Author/Authors :
Jin، Zian نويسنده Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China , , Sun، Tao نويسنده , , Xia، Xueshan نويسنده Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China , , Wei، Qiujiang نويسنده Kunming Biomed International Company, Kunming, China , , Song، Yuzhu نويسنده Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China , , Han، Qinqin نويسنده Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China , , Chen، Qiang نويسنده The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Science, Nankai University, Tianjin 300071, China , , Hu، Juan نويسنده Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China , , Zhang، Jinyang نويسنده Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China ,
Abstract :
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. 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. 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. 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 × 106, 2 × 105, 2 × 105, 2 × 103 and 2 × 102, respectively. The 1H3 and 4E3 belonged to the IgG2b subclass, while 3E7, 3F8 and 4B6 belonged to the IgG1 subclass. 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.
