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The surface of the herpes simplex virus type 2 (HSV-2) has twelve kinds of membrane glycoproteins.Of those,glycoprotein D (gD) is the most important candidate antigen for vaccine development.So far,stable overexpression of gD in eukaryotic cells has not been reported.The DNA sequence of the extracellular fragment (1-286 aa,epitope-rich region) ofgD was optimized,synthesized chemically,and cloned into the plasmid vector pMD902.The recombinant plasmid pMD902-gD was stably transfected into CHO-DG44 cells,and cell lines with high levels of expression of the recombinant protein were established.The gD protein was purified efficiently using an anion exchange column and a Sephadex G-25 desalting column.The yield of the purified gD protein was 57 mg/L of serum-free culture medium,and its purity was determined to be about 99% by HPLC analysis.The antigenicity and immunogenicity of the purified gD protein were measured by ELISA and by immunizing BALB/C mice with it.The purified gD protein not only reacted with the anti-HSV-2 positive sera but also induced strong,specific humorai and cellular immune responses in mice.Our results lay the foundation for preparation of large amounts of the gD protein that are necessary for HSV-2 vaccine development.