Cloning And Expression Of SARS-CoV M Protein Gene Fragment And Characterization Of Monoclonal Antibody Against The Expressed Protein

SARS emerged as an acute infectious disease characterized by fever and severe pneumonia infection. On April 16, 2003, WHO confirmed that a new coronavirus named SARS-CoV was the cause agent of SARS. It was demonstrated that SARS-CoV was a new coronavirus which had not been found in human and animal and no cross-reaction with human susceptible strains HCoV-OC43 and HCoV-229E. The objective of this paper was to express S protein fragment, M protein fragment in E.coli, analyze the antigenicity and immunogenicity of the expressed proteins, and to characterize the specific monoclonal antibody (mAb) against M protein fragment, which laid a foundation for constructing an ELISA diagnostic assay for SARS-CoV. 1. The cloning and expression of S protein, M protein coding gene fragments of SARS-CoV and analysis of their antigenicityN-terminal 501bp of S coding gene fragment and N-terminal 129bp of M coding gene fragment of SARS-CoV were synthesized chemically to construct recombinant prokaryotic expression plasmids pGEX-6p-l+SARS-S and pGEX-6p-l+SARS-M. Recombinant plasmids were transformed to E.coli BL21 competent cell followed by restriction enzyme digestion and nucleotidesequence analysis. E.coli BL21 containing recombinant plasmids were induced with isopropyl- 3 -D-thiogalactoside (IPTG), and then SDS-PAGE revealed that both S and M fusion proteins were expressed correctly. Western blot analysis demonstrated that the expressed M fusion protein fragment was highly antigenic by using convalescent serum from SARS patient, but not GST alone and S fusion protein. By means of Glutathione Sepharose 4B affinity chromatography, M fusion proteins were purified effectively, and then the purified M fusion proteins were used as antigen to coat 96-well plates to construct ELISA diagnostic assay for SARS. And it was confirmed that the purified M fusion protein could serve as an ELISA antigen.2. Preparation and characterization of mAb against SARS-CoV M protein fragmentThe purified M fusion protein was further employed to immunize BALB/c mice and one hybridoma cell line (3H9) stably secreting mAb against M protein was subsequently obtained. The mAb was shown to be IgG2a with the Kappa type of light chain and could react specially with whole proteins of SARS-CoV coated on the 96-well plates by using Indirect ELISA assays.3. The localization of B-cell antigen epitope of M protein fragment and analysis of its mAb recognizing regionBased on PCR techniques, two recombinants, which expressed part-overlapping truncated protein (Ml, M2) of M protein fragment in E.coli...