Eukaryotic Expression And Identification Of Mouse/Human Chimeric IgG Against Shiga Toxin2

Since1982, Enterohemorrhagic Escherichia coli (EHEC) O157:H7has been recognized as pathogenic bacteria for more than20years, which caused food poisoning outbreak spreading through all over the world on a different scale, including China. EHEC O157:H7has been increasingly taken into account on national health prevention organizations’agenda because it can lead to serious human gastrointestinal disease and serious complications such as hemolytic urinary tract syndrome (HUS). Shiga toxin type II(Stx2) which is produced by EHEC O157:H7is now considered as the most important virulence factor. Currently, there is no effective therapy or prophylaxis for the infection of EHEC O157:H7. The use of certain antibiotics is controversial, which appears to increase the risk of HUS development.We have previously developed a mouse monoclonal antibody called S2C4which belongs to the immunoglobulin G1subclass and has a K light chain, and reacting with the A subunit of Stx2and without binding to Stx2B subunit or to Stxl. Its neutralizing activity against Stx2and its variants including Stx2c and Stx2vha has been demonstrated by a series of experiments. Nonetheless, S2C4derives from mouse may cause clinical problems as a result of formation of human antimurine antibodies and can not effectively activating the body’s biological effects. Besides, other pharmacodynamic effects which probably in the lignt of its short half-life have hampered its efficacy in human body. Human-mouse chimeric antibody, although there is still about30%of mouse-derived ingredients, but90%of the HAM A response against the antibody constant region, chimeric antibodies can effectively reduce the HAMA response to the human body. Therefore, in this study, we raise the human mouse chimeric antibody cS2C4-IgG, whose identification of immunological activity and neutralization activity against Stx2in vitro and in vivo experiments need to detect. That will provide the basis for a range of diseases due to EHEC infection for further effective treatment.Aims1. To construct eukaryotic expression vector pAc-k-CH3-Vκ-VH, and express human mouse chimeric antibody cS2C4-IgG in Bac-to-Bac baculovirus expression system.2. To analysis the characterization of cS2C4-IgG and observe the neutralization activity against Stx2in vitro and in vivo experiments.Methods1. Eukaryotic expression vector pAc-k-CH3-Vκ-VH was constructed by cloning the variable region genes from the neutralizing antibody-secreting hybridoma S2C4against EHEC Shiga toxin Ⅱ and was transfected into Sf9cells. Human mouse chimeric antibody cS2C4-IgG was obtained from infected Sf9cell culture supernatant samples which was purified by an affinity chromatography column Protein A.2. The characterization of cS2C4-IgG was identified by SDS-PAGE. Western Blot、immunofluorescence assays and ELISA.3. Compared to S2C4, the neutralization activity against Stx2of cS2C4-IgG was detected in the vitro vero cell experiments. The survival rates of vero cell were recorded after post-inoculation24h and calculated the cell viability until post-inoculation96h.4. Compared to S2C4, the neutralization activity against Stx2of cS2C4-IgG was detected in the vivo mouse experiments. The survival rates of the mice were recorded every day and calculated the cell viability until the post- inoculation day12.Results1. The mature VH and Vκ of mAb S2C4encoded sequences contained were354bp and330bp, respectively. Eukaryotic expression vector pAc-k-CH3-Vκ-VH was constructed and transfected. High purity cS2C4-IgG was obtained.2. It was demonstrated that cS2C4-IgG could effectively bind to Stx2by Western Blot、immunofluorescence assays. In addition, cS2C4-IgG could bind to the A subunit of Stx2and its cleavage fragments A1through Western Blot. The purified cS2C4-IgG remained specific antigen binding activity which was detected by ELISA.3. Both cS2C4-IgG and S2C4could neutralize the5TCID50Stx2and protect the vero cells. The higher doses of antibodies used, the higher neutralization rate on vero cells was. The cS2C4-IgG and S2C4concentration required to obtain100%neutralization were1250μg/L (62.5ng/50μL). The IC50of cS2C4-IgG was at a dose of39μg/L (1.9ng/50μL), The IC50of S2C4was at a dose of58.5μg/L (2.925ng/50μL)4. In the mouse toxin neutralization assays, cS2C4-IgG protected the mice from83%to100%at each dose ranging from3.8～30μg/mouse while without protection at a dose of1.9μg/mouse. S2C4protected the mice from91%to100%at each dose ranging from3.8～30μg/mouse while without protection at a dose of1.0μg/mouse.Conclusions1. Human mouse chimeric antibody cS2C4-IgG was successfully obtained in Bac-to-Bac baculovirus expression system. CS2C4-IgG could bind to the A subunit of Stx2, which remained its mAb S2C4’s binding activity with Stx2.2. Compared to S2C4, cS2C4-IgG had similar neutralization against Stx2on vero cell experiments, while cS2C4-IgG had similar protection against Stx2in the mouse toxin neutralization assays. It might point that cS2C4-IgG could be used for treatment of EHEC O157:H7infection.