摘要
利用化学合成技术合成H5亚型猪流感病毒(SIV)的HA1基因,克隆到原核表达载体pET-28a(+),构建了重组表达质粒pET28a-HA1/H5,用重组质粒转化表达菌株E.coli BL21(DE3),经IPTG诱导表达,以镍螯合层析纯化表达蛋白;通过优化反应条件,建立了基于HA1重组蛋白的H5亚型SIV抗体间接ELISA(iHA1-ELISA)检测方法。SDS-PAGE分析结果显示,在大肠杆菌中成功表达了分子质量为45ku的His-HA1融合蛋白,表达蛋白以包涵体形式存在;Western-blot分析结果显示,表达蛋白能被抗His单克隆抗体和抗H5亚型SIV阳性血清特异识别。优化iHAI-ELISA中HA1/H5重组蛋白的包被浓度为0.31μg/mL,待检血清的最佳稀释度为1∶100。特异性测定结果显示,iHA1-ELISA方法不与H1、H3、H9亚型猪流感病毒抗体以及猪圆环病毒、猪生殖与呼吸综合征病毒阳性血清发生交叉反应;iHA1-ELISA方法与血凝抑制试验的符合率达93.5%。表明利用重组HA1蛋白建立的检测H5亚型SIV抗体的ELISA方法具有良好的特异性和敏感性。
HA1 gene of swine influenza virus（SIV） H5 subtype was synthesized and subcloned into pET-28a（＋） vector to form recombinant plasmid pET28a-HA1 / H5. After being transformed with the recombinant plasmid,Escherichia coli BL21（DE3） was induced by IPTG, and the expressed protein was purified by a Ni-chelated chromatography. After reaction conditions for an indirect ELISA were optimized,the indirect ELISA based on the recombinant HA1 protein（iHA1-ELISA） was developed for detecting SIV H5 subtype specific antibody. SDS-PAGE analysis showed that a fusion protein of His-HA1 with a molecular mass of approximately 45 ku was expressed successfully in inclusion bodies in E. coli. Western-blot analyses showed that the expressed protein was recognized specifically by anti-His monoclonal antibody as well as SIV H5 subtype positive serum. In the optimized iHAI-ELISA, the HA1 protein was coated at 0.31 μg/mL and swine serum samples were diluted at 1 ： 100 for SIV-antibody detection. The specificity test showed that iHA1-ELISA had no cross reaction with the antibodies against swine influenza virus subtypes H1, H3 and H9,and the positive sera against porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus sera. The accuracy of iHA1-ELISA was 93.5% compared with the classical hemagglu tination inhibition assay. These results indicated that the iHA1-ELISA based on the recombinant HA1 pro rein possessed good specificity and sensitivity for detecting SIV H5 subtype antibody.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2008年第9期762-766,共5页
Chinese Veterinary Science
基金
浙江省重大科技攻关计划项目(200C12021-1)
