The Expression And Purification Of Human S100A4and The Preparation Of Human S100A6Monoclonal Antibody

The liver is the largest human digestive organ, which is mainly responsible formaintaining normal metabolism. Liver is easily injured for the reason of participating infood digestion, resulting in acute liver failure, hepatic fibrosis and so on. The highincidence and mortality of these liver diseases draw the attention of the public.In order to find novel strategies and new drugs for the treatment of liver injury, weused gene chips to detect the gene expression profiles in the murine models ofCCL4-induced liver damage and follow-on self-regeneration. The expression of S100family members was altered with characteristics suggesting their involvement in the liverresponse to CCL4.In the present study, we focus on the S100family, and primarily study the prokaryoticexpression, purification and activity assay of recombinant human S100A4, and thepreparation of S100A6monoclonal antibody.S100A4is a member of S100protein family, which exerts functions in cellproliferation, differention and cell damage repair. It is also reported that S100A4isassoicated with cancer metastasis. To facilitate further studies of S100A4, we developed astrategy for expressing and purifying recombinant human S100A4protein using E.coliexpression system and affinity chromotography methods. This strategy is simple andefficient for purifying recombinant S100A4with high purity and low endotoxincontamination. Furthermore, in vitro studies validated the biological activity of the purified S100A4since it could enhance the viability of A375cells. We hope that thisstrategy could facilitate the physiological and pathological research of S100A4and havereference values for the expression and purification of other S100family members.In order to analyze hS100A6’s role in liver fibrosis, we aimed to develop aneutralizing antibody against human S100A6. Therefore, we generated mouse monoclonalanti-human S100A6antibodies by immunization Balb/c with recombinant full lengthhuman S100A6protein which purity>97%. Lymphocytes isolated from the spleen ofimmunized mice were fused with the mouse myeloma SP2/0, and hybridoma supernatantswere tested in enzyme-linked immunosorbent assays (Elisas) for binding to recombinanthuman S100A6. The limited dilution method is screened for the secretion of specificanti-human S100A6antibody hybridoma strain and obtained two stable monoclonalantibody secretion of mouse anti-human S100A6, named as5-1G-6G. For purification ofmAbs, hybridomas were intraperitoneal injected into Balb/c mice inducing ascites andantibodies were purified with protein G-Sepharose. We designed a set of primers for thePCR amplification for mouse VL and VH coding DNA sequence and demonstrated thatthe antibodies can be used effectively to neutralize human S100A6. Ultimately weprepared two mouse anti-hS100A6monoclonal antibody successfully and established thefoundation of detecting hS100A6protein. We conclude that this antibody can be useful toboth target and analyze murine liver fibrosis in which S100A6may be involved.