Preparation And Characterization Of Monoclonal Antibodies To Nucleoprotein Of Ebola Virus And Marburg Virus

The nucleoprotein(NP) genes of Ebola Virus (EBOV) and Marburg Virus (MARV) (enp, mnp) were cloned into an expression vector pET-32a(+), respectively; moreover, the sequence of sbp tag was added to 3' terminus of mnp and inserted into an expression vector pET-20b(+). Subsequently, the resulting recombinant vectors pET-32a(+)-enp, pET-32a(+)-mnp, and pET-32a(+)-ms were transformed into E.coli BL21(DE3) and recombinant proteins were expressed with high efficiency after induction with IPTG, respectively. The fusion proteins EBOV-NP-Trx-His (ETH), MARV-NP-Trx-His (MTH), and MARV-NP-SBP (MS) had apparent molecular weight of about 116 kDa, 108 kDa, and 98 kDa as indicated by SDS-PAGE analyses, respectively. The Western blot analyes showed that ETH and MTH had specific reactivity with anti-His-tag monoclonal antibody (mAb), and MTH, MS had specific reactivity with anti-MNP mAb.MAbs specific to ENP and MNP were developed by fusing Sp2/0 myeloma cells and spleen cells of BALB/c mice immunized with the recombinant ETH or MTH purified by Ni-NTA colomn.Then hybridoma cell lines were harvested using indirect ELISA and cloned by limited dilution: 3G8, 4B4, and 4C12 directed to ENP; 1H4, 2G1, and 3B5 directed to MNP. All the mAbs belonged to IgG1κexcept that mAb 3G8 belonged to IgG2aκ. ELISA titers of supernatant culture and ascites of 3G8, 4B4, 4C12, 1H4, 2G1, and 3B5 were 1: 2.560×10~3, 1: 2.560×10~3, 1: 2.560×10~3, 1: 1.640×10~3, 1: 1.640×10~3, 1: 1.280×10~4and 1: 1.638×10~7, 1: 8.192×10~6, 1: 8.192×10~6, 1: 1.024×10~6, 1: 4.096×10~6, 1: 8.192×10~6, respectively. The Western blot analyses suggested that the mAbs against ENP did not show cross-reaction to MTH, and mAbs against MNP without cross-reaction to ETH; moreover, the 6 mAbs had no reactivity with His-tag and Trx-tag. The concentrations of mAbs 3G8, 4B4, 4C12, 1H4, 2G1, and 3B5 were 4.584 mg/mL, 4.822 mg/mL, 10.000 mg/mL, 0.713 mg/mL, 1.166 mg/mL, and 3.702 mg/mL after purification by octanoic acid/ammonium sulfate precipitation assay. The affinity assay indicated that the affinity constants (Kaff) of mAbs 3G8, 4B4, and 4C12 with ETH were 2.023×10~9 L/mol, 1.633×10~9 L/mol, and 1.607×10~9 L/mol, respectively; the affinity constants (Kaff) of mAbs 1H4, 2G1, and 3B5 with MTH were 1.100×10~9 L/mol, 1.235×10~9 L/mol, and 1.408×10~9 L/mol, respectively. The epitopes of the mAbs recognized was preliminaly analysis by additivity index. The results show that mAbs 3G8 and 4B4 or 4C12 may recognize different epitopes, while mAbs 4B4 and 4C12 recognized the same epitopes; moreover, mAbs 1H4, 2G1, and 3B5 recognized the same epitopes. The mAbs were prepared successfully and may lay foundations for diagnosis of EBOV and MARV.