Construction And Characteristics Of PH-responsive Hepatitis B Virus-like Particles

Hepatitis B virus-like particles(HBV VLPs)are produced by the self-assembly of hepatitis B virus core protein(HBc)in vitro.They are icosahedral hollow particles without virus genetic material,and have a particle size of 30-34 nm.Due to its nano-structure and good biocompatibility,VLPs have good potential applications in vaccine development,disease diagnosis,drug delivery and nano-materials.However,the application was restricted by its low self-assembly efficiency and stability.Therefore,in this paper,the pH responsive HBc VLPs were constructed by modifying HBc with charged groups to improve its self-assembly efficiency and stability.By inserting 8 arginine or 8 aspartic acid into the dominant immune region of wild-type HBc(wt HBc),the HBc mutant with extra positive charge(m+HBc)or extra negative charge(m-HBc)was obtained to introduce charge groups on the surface of HBc VLPs.The recombinant plasmids p ET-28a-wt HBc,p ET-28a-m+HBc and p ET-28a-m-HBc were constructed and transferred into E.coli for expression to obtain the target proteins.The optimal expression condition was determined as: 1.0 mmol/L IPTG as revulsant,inducing 10 h at 28?.Then the HBc was purified by affinity chromatography and gel chromatography,followed by the investigation on the self-assembly and stability.Using the equimolecular mixture of m+HBc and m-HBc,successful self-assembly was observed to VLPs nanoparticles with a particle size of 30.44±2.23 nm,which was consistent with wt HBc VLPs.The assembly process and assembly stability were affected by ionic strength and pH values.The change on the stability of VLPs in different buffers was characterized by size exclusion chromatography,atomic force microscope and dynamic light scattering.It could be found that the wt HBc VLPs could exist in solutions containing 0.3-1.0 mol/L Na Cl.The optimum stable pH value was 7.4.Deviation from this pH value made its stability reduced.Significant decrease of stability was observed when pH value below 5.5,which was only 64.2% of that at pH7.4.For m HBc VLPs,no obvious difference of stability was observed at various pH values,and relative stability more than 88.3% was observed at the tested pH range of5.5?9.5.The pH responsiveness of m HBc VLPs indicated that the improved tolerance of HBc VLPs to pH changes could be obtained by the introduction of extra electrostatic interactions through charged amino acid residues,and thus enhanced stability of VLPs was achieved,which was beneficial to its practical application.