Knowledge Management System Of Institute of process engineering,CAS
病毒样颗粒（Virus like particles，VLP）是多个蛋白质亚基组装而成的纳米颗粒，有望发展成为各种疫苗抗原、佐剂或药物载体，是近年来生物制药工程领域的热点之一。本文以乙肝核心抗原病毒样颗粒（Hepatitis B core antigen virus-like particles，HBc-VLP）为研究对象，在已有的蛋白质研究方法之上，对其进行颗粒学研究，建立了适合其批量制备的纯化工艺，探索了其作为疫苗抗原和药物载体的应用。主要研究结果如下：（1）分析了HBc-VLP颗粒表面结构性质，利用其表面强疏水性的特点，建立了基于疏水层析的批量制备HBc-VLP的技术。以疏水层析纯化为核心，以60oC加热30min预处理为前级分离，以凝胶过滤层析为后级精制，从发酵培养后破菌上清液中分离纯化HBc-VLP，总收率41.92%，纯度大于99%，是迄今为止文献上报道的全套HBc-VLP分离纯化工艺。先后进行6批次发酵液的层析纯化，产品收率和纯度重现性良好。（2）根据结构同源性模拟计算了HBc-VLP与三种衍生物颗粒表面疏水值的差异，优化了疏水层析条件，采用（1）中建立的HBc-VLP纯化工艺，成功地从发酵后细胞破碎上清液中纯化出高纯度的M2e-HBc、NP-HBc和OVA-HBc。其中，三种衍生物M2e-HBc、NP-HBc和OVA-HBc均以HBc-VLP为骨架，分别通过基因融合的方式插入了流感病毒基质蛋白M2e、流感病毒核蛋白NP和模型抗原卵清蛋白OVA三种抗原表位。（3）从颗粒变化的角度对纯化工艺中的前处理步骤进行了考察，发现当加热处理菌体破碎液沉淀宿主蛋白时，HBc-VLP在高于70℃处理后颗粒尺寸和分子量都有增加，而低于70℃处理就没有这种现象发生。仔细分析发现，HBc-VLP具有热敏可逆膨胀性，温度升高，亚基与亚基间孔道扩张，颗粒尺寸增大；但温度恢复常温后，尺寸也能复原。但是当温度大于70℃后，孔道扩张足够大，使得细胞破碎上清液中大量杂质蛋白能够通过孔道，而VLP是中空的颗粒，进入颗粒内部的杂质蛋白在颗粒内部积累，并在热处理后仍然驻留在颗粒内部，导致VLP尺寸增加和分子量增大。（4）建立了两步加热预处理的策略。第一步在60℃加热30 min以沉淀去除菌体破碎上中大部分杂质蛋白，第二步升高到70℃加热30 min进一步沉淀残留的杂蛋白。两步加热的策略有效避免了一次升高到70℃出现的大量杂质进入颗粒的问题，HBc-VLP收率达到85.8%，纯度74.7%。耦合一步疏水层析精制使纯度达到99.0%，整个工艺收率77.7%。新工艺用于HBc-VLP的衍生物的纯化亦获得成功，M2e-HBc、NP-HBc和OVA-HBc的纯度都达到97%以上，收率均在50%以上。（5）根据上面HBc-VLP在高温下颗粒尺寸膨胀、亚基之间孔道加大的发现，设计了高温装载抗原的应用过程。将纯化后的M2e-HBc（表面融合流感基质蛋白M2e）作为流感疫苗的候选，通过加热法使流感病毒核蛋白抗原表位多肽（NP）进入到M2e-HBc颗粒中，构建了一种表面、内部都具有抗原的流感疫苗。免疫小鼠后，ELISA法检测针对M2e/NP的抗体，抗体水平较对照组有明显提升；A/FM/1/47 (H1N1)流感病毒进行攻毒实验，内外双抗原流感疫苗保护率能够达到100%。（6）将加热装载法拓展到装载抗癌药物的应用上，以纯化的HBc-VLP为抗癌药物阿霉素（DOX）的载体，通过70℃加热90 min实现每个HBc-VLP分子装载4248个DOX，优于传统方法的装载效率。利用HBc-VLP多对二硫键对谷胱甘肽敏感的特性，实现阿霉素在肿瘤细胞内的控释。HBc-VLP表面化学修饰RGD靶向肽，达到特异性靶向的目的，取得了比传统给药更好的抑瘤效果。;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, 60oC 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.
|李正军. 基于乙肝核心抗原病毒样颗粒及其衍生物颗粒性质的纯化和应用过程设计[D]. 中国科学院大学,2018.|
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