Preparation Of Monoclonal Antibodies And Screening Epitopes For Human Papillomavirus Subtype 45 L1 Protein

Cervical cancer is one of the most common cancers among female patients in the world,with high morbidity and mortality.The occurrence of this disease is due to the infection of high-risk types of human papillomavirus?HPV?.At present,more than 200subtypes of HPV have been found,among them,HPV45 is one of the high-risk types and closely related to the occurrence of cervical cancer.The L1 protein is a major structural protein of HPV viral capsid,which can induce the organism to produce neutralizing antibodies.Therefore,the preparation of monoclonal antibodies?m Abs?against HPV45 L1 protein and the identification of L1 protein epitopes are of great significance for the prevention and detection of the disease.The optimization of expression conditions of SUMO-HPV45 L1 protein were studied,and the L1 protein was purified by Ni-NAT affinity chromatography,the results indicated that the best conditions to express soluble L1 protein were added 0.1 mmol/L IPTG?Isopropyl?-D-thiogalactoside?and induced at 16?for 18 h,SDS-PAGE results indicated that the purity of the target protein was more than 90%,and the L1 protein could specifically be reacted with anti-His monoclonal antibody when analyzed by Western-blot.Two mice were immunized with the purified protein,and the serum titer can reach 1:1.024×10⁵.Monoclonal antibodies were prepared by hybridoma technology.Four positive hybridoma cell lines were screened,which were named 2F5,3B7,3C11,3G11respectively.The cell line 3C11 with the highest titer was selected to prepared ascites by in vivo-induced method,and purified by caprylic acid-ammonium sulfate method.Indirect ELISA results showed that the titer of the purified antibody of 3C11 was1:2.56×10⁵,the affinity constant was 1.4×10⁹ L/mol,and the antibody has not cross-reaction with HPV18/31/53/58 subtypes.The purified 3C11 monoclonal antibody was used as the target,and biopanning was performed by a 12-mer phage peptide library.12 positive phage clones that reacted with monoclonal antibody 3C11 were selected and sequenced,then analyzed by DNASTAR software and identified by Dot-ELISA and indirect competitive ELISA.The results showed that“³⁸¹VPNTYD³⁸⁶”was a linear epitope of L1 protein.In this study,monoclonal antibody against HPV45 L1 protein was successfully prepared,and a linear epitope of L1 protein was selected.This provides a convenient method for the immunoassay of L1 protein,lays the foundation for further analysis of the structure and functional relationship of L1 protein,and it is of great significance for studying the antigen-antibody interaction.