牛γ 干扰素的表达及其抗病毒活性测定

Expression and antiviral assay of bovine interferon-γ

摘要:

通过RT-PCR 从经ConA 刺激诱导的奶牛脾脏淋巴细胞总RNA 中扩增出牛γ 干扰素 (BoIFN-γ) cDNA，克隆到真核载体pVAX1 中， 测序结果显示pVAX1 中的插入序列BoIFN-γ 基因与已报道序列一致。用重组质粒pVAX1-BoIFN-γ 转染COS-7 细胞并进行间接免疫荧光试验鉴定，结果显示BoIFN-γ 在COS-7 细胞中得到成功表达。将BoIFN-γ 基因克隆到原核

Abstract:

Bovine interferon-γ (BoIFN-γ) 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-BoIFN-γ was transfected into COS-7 cells mediated by lipofectine, indirect immunofluorescent assay analysis confirmed that rBoIFN-γ was expressed in COS-7 cells. BoIFN-γ 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. BoIFN-γ precursor gene (with signal peptide) was cloned into transfer vector pFastBac^TM1, and transformated into DH10Bac E. coli cells. By site-specific transposition, BoIFN-γ 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-BoIFN-γ was expressed successfully in Baculovirus vector system. The antiviral activities of rHis-BoIFN-γ, rGST-BoIFN-γ and rBac-BoIFN-γ were up to 8.389×10⁷ U/mg, 6.554×10⁵ U/mg and 4.096×10⁴ 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-γ in quantity and provide a useful method for the clinical practice and research of BoIFN-γ.