title: Overexpression and purification of HSV-2 glycoprotein D in suspension CHO cells with serum-free medium and immunogenicity analysis.
abstract: Glycoprotein D (gD2) is the most important candidate antigen for herpes simplex virus type 2 (HSV-2) vaccine development. Establishment of a stable eukaryotic cell line to overexpress gD2 and an efficient purification process to purify is essential for the development of subunit vaccine against HSV-2. The DNA sequence of the extracellular epitope-rich fragment of gD2 was optimized, chemically synthesized, and cloned into plasmid pMD902. The recombinant plasmid pMD902-gD was stably transfected into CHO-DG44 cells, and cell lines with high levels of expression of gD2 were established. The recombinant gD2 was purified efficiently using an anion exchange column and a Sephadex G-25 desalting column. The yield of the purified gD2 was 57 mg/L of serum-free culture medium, and its purity was determined to be about 95% by HPLC analysis. Finally, the immunogenicity of the purified gD2 was measured and it induced strong and specific humoral immunity and higher level of cellular immune response than gD2 expressed in prokaryotic cells. We established a stable, secretory, and high-yield gD2-expression cell line and an easy and efficient gD2-purification process, which lays the foundation for preparation of large amount of gD2 that is essential for HSV-2 subunit vaccine development.
authors.affiliation: Huadong Research Institute for Medicine and Biotechniques, Nanjing, People's Republic of China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Qi
authors.affiliation: Huadong Research Institute for Medicine and Biotechniques, Nanjing, People's Republic of China.|Department of Biochemistry and Molecular Biology, School of Preclinical Medicine, Nanjing Medical University, Nanjing, People's Republic of China.
authors.forename: Yueyue
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Huadong Research Institute for Medicine and Biotechniques, Nanjing, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Pan
authors.affiliation: Huadong Research Institute for Medicine and Biotechniques, Nanjing, People's Republic of China.
authors.forename: Suqin
authors.initials: S
authors.lastname: Li
authors.affiliation: Huadong Research Institute for Medicine and Biotechniques, Nanjing, People's Republic of China.
authors.forename: Bingjun
authors.initials: B
authors.lastname: Li
authors.affiliation: Huadong Research Institute for Medicine and Biotechniques, Nanjing, People's Republic of China.|Department of Biochemistry and Molecular Biology, School of Preclinical Medicine, Nanjing Medical University, Nanjing, People's Republic of China.
authors.forename: Mingjie
authors.initials: M
authors.lastname: Pan
authors.affiliation: National Institutes for Food and Drug Control, Beijing, People's Republic of China.
authors.forename: Shumin
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Huadong Research Institute for Medicine and Biotechniques, Nanjing, People's Republic of China.
authors.forename: Yuexi
authors.initials: Y
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D007111:Immunity, Cellular / Q000276:immunology; D056724:Immunity, Humoral / Q000276:immunology; D013535:Suspensions; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D014765:Viral Vaccines / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D016895:Culture Media, Serum-Free; D013535:Suspensions; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C040570:glycoprotein D-herpes simplex virus type 2
keywords: eukaryotic expression; glycoprotein D; herpes simplex virus type 2; purification; subunit vaccine