| Hantavirus belongs to Hantavirus genus which is a member of Bunyaviridaefamily. Based on the differences of antigenicity and genetic structure, Hantaviruscan be categorized to more than10types. Hantavirus could cause two types ofacute infectious diseases, one is known as hemorrhagic fever with renalsyndrome （HFRS）, which presents hyperpyrexia, hemorrhage and renaldysfunction as its main clinical features; the other one, which is mainlycharacterized by hyperpyrexia, diffuse lung inflammation and acute respiratoryfailure, is called hantavirus pulmonary syndrom（eHPS）. So far, no report on HPScases has found in China. However，with the wide epidemic range, high incidenceand high mortality, the epidemic situation of HFRS in China is most severe inworldwide. In addition, Hantavirus is also a potential biological warfare agents,so it may be used by terrorists. Currently, as no specific drug targeted HFRS iseffective for the clinical treatment, then it will be a long-term and arduous task inthe prevention and treatment of HFRS in the future. Active immunization using vaccines and antibody therapy are the mostimportant protective and therapeutic measures to viral infectious diseases. Inrecent years, inactivated vaccines against Hantavirus have been developed.However, the immune response induced by the currently applied vaccines is notgood enough, based on the results of the trials. The monoclonal antibodies（mAb）with neutralizing activity could effectively inhibit or block viral infection in vivoand in vitro. As early as the mid-1980s in the last century, an anti-Hantavirusmurine monoclonal antibody with higher neutralizing and protective activity, hadbeen screened in our laboratory. Under the support of a grant from the NationalHigh Technology Research and Development Program of China, we successfullydeveloped an anti-Hantavirus monoclonal antibody for HFRS treatment, whichhas now completed its clinical trials. As the potential immunogenicity to human,the murine mAb could cause some side effects such as hypersensitivity. Therefore,in this study, we intends to construct a humanized antibody on the basis ofanti-Hantavirus murine monoclonal antibody3G1. We hope this chimericantibody could be a new therapeutics for HFRS treatment.1. The eukaryotic expression vectors encoding anti-HTNV mouse/humanchimeric antibody were successfully constructed.We rebuild the eukaryotic expression vectors pCI-neo-M and pCI-M byreplacing their promotors MARs-mCMV with ease2.1-cmv5respectively, therebuilt vectors named as pCI-neo-ease2.1-cmv5-M and pCI-ease2.1-cmv5-M.The genes of chimeric antibody consisting of the genes of light chain and heavychain variable region from anti-Hantavirus monoclonal antibody3G1and thegenes of constant region from human IgG were successfully cloned into theabove vectors respectively.2. The CHO-K1cell line stably expressing anti-Hantavirus mouse/human chimeric antibody was established.The two vectors expressing light chain and heavy chain of chimericantibody were cotransfected into wildtype CHO-K1cells. After selection byG418and MTX, the cell line stably and high effectively expressing chimericantibody was established. The expression of chimeric antibody was determinedby ELISA.3. The biological activity of anti-Hantavirus mouse/human chimericantibody was identified.Collecting the supernatant of the cell culture and purifying by protein A,we got the chimeric antibody, which was characterized by SDS-PAGE andwestern-blot. The chimeric antibody had a good neutralizing activity similar tothe parent monoclonal antibody by in vitro neutralization tests. Our study laidthe foundation for the further research and clinical application of theanti-Hantavirus mouse/human chimeric antibody. |
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