Preparation Of Monoclonal Antibodies Against NS1 Protein Of West Nile Virus And Identification Of B-cell Epitopes

West Nile Fever (WNF) is an acute zoonoses caused by West Nile virus (WNV). WNV belongs to the family Flaviviridae, genus Flavivirus, and it has been serologically classified into the Japanese encephalitis virus serocomplex, which includes Japanese encephalitis virus (JEV), Saint-Louis encephalitis virus (SLEV) and Murray Valley encephalitis virus (MVEV). WNV is distributed with JEV, SLEV or MVEV in many regions of the world, and there is serologic cross-reactivity between them. Thus, it is necessary to develop a diagnostic method which can differential diagnosis the viruses of Japanese encephalitis virus serocomplex.For investigating the NS1 protein of West Nile virus (WNV) NY99 strain, the NS1 gene amplified by PCR was cloned into prokaryotic expression vector pMAL-c2X and donor vector pFastBac HTA of eukaryotic expression system respectively. The results of enzyme digestion analysis and sequencing showed that the recombinant plasmid pc2X-NS1 and pFast-NS1 were constructed successfully.The pc2X-NS1 was transformed into E. coli TB1 competent cells, then induction was exerted on TB1 with IPTG at 16℃. It was demonstrated by SDS-PAGE that a soluble fusion protein of 90 ku was expressed. NS1 fusion protein was purified by the Amylose Resin Column. Western blot and indirect enzyme-linked immunosorbent assay (ELISA) showed that the purified NS1 fusion protein retained good antigenicity and specificity.The recombinant donor vector pFast-NS1 was transformed into DH10Bac competent cells which contained Bacmid. Through the homologous recombination between donor vector and Bacmid, a recombinant Bacmid Bac-NS1 containing the interest gene was obtained. The Bac-NS1 was transfected into Sf9 insect cell, then the recombinant Baculovirus BacV-NS1 was generated. SDS-PAGE analysis indicated that Sf9 insect cells infected with BacV-NS1 would express NS1 fusion protein as inclusion body form. The inclusion body was degenerated and dissolved by 8M urea, then it was purified with the Ni+NTA column including His-Bind Resin. Western blot analysis showed that the purified NS1 fusion protein retained good antigenicity.For NS1 protein McAb preparation, BALB/c mice were immunized intraperitoneally with the purified NS1 protein expressed by prokaryotic expression system. Then the spleen cells of immunized mouse were fused with SP2/0 myeloma cells by the PEG regent. Meanwhile, an indirect ELISA using the purified NS1 protein derived from eukaryotic expression system as antigen was developed to screen antibody-producing hybridomas. Consequently, four McAbs, named as WN-1C10, WN-3D10, WN-3C7 and WN-3F1, were prepared. Western blot analysis showed that WN-1C10, WN-3D10 and WN-3C7 were able to react with WNV-NS1 protein specifically, and the three McAbs would not react with JEV-NS1 protein. However, WN-3F1 was able to react with both of them.In order to identify the B-cell epitope, two consensus sequences, YDEGR & DRYKYY, were screened from a random 12-peptide library by biopanning using WN-1C10 and WN-3D10 respectively. The motifs showed high similarity with the two amino acid sequence 268WDEGR272 and 30DRYKYY35 of WNV-NS1 protein. Then a series of short peptides were synthetized according to the theory of peptide screening. Using Western blot and indirect ELISA analysis, two linear B-cell epitopes on WNV-NS1 protein,which are 265QGPWDEGRVE274 and 27AWMDRYKYYP36, were identified finally.The above McAbs and B-cell epitopes are helpful for further structural and functional research on WNV-NS1 protein and development of differential diagnosis for WNV from the Japanese encephalitis virus serocomplex.