[Expression of Epstein-Barr virus membrane antigen in CHO cell]

A recombinant plasmid, pCMV/MA, was generated by isolating the Epstein-Barr Virus(EBV) membrane antigen(MA) gene with anchor sequence removed from pSV40/MA and cloning it into pcDNA3, with MA gene and Neomycin gene under the control of CMV promoter and SV40 promoter respectively. CHO cells were transfected with pCMV/MA using liposome and then grown in DMEM medium in the presence of G418. Two clones highly expressing MA were obtained. EBV-MA was purified from the medium of CHO cells by ammonium sulfate precipitation. Western-blot showed that the molecular weight of proteins expressed were about 340 kD and 220 kD. It could specifically react with anti-MA monoclonal antibody in indirect immunofluorescent and immunodot assay. The yield of MA was estimated to be 1.9 micrograms/ml per day by SDS-PAGE scanning and Lowry methods. The optimal culture condition of the CHO cells was to use medium supplemented with 10% serum until 80% cell confluence, then with 2%-5% serum and collected medium continually. The genetic stability of the cells was confirmed by freeze--revival and culture without G418. The purified MA was used to immunize mice and the geometric mean titer of the specific antibody was 1:180, while the positive rate of antibody was 100%.