Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies

PLOS ONE, Dec 2019

This study describes the identification of one linear B-cell epitope on TMUV NS1 protein with monoclonal antibody (mAb) 3G2 by indirect enzyme-linked immunosorbent assay (ELISA). In this study, NS1 protein was expressed in prokaryotic expression system and purified. One mAb against NS1 protein was generated from Balb/c mice immunized with recombinant protein NS1. A set of 35 partially-overlapping polypeptides covering the entire NS1 protein was expressed with PGEX-6P-1 vector and screened with mAb 3G2. One polypeptide against the mAb was acquired and identified by indirect ELISA and western-blot. To map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. A series of truncated oligopeptides were expressed and purified. The minimal determinant of the linear B cell epitope was recognized and identified with mAb 3G2. The accurate linear B-cell epitope was 269DEKEIV274 located in NS1 protein. Furthermore, sequence alignment showed that the epitope was highly conserved and specific among TMUV strains and other flavivirus respectively. The linear B-cell epitope of TMUV NS1 protein could benefit the development of new vaccines and diagnostic assays.

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Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies

July Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies Jinfeng Ti 0 1 Zhijie Li 1 Xiuli Li 0 1 Yunjian Lu 0 1 Youxiang Diao 0 1 Fang Li 1 0 Zoology Institute, Shan Dong Agricultural University , Shan Dong province, Tai'an, China , 2 Shandong Vocational Animal Science and Veterinary College , Shan Dong province, Weifang , China 1 Editor: Yongchang Cao, Sun Yat-Sen University , CHINA This study describes the identification of one linear B-cell epitope on TMUV NS1 protein with monoclonal antibody (mAb) 3G2 by indirect enzyme-linked immunosorbent assay (ELISA). In this study, NS1 protein was expressed in prokaryotic expression system and purified. One mAb against NS1 protein was generated from Balb/c mice immunized with recombinant protein NS1. A set of 35 partially-overlapping polypeptides covering the entire NS1 protein was expressed with PGEX-6P-1 vector and screened with mAb 3G2. One polypeptide against the mAb was acquired and identified by indirect ELISA and western-blot. To map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. A series of truncated oligopeptides were expressed and purified. The minimal determinant of the linear B cell epitope was recognized and identified with mAb 3G2. The accurate linear B-cell epitope was 269DEKEIV274 located in NS1 protein. Furthermore, sequence alignment showed that the epitope was highly conserved and specific among TMUV strains and other flavivirus respectively. The linear B-cell epitope of TMUV NS1 protein could benefit the development of new vaccines and diagnostic assays. - Data Availability Statement: All relevant data are within the paper. Introduction Tembusu virus infection in ducks is caused by Tembusu virus (TMUV), which was first reported in April 2010 in China[ 1 ]. The virus can infect more varieties of ducks such as Beijing duck, golden duck, Shaoxing duck, Cherry Valley, Campbell ducks, Jinyun duck, etc. Laying ducks infected mainly demonstrated drops in egg production, follicular rupture and bleeding, and yolk peritonitis. Ducklings mainly displayed standing instability and paralysis, retarded growth, with 10 to 30% mortality rates [ 2, 3 ]. Other birds such as Chickens, geese, sparrows, etc were also infected and displayed obvious clinical signs [4±6]. So far, the disease has resulted in a great economic loss to the poultry industry and caused wide public concern. There is no specific treatment available for TMUV and the vaccination is an effective way to prevent TMUV infection in waterfowl. The inactivated vaccines and live attenuated vaccines against Shandong Agriculture Significant Application and Technology Innovation Project of 2016 to Youxiang Diao, and the Shandong Science and Technology Plan Program of College and University (J16LF58) to Dr. Jinfeng Ti. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. TMUV have been successfully developed and already used in clinical production[ 7, 8 ]. But the live attenuated vaccines could display disadvantages of reversion of virulence and spread, and the inactivated vaccines didn't display an effective cellular immunity. So the development of a new type of vaccine is very urgent. TMUV is a mosquito-borne flavivirus which belongs to the Ntaya virus group within flavivirus genus, flaviviridae family [ 9 ]. TMUV genome encodes a single polyprotein which is cleaved into three structural proteins (C, prM and E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) [ 3 ]. Among them, NS1 protein is a glycoprotein which is present in cell-surface or in the form of cell-associated protein [ 10 ]. NS1 protein possesses several unusual and interesting performances which is closely related to the membrane function and indispensable in the early viral replication, assembly and release of the virus [ 11 ]. NS1 protein contains multiple protective T-cell and B-cell epitopes which can induce both humoral and cell-mediated immunity without the risk of antibody-dependent enhancement [ 12, 13 ]. The B-cell epitopes mainly refer to multiple continuous amino acid residues on the protein surface or the spatial conformation of discontinuous amino acid residues. Accurate analysis of the epitopes on NS1 protein is critical for further understanding the mechanism of NS1-mediated immune protection. In recent years, epitope-based marker vaccines have increasingly attracted wide attentions in public [14]. The identification of linear epitopes on NS1 protein would be conducive to the development of epitope-based marker and subunit vaccines, preparation of protective antibodies and understanding protein functions [ 15 ]. It has been demonstrated that NS1 protein of flavivirus has more virus-specific epitopes than cross-reactive ones in contrast to E protein which has more cro (...truncated)


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Jinfeng Ti, Zhijie Li, Xiuli Li, Yunjian Lu, Youxiang Diao, Fang Li. Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies, PLOS ONE, 2017, Volume 12, Issue 7, DOI: 10.1371/journal.pone.0181177