| 朱函坪,姚苹苹,徐芳,翁景清,谢荣辉,陆群英,朱智勇,严杰.汉坦病毒N蛋白的重组表达及其用于IgM直接捕捉ELISA的建立和应用[J].Chinese journal of Epidemiology,2007,28(7):692-696 |
| 汉坦病毒N蛋白的重组表达及其用于IgM直接捕捉ELISA的建立和应用 |
| Study on the recombinant expression of Hantaan virus protein N and the establishment and application of rNP-IgM direct capture ELISA |
| Received:December 15, 2006 Revised:June 28, 2012 |
| DOI： |
| KeyWord: 汉坦病毒 N蛋白 重组表达 IgM捕捉ELISA |
| English Key Word: Hantavirus Nueleocapsid protein Recombinant expression IgM antibody Capture enzyme-linked immunosorbent assay |
| FundProject:浙江省科技厅重点科研资助项目(2005C23014);浙江省医药卫生科学研究基金资助项目(2006B020) |
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| Abstract: |
| 目的 克隆并表达汉坦病毒(HV)Z10株(HV-Z10)N蛋白(NP)编码基因,建立基于辣根过氧化酶(HRP)标记重组NP(rNP)的rNP-IgM直接捕捉ELISA,检测肾综合征出血热(HFRS)患者血清并评价其检测效果.方法 PCR扩增HV-Z10株NP编码基因,基因工程方法构建NP编码基因原核表达系统pET28a-Z10N-E.coli BL21DE3.SDS-PAGE了解rNP表达情况,离子交换法和Ni-NTA亲和层析法提纯rNP.Western blot检测rNP的特异性免疫反应性.建立HRP标记rNP-IgM直接捕捉ELISA检测HFRS患者血清样品,并与常规HV-IgM间接捕捉ELISA进行比较.结果 pET28a-Z10N-E.coli BL21DE3高效表达rNP.提纯rNP SDS-PAGE显示单一蛋白条带,HV-IgG能有效识别rNP并与之结合.94.73%(90/95)HFRS患者血清样品rNP-IgM直接捕捉ELISA检测结果阳性,HV-IgM间接捕捉ELISA阳性率为92.63%(88/95).两种IgM捕捉ELISAs检测的HFRS患者血清样品A450值分布及对多份不同稀释度血清检测的A450均值变化均相似.结论 成功构建了HV-Z10株NP编码基因高效原核表达系统.所建立的rNP-IgM直接捕捉ELISA可作为HFRS简便、安全、敏感、特异的血清学诊断新方法. |
| English Abstract: |
| Objective To clone the gene encoding nucleocapsid protein(NP) of hantavirus strain Z10(HV-Z10),to construct its prokaryotic expression system as well as to establish a rNP-IgM direct capture ELISA based on HRP-labeled recombinant NP(rNP),in order to detect serum samples of patients suffering from hemorrhagic fever with renal syndrome(HFRS) and to evaluate the effects of detection. Methods Gene encoding NP of strain HV-Z10 was amplified by PCR and then its prokaryotic expression system pET28a-Z10N-E,coli BL21DE3 was constructed,using routine genetic engineering method.SDS- PAGE was applied to measure the expression of rNP and ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product.Western blot assay was used to determine the specific immuno-reactivity of rNP while HRP-labeled rNP-IgM direct capture ELISA was established to detect the serum samples from 95 cases of confirmed HFRS patients.The detection effect was compared with that by routine HV-IgM indirect capture ELISA method.Results pET28a-Z10N-E,coli BL21DE3 was able to express rNP with high efficiency.The purified rNP only showed a single protein fragment in the gel after SDS-PAGE.HV-IgG could efficiently recognize rNP and hybridize with the recombinant protein.94.73% (90/95) of HFRS patients' serum samples were positively confirmed by rNP- IgM direct capture ELISA,while a positive rate of 92.63% (88/95) in the same samples was confirmed by HV-IgM indirect capture ELISA.The distributions of A450) values of the serum samples detected by the two IgM capture ELISAs as well as the changes of the A450) mean values from several serum samples with different dilutions were similar.Conclusion We successfully constructed a high efficient prokaryotic expression system of NP encoding gene of hantavirus strain HV-Z10.The rNP-IgM direct capture ELISA that established in this study could be used as a new serological test for HFRS diagnosis because of its simplicity,safety,with high sensitivity and specificity. |
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