title: [Expression and antiviral assay of bovine interferon-gamma].
abstract: Bovine interferon-gamma (BoIFN-gamma) gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA of bovine spleen lymphocytes stimulated with ConA. The products of RT-PCR were cloned into pVAX1 vector, positive recombinant clone was identified by restriction enzyme digestion and sequencing. The recombinant plasmid pVAX1-BolFN-gamma was transfected into COS-7 cells mediated by lipofectine, indirect immunofluorescent assay analysis confirmed that rBoIFN-gamma was expressed in COS-7 cells. BoIFN-gamma gene (without signal peptide) was cloned into pET-30a(+) and pGEX-6p-1 vector, and transformed into the Escherichia coli cells. After optimizing the induction condition, SDS-PAGE analysis showed that the expression products were all found in soluble form and had a molecular weight of 23 kDa and 43 kDa respectively. BoLFN-gamma precursor gene (with signal peptide) was cloned into transfer vector pFastBac 1, and transformated into DH10Bac E. coli cells. By site-specific transposition, BoIFN-gamma gene was integrated into shuttle vector Bacmid, and transfected into the Sf9 insect cells mediated by lipofectine to produce recombinant baculovirus. Indirect immunofluorescent assay analysis confirmed that rBac-BoLFN-gamma was expressed successfully in Baculovirus vector system. The antiviral activities of rHis-BoIFN-gamma, rGST-BoIFN-gamma and rBac-BoIFN-gamma were up to 8.389 x10(7) U/mg, 6.554 x10(5) U/mg and 4.096 x 10(4) U/mL respectively, which were analyzed in MDBK/VSV system. A sandwich ELISA was established using monoclonal antibodies 3E6 and 5G4, which can detect BoIFN-gamma in quantity and provide a useful method for the clinical practice and research of BolFN-gamma.
authors.affiliation: Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou 225009, China.
authors.forename: Zhengzhong
authors.initials: Z
authors.lastname: 'u
authors.forename: Xiang
authors.initials: X
authors.lastname: Chen
authors.forename: Fengli
authors.initials: F
authors.lastname: Shan
authors.forename: Chuang
authors.initials: C
authors.lastname: Meng
authors.forename: Lin
authors.initials: L
authors.lastname: Sun
authors.forename: Jinlin
authors.initials: J
authors.lastname: Huang
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Pan
authors.forename: Shizhong
authors.initials: S
authors.lastname: Geng
authors.forename: Xinan
authors.initials: X
authors.lastname: Jiao
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D014162:Transfection
chemical_list: D000998:Antiviral Agents; D011994:Recombinant Proteins; D007371:Interferon-gamma