title: Optimized Expression, Purification of Herpes B Virus gD Protein in Escherichia coli, and Production of Its Monoclonal Antibodies.
abstract: BACKGROUND
  Herpes B virus (BV) is a zoonotic disease caused by double-stranded enveloped DNA virus with cercopithecidae as its natural host. The mortality rate of infected people could be up to 70% with fatal encephalitis and encephalomyelitis. Up to now, there are no effective treatments for BV infection. Among the various proteins encoded by monkey B virus, gD, a conserved structural protein, harbors important application value for serological diagnosis of frequent variations of the monkey B virus.
  
  
  OBJECTIVE
  This study aimed to expressed the gD protein of BV in Escherichia coli by a recombinant vector, and prepare specific monoclonal antibodies against gD of BV to pave the way for effective and quick diagnosis reagent research.
  
  
  METHODS
  The gD gene of BV was optimized by OptimWiz to improve codon usage bias and synthesis, and the recombinant plasmid, pET32a/gD, was constructed and expressed in E. coli Rosetta (DE3). The expressed fusion protein, His-gD, was purified and the BALB/c mice were immunized by this protein. Spleen cells from the immunized mice and SP2/0 myeloma cells were fused together, and the monoclonal cell strains were obtained by indirect enzyme-linked immunosorbent assay (ELISA) screening, followed by preparation of monoclonal antibody ascetic fluid.
  
  
  RESULTS
  The optimized gD protein was highly expressed in E. coli and successfully purified. Five monoclonal antibodies (mAbs) against BV were obtained and named as 4E3, 3F8, 3E7, 1H3 and 4B6, and with ascetic fluid titers of 2 × 10(6), 2 × 10(5), 2 × 10(5), 2 × 10(3) and 2 × 10(2), respectively. The 1H3 and 4E3 belonged to the IgG2b subclass, while 3E7, 3F8 and 4B6 belonged to the IgG1 subclass.
  
  
  CONCLUSIONS
  The cell lines obtained in this work secreted potent, stable and specific anti-BV mAbs, which were suitable for the development of herpes B virus diagnosis reagents.
authors.affiliation: Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China.
authors.forename: Zian
authors.initials: Z
authors.lastname: Jin
authors.affiliation: Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Sun
authors.affiliation: Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China.
authors.forename: Xueshan
authors.initials: X
authors.lastname: Xia
authors.affiliation: Kunming Biomed International Company, Kunming, China.
authors.forename: Qiujiang
authors.initials: Q
authors.lastname: Wei
authors.affiliation: Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China.
authors.forename: Yuzhu
authors.initials: Y
authors.lastname: Song
authors.affiliation: Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China.
authors.forename: Qinqin
authors.initials: Q
authors.lastname: Han
authors.affiliation: Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Hu
authors.affiliation: Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China.
authors.forename: Jinyang
authors.initials: J
authors.lastname: Zhang
keywords: Herpes B Virus (BV); Monoclonal Antibody; Optimized Expression; Protein Purification; gD Protein