Preparation Of Monoclonal Antibodies Against NS1’ Protein In Japanese Encephalitis Virus And Identification Of Its B-Cell Epitopes

As one of the important zoonotic pathogens, Japanese encephalitis virus can cause abortion or stillbirth in pregnant sows, death in piglets, orchitis without fertility in boars and acute encephalitis in humans, which is a severe threat to public health and husbandry. JEV is a single-stranded positive-sense RNA, belonging to Flaviviridae flavivirus. An open reading frame in the genome can encode three structural proteins and seven non-structural proteins. NS1’ protein, produced by ribosomal frameshifting, is found in early stage of viral replication and related to viral assembly and release. In this study, via hybridoma technology, four monoclonal antibodies (McAb) against JEV NS1’protein were obtained and one linear epitope was identified in NS1’protein. It provides the foundation for research and diagnosis of JEV in the future.The main contents were as following:1. Prokaryotic expression and antigenic analysis of NS1’ proteinTo express the NS1’ protein resulted from ribosomal frameshifting in Japanese encephalitis virus, the NS1’ gene was amplified by RT-PCR and cloned into a prokaryotic expression vector pET-32a. After identification of the recombinant plasmid pET-32a-NS1’, NS1’protein was expressed in E.coli BL21(DE3) cells by induced with IPTG. The fusion protein with a molecular weight of 25KDa was visible on the SDS-PAGE gel and purified with His-Bind Purification Kit. Correctness and antigenicity of expressed fusion protein were identified by Western-blotting analysis. All results indicated that the NS1’ protein has the potential to distinguish between wild strain infection and vaccine immunization in clinical practice.2. Preparation of monoclonal antibodies against NS1’ proteinTo prepare monoclonal antibodies against NS1’ protein in JEV, the synthesized peptide was used to immunize BALB/c mice. The stimulated spleen cells were fused with myeloma cells of SP2/0. Four hybridoma cell lines secreting McAbs against NS1’ protein were screened by indirect enzyme-linked immunosorbent assay (ELISA) and named as 2E10, 3B2,4G9,5E6, respectively. The titers of the four McAbs in the supernatant of cell culture were 1:10240,1:10240,1:5120 and 1:10240, respectively. Correspondingly, the titers in ascites were all 1×107. The isotype of 2E10 belongs to IgG2b/K; 3B2,4G9 and 5E6 belong to IgG1/K. Western-blotting and IFA showed that the four McAbs reacted with JEV NJ2008, but did not react with JEV SA14-14-2. The obtained McAbs laid a foundation for the further study of diagnosis and antigen epitope analysis of JEV.3. Preliminary identification of B-cell Epitopes of NS1’proteinIn this study,2 truncated NS1’ gene fragments were designed and amplified by PCR. These fragments were cloned into vector pET-32a and expressed in E.coli BL21 (DE3) cells. Moreover, we synthesized six peptides. Identification of ELISA and Western-blotting indicated that four McAbs (2E10,3B2,4G9,5E6) recognized the linear epitope 10GHPGGPSQEVDG21. The result might promote the future investigation into the antigen structure and function of NS1’ and facilitate the development of diagnostic methods for JEV infection.In summary, the obtained McAbs laid a foundation for the epidemiological surveys and diagnosis of JEV. Moreover, the epitope 10-21aa will facilitate future investigation of function and structure of NS1’ protein.