Construction And Expression Of A Human-mouse Chimeric Antibody Against Candida Albicans

Clinical relevance of Candida albicans diseases has increased enormously in recent years, mainly because of an increasing popμlation of immunocompromised hosts, including individuals infected with HIV, transplant recipients and patients with cancer. More attention has to be paid to fungal infective disease. The incidence of Candidiasis is highest among fungal infective diseases[1] .Some reseach in the world indicate that hospital fungal infection has been increasing in recent decades which mostly caused by Candida albicans with a high mortality [2]. Treatment of disseminated C. albicans infections is still difficμlt . There is still no safe and effective antifungal drμg in the clinic because of toxicity and drμg-resistance[3] .With the developing of engineering antibody technology,mouse-human chimeric antibody technology may set the stage for the new approach to the treatment of fungal infections[4,6]. Chimeric antibody is more mature than other humanized antibody .It has low immunogenicity and high affinity and specificity to revalent antigen.It also has the more powerfμl biological function and longer serum half-life than murine monoclonal antibody. It's constant fragmemt can be shifted to other IgG isotype in order to recruit more human immune effector functions.Since Morrison[4] first successfμlly construct the mouse-human chimeric antibody in 1984,there are lots of chimeric antibodies haven been used in clinical application.A lot of clinical trials have proved the safety of the chimeric antibodies and the better affinity and specificity they can maitain than shaping antibodies.Reseach on treatment of fungal infection is going on. Engineering antibody technololy is a new strategy[7].reseach in other lab show us that momoclonal antibodies against Candida albicans have powerfμl functions agaist Candida albicans,which show us the bright prospect in clinical application[8,9].Our reseach group gained monoclonal antibody 3B4 and found it has definite function against Candida.albicans[10].The study is to humanized the murine monoclonal antibody by engineering technique.Link murine variable region to constant region of human,Construct the eukaryotic expression vector of a human-mouse chimeric antibody against C.albicans and express it in eukaryotic cells.This is a basic work for the further study in the function of the chimeric antibody and clinical application.1.Construction of eukaryotic expression vector of human-mouse chimeric antibody against C.albicans.Variable region genes of a mouse hybridoma cell line 3B4 against C. albicans were cloned from plasmid pMDT-V2H and pUC-VL separately, and were inserted into the chimeric antibody eukaryotic expression vector pMH-CA. The resμlt showed that the coding sequences of VH.and VL were correct .The restriction enzyme digestion analysis confirmed that eukayotic expression vector against C.albicans was successfμlly constructed. 2. Eukaryotic expression of human-mouse chimeric antibody against C.albicans.The constructed vector was transiently transfected into J558L cells by electroporation.The expression of the chimeric antibody was firstly proved by RT-PCR, and the specificity of the chimeric antibody was confirmed by ELISA, which show us that the chimeric antibody has the binding specificity to both self-antigen keratin and C. albicans.In summary, we successfully constructed the human-mouse chimeric antibody against Candida albicans.After being transiently transfected in J558L cells, the activities of the product were confirmed.These results are helpful for the further reseach on its function and clinical application.