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禽流感野毒感染禽与疫苗免疫禽NS1-ELISA
鉴别方法的建立
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孙进华1，2，张雅为1 ，李士平2，王君伟1*
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（1.东北农业大学 动物医学院，黑龙江 哈尔滨150030；
2.东北农业大学 动物科技学院，黑龙江 哈尔滨150030）
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摘要：
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分别构建了重组禽流感病毒H9N2亚型、H5N1亚型NS1基因原核表达菌株，IPTG诱导表达，表达的蛋白用Ni+-NTA Agarose纯化，Western-blotting检测两种蛋白的活性，并进行交叉反应检测；以H5N1亚型禽流感病毒NS1蛋白为包被抗原，建立了间接ELISA检测方法，并优化各项反应条件。高效表达了H9N2和H5N1亚型NS1蛋白，融合蛋白的分子质量均为30ku左右；Western-blotting检测均有特异性条带，并且存在明显的交叉反应。选择重组的H5N1亚型NS1蛋白建立了间接ELISA检测方法，最佳抗原包被浓度为1.325μg/mL，血清稀释度为1∶400。结果表明，以H5N1亚型禽流感病毒NS1蛋白为包被抗原的ELISA检测方法可区分禽流感病毒感染禽与疫苗免疫禽。 |
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关键词：
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禽流感病毒；非结构蛋白；ELISA |
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中图分类号：S 855.3 文献标识码：
A 文章编号：1673-4696(2008)05-0412-05 |
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Establishment of an NS1-ELISA for differentiating poultry infected
from those vaccinated |
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SUN Jin-hua1,2 ，ZHANG Ya-wei1，LI
Shi-ping2，WANG Jun-wei1 |
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（1.College of Veterinary Medicine，Northeast Agricultural University,Harbin 150030，China；
2.College of Animal Science and Technology，Northeast Agricultural University，Harbin 150030，China） |
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Abstract: The prokaryotic expression strains expressing AIV recombinant NS1 genes of H5N1 and H9N2 subtype was constructed.The recombinant strains were induced for different time and at different concentrations of IPTG.The expressed proteins were detected by SDS-PAGE and Western-blotting tests.H5N1-NS1 protein was purified by Ni+-NTA Agarose and used to establish an indirect ELISA.The fusion protein was specifically expressed.The SDS-PAGE test indicated that the molecular mass of the fusion protein was approximately 30ku in mass as expected.The expressed proteins could react with the polyclonal antibodies in Western-blotting test and the cross reaction existed between H5N1 NS1 and H9N2 NS1 protein.An indirect ELISA was developed by using the purified H5N1-NS1 fusion protein as the coating antigen.The optimal working concentration of NS1 protein was 1.325μg/mL and the dilution of serum sample was 1∶400 in the ELISA.The results showed that the developed indirect NS1-ELISA was sensitive and specific for differentiating infected poultry from those vaccinated. |
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Key words: avian influenza virus；nonstructural protein；ELISA |
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