The Expression Of Human Papillomavius 33 Subtype L1 Protein And Identification Of Immunogenicity

Cervical cancer is one of the most common malignant tumors in women,and the incidence of which is second only to breast cancer.Human papillomavius(HPV),belongs to the family of Papoviridae,is a non-enveloped DNA virus,the infection of which is intimately linked to cervical cancer.HPV has obvious species specificity,which mainly infects the skin and mucous membrane tissue of the human,and cause corresponding parts epithelial hyperplastic lesions.The major capsid protein L1 of HPV can self-assemble into virus-like particles(VLPs)in vitro which similar to the natural virus could induce the production of the neutralizing antibody.The VLPs prophylactic vaccine could be successful in preventing HPV infection.Currently,the listed HPV preventive vaccines are genetic engineering subunit vaccine which comes from insect cells or yeast cells.Due to the complex production process and the high immune cost,the study on a new cheaper HPV L1 protein preparation is the hotspot in the research of preventive HPV vaccine.In order to find a cheaper method for preparing HPV L1 protein,we use rice endosperm expression system and Escherichia coli expression system to express HPV33-L1 protein respectively.Combining the advantages of rice endosperm bioreactor,The target gene of HPV33 L1 was sub-cloned to recombinant plant expression vector for expressing the HPV33 L1 protein in rice endosperm expression system expression.Simultaneously,we successfully constructed prokaryotic expression vector PE-HPV33-L1 which contained HPV33 L1 gene,and then transform the recombinant plasmids into E.coli BL21 competent cell with chemical transformation.Finally,the optimization of expression conditions,such as concentration of IPTG,temperature and induced expression time,were studied.The soluble yeild of the recombinant protein L1 in Escherichia coli is up to 240 mg/L when induced with 0.3 mmol/L IPTG at 18 ℃ and expression for 16 h.The purity of target protein L1 purified by Ni-NTA was 90%.The analysis of TEM showed that L1 protein can spontaneously assemble into VLPs.The results of ELISA showed that the mice immunized with HPV33 VLPs could effectively produce high titer specific antibodies(1:89600),which showed that the HPV33 L1 VLPs obtained from Escherichia coli could effectively stimulate the body to produce high titer specific antibody,which lay the foundation for the development of HPV33 vaccine.