Prokaryotic Expression of NS3 Protein of Duck tembusu virus and Establishment and Application of Its Indirect ELISA Detection Method
AN Er-Li1,2,3,4, JI Xin-Qin1,2,*, RUAN Yong1,3,4,*, CHEN Ze-Hui1,2,3,4, CAO Wei1,2,3,4, WANG Han-Han1,2,3,4, LUO Xiao-Yu1,2,3,4, LONG Dan-Dan1, CHEN Jia-Qi1,2, YANG Chun-Pei5, WU Zong-Hao5, WANG Li-Juan5, YAO Bi-Qiong5
1 Faculty of Animal Sciences, GuiZhou University, Guiyang 550025, China; 2 Guizhou Institute of Animal Disease, GuiZhou University, Guiyang 550025, China; 3 Key Laboratory of Plateau Mountain Animal Genetics, Breeding and Reproduction, Ministry of Education, Guizhou University, Guiyang 550025, China; 4 Key Laboratory of Animal Genetics, Breeding and Reproduction, Guiyang 550025, China; 5 County Animal Disease Prevention and Control Center, Sansui 556500, China
Abstract To develop an indirect ELISA method for rapid detection of Duck tembusu virus (DTMUV) antibodies, in this study, the prokaryotic recombinant expression vector pET-28a-NS3 was constructed, and NS3 protein obtained through induced expression was used as the coated antigen. After optimizing a series of conditions, an indirect ELISA method for detecting DTMUV antibodies was established. Antibody analysis was performed on 30 serum samples for challenge test and 150 collected serum samples by the established ELISA method. The results showed that the intra-assay coefficient of variation and the coefficient of variation (CV) of the indirect ELISA method was 1.49%~4.31% and 1.22%~6.07%, respectively, which met the national standard. DTMUV antibody could be specifically detected by this method. And the indirect ELISA method had no cross-reaction with Avian influenza virus (AIV) H5 subtype, AIV H7 subtype, Newcastle disease virus (NDV), Duck Circovirus (DuCV) and Duck hepatitis A virus (DHAV-1) positive sera. The coincidence rate of positive and negative detection numbers of challenge test samples and commercial kit was 100%, and the coincidence rate of positive and negative detection numbers of farm samples and commercial kit was 97.33%. In summary, the DTMUV NS3 protein antibody indirect ELISA method established in this study has good specificity and repeatability, and provides a rapid and effective detection method for the investigation of DTMUV epidemiological situation.
AN Er-Li,JI Xin-Qin,RUAN Yong, et al. Prokaryotic Expression of NS3 Protein of Duck tembusu virus and Establishment and Application of Its Indirect ELISA Detection Method[J]. 农业生物技术学报, 2024, 32(10): 2437-2446.
[1] 董嘉文, 李林林, 孙敏华, 等. 2017. 鸭坦布苏病毒JM株E蛋白的截断表达及间接ELISA方法的建立[J]. 中国动物传染病学报, 25(01): 12-18. (Dong J W, LI L L, Sun M H, et al.2017. Truncated expression of E protein of duck Tambusu virus JM strain and establishment of indirect ELISA method[J]. Chinese Journal of Animal Infectious Diseases, 25(01): 12-18.) [2] 姬希文, 闫丽萍, 颜丕熙, 等. 2011鸭坦布苏病毒抗体间接ELISA检测方法的建立[J]. 中国预防兽医学报, (08): 630-634. (Ji X W, Yan L P, Yan P X, et al. 2011. Establishment of indirect ELISA method for detection of Tambusu virus antibody in duck[J]. Chinese Journal of Preventive Veterinary Medicine, (08): 630-634.) [3] 林霄, 贺煜, 程安春, 等. 2020. 鸭坦布苏病毒NS3蛋白的原核表达及其多克隆抗血清的制备[J]. 四川农业大学学报, 38(05): 619-623. (Lin X, He Y, Cheng A C, et al.2020. Prokaryotic expression of duck Tambuso virus NS3 protein and preparation of polyclonal antiserum[J]. Journal of Sichuan Agricultural University, 38(5): 619-623.) [4] 苏观志. 2018. 鸭坦布苏病毒NS1蛋白的原核表达及其间接ELISA检测方法的建立[D]. 硕士学位论文, 安徽农业大学, 导师: 王桂军, 于圣青, pp. 12-31. (Su G Z.2018. Prokaryotic expression of duck Tambuso virus NS1 protein and establishment of indirect ELISA detection method[D]. Thesis for M. S., Anhui Agricultural University, Supervisor: Wang G J, Yu S Q, pp. 12-31.) [5] Assenberg R, Mastrangelo E, Walter T S, et al.2009. Crystal structure of a novelconformational state of the flavivirus NS3 protein: Implications for polyproteinprocessing and viral replication[J]. Journal of Virology, 83: 12895-12906. [6] Aydin S.2015. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA[J]. Peptides, 72: 4-15. [7] Chua J J, Ng M M, Chow V T.2004. The non-structural 3 (NS3) protein of dengue virus type 2 interacts with human nuclear receptor binding protein and is associated with alterationsin membrane structure[J]. Virus Research, 102(2): 151-163 [8] Clum S, Ebner K E, Padmanabhan R.1997. Cotranslational membrane insertion of theserine proteinase precursor NS2B-NS3 (Pro) of dengue virus type 2 is requiredfor efficient in vitro processing and is mediated through the hydrophobic regions of NS2B[J]. The Journal of Biological Chemistry, 272: 30715-30723. [9] Du Pont K E, McCullagh M, Geiss B J.2022. Conserved motifs in the flavivirus NS3 RNA helicase enzyme[J]. Wiley Interdiscip Review RNA, 13(2): e1688. [10] Gebhard L G, Kaufman S B, Gamarnik A V.2012. Novel ATP-independent RNA annealingactivity of the dengue virus NS3 helicase[J]. PLOS ONE, 7(4): e36244. [11] Homonnay Z G, Kovács E W, Bányai K, et al.2014. Tembusu-like flavivirus (Perak virus) as the cause of neurological disease outbreaks in young Pekin ducks[J]. Avian Pathology, 43(6): 552-560. [12] Kono Y, Tsukamoto K, Abd Hamid M, et al.2000. Encephalitis and retarded growth of chicks caused by Sitiawan virus, a new isolate belonging to the genus Flavivirus[J]. The American Journal of Tropical Medicine and Hygiene, 63(1-2): 94-101. [13] Leung D, Schroder K, White H, et al.2001. Activity of recombinant dengue 2 Virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors[J]. The Journal of Biological Chemistry, 276: 45762-45771. [14] Liu H J, Kuo L C, Hu Y C, et al.2002. Development of an ELISA for detection of antibodies to avian reovirus in chickens[J]. Journal of Virological methods[J]. 102(1-2): 129-38 [15] Li K, Phoo W W, Luo D.2014. Functional interplay among the flavivirus NS3 protease. helicase, and cofactors[J].Virologica Sinica, 29(2): 74-85. [16] Li L, Li H, Tian Q, et al.2022. Expression and purification of soluble recombinant β-lactamases using Escherichia coli as expression host and pET-28a as cloning vector[J]. Micobial Cell Factories, 21(1): 244. [17] Luo D, Vasudevan S G, Lescar J.2015. The flavivirus NS2B-NS3 protease-helicase as a target for antiviral drug development[J]. Antiviral Research, 118: 148-158. [18] Morozova O V, Maksimova T G, Bakhvalova V N.1999. Tick-borne encephalitis virus NS3 gene expression does not protect mice from homologous viral challenge[J]. Viral Immunol, 12(4): 277-80. [19] Platt G S, Way H J, Bowen E T.1975. Arbovirus infections in Sarawak, October 1968-February 1970 Tembusu and Sindbis virus isolations from mosquitoes[J]. Annals Tropical Medicine Parasitology, 69(1): 65-71. [20] Simmons M, Porter K R, Hayes C G, et al.2006. Characterization of antibody responses to combinations of a Dengue virus type 2 DNA vaccine and two Dengue virus type 2 protein vaccines in Rhesus macaques[J]. Joural Virology, 80(19): 9577-9585. [21] Tabatabaei M S, Ahmed M.2022. Enzyme-linked immunosorbent assay (ELISA)[J]. Methods in Molecular Biology, 2508: 115-134. [22] Thontiravong A, Ninvilai P, Tunterak W, et al.2015. Tembusu-related flavivirus in ducks, Thailand[J]. Emerging Infectious Diseases, 21(12): 2164-2167. [23] Wang Y, Zhang S, Tang Y, et al.2019. Screening of Duck tembusu virus NS3 interacting host proteins and identification of its specific interplay domains[J]. Viruses, 11(8): 740. [24] Yang S H, Zhao S J, Liu J, et al.2008. Expression of plant monellin in Escherichia coli induced by lactose[J].China Biology, 28(3): 53-58. [25] Zhao J, Zhang T, Chen G, et al.2022. Non-structural protein 3 of duck Tembusu virus induces autophagy via the ERK and PI3K-AKT-mTOR signaling pathways[J]. Frontiers Immunology, 13: 746890. [26] Zhong J, Guan J, Zhou Y, et al.2019. Genomic characterization of two Orf virus isolates from Jilin province in China[J]. Virus Genes, 55(4): 490-501.
