【摘要】 目的表达H5N1亚型禽流感病毒（AIV）NS1蛋白,用于AIV感染与注射灭活疫苗鸡的鉴别诊断和NS1蛋白功能研究。方法采用RT_PCR方法对H5N1亚型AIVNS1基因进行扩增,将PCR产物克隆于pGEM_Teasy载体,将该基因插入pGEX₄T₁中构建NS1基因原核表达载体,转化BL21大肠杆菌后,在IPTG诱导下表达NS1蛋白,Westernblot鉴定表达NS1蛋白。结果成功克隆H5N1亚型AIV的NS1基因,其核苷酸序列长度为690bp,编码230个氨基酸残基。构建NS1基因原核表达载体在大肠杆菌内表达出约51×103的NS1融合蛋白。Westernblot鉴定表明表达NS1蛋白与H7N2AIV感染鸡血清有反应性。结论在大肠杆菌中成功表达了H5N1亚型AIVNS1基因蛋白,具有与感染H7N2亚型AIV阳性血清反应原性。更多还原

【Abstract】 Objective To express nonstructural protein NS1 gene of H5N1 subtype AIV for differentiation of the infected and vaccinated chicken. It could be useful for the further study of NS1 gene function. Method The NS1 gene of H5N1 subtype AIV was amplified by RT_PCR and cloned to pGEM_Teasy vector. The NS1 gene expressing plasmids was constructed by inserting the target gene fragments into pGEX₄T₁ vectors. The expression proteins were indued by IPTG and analysed by Western blot. Results NS1 gene of H5N1 subtype AIV was successfully cloned and sequenced. This gene encoded 230 aa. The constructed NS1 gene expressing plasmids was tansformed into E.coli BL21 competent cells. The NS1 fusion protein was expressed. Its molecular weight was 51×10 3. Expression NS1 fusion protein was reaction with H7N2 AIV positive serum. Conclusion The NS1 gene of H5N1 AIV was expressed in E.coli BL21 Expression NS1 fusion protein shared reaction with AIV positive serum.更多还原