title: Expression and purification of the nucleocapsid protein of Schmallenberg virus, and preparation and characterization of a monoclonal antibody against this protein.
abstract: Schmallenberg virus (SBV) is a novel orthobunyavirus that primarily infects ruminants such as cattle, sheep and goats. The nucleocapsid (N) protein of SBV has been shown to be an ideal target antigen for serological detection. To prepare a monoclonal antibody (mAb) against the N protein, the full-length coding sequence of the SBV N gene was cloned into pET-28a-c(+) and pMAL-c5X vectors to generate two recombinant plasmids, which were expressed in Escherichia coli BL21 as histidine (His)-tagged (His-SBV-N) and maltose-binding protein (MBP)-tagged (MBP-SBV-N) fusion proteins, respectively. After affinity purification of His-SBV-N with Ni-NTA agarose and MBP-SBV-N with amylose resin, His-SBV-N was used to immunize BALB/c mice, while MBP-SBV-N was utilized to screen for mAb-secreting hybridomas. Six hybridoma cell lines stably secreting mAbs against N were obtained. Clone 2C8 was selected for further study because of its rapid growth characteristics in vitro and good reactivity with recombinant SBV N proteins in enzyme-linked immunosorbent assays. The epitope recognized by 2C8 is located at amino acids 51-76 of the SBV N protein. Western blot analyses showed that 2C8 reacts with both recombinant SBV N proteins and SBV isolates. It is also cross-reactive with the N proteins of genetically related Shamonda, Douglas and Akabane viruses, but not with the Rift Valley fever virus N protein. The successful preparation of recombinant N proteins and mAbs provides valuable materials that can be used in the serological diagnosis of SBV.
authors.affiliation: Institute of Animal Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100029, China.
authors.forename: Yongning
authors.initials: Y
authors.lastname: Zhang
authors.forename: Shaoqiang
authors.initials: S
authors.lastname: Wu
authors.forename: Jianchang
authors.initials: J
authors.lastname: Wang
authors.forename: Kerstin
authors.initials: K
authors.lastname: Wernike
authors.forename: Jizhou
authors.initials: J
authors.lastname: Lv
authors.forename: Chunyan
authors.initials: C
authors.lastname: Feng
authors.forename: Jihong
authors.initials: J
authors.lastname: Zhang
authors.forename: Caixia
authors.initials: C
authors.lastname: Wang
authors.forename: Junhua
authors.initials: J
authors.lastname: Deng
authors.forename: Xiangfen
authors.initials: X
authors.lastname: Yuan
authors.forename: Xiangmei
authors.initials: X
authors.lastname: Lin
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002044:Bunyaviridae Infections / Q000662:veterinary / Q000821:virology; D002417:Cattle / Q000821:virology; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D005260:Female; D006041:Goats / Q000821:virology; D051379:Mice; D008807:Mice, Inbred BALB C; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D016851:Orthobunyavirus / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012756:Sheep / Q000821:virology
chemical_list: D000911:Antibodies, Monoclonal; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins
keywords: Epitope; Monoclonal antibody (mAb); Nucleocapsid (N) protein; Schmallenberg virus (SBV)