Humanization Of EV71 3D Protein Specific IgA Monoclonal Antibody

Enterovirus 71(EV71)is one of the major pathogen of hand,foot,and mouth disease(HFMD),which commonly infect infants and children,lead to hand,foot and mouth disease,and may also induce encephalitis and other serious symptoms,morbidity and mortality are very high.Research shows that EV71 usually transmit via the fecal-oral route,and replicate in the lymphoid tissues of the oropharyngeal.So epithelial cell of pharyngeal is the initial target cell for EV71 infection and replication.The virus-specific IgA is an important factor of mucosal epithelial cells that against pathogen infection.It can be combined with p IgR of epithelial cells,and then transferred into the cells,thus play the role of intracellular neutralization,reducing the viral replication.The 3D polymerase(3Dpol)as an RNA-dependent RNA polymerase(RdRp)of EV71,which is essential for viral genomic transcription and RNA synthesis.According to this,we prepared a series of monoclonal antibodies against EV71 3Dpol and obtained a murine monoclonal IgA antibody-3A12-IgA,which has high efficiency inhibition of EV71 replication.In our research,we adopted the chimeric antibody method to modify the 3A12-IgA,and explored the eukaryotic expression and preparation of recombinant IgA antibody.First,we constructed the eukaryotic expression plasmids of humanized IgA antibody(RH3A12-IgA)respectively,which include the recombinant eukaryotic expression plasmid of the recombinant IgA heavy chain(VH-p FUSEss-CHIg-hA1),the recombinant eukaryotic expression plasmid of the recombinant IgA light chain(VL-pFUSE2ss-CLIg-hk),and the eukaryotic expression plasmid of human J chain(IgJ-pcDNA3.1(+)).Second,establish the ELISA,Western Blot and other methods by co-transfected the CHO-K1 cells with three plasmids expressing the heavy chain,light chain and J chain(VH-pFUSEss-CHIg-hA1,VL-pFUSE2ss-CLIg-hk,IgJ-pcDNA3.1(+)),collect the supernatant to detect the content and biochemical function of the recombinant antibody.Thirdly,optimize the expression system of RH3A12-IgA the signal peptide of heavy chain and light chain,the transfection ratio of 3 plasmids,the expression of cell line and the addition of Kozark sequence,determine the expression system parameters,and the production of recombinant in supernatant was up to 4.4μg / ml.Fourthly,mass express the recombinant antibody RH3A12-IgA in 293 T cells,and purified by Kappa-select affinity chromatography.Using Transwell system to detect the biological activity of the recombinant antibody.The results showed that the recombinant antibody could be efficiently transported by Vero-p IgR cells and had a certain neutralizing effect.In conclusion,we constructed the eukaryotic expression plasmid of heavy chain,light chain and J chain of humanized RH3A12-IgA,established the 293 T cell expression system.The purified humanized recombinant antibody RH3A12-IgA was prepared effectively,demonstrating the biological activity of the recombinant antibody.The results laid a foundation for the humanization of murine IgA monoclonal antibody and its preparation,which have important potential application significance.