猪α干扰素与白介素-18融合基因的克隆、原核表达及其抗病毒活性的初步研究  

Cloning and Prokaryotes Expression of Porcine Interferon-αand Interleukin-18 Fusion Gene and Detection of its Antiviral Activity

作　　者：张盼盼[1] 何静[1] 康银峰[1] 向斌[1] 任涛[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《中国畜牧兽医》2014年第6期29-33,共5页China Animal Husbandry & Veterinary Medicine

基　　金：公益性行业(农业)科研专项(201303033);广东省科技计划项目(2012A020800006)

摘　　要：为探索猪α干扰素(poIFN-α)与白介素-18(poIL-18)融合基因的抗病毒效果,本研究通过融合PCR,以质粒pMD18-T/poIFN-α和pMD18-T/poIL-18为模板扩增猪IFNα-IL18(poIFNα-IL18)融合基因。将poIFNα-IL18融合基因插入原核表达载体pET-28b(+)构建重组表达质粒pET-28b/poIFNα-IL18,转化大肠杆菌BL21感受态细胞进行诱导表达,SDS-PAGE分析重组表达蛋白;经镍琼脂糖凝胶柱亲和层析纯化后,用微量细胞病变抑制法在PK-15/VSV系统上进行抗病毒活性测定。结果表明成功构建重组表达质粒pET-28b/poIFNα-IL18;SDS-PAGE可检测到分子质量为37ku左右的蛋白条带,与目的蛋白大小相近;经镍琼脂糖凝胶柱亲和层析纯化,获得高纯度重组蛋白;用微量细胞病变抑制法在PK-15/VSV系统上检测到重组蛋白poIFNα-IL18比单一蛋白poIFN-α和poIL-18的抗病毒活性显著,其抗水疱性口炎病毒(vesicular stomatitis virus,VSV)活性分别为1.03×105、1.28×104及0.11×104 U/mg。In order to explore the antiviral effect of porcine interferon-α and interteukin-18 fusion gene （poIFN-α-IL-18）, poIFN-α-IL-18 was amplified from pMD18-T/IFN-α and pMD18-T/IL-18 by using fusion-PCR in this study. The recombinant expression plasmid pET-28b/IFNα-IL18 was constructed by inserting poIFNα-IL18 into vector pET-28b（＋） separately. SDSPAGE was used for analyzing the protein after induced by IPTG. The protein was purified by Ni^2＋-NTA. Antiviral activities of the purified protein were tested by cytopathogenic effect inhibition assay. Results showed that the recombinant expression plasmid pET-28b/IFNα-IL18 was successfully constructed. SDS-PAGE could detect the protein bands about 37 ku. After Ni^2＋- NTA affinity chromatography purification, the activity of purified fusion protein against astrovirus was better than the single protein. Antiviral activities of the purified proteins, poIFN-α-IL-18, poIFN-a and poIL-18, were about 1.03 ×10^5 , 1.28 × 10^4 and 0.11 × 10^4 U/mg.

关 键 词：融合基因 克隆 表达 抗病毒活性 

分 类 号：S859.79[农业科学—临床兽医学]