title: Purification of HRSV F protein from a eukaryotic expression vector and             establishment of a sandwich ELISA method.
abstract: In order to increase the expression of the fusion (F) protein and lay a foundation for the construction of a genetically engineered vaccine and rapid clinical detection, the F protein of the human respiratory syncytial virus (HRSV) was expressed and purified, and a sandwich enzyme-linked immunosorbent assay (ELISA) method was established. The F1 fragment of the HRSV F protein was amplified following reverse transcription, and was then combined with the vector and transformed into eukaryotic cells. The recombinant protein was induced and purified. The purified protein was used to immunize mice to produce antiserum and establish indirect ELISA. The established method was tested and verified by analyzing 100 samples using gold immunochromatography (GICA). The F1 fragment of the F gene was successfully amplified, the DNA (+) recombinant was selected, and a protein of molecular weight approximately 45,000 was obtained after the induction. The optimal reaction conditions and working concentration of ELISA were determined. The optimal concentration of mice anti-F1 IgG is 3.2 µg/ml, the best reaction time of the samples is 70 min at 37 ˚C, and the working concentration of the rabbit anti‑mouse IgG is 1:6,000. Compared with the GICA method, the sample's positive co-efficient of variation was 3.2-8.6%, and the negative co-efficient of variation was 5.1-8.3%. These were <10%, indicating that the ELISA method was reproducible. The F1 protein can be greatly expressed in transfected eukaryotic cells, and the purified F1 protein has good immunogenicity. The antiserum produced by the purified recombinant protein can be precisely detected using the ELISA detection method described in this study.
authors.affiliation: The Second Hospital of Jilin University, Changchun, Jilin 130041, PR China.
authors.forename: Hong
authors.initials: H
authors.lastname: Bao
authors.forename: Ting
authors.initials: T
authors.lastname: Yu
authors.forename: Yufen
authors.initials: Y
authors.lastname: Jin
authors.forename: Chune
authors.initials: C
authors.lastname: Wang
authors.forename: Yanlei
authors.initials: Y
authors.lastname: Li
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D018357:Respiratory Syncytial Virus Infections / Q000175:diagnosis; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000276:immunology; D014760:Viral Fusion Proteins / Q000032:analysis* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
chemical_list: C526850:F protein, human respiratory syncytial virus; D014760:Viral Fusion Proteins