鸡α干扰素/白细胞介素2基因的融合表达及活性研究  

Study on the Expression and Bioactivity of Chicken Interferon α/Chicken Interleukin-2 Fusion Protein in vivo and in vitro

作　　者：闫若潜[1] 谢彩华[1] 吴志明[1] 张志凌[1] 刘光辉[1] 刘梅芬[1] 

机构地区：[1]河南省动物疫病预防控制中心,郑州450008

出　　处：《中国农业科学》2010年第3期594-604,共11页Scientia Agricultura Sinica

基　　金：河南省科技攻关计划资助项目(082102130008)

摘　　要：【目的】研究高效广谱鸡基因工程重组复合抗病毒制剂以对鸡的病毒性疾病进行防治。【方法】采用重叠延伸PCR(splicing by overlap extension-PCR,SOE-PCR)方法通过一基因柔性接头将鸡α干扰素(chicken interferon alpha,ChIFN-α)与鸡白细胞介素2(chicken interleukin-2,ChIL-2)基因构建成ChIFN-α-linker-ChIL-2嵌合基因并克隆入pGEM-T Easy载体,将嵌合基因亚克隆入pQE-30表达载体中进行原核表达。通过尿素变性、低浓度蛋白复性液复性、PBS溶液透析等步骤对表达的重组融合蛋白(rChIFN-α-linker-ChIL-2)进行纯化。采用细胞病变抑制法检测rChIFN-α-linker-ChIL-2蛋白在细胞上抑制水泡性口炎病毒(VSV)和传染性法氏囊病毒(IBDV)增殖活性。采用ChIL-2ELISA试验方法检测rChIFN-α-linker-ChIL-2蛋白与抗ChIL-2单抗发生特异性免疫反应的活性。分别测定rChIFN-α-linker-ChIL-2蛋白在SPF鸡胚上对新城疫病毒(NDV)和禽流感H9N2亚型病毒(AIV H9N2)的抑制活性以测定其在鸡胚内抗病毒活性。分别测定rChIFN-α-linker-ChIL-2蛋白在鸡体内对NDV活疫苗和灭活疫苗的免疫增强作用以测定其在鸡体内的活性。【结果】成功构建并克隆了ChIFN-α-linker-ChIL-2嵌合基因。嵌合基因在大肠杆菌中表达的rChIFN-α-linker-ChIL-2蛋白分子量大小约35.9kD,蛋白经纯化后纯度在96%以上。rChIFN-α-linker-ChIL-2蛋白在CEF细胞上具有明显抗病毒活性,其抗VSV和IBDV活性明显高于单一rChIFN-α的抗病毒活性;rChIFN-α-linker-ChIL-2蛋白可以和抗ChIL-2单抗发生特异性免疫反应。IFN-α活性单位为200IU的rChIFN-α-linker-ChIL-2蛋白在SPF鸡胚内可明显降低NDV和AIV H9N2所引起的鸡胚死亡和胚体出血,并能显著延长鸡胚存活时间,但过高剂量的rChIFN-α-linker-ChIL-2蛋白抑制鸡胚死亡和出血能力有所下降。合适剂量的rChIFN-α-linker-ChIL-2蛋白在鸡体内具有显著的抗病毒活性和免疫增强活性。【结论】rChIFN-α-li[Objective] The aim of this study was to explore a high efficient chicken gene engineering antiviral agent to prevent and control the chicken viral disease. [Method] The recombinant chimeric gene of ChlFN-α-linker-ChlL-2 constructed by chicken interferon a （ChlFN-a） gene linked chicken interleukin-2 （ChlL-2） gene via a glycine-rich linker by SOE-PCR （splicing by overlap extension-PCR） method was cloned into pGEM-T Easy vector and subsequently sub-cloned into prokaryotic expression vector pQE30. The recombinant ChlFN-α-linker-ChlL-2 （rChlFN-α-linker-ChlL-2） protein was expressed in E.coli JM109 and purified with the innovated recombinant fusion protein purification method and denatured by 8 mol·L^-1 urea, refolded by a self-innovative renaturation buffer and dialyzed by PBS buffer, etc. The antiviral bioactivities of rChlFN-α-linker-ChlL-2 protein were tested by inhibiting the 50 percent appearance of cytopathic effect （CPE） of vesicular stomatitis virus （VSV） and infectious bursal disease virus （IBDV） on chicken embryo fibroblast （CEF） cell lines. The ChlL-2 bioactivities of rChlFN-α-linker-ChlL-2 protein were estimated by the method of chicken IL-2 ELISA assay for detection of the specific immunoactivity of ChlL-2 protein. The antivirus bioactivity of rChlFN-α-linker-ChlL-2 protein was evaluated by inhibiting the reproduction of NDV and AIV （H9N2） in chicken embryos, respectively. The bioactivity of rChlFN-α-linker-ChlL-2 protein in chicken was estimated by detecting the NDV hemagglutination inhibition （HI） antibody titer induced by NDV oil-adjuvant inactivated vaccine and attenuated live vaccine, respectively. [Result] The chimeric gene of ChlFN-α-linker-ChlL-2 was successfully constructed and cloned into pGEM-T Easy vector, respectively. The rChlFN-α-linker-ChlL-2 protein was abundantly fusion expressed in E.cali and successfully purified with a molecular mass of about 35.9 kD and purity more than 96% on SDS-PAGE, which indicated that the correct rChlFN-α-link

关 键 词：鸡α干扰素 鸡白细胞介素2 嵌合基因 融合表达 活性 

分 类 号：Q78[生物学—分子生物学]