A Rational Design Of Urification And Application Processes Based On The Particulate Characteristics For Hepatitis B Core Antigen Virus-like Particle And Its Derivatives

Virus-like particles(VLP),which are assembled from a number of protein subunits,are expected to be used as vaccine antigens,adjuvants or drug carriers.It is one of the hot topics in the field of biopharmaceutical engineering in recent years.In this thesis,the particuology aspect of hepatitis B core antigen virus-like particles(HBc-VLP)was studied in corporation with the existing protein research methods,and a purification process suitable for batch preparation of HBc-VLP was established.Its application as vaccine antigen and drug carrier was also explored.The main results and findings are as follows:(1)The surface structure and properties of HBc-VLP particles were analyzed.Based on the characteristics of strong hydrophobicity on the surface,a batch preparation technique of HBc-VLP based on hydrophobic interaction chromatography was established.With hydrophobic interaction chromatography as the core,60℃ heating pretreatment for 30 min as the front step separation,gel filtration chromatography as the later stage of purification,the total yield of HBc-VLP from the cell disruption supernatant of fermentation.was 41.92%and the final purity was above 99%.It is a complete set of HBc-VLP separation and purification process reported in the literature so far.The purification process was repeatedly operated in 6 batches fermentation broth,with good reproducibility of yield and purity.(2)According to the structural homology,the difference of surface hydrophobic value between HBc-VLP and its derivates was calculated,and the hydrophobic interaction chromatography conditions were optimized.By using the HBc-VLP purification process established in(1),the high purity M2e-HBc,NP-HBc and OVA-HBc were successfully purified from the cell disruption supernatant of fermentation.The derivatives of HBc-VLP,including M2e-HBc,NP-HBc and OVA-HBc,were constructed by gene fusion into HBc-VLP cloning and expression system,using influenza virus matrix protein 2 ectodomain(M2e),influenza virus nucleoprotein(NP)and ovalbumin(OVA)as insertion antigen epitope.(3)The pretreatment step in the purification process was investigated from the point of view of particle change.It was found that the particle size and molecular weight of HBc-VLP increased after heat treatment above 70℃ when the host cell protein was precipitated by the heat treatment.However,there is no such phenomenon happening if the treatment temperature was below 70℃.After careful analysis,it was found that HBc-VLP had the characteristics of reversible thermal expansion in size.High temperature would cause the pores between the protein subunits to expansion.When the temperature was higher than 70℃,the pore expansion was large enough to enable the entry of large amount of impurity proteins in the cell disruption supernatant of fermentation,because the VLP has a hollow interior.These impurities would stay inside of the VLP,increasing the size and molecular weight when the heat treatment was complete.(4)The strategy of two-step heating pretreatment was established.The first step was heated up to 60℃ for 30 min to precipitate and remove most of the impurity proteins in the cell disruption supernatant,and the second step was raised to 70℃ for 30 min to further precipitate the residual impurity proteins.The two-step heating strategy effectively avoids the problem of impurities entering the particles which appear at the temperature above 70℃.The yield of HBc-VLP is up to 85.8%and the purity is 74.7%.Coupling hydrophobic interaction chromatography,the final purity reached 99%and the whole process yield was 77.7%.The new process was also successfully used to purify the derivatives of HBc-VLP.The purities of M2e-HBc,NP-HBc and OVA-HBc were over 97%and the yields were over 50%.(5)Based on the discovery of the expanded particle size of HBc-VLP at high temperature and the enlarged pore between the subunits,a novel application process of antigen loading to the VLP by high temperature was designed.The purified M2e-HBc(surface-fused influenza matrix protein M2e)was used as a candidate for influenza vaccine.The influenza virus nucleoprotein epitope peptide(NP)was successfully incorporated into the hollow cores of M2e-HBc particles by heat treatment.In this way,a dual-antigen influenza vaccine was constructed with a surface antigen and an internal antigen.After immunizing mice,the antibody against M2e/NP was detected by ELISA method,and the level of antibody was significantly higher than that of control group,and the protective rate of the dual-antigen influenza vaccine was 100%when A/FM/1/47(H1N1)influenza virus was tested.(6)The heating loading method was extended to the application of loading anticancer drugs.Purified HBc-VLP was used as the carrier of doxorubicin(DOX),and 4248 DOX per HBc-VLP molecule was loaded by heating up to 70℃ for 90 min,which was superior to the traditional loading method.The HBc-VLP’s multi-disulfide bonds were used to realize the controlled release of doxorubicin to the tumor cells.The target peptide of RGD was also chemically conjugated on the surface of HBc-VLP to achieve the specific targeting.The inhibitory effect of doxorubicin was superior to the conventional drug delivery method.