摘要
根据已知H5N1亚型禽流感病毒NP基因序列设计、合成PCR克隆引物。自H5N1阳性病料中提取总RNA,反转录后采用高可信度DNA聚合酶,经PCR扩增NP基因,采用Invitrogen定向表达系统进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组NP蛋白,分子质量约66.0ku。采用单克隆抗体和阳性血清经ELISA、免疫印迹分析重组蛋白的免疫反应性。结果表明:所获得的重组NP蛋白在ELISA分析中均能与阳性血清和单克隆抗体发生特异性结合,但在免疫印迹分析中仅与阳性血清发生特异性结合。
A pair of primer was designed and synthesized according to nucleopmtein（NP） gene sequences of H5N1 subtype of avian influenza virus. Total RNA was extracted from positive clinical sample of H5 N1 subtype of avian influenza virus NP gene was amplified by reverse transcriptase - polymerase chain reaction using high proofreading polymerase （ PyobestTM DNA Polymerase）, and expressed using Invitrogen championTM pET directional TOPO expression system. Recombinant protein containing polyhistidine （6xHis） tag in N -terminal was ＇about 66. 0 ku in size, had been obtained and purified. Its immunoreactivity was analyzed by ELISA and western blot using MAb and positive serum. The results showed recombinant NP protein could bind to both positive serum and MAb with specificity in ELISA, but only bind to positive serum in western blot
出处
《黑龙江畜牧兽医》
CAS
北大核心
2007年第2期19-22,共4页
Heilongjiang Animal Science And veterinary Medicine
基金
云南省科技攻关项目(2004NG-01)
农业部948项目(2004-Z41)
关键词
禽流感病毒
H5N1亚型
核蛋白
表达
avian influenza virus
H5 N 1 subtype
nueleoprotein
expression
