Preparation Of Monoclonal Antibodies Against NS1 Protein Of Japanese Encephalitis Virus And Identification Of B-cell Epitopes

Japanese encephalitis virus (JEV), being a member of the family Flaviviridae, is usually infected to the human body and animal through mosquitoes and then may cause acute viral encephalitic and neurologic disease. 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.. Thus, it is necessary to develop a diagnostic method which can differential diagnosis the viruses of Japanese encephalitis virus serocomplex.Our study was Codon optimization to improve the Japanese encephalitis virus NS1protein Gene,then, Synthesized by the biotechnology company. The NS1 gene was cloned into prokaryotic expression vector pET-30 and pMAL-c5X. The results of enzyme digestion analysis and sequencing showed that the recombinant plasmid pET-30-opti-NS1 and pc5X-opti-NS1 were constructed successfully.The pET-30-opti-NS1 was transformed into E. coli BL21 competent cells. After IPTG induction, the fusion protein was purified and used to immunize BALB/c mice. After the procedure of immunization, cell fusion and selection, 16 strains of hybridoma were generated. IFA suggested that eight of these mAbs recognized native NS1 protein. The pc5X-opti-NS1 was transformed into E. coli ER2523 competent cells. After then induction, It was demonstrated by SDS-PAGE that a soluble fusion protein of 80ku was expressed. Western blot and indirect enzyme-linked immunosorbent assay (ELISA) showed that the purified NS1 fusion protein retained good antigenicity and specificity. Meanwhile, an indirect ELISA using the purified MBP-NS1 protein as antigen was developed to screen antibody-producing hybridomas.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. The GST fusion proteins were expressed after IPTG induction and screened by indirect ELISA. Results showed that NS1 were recognized by the mAbs . So we deduce that the epitope of mAb100816-10F7,100816-8B5,100910-1E8,100816-7G1,100816-4E3,100910-6D8. ELISA and Western blot indicated that ⁵AIDITRK¹¹,⁷²RDELNVL⁷⁸,²²⁷ETHTLW²³²,²⁵¹KSKHNRREGY²⁶⁰,²⁶⁹DENGIVLD²⁷⁶,³⁴¹DETTLVRS³⁴⁸ are the minimal unit of this epitopes...