Monoclonal Antibody Preparation And Epitope Identification Of EIAV Accessory Protein Rev

Equine infectious anemia virus (EIAV) is one of the lentivirus of the retrovirus family and is the pathogen of equine infectious anemia (EIA),one kind of infectious diseases that damages equine seriously.The lentivirus could continuously evolve and escape from the host’s immune surveillance.It is a important factor of impacting directly the research progress of vaccine development.The EIA vaccine was first successfully developed and applied.EIAV is regarded as the important model to research the immune mechanism of the lentivirus infection.Rev,as an accessory protein of EIAV,mainly transports mRNAs which are unfinished-spliced towards the outside of the nuclear in the late phase of virus replication to promote the assembly of the virus.So Rev plays an important role in the replication of EIAV.The latest studies indicate that Rev could form different sub-types through mutation during different clinical phases of infection,therefore the research on the structure and function of Rev would provide important research date for developing the vaccines and drugs about lentivirus.Monoclonal Antibody was a tool for studying the function of protein.In this study, the target gene of Rev was obtained by PCR,and then connected to the prokaryotic expression vector Pet30a through double enzyme digestion.After the prokaryotic expression and protein purification, the high-pure Rev with the immunogenicity was obtained.The pure Rev protein was used to immunize the8weeks old BALB/c mouse and one hybridoma cell strain secreting the antibody against Rev was acquired through cell fusion technique,which was named2D9. The results of Western Blot, indirect ELISA and IF A indicated that this antibody had relatively high sensitivity and specificity. B-cell epitope of2D9was indentified through peptide scanning technique which indecated the correspondent epitope was71QHTPSRRDRWIRGQ84locating in the RNA binding area (RBD) of Rev.The antibody not only provides a basic tool for researching the protein structure and function of Rev,but also lays an important foundation for researching the interaction mechanism between Rev and mRNA.