AAV-Mediated Functional Study Of Anti-Rabies Virus Intracellular Antibodies And Their PROTAC Molecules

Rabies virus encodes five structural proteins,including nucleoprotein(N),phosphoprotein(P),matrix protein(M)glycoprotein(G)and transcriptase major protein(L),and is a single stranded negative-stranded RNA virus without segmentation.Once a person is infected with rabies virus and develops clinical symptoms,the mortality rate can be 100%.thus,it is an important disease threatening human health,which makes the development of drugs for rabies urgent.Intracellular antibodies are recombinant antibody fragments that can be expressed intracellularly and are one of the new strategies for immunotherapy.Their gene fragments can be delivered by vectors to cells,expressed,and then bound to antigens in the cell to inhibit target protein activity Single chain antibodies are the most common form of antibody fragments that constitute intracellular antibodies,and there has been much evidence that intracellular antibodies have great clinical potential.Therefore,in this experiment,intracellular antibodies against N and G proteins of rabies virus and their PROTACs were constructed.In order to make the intracellular antibodies and their PROTACs can be secreted and expressed in vivo for a long time,adeno associated virus was chosen as the vector in this experiment,in order to be able to achieve the purpose of rabies prevention.Part Ⅰ:The pLVX-Puro-CVS-G recombinant plasmid was constructed using molecular cloning techniques,and the positive clones were transformed and analyzed by DNA sequencing.The successfully sequenced bacterial broth was amplified,and the expression of pLVX-Puro-CVS-G protein was verified by Western blot as well as indirect immunofluorescence assays,followed by screening with puromycin to N2a cell lines stably expressing CVS-G.Part Ⅱ:Preparation of anti-rabies virus monoclonal antibody.BALB/c mammary mouse brain tissue grinding supernatant of inactivated CVS strain of RABV was used as antigen to immunize mice,and after boosted immunization,mouse spleen cells and SP2/0 cells were fused and positive clones were detected by indirect immunofluorescen-ce assay(IFA),and after three times of positive screening with subcloning,specific antibodies against rabies virus were obtained.The monoclonal cells were identified by Western blot assay,enzyme linked immunoassay and immunofluorescence,and the neutralization activity of monoclonal antibodies was detected by cell ELISA and fluorescent antibody neutralization assay,and the recognition effect of monoclonal antibodies on rabies virus particles was detected by applying immuno-PCR.Finally,four anti-RABV monoclonal antibodies were obtained,among which,three recognized the G protein of rabies virus,and all three antibodies had the ability to recognize the virus particles as well as neutralize the virus,which laid the foundation for the subsequent experiments as well as provided the necessary antibody tools for the immunoassay experiments.Part Ⅲ:Preparation and overexpression validation of antirabies virus intracellular antibody and its PROTAC molecule,based on the antirabies virus N protein monoclonal antibody 1N1 obtained from the previous laboratory screening and the antirabies virus glycoprotein monoclonal antibody 1B1 and 1D4 obtained from the current test screening.The monoclonal antibody heavy chain and light chain variable region genes were cloned and linked by Linker,and pAAV-MCS was used as the expression vector.Intracellular antibodies pAAV-scFv1N1-Myc,which expresses antiabies virus N protein,and intracellular antibodies pAAV-scFv1B1-Myc and pAAV-scFv1D4Myc,which expresses anti-rabies virus G protein,were constructed by adding a Myc tag to the Cterminus of the light chain.Meanwhile,based on the protein degradation targeting chimera technology,the PROTAC sequence was added to the cterminus of the intracellular antibody in order to degrade the virus and viral proteins.The constructed and sequenced intracellular antibodies and their recombinant plasmids with PROTAC sequences were transfected into N2a cells and 293T cells by liposome transfection reagent,and the expression of intracellular antibodies and intracellular antibody molecules with PROTAC sequences were successfully verified by immunofluore cence and WB.Part Ⅳ:Laser confocal and co-immunoprecipitation verified at the cellular level that both the constructed intracellular antibodies and their PROTACs molecules can interact with viruses and viral proteins.The CVS strain of RABV was infected with viral titers in the N2a cell supernatant expressing intracellular antibodies and their PROTACs molecules,and it was proved that intracellular antibodies and their PROTACs molecules had a significant inhibitory effect on the virus at 24 h.Subsequently,blood brain barrier permeable recombinant adeno associated viruses(rAAVscFv1N1-Myc、rAAV-scFv1N1-Myc、rAAV-scFv1B1-Myc、rAAV-scFv1B1-Myc、rAAVscFv1B1-Myc、rAAV-scFv1B1-Myc-PROTAC、rAAV-scFv1D4-Myc、rAAV-scFv1D4-MycPROTAC),meanwhile,a negative control group(rAAV-MCS)was set.Finally,the purification and titer determination of the virus were carried out,and the purified recombinant adeno associated virus was injected into mice with 1011 copies/mouse through the tail vein using BALB/c mice and C57/BL6 mice as models,and the mice injected with recombinant adeno associated virus were challenged and the survival of mice was observed two weeks later.In this study,a N2a cell line expressing CVS-G was constructed,and an anti-RABV monoclonal antibody was successfully prepared,and on this basis,intracellular antibody and its PROTACs were prepared.Using recombinant adeno associated virus that can penetrate the bloodbrain barrier as the vector,intracellular antibodies and their PROTACs molecules targeting N and G proteins were expressed,and recombinant adeno associated viruses with blood brain barrier penetration ability were packaged,and their antirabies virus activity was evaluated in vivo and in vitro,which provided a new idea for rabies immunotherapy.