Jin Zian, Sun Tao, Xia Xueshan, Wei Qiujiang, Song Yuzhu, Han Qinqin, Chen Qiang, Hu Juan, Zhang Jinyang
Research Center of Molecular Medicine of Yunnan Province, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China.
Kunming Biomed International Company, Kunming, China.
Jundishapur J Microbiol. 2016 Mar 12;9(3):e32183. doi: 10.5812/jjm.32183. eCollection 2016 Mar.
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 × 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.
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.
B病毒（BV）是一种由双链包膜DNA病毒引起的人畜共患病，以猕猴科动物为自然宿主。感染人类的死亡率可达70%，可引发致命性脑炎和脑脊髓炎。目前，尚无针对BV感染的有效治疗方法。在猴B病毒编码的多种蛋白质中，gD作为一种保守的结构蛋白，对于猴B病毒频繁变异的血清学诊断具有重要应用价值。
本研究旨在通过重组载体在大肠杆菌中表达BV的gD蛋白，并制备针对BV gD的特异性单克隆抗体，为有效快速诊断试剂的研发奠定基础。
利用OptimWiz对BV的gD基因进行优化，以改善密码子使用偏好性并进行合成，构建重组质粒pET32a/gD，并在大肠杆菌Rosetta(DE3)中表达。对表达的融合蛋白His-gD进行纯化，并用该蛋白免疫BALB/c小鼠。将免疫小鼠的脾细胞与SP2/0骨髓瘤细胞融合，通过间接酶联免疫吸附测定（ELISA）筛选获得单克隆细胞株，随后制备单克隆抗体腹水。
优化后的gD蛋白在大肠杆菌中高表达并成功纯化。获得了5株抗BV的单克隆抗体（mAb），分别命名为4E3、3F8、3E7、1H3和4B6，腹水效价分别为2×10(6)、2×10(5)、2×10(5)、2×10(3)和2×10(2)。1H3和4E3属于IgG2b亚类，而3E7、3F8和4B6属于IgG1亚类。
本研究获得的细胞株分泌高效、稳定且特异性的抗BV mAb，适用于B病毒诊断试剂的开发。
