| Japanese encephalitis (JE), a serious zoonosis, is widely distributed in most of East and South-east Asia and partly in Oceania. JE is caused by Japanese encephalitis virus (JEV), the NS1 protein of JEV play roles in viral RNA replication and is able to induce protective immunity without neutralization antibody, but those definite mechanisms are less clear. Except JEV, the Japanese encephalitis virus serocomplex of the family Flaviviridae includes West Nile virus (WNV), Saint Louis encephalitis virus (SLEV) and Murray Valley encephalitis virus (MVEV). These viruses have a similar ecology, it is very common that two or more of these flaviviruses co-circulate in some regions of the world, the initial symptoms of most of these viral infections are similar to each other. Although the plaque reduction neutralization test (PRNT), virus isolation and reverse transcription-polymerase chain reaction (RT-PCR) are generally conducted to achieve laboratory diagnosis, these methods are time-consuming or involve manipulation of live virus which requires high level of biosafety laboratory, and not amenable to testing large numbers of specimen. Hence the development of a new serodiagnosis method is necessary.In this study, the NS1 gene of JEV SA14-14-2 strain was cloned to pET-30 vector and transformed into Escherichia coli BL21 cells for expression. After IPTG induction, the fusion protein was purified and used to immunize BALB/c mice. After the procedure of immunization, cell fusion and selection, 4 strains of hybridoma were generated. IFA suggested that three of these mAbs recognized native NS1 protein. Then a library of 51 peptides spanning the entire NS1 protein was designed for the purpose of mapping epitopes broadly. Each peptide was 16 amino-acid in length and adjacent peptides had 8 amino-acid residues in common; To express these polypeptides, we synthesized complementary oligonucleotide pairs encoding each peptide, a BamHI site and XhoI site were added to the 5' and 3' ends of coding sequence, respectively. Then the complementary oligonucleotide pairs were annealed and cloned into the BamH I and Xho I sites of pGEX-6p-1. The GST fusion proteins were expressed after IPTG induction and screened by indirect ELISA. Results showed that NS1-18 and NS1-19 were recognized by the mAbs 1H6 equally whereas others were not. So we deduce that the epitope of mAb 1H6 locates in the overlapping domain of NS1-18 and NS1-19. This region and 10 peptides with deletions were expressed to identify the epitope precisely. ELISA and Western blot indicated that 146EHRAW150 is the minimal unit of this epitope, and it can be recognized by positive serum from pigs.To investigate the homology of the epitope among flavivirus, sequence alignment of the epitope and other peptides from the homologous region of NS1 protein of 39 JEV strains was completed. It was found that this linear epitope is totally conserved among these JEV strains. Then other 162 flavivirus strains, including the members of JE serocomplex of WNV. SLEV and MVEV and another three antigenically related flavivirus, Dengue virus(DENV, type 1～4), Yellow Fever virus (YFV) and Tick-borne encephalitis virus (TBEV) were selected for alignment analysis. Alignment result showed that this epitope is less conserved among other flavivirus strains. According to the alignment result, 12 clones represent the same domains as this JEV-NS1 epitope on other flavivirus NS1 protein were obtained and screened with Western bolt, it was found that all the homologous sequences of other 162 flavivirus strains were not reactive with mAb 1H6. The data obtain in current study indicated that this epitope is a JEV-specific epitope.This is the first time to identify a virus-specific epitope on NS1 protein of JEV. It is valuable for the development of a convenient, efficient and large amount samples amenable differential diagnosis method. And also provides useful information for the research of a new sub-unit vaccine against JE and the study of the virus-antibody interactions at a molecular level. |
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