Production And Evaluation Of The L1 Protein Of Human Papillomavirus Type 58

Cervical cancer is the second malignancy,which has the higher morbidity and mortality after breast cancer.Human papillomavirus(Human papillomavirus,HPV)infection is the main cause of cervical cancer and the researches show that more than 95% of cervical cancer is highly associated with HPV infection.HPV infection has regional epidemics and HPV16 and 18 are two common subtypes closely related to cervical cancer.The HPV58 subtype has a higher incidence in East Asia,Central America and South America.The HPV-L1 protein is the main protein capsid of the virus and can be assembled into virus-like particles(Virus-like particles,VLPs)that can stimulate the body to produce long-term neutralizing antibodies,which is commonly used in the development of prophylactic vaccines.Currently,the three listed vaccines were using yeast or insect baculovirus systems to express recombinant VLPs vaccine.These vaccines have some defects,such as high cost and low yield,those limit the promotion and widely use of prophylactic vaccines in developing countries,therefore new and more effective and cheaper vaccine is still the hot spots of current cervical cancer prevention and control.The aim of this study was to seek a much more inexpensive method for obtaining HPV58-L1 protein and lay the foundation for the study of HPV58 VLPs vaccine.In this study,we successfully constructed the prokaryotic expression vector p E-S-HPV58-L1 and transformed the recombinant vector into E.coli BL21.Optimized the induction conditions,such as IPTG concentration,induction time and induction temperature,finally we chose 0.3 mmol of the IPTG concentration,18 ?of the temperature,and 12 h of the expression time as the best conditions to express soluble HPV58-L1 protein,and its expression level was about to 100 mg/L;After purification by Q Sepharose Fast Flow anion exchange chromatography,most of the primitive proteins expressed by E.coli could bind to the Q column,while the target protein HPV58-L1 only a little bind to the Q column,with the purity was about 70%of the whole protein;The purified HPV58-L1 protein was divided into high and low dose groups to immune the mice at 0 d,28 d and 56 d respectively and the results showed that both the high dose group and the low dose group had specific antibodyproduction and a significant difference compared with PBS immunization group and the serum titer was up to 1: 25600;Along with the increasing number of immunization,the antibody level in mice was increasing,and the antibody level was still higher in 84 d after the first immunization.Animal experiments results showed that HPV58-L1 protein expressed by E.coli had a good immunoprotective effect and laid the foundation for the development of HPV-related vaccine.At the same time,we made use of the advantages of the rice endosperm bioreactor to construct the p CAMBIA1300-p MP3-HPV58-L1 plant expression vector by introducing the HPV58-L1 gene into the plant expression system.Though Agrobacterium tumefaciens-mediated transformation used to transfect the rice callus and the rice callus though screened,differentiated and rooted,we obtained the transgenic rice.Finally though PCR identification,we obtained twenty-eight strains of transgenic rice containing HPV58-L1 gene,which laid the foundation for further preparation of plant source HPV58-L1 vaccine.