摘要
研究鸡白细胞介素-2(chicken interleukin-2,ChIL-2)的免疫佐剂功能,构建ChIL-2与新城疫病毒F蛋白多抗原表位(NDV-F)的嵌合基因,以对鸡新城疫疫病进行防治。采用重叠延伸PCR方法通过基因柔性接头将ChIL-2基因和NDV-F多抗原表位基因构建成ChIL-2-linker-NDV-F嵌合基因并克隆入PET-32a载体,经测序鉴定后,转化BL21大肠杆菌,IPTG诱导表达6×His融合蛋白,Ni2^+亲和柱纯化,表达产物经SDS-PAGE、Western blot和间接ELISA检测和鉴定。结果表明,实验成功构建并克隆了ChIL-2-linker-NDV-F嵌合基因,嵌合基因在大肠杆菌中表达的ChIL-2-linker-NDV-F融合蛋白分子量约为48kD,表达量约占菌体蛋白总量的45%,纯化后的ChIL-2-linker-NDV-F融合蛋白能与感染NDV的鸡血清发生反应。上述结果证明ChIL-2-linker-NDV-F嵌合基因在原核细胞中能有效表达,且融合蛋白具有较强的特异性和免疫原性。
To explore the immune adjuvant effects of chicken IL-2 （ChlL-2） , a recombinant plasmid with ChIL-2 and multi-antigen epitopes from F gene of Newcastle disease virus （NDV-F） was built and expressed in prokaryotic cells to prevent and control the chicken ND disease. The recombinant chimeric gene of ChlL-2-linker-NDV-F constructed by chicken ChlL-2 gene linked multi-antigen epitopes gene of NDV F protein via a serine-rich linker by overlap-PCR method was cloned into prokaryotic expression vector PET-32a. After identification by sequencing, the recombinant ChlL-2-inker-NDV-F protein was expressed in E. coli BI21 through IPTG and purified with the Ni2^＋ affinity column. The expressed ChlL-2-linker-NDV-F protein was analyzed by SDS-PAGE, Western-blot and indirect ELISA, respectively. The chimeric gene of ChlL-2-linker-NDV-F was successfully constructed and cloned into PET-32a vector respectively. The expressed ChlL-2-linker-NDV-F protein was shown by a major band with a expected molecular weight about 48 kD on SDS-PAGE and Western-blot and accounted for almost 45 % of the total bacteria proteins, Serological assay by indirect ELISA showed that it reacted strongly and specifically with chicken serum of NDV infection. These results indieated that the chi- meric gene encoding Chll,-2-linker-NDV-F could effectively express in prokaryotic cells and the expressed protein had high specifieity and good irnmunogenicity.
出处
《激光生物学报》
CAS
CSCD
2011年第2期230-235,共6页
Acta Laser Biology Sinica
基金
国家自然科学基金项目(30671537)
安徽农业大学博士启动基金项目(yj2009-26)
