Preparation And Identification Of Monoclonal Antibodies Specific For Chikungunya Virus Envelope 1 Protein

Chikungunya virus(CHIKV),a member of the Togaviridae Alphavirus,which is transmitted mainly through the bite of Aedes mosquitoes and causes Chikungunya fever.The main symptoms of patients including hyperpyrexia,skin rash,headache and polyarthralgia.It also can progress to joint deformities or even death in some patients.In terms of the lack of diagnostic reagents for Chikungunya virus antigen and the difficulties in vaccine development in China,this study aims to prepare the envelope 1 protein(E1 protein)of Chikungunya virus and the specific monoclonal antibodies of the El protein,and then provide the technical reserves for the establishment of antigen diagnostic methods for Chikungunya virus.The research is mainly conducted through three parts as follows.Ⅰ.Cloning and expression of recombinant Chikungunya virus El proteinAfter synthesized by Sangon Biotech(Shanghai)Co.,Ltd.,the full-length Chikungunya virus E1 gene was cloned into the prokaryotic expression vector pET-21b(+)with 6 histidine tags.CHIKV-E1 protein was expressed in the form of inclusion bodies by optimizing the expression conditions of recombinant Chikungunya virus E1 protein.And then soluble recombinant CHIKV-E1 protein was successfully obtained by optimizing the washing,purification and refolding conditions of inclusion bodies.It was confirmed that the recombinant E1 protein can react with anti-his monoclonal antibody through Western blot(Wb),and it did not cross-react with the monoclonal antibodies of those including dengue virus NS1 protein,dengue virus EDⅢ protein and West Nile virus NS1 protein used in this study.Which can be use for development of monoclonal antibodies against E1 protein of Chikungunya virus.Ⅱ.Preparation of specific monoclonal antibodies against E1 protein of Chikungunya virusOn the basis of obtaining the soluble recombinant Chikungunya virus E1 protein,Balb/c mice were immunized by injecting with the recombinant E1 protein and the whole Chikungunya virus with recombinant E1 protein alternatively.The spleen cells of immunized mice with higher antibody titer in serum were fused with myeloma cells of mice to prepare the monoclonal antibodies.First of all,the recombinant Chikungunya virus E1 protein was used as the coating antigen to screen positive clones by indirect enzyme-linked immunosorbent assay(ELISA).Next,and positive clones that were confirmed by indirect ELISA were retested by indirect immunofluorescence assay(IFA),of which the Chikungunya virus-infected cells were used as antigens.Then,for the hybridoma cell line that was retested positive,subcloning 2-3 times by the limiting dilution method.Finally,we obtained seven strains of hybridoma cell lines that can secrete specific monoclonal antibodies against Chikungunya virus E1 protein stably.Ⅲ.Characterization of monoclonal antibodies specific to the E1 protein of Chikungunya virusThe Ig subclass of seven monoclonal antibodies was identificated by the indirect ELISA,the result in which showed that there were five strains of IgGl and two strains of IgG2a.It was confirmed by the indirect ELISA and indirect IFA that there were five strains of monoclonal antibodies that have cross-reactivity with dengue virus or the N protein of the SARS-CoV-2.Two out of the seven monoclonal antibodies were specific for the E1 protein of Chikungunya virus without cross-reactivity with the yellow fever virus,Japanese encephalitis virus,nucleocapsid protein of SARS-CoV-2 and dengue virus.It was further confirmed by the Wb assay that these monoclonal antibodies specifically recognized recombinant El protein of Chikungunya virus with a molecular weight of 40～50 kDa as expected.In conclusion,the soluble recombinant E1 protein of Chikungunya virus was successfully expressed,purified and refolded.Seven strains of specific monoclonal antibodies against El protein were prepared by immunizing mice with recombinant E1 protein.Five strains of the monoclonal antibodies have different degrees of cross-reactivity with dengue virus or the nucleocapsid protein of SARS-CoV-2,and two strains of them are specific to the E1 protein of Chikungunya virus without cross-reactivity with the yellow fever virus,Japanese encephalitis virus and dengue virus,which are expected to be used in the early diagnosis of Chikungunya fever and the development of therapeutic antibodies for Chikungunya virus.