坦布苏病毒NS1蛋白的表达及ELISA检测方法的建立

Expression of NS1 Protein of Tembusu Virus and Development of Indirect ELISA Assay

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4.16μg·μL^(-1)。通过方阵试验,确定了NS1蛋白的最佳包被质量浓度是100ng·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1h;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。

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 indi- rect ELISA method was established and optimized. TMUV NS1 gene sequence was cloned into pET-28a （q-） 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 pro- tein 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 op- timized and the optimized conditions were as follows. The best incubation condition was overnight at 4 ＊C ;the secondary antibody was diluted by 5 000 times and incubated for 1 h at 37℃ ;the criti- cal 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 collect- ed from Shandong province were detected. The coincidence was above 93.5 % between the resultsof ELISA and conventional neutralization test. Serums in ducks infected with TMUV were detec- ted 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 de- tection of TMUV.