徐正中，陈祥，单锋丽，孟闯，孙林，黄金林，潘志明，耿士忠，焦新安

扬州大学 江苏省人兽共患病学重点实验室，扬州 225009

牛γ干扰素的表达及其抗病毒活性测定

徐正中*，陈祥*，单锋丽，孟闯，孙林，黄金林，潘志明，耿士忠，焦新安

扬州大学 江苏省人兽共患病学重点实验室，扬州 225009

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基因克隆到原核表达质粒pET-30a(+)、pGEX-6p-1后，分别转化重组表达菌BL21(DE3)、BL21后，通过对表达条件的优化，SDS-PAGE分析表明两种重组蛋白均可实现可溶性表达，大小分别为23 kDa和43 kDa。将含信号肽BoIFN-γ基因克隆到转座载体 pFastBacTM1中并转化 DH10Bac，通过位点特异性转座将 BoIFN-γ基因整合到穿梭载体Bacmid中并通过脂质体转染Sf9昆虫细胞，产生重组杆状病毒rBac-BoIFN-γ，重组病毒传代扩增感染Sf9细胞后，通过间接免疫荧光试验证实 BoIFN-γ在杆状病毒系统中获得表达。使用 MDBK/VSV细胞系统测定 rHis-BoIFN-γ、rGST-BoIFN-γ以及 rBac-BoIFN-γ的抗病毒活性，其效价分别达到 8.389×107U/mg、6.554×105U/mg、4.096×104U/mL，3种具有较高的抗病毒活性重组牛γ干扰素的获得为其开发应用提供了重要的生物材料。同时使用单克隆抗体5G4和3E6，建立了BoIFN-γ的双抗夹心ELISA检测方法并绘制了标准曲线，可定量分析BoIFN-γ的抗病毒活性，为其临床应用及相关研究提供了重要的方法。

牛γ干扰素，大肠杆菌系统，杆状病毒系统，抗病毒活性，ELISA

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 wasexpressed 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 pFastBacTM1, 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 8.389×107U/mg, 6.554×105U/mg and 4.096×104U/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.

Keywords:bovine interferon-γ, Escherichia coli, baculovirus vector system, antiviral activity, ELISA

γ干扰素 (IFN-γ) 主要是由有丝分裂……