Preparation And Identification Of Monoelonal Antibodies Against Nucleoprotein Of Four Subtypes Ebolavirus

Objective:To generete the monoclonal antibodies against artificial synt-hetic peptide derived from Ebolavirus nucleoprotein domain of Zaire,Sudan,Ta?Forest and Reston by Hybridoma technology,the mAbs may lay foundation for the establishment of EBOV rapid detection.Methods:The amino acid sequences of ZEBOV-NP,RESTV-NP,TAFV-NP and SUDV-NP were analyzed by DNAstar protean and BLAST to acquire the specific peptides.The synthesized peptides were linked with KLH,and inoculated into BALB/c mice.then the Spleen cells of immunized BALB/c mice were fused with SP2/0 myeloma cell.the Positive hybridoma cell lines were harvested using indirect ELISA and cloned by limited dilution.the recombinant plasmids pFlag-CMV-Z-NP,pFlag-CMV-R-NP,pflag-CMV-T-NP,pflag-CMV-S-NP were construct-ed respectively and transfected into 293T cells to produce recombinant NP.The fusion proteins with Flag tag were detected by Western blot.Subsequently,the recombinant NPs obtained in the expression were used as antigen to examine antibody specificity by Western blot.Results:We obtained the specific peptides of NPs of four species Ebolavirus(ZEBOV-NP,aa611-630:YRDHSEKKELPQDEQQDQDH;SUDV-NP,aa631-644:QGSESEALPINSKK;RESTV-NP,aa630-643:TSQLNEDPDIGQSK;TAFV-NP,aa630-643:NQVSGSENTDNKPH).then,using artificial synthetic peptide as Immunogen,we obtained 12strains hybridoma secreting anti-ZNP,6 strains hybridoma secreting anti-RNP,6 strains hybridoma secreting anti-TNP and 9 strains hybridoma secreting anti-SNP by Hybridoma technology.Isotyping of 33 strains clo-nes revealed Most of them were IgG1,apart from 4 strains belonged to Ig M,3 strains belonged to IgG2a and 5 strains belonged to IgG2b.The titer of aseites were between 1:10~5~1:10~6.The double enzyme digestion and DNA sequencing showed that The recombinant plasmids pFlag-CMV-Z-NP、pFlag-CMV-R-NP、pflag-CMV-T-NP、pflag-CMV-S-NP were successfully constructed.the recombinant NPs(Flag-ZEBOV-NP,Flag-RESTV-NP,Flag-TAFV-NP and Flag-SUDV-NP)were successfully expressed.The Western blot analyes showed that the positive bands about100~120 kDa was detected.we us the recombinant NPs as antigen to identify specificity of mAbs by Western blot,the result showed there were three anti-ZNP mAbs(ZNP3F10,ZNP15F11,ZNP14E2),six anti-RNP mAbs(RNP9E5,RNP10C12,RNP15B12,RNP2A10,RNP7E7,RNP10F9),six anti-TNP mAbs(TNP1H3,TNP4B10,TNP10C1,TNP2-A10,TNP8D3,TNP8G9)and four anti-SNP mAbs(SNP11G9,SNP1G3,SNP12F12,SNP3F12)can specifically reacted with recombinant NPs of ZEBOV,RESTV,TAFV and SUDV respectively and had no crossreacti-vities to other 3 subtypes of EBOV-NP.Conclusion:the specified mAbs of anti-ZNP,anti-RNP,anti-TNP and anti-SNP were produced.These mAbs may provides scientific basis for monitoring and diagnosis of EBOV.