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非洲马瘟病毒VP7基因拼接、表达及重组ELISA方法的建立与初步应用 被引量:11

Development and Application of the Recombinant ELISA Based on the Assembling and Expression of VP7 Gene of African Sickness Virus
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摘要 采用人工拼接的方式拼接了非洲马瘟病毒(AHSV)含有绝大多数线性抗原表位的VP7编码基因片段,克隆于pET-30a构建重组质粒pET-30a-VP7,将pET-30a-VP7转化BL21(DE3),经1.0 mmol/L IPTG诱导,外源基因以包涵体的形式获得高效表达。通过Dot-ELISA以及ELISA试验证明表达产物具有良好的反应原性。以纯化后表达产物作为诊断抗原包被酶标板建立了检测AHSV抗体的间接ELISA方法。结果表明,抗原的最佳包被浓度为0.25μg/mL,血清的最佳稀释度为1∶40,待检血清阳性临界值初步定为0.25。用此方法和商品化ELISA试剂盒检测了184份血清样品,结果完全符合。 A DNA fragment, which encodes the most linear epitopes of VP7 of African sickness virus(AHSV), was assembled artificially, then cloned into pET-30a. Target protein expressed at very high level as inclusion body after the recombinant pET-30a-VP7 plasmid was transformed into BL21(DE3), and induced with 1.0 mmol/L IPTG. The indirect ELISA for detecting AHSV VP7 protein antibody was established after the reaction activity of the recombinant protein was determined by Dot-ELISA and ELISA. The results obtained indicated that the optimum antigen concentration was 0.25μg/mL, and optimum serum dilution was 1 : 40. The cut-off value was determined as 0. 25. 184 serum samples were detected by this method and commercial ELISA kit and the results were all negative.
出处 《畜牧兽医学报》 CAS CSCD 北大核心 2008年第11期1548-1553,共6页 ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金 奥运科技(2008)行动计划专项(2004BA904B06)
关键词 非洲马瘟 基因拼接 重组ELISA african horse sickness gene assembling recombinant ELISA
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参考文献7

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