Study On Recombinant Human Antibody And Intrabody On Rabies Virus Phosphoprotein

With the phagemid pComb 3H vector system, the blood lymphocytes were isolated from five donors immunized with rabies vaccine. And total RNA was extracted and cDNA were synthesized, Fab antibody light chain segments were amplified with the Fab antibody primer pairs. The Fab antibody light chain segments were firstly cloned into the phagemid vector pComb 3H digested with enzymes Sac I/Xba I, and then heavy chain ones cloned in to the light chain library pool with Xho I/Spe I. Finally the recombinant Fab libraries were generated.After packaging of phage library, the three round screening procedure was performed by coating with purified CTN and aG virions overnight at 4℃. After last round of panning, over 200 randomly selected clones of each library were picked, incubated and induced to express crude Fab antibody. The bacterial supernatant containing Fab antibody was tested by indirect enzyme-linked immunosorbent assay with purified CTN virions and anti-human Fab antibody to confirm expression level and binding specificity. The Fab antibody genes of double positive clones were isolated, sequenced and aligned to V-BASE database. Sequence analysis of all these positive Fab clones revealed 2 unique sequence, represented by clone 1A2 and 1G4, both shared the same VH and similar VL chain genes by aligning the sequences to VBASE2 database. The selected Fab clones were further converted to IgG antibody and expressed in HEK293 cells. IgG antibody RV1A2 were confirmed by immunoflurescet assay (1FA), ELISA and western blotting. Further research of anti-RVP antibody RV1A2-IgG was performed by Rapid Fluorescence Focus Inhibition Test (RFFIT), and obviously RV1A2 had no neutralizing activity. RV-P involves binding to NP and LP, encapsidating viral genome RNA, assisting RNA-dependent RNA polymerase in the process of transcription and translation.After analysis of RV-P sequence, epitope mapping was performed using truncation mutations, and with the full-length RV-P as template, The N-terminal truncation mutations of RV-P with a His tag were generated by deletions including the in-frame ATG at the 5’ end of the RV-P gene (P2(△N19), P3(△N52), P4(A△N68), VR-RV P5(△N82)). Five C-terminal RV-Ps were truncated from C-terminal at every 20 amino acids first (P△C20, P△C40, P△C60, P△C80, P△C100), and then another four C-terminal truncation mutations named P△C23, P△C27, P△C31, P△C36 were generated. After transient transfection and western blotting, result showed all truncated derivatives were expressed by confirmation of interacting with anti-His antibody, the human antibody RV1A2 recognized the full length of RV-P(P1), also the natural shorted RV-P P2, P3, P4 and P5 which the RV-P were synthesized from the ATGs at the positions of 19,52,62 and 68 nucleo acid of N-terminal; Truncations from C-terminal, we found that RV1A2 only reacted with 20 amino acids (P△C20) but none of other truncations (P△C40, P△C60, P△C80, P△C100), therefore, the linear epitope of RV1A2 was limitedin the region from △C20 to △C40. Further, we narrowed the deletions of each 4 amino acid from △C20 to △C40 of C-terminal, we found that RV1A2 only recognized truncation of P△C36, indicating the linear epitope site ranging from 262 to 266 (VLGWV) of C-terminal. The crystal structure 1VYI of RV-P was found in PDB. From this docking model, the linear epitope of "VLGWV" ranging from aa 262-266 of RV-P C-terminal was confirmed, while the epitope showed a three-dimensional form, only W265 was on the surface of flat face of the "half-pear". Therefore we presume that the position W265 may be a critical amino acid to contribute to the "W-hole". Fom a previous publication for Rabies virus gene revolution analysis, we found that the epitope of "VLGWV" (aa262-266) recognized by the antibody RV1A2 was well conserved.The RV1A2 scFv genes with a HIS-tag were cloned into a intracellular cytoplasm expression vector PEF/myc/cyto to make 80% cells express intrabody. the cells were infected with Rabies virus Strain CVS-11. The infected cells and supernatants of day 1-4 were collected and detected. We observed a viral suppression phenomenon performed by expression of intracellular antibody ScFv-RV1A2 in MNA cells. Result showed this inhibition of RV replication through intracellular function might be achieved by blocking the key target, the conserved linear epitope comprised of RV-P C-terminal residues 262-266 (VLGWV). Our evaluation of RV1A2 on inhibiting RV propagation is preliminary. The role and mechanism of RV1A2 and/or its recombinant derivatives need to be further investigated on inhibition of RV propagation. In addition, it will be a great challenge in intrabody theraputic research:effective penetration of membrane barriers, particularly across the highly resistant blood-brain barrier and the intracellular targeting.