Preparation of monoclonal antibodies against the envelope protein extracellular domain of Zika virus in mice
Objective To prepare monoclonal antibodies against the envelope protein extracellular domain(Eecto)of Zika virus(ZIKV)in mice.Methods A prokaryotic expression plasmid,pET28a-ZIKV-Eecto of ZIKV Eecto,was constructed,transformed into Escherichia coli BL21 and induced by isopropyl β-D-thiogalactoside(IPTG).The recombinant Eecto protein was expressed in the form of inclusion bodies,and purified proteins were obtained through denaturation,renaturation and ultrafiltration.After three rounds of immunization with the Eecto protein,the serum of BALB/c mice was obtained and the titer of polyclonal antibodies in serum was determined.The reactivity of polyclonal antibodies was analyzed with Western blotting and immunofluorescence assay in HEK293T cells expressing the ZIKV prME.Spleen cells from mice with higher antibody titers were prepared and fused with SP2/0 myeloma cells.The hybridoma cells secreting antibodies were screened through the limited dilution method,and the ascites containing antibody were harvested for titer measurement and subclass analysis.The Eecto from the envelope proteins of Japanese encephalitis virus(JEV),Yellow fever virus(YFV),Dengue virus(DENV1-4),and Tick borne encephalitis virus(TBEV)were coated and used to analyze the cross-reactivity of ZIKV monoclonal antibodies by ELISA.Further specificity analysis was conducted on antibodies with high titers and strong specificity.Results The plasmid pET28a-ZIKV-Eecto was successfully constructed.The purified Eecto protein was obtained with good immunogenicity.Four monoclonal antibodies were prepared and screened,namely 1D6,4F11,4H7,and 4F8.Among them,1D6,4H7,and 4F8 are IgG(K)type antibodies,and 4F11 is an IgM(K)antibody.The ascitic fluid titer of 1D6 was higher than 1:108.Antibodies 1D6 and4H7 are ZlKV-specific and showed no cross-reactivity with other Flaviviruses.Conclusion The mice monoclonal antibodies against ZIKV-Eecto are produced successfully,which will provide experimental materials for the establishment of ZIKV detection methods and the study of its pathogenesis.
Zika virus(ZIKV)envelope protein extracellular domain(Eecto)prokaryotic expressionmonoclonal antibody
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