title: Prokaryote expression of HPeV-1 VP1 protein, production of VP1 polyclonal antibody and the development of an ELISA.
abstract: The VP1 protein of human parechovirus (HPeV) plays a critical role in receptor binding based on its functional arginine-glycine-aspartic acid (RGD) motif region. Currently, only the neutralisation assay is used for seroepidemiological surveys of HPeVs. In the present study, the VP1 gene of HPeV-1 was cloned into the vector pET28a(+) to express the His-tagged VP1 protein in the bacterium Escherichia coli Rosetta. The recombinant protein was purified from inclusion bodies by Ni(+)-NTA affinity chromatography under denaturing conditions, followed by a refolding process in gradient urea. The identity and antigenicity of the His-tagged protein was confirmed by Western blotting using an anti-His monoclonal antibody and human HPeV-1-positive serum respectively. Polyclonal antibodies against the His-tagged VP1 protein were raised in rabbits by standard procedures, and the reactivity and specificity were tested by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. An indirect ELISA was developed based on the fusion protein VP1, and evaluated in order to facilitate the detection of antibodies in persons who had been infected naturally with HPeVs. A serological survey was performed using the assay amongst children in the Shanghai region of China; the seropositivity rate was found to be about 73%.
authors.affiliation: Zoonosis and Comparative Medicine Group, Department of Animal Science, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Yu
authors.forename: Tongling
authors.initials: T
authors.lastname: Shan
authors.forename: Shaofeng
authors.initials: S
authors.lastname: Zhu
authors.forename: Caixia
authors.initials: C
authors.lastname: Zhu
authors.forename: Xiuguo
authors.initials: X
authors.lastname: Hua
authors.forename: Chunmei
authors.initials: C
authors.lastname: Wang
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood* / Q000302:isolation & purification; D015153:Blotting, Western / Q000379:methods; D002648:Child; D002675:Child, Preschool; D002681:China / Q000453:epidemiology; D002846:Chromatography, Affinity; D019411:Clinical Laboratory Techniques / Q000379:methods*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007223:Infant; D030061:Parechovirus / Q000235:genetics / Q000276:immunology*; D010850:Picornaviridae Infections / Q000175:diagnosis* / Q000453:epidemiology; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D016036:Seroepidemiologic Studies; D015678:Viral Structural Proteins / Q000235:genetics / Q000276:immunology*
chemical_list: D000914:Antibodies, Viral; D011994:Recombinant Proteins; D015678:Viral Structural Proteins