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

Abstract

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.

CLC Number:

S858.325.3

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

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