Expression Purification And Immunogenicity Analysis Of Papillomavirus Subtype 58 L1 Protein

Cervical cancer(CC)is one of the most common female cancers,the long-term infection of high-risk human papillomavirus(HR-HPV)is a necessary condition for women to develop cervical cancer.HPV is a ds DNA virus,the capsid is an icosahedral spherical structure which make up of L1 protein and L2 protein,the L1protein is the main capsid protein of capsid.The L1 protein can be spontaneously assembled in vitro under suitable conditions,five L1 monomer proteins can aggregate to form a pentamer,and 72 pentamers can assemble into virus-like particles(VLP),the body after VLP immune stimulation can be induced to produce neutralizing antibodies,which is a good platform for developing prophylactic HPV vaccines.Currently,the HPV prophylactic vaccines that have been approved for marketing are mostly genetically engineered subunit vaccines derived from insect cells or yeast cells,with relatively high preparation costs and limited virus types that can protect against,have low penetration rates in developing countries and underdeveloped regions.HPV58 is one of HR-HPV which is more prevalent in East Asia than other place in the world,this study is aims to express soluble L1 protein using the E.coli expression system and analyze its immunogenicity to find an alternative candidate method for the development of broad spectrum HPV prophylactic vaccines.In this study,the optimized L1 gene sequence was cloned into pE-SUMO to construct the recombinant plasmid pE-SUMO-L1,and then pE-SUMO-L1 was transformed into DH5?and BL21(DE3)respectively.After optimizing the expression conditions of the protein,the L1 protein with high soluble expression was obtained,after SUMO-tag removed,the antigen epitope presentation of the protein was verified by Hemagglutination assays(HA),Balb/c mice was immunized withe the L1 protein and the serum titer was determined by ELISA,the Indirect Immunofluorescence Assay(IFA)was performed,all of this work has accumulated experience for the development of broad spectrum HPV prophylactic vaccines.The recombinant plasmid pE-SUMO-L1 was successfully construced and the optimal conditions to express SUMO-L1 protein were as follows:0.6 mmol/L IPTG and 16?for 14 h.the SUMO-L1 has a high-throughput soluble expression about110 mg/L.After purified by Ni-NTA affinity column,the L1 protein was reaction with Ulp1 protease to remove the SUMO-tag,the result of HA showed that the hemagglutination activity was similar to natural virus with the titer 1:16.After immunizing Balb/c mice with L1 protein,the titer of immune serum was determined,the results showed that specific antibodies were produced in both 20?g group and 5?g group,the serum titers of mice could reach 1:1.024×10~5.Significant differences was observed between immunization group and PBS group,but there was no significant differences between 20?g group and 5?g group.The results of IFA showed that the antibodies produced by mice which immunization with L1 protein can react with the L1 protein expressed by the eukaryotic system.All above indicated that the L1 protein obtained in this study had good immunogenicity,and this work has accumulated experience for the development of broad spectrum HPV prophylactic vaccines.