Prokaryotic Expression And Purification Of Recombinant HPV16 L1 Capsid Protein

Persistent infection human papillomavirus can induce almost all cervical cancers,and about 55%of cervical cancers cases are caused by HPV16.Therefore,the development of prophylatic HPV16 vaccines have broad prospects.At present,the important direction of prophylatic HPV vaccine is VLPs vaccine.In this study,HPV16 L1 protein was expressed by E.coli.HPV16 L1 was assembled VLPs in vitro,which has good immunogenicity.The genome of HPV16 L1 was obtained from Gen Bank.According to the preferred codon of Escherichia coli,the HPV L1 gene sequence was optimized and synthesized.We obtained the expression vector p ET30a-16 L1 by gene recombination.We transformed p ET30a-16 L1 into BL21(DE3)cells.E.coli was induced to express HPV16 L1 protein.In order to obtain higher protein expression,we optimized the expression conditions.We determined that the protein expression conditions were as follows:the concentration of the inducer IPTG was 0.1 mmol/L,and the expression was induced at 30°C.We used high-density fermentation tanks to cultivate engineered bacteria,and collected 2.56 kg of bacteria by centrifugation.The E.coli was broken by a high-pressure homogenizer.We optimized the high-pressure homogenization conditions.The results showed that the cells were completely broken by homogenized 4 times at 800(±50)bar.In order to improve the recovery rate of protein,HPV L1 protein was separated and purified by four cation exchange chromatography media:Nuvia^TM S,UNOsphere^TM rapid S,POROS^TM 50 HS and POROS^TM XS.The results showed that the recovery of the protein from POROS?XS was 37%,higher than other chromatographic media.We optimize the POROS?XS chromatographic parameters by design of experiment.The loading flow rate of 5 m L/min and the buffer system p H 8.0 were used for chromatographic purification.The protein recovery was increased to 49.1%after optimization.In order to obtain protein with higher purity,four hydrophobic chromatography media,Hexyl-650C,Phenyl-600M,Butyl-600M,POROSTM Ethyl,were used to further separate and purify the protein.POROS^TM Ethylwas finally selected.After purified by POROS?XS and POROS^TM Ethyl,the protein concentration was 1.0 mg/m L,the purity was 97%,the protein recovery rate was 46.23%,the endotoxin content was less than 12.5 EU/mg,the host residual protein was 6.00 ng/mg,and the host residual DNA was 3.30 ng/mg.The HPV16 L1 protein was dialyzed and assembled to form VLPs.The morphology and size of HPV16 VLPs were identified by particle size analyzer and transmission electron microscope.The results show that particles were about 60 nm,which had similar shapes with natural virus.Pseudovirus neutralizing antibody evaluates the level of antibodies induced by VLPs in mice.After 6 weeks of immunization,the average Log10 of serum neutralizing antibody titer was 4.01.The VLPs assembled by the protein have good immunogenicity.In this study,HPV16 VLPs were obtained by the E.coli expression system.the chromatographic purification process was optimized.This study established a foundation for the research and development of virus-like particle vaccine.