鸡γ-干扰素的可溶性表达及纯化产物的抗NDV活性  

Soluble expression of chicken interferon-gamma and activity of purified expression product against NDV

作　　者：代丽[1] 单安山[1] 孙进华[1] 尹佳佳[1] 李远见[1] 

机构地区：[1]东北农业大学动物营养研究所,哈尔滨150030

出　　处：《东北农业大学学报》2011年第9期37-42,共6页Journal of Northeast Agricultural University

基　　金：国家自然科学基金(30671527)

摘　　要：将鸡γ-干扰素(chIFN-γ)基因先克隆,接着通过大肠杆菌表达后进行产物纯化与活性检测。通过PCR方法以带有信号肽的重组质粒PMD-18T-preIFN-γ为模板,扩增无信号肽chIFN-γ基因片段,克隆至原核表达载体pProEXTM HTa,构建重组表达质粒pProEXTM HTa-chIFN-γ,在大肠杆菌BL21(DE3)中经IPTG诱导表达,然后利用Novagen蛋白纯化试剂盒进行天然纯化可溶性蛋白,并进行SDS-PAGE、Western blot鉴定,同时利用CEF-NDV系统测定抗病毒活性。结果表明,459 bp的chIFN-γ成熟蛋白编码基因被成功克隆,同时chIFN-γ蛋白在大肠杆菌中也获得了成功表达,分子质量约为20 ku,能与抗His的单克隆抗体发生特异性反应。表达蛋白一部分形成包涵体,另一部分以可溶形式存在,可溶性蛋白经镍柱在天然条件下的纯化得率约为1.9 mg·mL-1。生物学活性试验表明,1 54稀释纯化的重组chIFN-γ(rchIFN-γ)孵育CEF细胞后能抵抗100TCID50的NDV(新城疫Ⅳ系弱毒苗)攻击。研究纯化获得的rchIFN-γ具有较好的抗病毒活性,为研制新型抗病毒干扰素制剂奠定基础。chIFN-γ mature protein gene was amplified by RT-PCR from recombinant PMD-18TpreIFN-γ and then cloned into the prokaryotic expression vector pProEXTMHTa.Recombinant expression plasmid of pProEXTMHTa-chIFN-γ was constructed and then transformed into the competent E.coli BL21（DE3） cells for expression with IPTG induction.Purified soluble rchIFN-γ proteins were obtained by His.Bind Purification Kit（Novagen） and identified by SDS-PAGE gel and Western blot assay.The antiviral activity was detected by CEF-NDV system.A 459 bp cDNA encoding chIFN-γ mature protein gene was cloned and successfully expressed in E.coli with approximate molecular weight of 20 ku,which could be recognized by anti-His mAb against chIFN-γ.The recombinant chIFN-γ proteins were expressed to form inclusion bodies with a portion of soluble protein.The soluble rchIFN-γ proteins were purified by Ni-NTA column under a native condition with the yield of 1.9 mg.mL-1.The purified recombinant chIFN-γ（rchIFN-γ） proteins by 1∶54dilution could resist 100 TCID50 NDV virus attack.There fore,the purified rchIFN-γ proteins by Ni-NTA column under a native condition had better antiviral activity,which will establish a basis for further developing new type antiviral interferon praeparatum.

关 键 词：鸡γ-干扰素 克隆 可溶性表达 病毒活性 

分 类 号：S826[农业科学—畜牧学]