Preparation For Antibody Against MxA Protein And Its Specificity

MxA is an exclusively interferon-I-inducible protein and is involved in the inhibition to multiplication of various viruses (mainly RNA viruses). Its expression level is positively correlated with endogenetic expression level of interferon-I. It was well known that challenged with viral pathogens, the IFN-defense system was activated and up regulated in the expression level of IFN-I in vivo. The MxA level indirectly reflects whether the organism is infected with viral pathogen or not. For identification and determination of MxA protein in vitro and in vivo by a standard immunological protocol, it is indispensable to prepare the monoclonal antibody against MxA protein. Although there were reports about preparation of monoclonal antibody to MxA oversea, it has not been commercially available and furthermore not been reported on its preparation in our country. It is important to prepare polyclonal and monoclonal antibodies highly specificity to MxA and to develop rapid test kit to MxA for the application to researchers, physicians and patients.We constructed the recombinant vector pET-32a (+)-MxA and highly expressed the fusion protein of MxA in E.coli BL-21(DE3) which accounted for 25% of total bacterial protein. Laboratory mice were immunized by peritoneal injection with MxA protein of 90% purification, isolated form SDS-PAGE gel and antiserum were gathered. The specificity of antiserum was detected with diversified MxAs used as antigen. The antiserum was validated to have a high specificity. We conjugated the splenocyte of the purified MxA immunized Balb/c mice with myeloma cell Sp2/0 with PEG4000, and successfully gained a clone of hybridoma, 3C2, which secreted monoclonal antibody against MxA. The monoclonal antibody was concentrated and purified from seroperitoneum by saturated ammonium sulfate precipitation method, desalination with Sephadex-G25 and affinity chromatography with protein G resin, and tested to evaluate its specificity and affinity by Western-blot, immuno -fluorescence, immunohistochemistry and immunoprecipitation. ELISA was carried out to estimate the titer of the purified monoclonal antibody. All the results showed that the monoclonal antibody had high specificity and titer. The antibodies, PcAb and McAb, are not only the basic material for MxA research, but also laid a foundation for the development of rapid test kit of MxA.