禽流感病毒神经氨酸酶基因在重组杆状病毒中的表达  

Expression of avian influenza virus neuraminidase in recombinant baculovirus

作　　者：孙晓东[1] 包红梅[1] 赵玉辉[1] 路冰[2] 孙佳善[1,2] 文雪霞[1] 熊永忠[1] 陈化兰[1] 王秀荣[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所,国家禽流感参考实验室/兽医生物技术国家重点实验室,黑龙江哈尔滨150001 [2]东北农业大学动物医学学院,黑龙江哈尔滨150030

出　　处：《畜牧与兽医》2012年第5期16-20,共5页Animal Husbandry & Veterinary Medicine

基　　金：哈尔滨市科技创新人才科研专项资金RC2011XK002029;公益性行业(农业)科研专向经费项目(200903055)

摘　　要：根据已知H5N1亚型禽流感病毒(AIV)神经氨酸酶(NA)基因序列设计并合成引物。从H5N1亚型病毒感染的鸡胚尿囊液中提取总RNA,反转录后采用高保真DNA聚合酶扩增NA基因,构建转移载体pFastBacHTA-NA,并与大肠杆菌DH10Bac的Bacmid质粒重组,构建重组转座质粒rBacmid-NA。在脂质体介导下将rBacmid-NA转染sf9昆虫细胞获得重组杆状病毒。在sf9昆虫细胞中表达NA蛋白,通过SDS-PAGE、Western blot和激光共聚焦检测蛋白。结果表明:表达的NA蛋白分子量约为53 ku,该蛋白能与H5N1亚型AIV血清发生特异性反应,证明NA蛋白表达正确,具有良好的免疫反应性。A pair of primers was designed according to the published neuraminidase （NA） gene sequence （ GenBank No. AF250362） of HSN1 subtype avian influenza virus （AIV）. Genomic RNA was extracted from the chick embryo allantoic fluid infected by H5N1 subtype AIV, and the NA gene was amplified by RT-PCR. Then the transfer vector pFastBacHTA-NA was constructed and recombined with Bacmid plasmid of Escherichia coli DH10Bac. Then the recombinant plasmid rBacmid-NA was constructed. After the rBacmid-NA was transfected into sf9 ceils, the recombinant baculovirus was harvested. The specific protein band of 53 ku was identified and could react with chicken sera against HSN1 subtype AIV. These results indicated that the NA protein was accurately expressed in sf9 cells and had a good immunoreactivity.

关 键 词：禽流感病毒 神经氨酸酶 重组杆状病毒 

分 类 号：S852.652[农业科学—基础兽医学]