摘要
以禽流感病毒A/chicken/Hubei/489/2004(H5N1)NA基因全长cDNA克隆pMD-NA为模板,PCR扩增第406位至第1 353位基因片段.克隆到pET-28a表达载体,构建重组质粒pET-28/ΔNA,转化大肠杆菌,用异丙基β硫代半乳糖苷(IPTG)诱导重组蛋白表达,SDS-PAGE和Western blot分析证实,截短的NA重组蛋白获得高效表达.采用SDS-PAGE纯化重组蛋白,免疫家兔,制备特异性神经氨酸酶(NA)抗体.ELISA及间接免疫荧光检测结果显示,该抗体效价在1∶1×105以上,能与在大肠杆菌和Vero细胞中表达的NA蛋白产生特异性反应.纯化的NA重组蛋白可用作建立禽流感检测方法的诊断抗原,所制备NA抗体可用于检测禽流感基因工程疫苗中的NA抗原组分.
In order to express the recombinant Neuraminidase(NA) protein of avian influenza virus Hubei strain,A/chicken/Hubei/489/2004 (H5N1),a truncated na gene fragment was amplified by PCR with pMD-NA plasmid (GenBank No. AY770078) as template. After digestion with EcoR I and Hind Ⅲ, the PCR product was cloned into pET-28a vector and the recombinant plasmid pET-28/ANA was analyzed by restriction enzyme digestion and sequencing, and then was transformed into E coli BL21-CondonPlus (DE3)-RIL. A truncated NA protein fused with the polyhistidine tag in N-terminal was expressed by the induction of IPTG and identified by SDS-PAGE and Western blot analysis. Polyclonal antibody against the NA protein was prepared after injected rabbit with the purified recombinant NA protein. The results from ELISA and indirect immunofluorescence indicated the antibody has high titer and excellent specificity for avian influenza virus H5N1 NA protein. These results might be contributive to develop the candidate NA subunit vaccine against avian influenza virus H5N1 subtype and detect virus infection.
出处
《武汉大学学报(理学版)》
CAS
CSCD
北大核心
2007年第4期481-484,共4页
Journal of Wuhan University:Natural Science Edition
基金
国家高技术研究发展计划(863)项目(2006AA02Z463)