Purification, Crystaliziation Of Broad Spectum Anti-H5 AIV Therapeutic Mab 13D4 And Prediction Of Its Epitope

Highly pathogenic avian influenza(AI),one of syndrome disease caused by influenza A virus H5N1,has recently spread to many countries by the migratory birds. It severely led heavy loss of national economy in these countries,and even caused fatal disease on human.Study on prevention and treatment of avian influenza has aroused extensive attention.Neutralization antibody becomes one of effective means to contral virus disease.Because avian influenza virus mutants frequently,especially in the hemagglutinin gene,most H5N1 mab only neutralize narrow spectrum of virus.Our research group has indentified a strain of broad spectum neutralization antibody,named 13D4,which binds to 41 representative strains of H5N1 virus from various clades.In this study,a easilyscaled -up and high-effective process with low cost was established to purify 13D4 and F(ab)'₂ fragments.The purity of final product was about 99%and the recoverity rate is about 60%.All the test ruesults fulfiled the requirements of PHARMACOPOEIA of PRC.In this study,the vapor diffusion technique was used to crystallize 13D4 Fab fragment,and sigle crystals suitable for X-ray diffraction were obtained in some conditions after screening and Optimization.The structures of 13D4 Fab fragment and HA1 from A/Ck/HK/Yu22/02 (H5N1) were modeled by homology modeling.Then 13D4 Fab was subject to dock with three HA structures found in PDB(PDB ID:ljsm,2ibx and 2fk0) and HA1 from A/Ck/HK/Yu22/02(H5N1).The binding patterns between the antibody and the HA were analyzed according to the docking results.The binding pattern of 13D4 Fab with the 4 HAs was used to search the broad neutral epitopes which the antibody binding to and the interacting polypeptideslocated in the antibody which may be made use of the reference of the anti-virus drug.The polypeptide located in 13D4 Fab fragment is Gln²⁷-Thr²⁸-Ser³⁰-Gly(31)-Leu⁵²-Gly⁵⁴-Ser⁵⁵-Asn⁵⁷-Thr¹⁰⁰-Thr¹⁰¹-Ala¹⁰²-Val¹⁰³.