文章摘要
卢士强,周 锋,姜兰兰,茌 静,冯东举,姚 堃.甲型流感病毒FM1非结构蛋白(NS1)在大肠杆菌表达及其ELISA检测方法建立[J].南京医科大学学报,2009,29(1):23~27
甲型流感病毒FM1非结构蛋白(NS1)在大肠杆菌表达及其ELISA检测方法建立
Prokaryotic expression of nonstructural protein(NS1) of murine influenza virus FM1 and its application in differentiating infected from vaccinated mice
投稿时间:2008-08-10  
DOI:10.7655
中文关键词: 鼠流感病毒  NS1  原核表达  ELISA
英文关键词: murine influenza virus  NS1  prokaryotic expression  ELISA
基金项目:
作者单位
卢士强 南京医科大学微生物与免疫学系,江苏 南京 210029 
周 锋  
姜兰兰  
茌 静  
冯东举  
姚 堃  
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中文摘要:
      目的:构建流感病毒FM1非结构蛋白NS1全长基因表达载体在大肠杆菌(E.coli)中表达,并将表达蛋白用于病毒感染和疫苗免疫小鼠的血清检测?方法:提取流感病毒鸡胚尿囊液RNA,RT-PCR扩增NS1全长基因,克隆到pET28a,转化E.coli BL21感受态细胞,经酶切鉴定及序列分析,筛选出阳性重组质粒pET28-NS1?异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白,表达蛋白在变性条件下经镍柱亲和层析纯化,并用Western blot法检测其抗原活性?以纯化的NS1蛋白为抗原建立了检测NS1抗体的ELISA(NS1-ELISA)方法分别对7?15和30天灭活疫苗免疫和病毒感染小鼠血清进行检测?统计分析各组吸光度(A)差异,并以灵敏度?特异度?阳性预测值?阴性预测值等指标对NS1-ELISA进行方法学评价?结果:重组质粒经PCR 及酶切鉴定为阳性, 核酸序列分析正确?SDS-PAGE 和Western blot 结果显示表达重组蛋白分子质量约为26 ku?NS1-ELISA法在第15~30天,感染组血清吸光度明显高于疫苗免疫组?结论:成功获得了NS1重组基因表达蛋白,建立的NS1-ELISA法可用于病毒感染和疫苗免疫血清的鉴别检测?
英文摘要:
      Objective:To construct a plasmid pET28-NS1 for expression of nonstructural protein(NS1) of murine influenza virus FM1 in E.coli and utilize the NS1 protein in serological detection for differentiating infected from vaccinated mice. Methods:The NS1 gene of murine influenza virus A/FortMonmouth/1/47(FM1) was amplified by RT-PCR and cloned into pET28a to construct a recombinant expression plasmid, named pET28-NS1. The constructed vector was identified by PCR, enzyme digestion and nucleotide sequences analysis after being transformed into E.coli BL21. Induced by IPTG, the recombinant protein was analysed by Western blot and purified with Ni+ affinity chromatography. Based on the purified recombinant protein, an indirect ELISA assay for detection of anti-NS1 protein antibody(NS1-ELISA) was developed. The sera of virus-infected mice and vaccinated mice were detected at 7,15 and 30 day, respectively, and data were analyzed by SPSS13.0. In the end, The new assay was methodologically evaluated by sensitivity, specificity, positive and negative predictive value. Results:The recombinant plasmid was constructed correctly. SDS-PAGE and western blot analysis showed that the recombinant protein was highly expressed by inclusion body and its molecular weight was about 26 ku as expected. NS1-ELISA indicated that the purified protein reacted with the sera of mice experimentally infected with FM1, but not with those of the mice immunized with the inactivated viruses. Conclusion:The result demonstrated that the NS1-ELISA assay could differentiate infected from vaccinated mice on the basis of anti-NS1 antibody.
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