坦布苏病毒NS1蛋白的表达及ELISA检测方法的建立

doi:10.11843/j.issn.0366-6964.2016.05.014

摘要/Abstract

摘要：

为了建立坦布苏病毒（TMUV）感染鸭群的抗体检测方法，以TMUV NS1的原核表达蛋白质作为包被抗原，建立了间接ELISA方法，并对该方法进行了检测条件的优化。将TMUV NS1全基因序列克隆至pET-28a(+)，利用BL21（DE3）表达NS1蛋白，纯化后其质量浓度为4.16 μg•μL^－1。通过方阵试验，确定了NS1蛋白的最佳包被质量浓度是100 ng•孔^-1，检测血清的最佳稀释倍数为40倍。随后对检测条件进行了优化，优化后的结果：抗原的最佳包被条件是4 ℃作用过夜；酶标二抗的最佳工作条件是稀释5 000倍，37 ℃作用1 h；阴阳血清的临界值为0.278。一系列试验验证了该检测方法具有很强的特异性、敏感性、重复性。对采集自山东各地的80份血清样品进行检测，其检测结果显示，该方法与经典的鸡胚中和试验检测结果的符合率为93.5%以上，且比传统的中和试验更敏感。对TMUV感染鸭群的血清进行检测，描述了TMUV感染后鸭群抗体水平的消长规律，为该病的防治提供重要的理论依据。以NS1蛋白为包被抗原的间接ELISA方法的建立为临床上TMUV的感染提供了一种新的抗体检测方法，尤其是在TMUV早期感染的检测方面有着更为广泛的应用前景。

关键词: 鸭坦布苏病毒, NS1蛋白, 间接ELISA, 检测方法

Abstract:

In order to establish a detection method about the antibodies to Tembusu virus (TMUV)，TMUV NS1 prokaryotic expression protein was used as a coating antigen and an indirect ELISA method was established and optimized.TMUV NS1 gene sequence was cloned into pET-28a (+) and the recombinant expression vector PET-28-NS1 was developed.pET-28-NS1 was transformed into BL21 (DE3) and the fusion protein NS1 was successfully expressed.The NS1 protein was purified with different concentrations of urea and the concentration of NS1 protein was 4.16 μg•μL^-1.According to phalanx test，the coating concentration of NS1 protein was 100 ng•cell^-1 and the detected serum was diluted by 40 times.The detection conditions were optimized and the optimized conditions were as follows.The best incubation condition was overnight at 4 ℃；the secondary antibody was diluted by 5 000 times and incubated for 1 h at 37 ℃；the critical value of serum was 0.278.It was verified by a series of tests that the ELISA method based on NS1 protein had a higher specificity，sensitivity and reproducibility.Eighty serum samples collected from Shandong province were detected.The coincidence was above 93.5% between the results of ELISA and conventional neutralization test.Serums in ducks infected with TMUV were detected by the ELISA assay and dynamic changes of antibody levels were described.Prevention and control measures of this disease can be developed according to antibody characteristics.A new method for detecting antibodies to TMUV is developed and will provide wide use in the early detection of TMUV.

Key words: duck Tembusu virus, NS1 protein, indirect ELISA, detection methods

中图分类号:

S858.325.3

提金凤，李志杰，李秀丽，张敏敏，张媛媛，刁有祥. 坦布苏病毒NS1蛋白的表达及ELISA检测方法的建立[J]. 畜牧兽医学报, 2016, 47(5): 970-977.

TI Jin-feng，LI Zhi-jie，LI Xiu-li，ZHANG Min-min，ZHANG Yuan-yuan，DIAO You-xiang. Expression of NS1 Protein of Tembusu Virus and Development of Indirect ELISA Assay[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(5): 970-977.

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