Preparation Of Monoclonal Antibody Against CHIKV Envelope Protein2(E2)

Chikungunya fever is amosquito-borne viral disease of humans, which ischaracterized by high fever, rash and polyarthralgia by Chikungunya virus infection.Most of the epitope site located in envelope protein2(E2), which is conserved indifferent genetype of CHIKV. So E2can be a biomarker of CHIKV in viremia period.Because of no effective vaccine or therapies, early diagnosis is very important forcontrolling the Chikungunya infection. Preparation of monoclonal antibody againstE2can be applied to the immune-diagonosis of CHIKV.Virus RNA of CHIKV was extracted from the serum from a Chikungunya feverpatient and the cDNA of E1and E2were obtained via reverse transcription PCR.After sequencing, recombinant plasmid pET22b-E2was successfully constructed andidentified by PCR and double digestion. Phylogenetic analysis of CHIKV on thesequence of E1, showing that the genotype of this viral strain belong toCentral/East/SouthAfrican genotype.Then recombinant plasmid pET22b-E2was transformed into E.coli BL21forrecombinant protein expression. The optimal temperature and concentration of IPTGfor induction were37℃and0.4mmol/L. SDS-PAGE and western blot showed thatthe protein were expressed in the form of inclusion body and the molecular weightwere about47kDa which is consistent with expectation. The inclusion body weredissolved in8M urea and purified by one-step affinity chromatography with a purityof85.3%. Then the inclusion body were renaturated in a soluble form by dialysis toremove the urea with dilution refolding.25.92mg protein in1L fermentation brothwas achieved with a refolding efficiency of76.41%. The fusion protein with a his-tagwere identified by western blot.Balb/c mice were immunized with recombinant E2protein. After4times ofimmunizations, the mice with high serum antibody titer of mouse’s serum weresacrificed and the spleen cells were harvested to fused with SP2/0cell. Hybridoma cells which were positive to E2were screened by ELISA with recombinant protein E2.Six cell strains, which were designated as11-11,10-3,2-2,3-3,6-6,9-1can stablysecreting anti-E2monoclonal antibodies after subcloning and screening. The titer ofsix mAbs in ascites was106,108,108,107,107,108respectively. The mAbs werepurified with a purity of95%and the titer was105,106,106,106,106,106respectively. The subclass of monoclonal antibodies was IgG1.The monoclonal antibodies were labelled by horseradish peroxidase (HRP) anddifferent coating and labeling mAb pairs were screened by double-antibody sandwichELISA. Two antibody pairs consisting of capture (mAb10-3,2-1) and detector(mAb11-11) showed a relatively higher sensitivity to E2in the ELISA platform. Thenthe concentrations of coating mAb and labeling mAb were optimized bydouble-antibody sandwich ELISA. Above two antibody pairs can detect E2at limit ofdetection of10ng/mL. All these material can be used in quick diagnosis forChikungunya virus infection.