carried out to detect the expression of rSraP_L-Lectin.3.Function detection of anti-SraP_L-Lectin antibody:The biological specificity of anti-SraP_L-Lectin was detected by Western blot and ELISA.The function of anti-SraP_L-Lectin in anti-infection of S.aureus was detected by adhesion and invasion of A549 cells,inflammatory cytokine induction assay,CCK8 assay,phagocytosis assay and antibody passive immunity experiment.Results:1.The recombinant plasmid pET28a-srap_L-Lectin was constructed without any mutations and the rSraP_L-Lectin was successfully expressed.SDS-PAGE and Western blot showed that the molecular weight of the rSraP_L-Lectin was about 30KDa.2.Anti-SraP_L-Lectin-Lectin antibody was successfully prepared and purified after immunizing mice.SDS-PAGE and Western blot showed that the heavy chain and light chain of anti-SraP_L-Lectin monoclonal antibody were bands of 55KDa and 25KDa respectively.3.Western blot and ELISA indicated that the anti-SraP_L-Lectin could specifically recognize the rSraP_L-Lectin-Lectin and in concentration-dependent.Anti-SraP_L-Lectin can effectively reduce the amounts of S.aureus in adhesion and invasion to A549 cells,down-regulate the expression of inflammatory cytokines of mouse macrophages,and reduce the amount of S.aureus in supernatant of macrophages and mouse blood.Conclusion:The study successfully prepared anti-SraP_L-Lectin antibody,and the antibody can specifically bind to rSraP_L-Lectin antigen in a concentration-dependent manner.In vitro and vivo experiments showed that treated with antibody can obviously attenuate the effect of S.aureus invasion and infection on the host,and effectively reduce the amounts of bacterias.In this study,the anti-SraP_L-Lectin monoclonal antibody obtained by genetic engineering technology followed by mice immune and cell fusion technology possesses potential value in the treatment of S.aureus infection,making a foundation for the development of a new effective strategy on anti-infection of S.aureus.