作　　者： ; ; ; ; ;

机构地区： 中国农业科学院哈尔滨兽医研究所农业部动物流感重点开放实验室

出　　处： 《中国兽医科学》 2007年第9期783-786,共4页

摘　　要： 用RT-PCR方法从H5N1亚型禽流感病毒(AIV)A/Duck/Zhejiang/12/00中获得NA基因,将目的基因定向克隆到原核表达载体pET-32a中,将序列测定和双酶切验证准确的阳性重组质粒转化大肠杆菌BL21,用IPTG诱导,经SDS-PAGE和Western-blot分析,结果显示,重组蛋白得到了可溶性表达,表达的蛋白质分子质量为33 ku;该蛋白可以与禽流感H5N1亚型阳性血清反应,具有良好的免疫原性;ELISA检测结果表明,用此纯化蛋白作为包被抗原检测H5N1亚型AIV神经氨酸酶抗体具有良好的灵敏性。 The NA gene of avian influenza virus（AIV） subtype H5N1 was amplified from the eDNA of AIV subtype H5N1 strain A/Duck/Zhejiang/12/00 by RT-PCR. The amplicon was cloned into pET-32a vector,and the constructed positive recombinant plasmid,which was identified by sequencing and digestion with EcoR Ⅰ ＋ Hind Ⅲ ,was transformed into competent cells BL21（DE3） and induced with IPTG. SDS- PAGE analysis showed that the expressed recombinant fusion protein was 33 ku in size. Western-blot anal- ysis showed that the protein,which had good immunogenicity,was able to react with positive sera against AIV subtype H5N1. The ELISA result demonstrated that the purified protein could be used to detect the antibody against NA of AIV subtype H5N1 and had good sensitivity.

关 键 词： 禽流感病毒 基因 克隆 原核表达

领　　域： [农业科学] [农业科学] [农业科学]