title: Expression, purification and characterization of two truncated peste des petits ruminants virus matrix proteins in Escherichia coli, and production of polyclonal antibodies against this protein.
abstract: Peste des petits ruminants virus (PPRV), the etiological agent of peste des petits ruminants, is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) gene is composed of 1483 base pairs, encoding a 335 amino acids M protein with a molecular weight of approximately 38kD. We have demonstrated previously that the full-length M protein was expressed at an extremely low level or not even expressed in Escherichia coli BL21 (DE3). In this study, the M protein was split into two truncated forms to be successfully expressed in E. coli at a high level using the pET30a (+) vector, respectively, by analysis of SDS-PAGE, western blot and MALDI-TOF-MS. The optimization of culture conditions led us to perform the recombinant protein induction with 0.2mM IPTG at 28°C for 12h, whereby both proteins nevertheless were expressed in the insoluble form. Therefore, both His-tagged proteins were purified under the denaturing condition using a commercially available kit. Balb/c mice were immunized with the complex of purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by the analysis of ELISA. The specificity of the prepared polyclonal antibodies was checked by western blot and immunofluorescence, revealing them with the desirable specificity against both non-denatured and denatured M proteins.
authors.affiliation: Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, No. 5333 Xi'an Road, Changchun, Jilin 130062, China.
authors.forename: Fuxiao
authors.initials: F
authors.lastname: Liu
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Wu
authors.forename: Lin
authors.initials: L
authors.lastname: Li
authors.forename: Zengshan
authors.initials: Z
authors.lastname: Liu
authors.forename: Zhiliang
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis / Q000097:blood / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D018107:Peste-des-petits-ruminants virus / Q000235:genetics* / Q000276:immunology; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility; D014763:Viral Matrix Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000914:Antibodies, Viral; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins
keywords: E. coli; Expression; Optimization; Polyclonal antibodies; Purification; Truncated PPRV M protein