Preparation And Application Of The Monoclonal Antibodies Against M And L Protein Of Rabies Virus

Rabies virus(RABV),the causative agent of rabies,ishighly neurovirulent for warm-blooded animals with a mortality rate of up to 100%.The RABV matrix protein(M)is required for virus particle assembly and budding and large protein(L)plays an important role in regulating the transcription and replication of viral genome.The study of antigenic epitopes is not onlyhelpful to the basic immunology research,but also important to the development of new epitope vaccine design and diagnostic reagents.At present,double antibody sandwich ELISA is often used for quantitative detection of RABV,Therefore,the development of a new RABV double antibody sandwich ELISA is of great significance for the clinical diagnosis of RABV.In this study,five monoclonal antibodies(MAbs),designated 3B9?4A1?2B11?2C1 and 4B11,against the RABV M protein were generated using a recombinant M protein and three monoclonal antibodies(MAbs)and designated 3F3?4D10 and 5A3,against the RABV L protein were generated using a recombinant L protein.A linear epitope 25PPYDD30 of M protein was identified.A double antibody sandwich ELISA method for detecting RABV based on M protein was established based on the M monoclonal antibody.It not only provides a powerful tool for the clinical diagnosis of RABV,but also lays a foundation for the further study of the related functions of M protein.The research contents were as follows.1.Prokaryotic expression and identification of rabies virus M proteinM gene was amplified by PCR using reverse transcription product of laboratory-preserved rabies virus CVS-11 strain as the template and cloned into pET-28a(+)expression vector.The correctly sequenced positive recombinant plasmid pET-28a-M was transformed into E.coli BL-21(DE3)competent cells.Prokaryotic expression of M protein was achieved after induction by lmM IPTG.The molecular weight of the recombinant M protein was about 27kDa,and proteins mainly exist in the form of inclusion bodies.Western blotting showed that the recombinant M protein could react with anti-His monoclonal antibody.The results showed that the M protein of rabies virus CVS-11 was successfully expressed in E.coli prokaryotic expression system.2.Establishment of thehybridoma secreting monoclonal antibodies against RABV M proteinBALB/c mice were immuned with the purified recombinanthis-M protein mice.Splenocytes of the mouse were with thehighest serum antibody titre collected and fused with mouse myeloma cells(SP2/0).Indirect ELISA and indirect immunofluorescence assays(IFA)were used for screening positivehybridomas.The positive clones were sub-cloned by limiting dilution for three more times and further characterized by IFA.Finally,fivehybridomas named 3B9,4A1,2B11,2C1 and 4B11 producing mAbs were generated.The isotype of MAbs was identified and 3B9,4A1 and 4B11 MAbs were subclass IgG2bK;2B11 was subclass IgG2aK and 2C1 was subclass IgA?.Positivehybridomas were used to immunize the BALB/c mice to produce ascites,the titer of ascites was 105-108.All MAbs can react with the RABV M protein by western blotting and IFA.In addition,all five MAbs reacted with the CVS-11 and WT-RABV but showed no reactivity against the HEP-Flury strain in indirect immunofluorescence and western blotting,suggesting that the antigenic epitopes identified by these five MAbs might be different in different strains.The monoclonal antibodies generated in our study laid a foundation for the detection of rabies virus and the analysis of antigen epitopes.3.Identification of B cell epitopes of Rabies Virus M ProteinFive truncated M gene fragments were predicted by online prediction software and amplified using the reverse transcription products of rabies virus CVS-11 strain as template.Then five truncated M gene fragments were cloned into pET-3 2a(+)vector and transformed into E.coli BL21(DE3)for recombinant truncated proteins expression.The range of B cell epitopes recognized by five monoclonal antibodies was determined to 23SAPPYDDLW32 by western blotting.Then 23SAPPYDDLW32 was truncated one by one residue from both ends,cloned into the pGEX-4T-1 vector and expressed in E.coli BL21(DE3).Truncated proteins with GST tag were tested by western blotting,and finally the fine epitope of M protein was located to 25PPYDDDD30.The 25PPYDDDD30 was cloned into pCMV-Flag vector and can be recognized by monoclonal antibodies after transfection by IFA.In addition,alignment of amino acid sequences and phylogenetic analysis of different RABV strains indicated that the variable epitope 25PPDGDD30 is only present in the HEP-Flury and variant Flury strains of clade III,while Y27D mutation within the epitope was found among the RABV strains distributed in three clades.however,a single mutation eliminated the reactivity of these five mAbs.The discovery of this epitope enriches the antigen epitope map of rabies virus and provides a basis for the study of the function of M protein.4.Establishment of the monoclonal antibody sandwich ELISA for detecting infectious bursal disease virusThe purified monoclonal antibody 3B9 was labeled withhorseradish peroxidase ashRP antibody and the purified monoclonal antibody 4A1 was coated,then double antibody sandwich ELISA was establishment for detecting RABV.The optimum reaction conditions were as follows:the coating concentration of monoclonal antibody 4A1 was 2 ug/mL,overnight at 4? was used as the coating condition,10%calf serum 37? was for blocking condition,the dilution of enzyme-labeled monoclonal antibody was 1:4000,and the reaction time of enzyme-labeled monoclonal antibody was lhour.The results showed that double antibody sandwich ELISAhad no cross-reaction with canine parvovirus and canine influenza virus.The lowest detection limit was 102 TCID50.The variantion coefficients of the intra-assay and inter-assay were less than 5%.The double antibody sandwich ELISA was specific,sensitive and stable and it has potential application for diagnosis and detecting RABV.5.Establishment of thehybridoma secreting monoclonal antibodies against RABV L proteinThe L gene was amplified by PCR using reverse transcription product of laboratory-preserved rabies virus CVS-11 strain as the template and was cloned into pET-32a(+)expression vector,and "the prokaryotic expression of L protein was achieved after induction by lmM IPTG.The molecular weight of the recombinant L protein was about 57kDa.BALB/c mice were immunized with purified recombinant L protein.Spleen cells of mice with thehighest serum titer were fused with myeloma cells(SP2/0).hybridoma was screened by indirect ELISA and IFA.Positive clones were subcloned three times by limited dilution method.IFA was used to verify the positive clones.Finally,three MAbs,3F3?4D10 and 5A3,which secrete L protein monoclonal antibodies stably were obtained.IFA and western blotting showed that the three monoclonal antibodieshad good specificity with rabies virus CVS-11 strain.