抗狂犬病毒G蛋白单链抗体scFv98H的纯化和复性工艺研究

发布时间：2018-09-05 05:43

【摘要】：狂犬病是致死率最高的急性传染病，是由狂犬病毒感染中枢神经系统导致的一种人畜共患病，具有急性、接触性、高致死性等特点。WHO建议采用暴露后预防（PEP）获得快速的免疫保护，防治狂犬病的发生。抗狂犬病毒血清起着重要的作用，然而，抗狂犬病人免疫球蛋白（HRIG）来源有限，存在潜在的血液疾病污染，抗狂犬病马免疫球蛋白（ERIG）容易引起过敏反应，存在批间差异等缺点，因此，单克隆抗体由于无血液污染和可连续生产等优点，被认为可能成为上述两种抗血清的理想替代品。但是，鼠源单抗容易引起人体极大副反应，基因工程抗体分子量大，需要真核系统表达，价格昂贵，因此限制单抗广泛应用。单链抗体具有原核表达、分子小、穿透力强、免疫原性低，易于基因操作等特点，比较适合大批量生产和具有很好的应用前景。本课题组谷铁军等人曾根据已进入临床的单抗鸡尾酒混合抗体CR57/CR4098，设计并制备得到相应的单链抗体FV57和FV4098，且这两株抗体在体外实验中都表现出对狂犬病毒较好的结合活性和中和活性，尤其是FV4098在小鼠攻毒实验中提供保护率相似于市售的HRIG和ERIG。因为FV4098基因序列的C端有His-tag，本论文称其为scFv98H。所以本论文将在前期表达载体构建，原核表达及大规模发酵的基础上，尝试建立一套适合scFv98H纯化和包涵体复性工艺，为其将来中试规模和产业化规模生产scFv98H奠定基础。本论文研究内容主要包括以下几个部分：（1）研究scFv98H分离纯化工艺并优化其条件。选择适合scFv98H纯化的Nuvia Q强阴离子交换层析介质。对Nuvia Q纯化scFv98H条件进行优化，主要考察起始缓冲液pH，上样流速，单位体积层析介质的载量。优化后的条件为：采用流穿模式纯化，20ml柱体积的最适pH7.2，最适上样流速5ml/min，最适载量80mg。流动相A为20mmol/LTris、8mol/L脲，pH7.2，流动相B为20mmol/LTris、8mol/L脲、1mol/L NaCl，pH9.0。复性后的scFv98H经过凝胶过滤进行精纯，通过scFv98H分子量选择合适的凝胶过滤层析介质Superdex75。scFv98H纯化条件为：流速为1.5ml/min，上样量不超过柱体积的5%，采用一步洗脱方式，流动相为0.1mol/L脲、10%甘油、1%甘氨酸、50mmol/L Tris、2mmol/L EDTA，pH9.0。通过最后一步精纯得到scFv98H的单体。经过纯化得到产品的纯度＞97%，蛋白回收率72%，内毒素残留量＜0.5EU/mg，DNA残留量3ng/mg。（2）研究scFv98H柱层析（柱上）复性工艺并优化其条件。选择适合scFv98H复性的Qxl强阴离子交换层析介质。对Qxl复性scFv98H条件进行优化，主要考察复性液流速，复性pH，复性载量，复性液中变性剂含量。优化后的条件为：20ml柱体积的Qxl柱上复性的最适流速1.5ml/min，最适pH9.0，最适载量100mg，复性液里变性剂含量0.1mol/L，复性方案：脲浓度8mol/l→0.1mol/l，时间3h，流速1.5ml/min；变性液为20mmol/l，8mol/l脲，pH9.0，复性液为0.1mol/L脲、10%甘油、1%甘氨酸、50mmol/L Tris、2mmol/L EDTA，pH9.0。通过Qxl柱上复性，scFv98H的蛋白回收率为65%，蛋白比活力为700IU/mg。（3）在上述研究基础上，确定了scFv98H纯化和复性工艺路线，进行了实验室规模scFv98H分离纯化和复性，得到的产品经SDS-PAGE和HPLC测定，纯度达到97%以上，总回收率为48.6%。对其进行鉴定分析，AB SCIEX TOF/TOF测得分子量为27681.4844Da，，Western blot显示具有良好的抗原性，小鼠异常毒性试验合格，快速荧光灶抑制试验（RFFIT）检测中和活性为610IU/mg，细菌内毒素残留量检测法（凝胶法-鲎试剂）检测内毒素含量＜0.5EU/mg，外源性DNA残留量测定法（荧光染色法）检测DNA残留量为3ng/mg。通过本论文的研究，建立了实验室规模的scFv98H分离纯化和复性工艺，研究结果为将来的中试规模和产业化规模生产scFv98H奠定基础。
[Abstract]:Rabies is the most lethal acute infectious disease, is caused by rabies virus infection in the central nervous system of a zoonosis, with acute, contact, high lethality and other characteristics. WHO recommends the use of post-exposure prevention (PEP) to obtain rapid immune protection against the occurrence of rabies. Anti-rabies virus serum plays an important role, of course. However, the source of anti-rabies immunoglobulin (HRIG) is limited, there is potential blood disease contamination, anti-rabies equine immunoglobulin (ERIG) is easy to cause allergic reactions, there are differences between batches and other shortcomings, therefore, monoclonal antibodies due to no blood contamination and continuous production advantages, is considered to be the ideal of the two antisera. Substitutes. However, murine monoclonal antibodies are prone to cause great adverse reactions in humans. Genetically engineered antibodies have high molecular weights, need eukaryotic expression and are expensive, thus limiting their wide application. Gu Tiejun and his colleagues have designed and prepared the corresponding single-chain antibodies FV57 and FV4098 according to the mixed monoclonal antibody CR57/CR4098 which has entered the clinic, and both of them showed good binding activity and neutralization activity to rabies virus in vitro, especially FV4098 attacked mice. The protective rate in toxicity test is similar to that of HRIG and ERIG. Because there is His-tag in the C-terminal of FV4098 gene sequence, this paper calls it scFv98H. So this paper will try to establish a suitable purification and inclusion body renaturation process for scFv98H on the basis of construction of expression vector, prokaryotic expression and large-scale fermentation. Mold and industrialization scale production scFv98H lay the foundation.
The main contents of this thesis are as follows:
(1) Study the separation and purification process of scFv98H and optimize the conditions. Select a strong anion exchange chromatography medium suitable for purification of scFv98H. Optimize the conditions of purification of scFv98H by Nuvia Q, mainly investigate the initial buffer pH, sample flow rate, unit volume of chromatography medium loading. The optimized conditions are: using flow through mode purification, 20 ml column. The optimum volume pH was 7.2, the optimum sample velocity was 5ml/min, the optimum loading was 80mg. The mobile phase A was 20mmol/LTris, 8mol/L urea, pH 7.2, the mobile phase B was 20mmol/LTris, 8mol/L urea, 1mol/L NaCl, pH 9.0.
The refolded scFv98H was purified by gel filtration. The optimum purification conditions of the gel filtration chromatography medium Superdex 75.scFv98H were selected by molecular weight of scFv98H. The flow rate was 1.5ml/min, the sample volume was not more than 5% of the column volume. The mobile phase was 0.1mol/L urea, 10% glycerol, 1% glycine, 50mmol/L Tris, 2mmol/L EDTA, pH value. 9.0. The monomer of scFv98H was purified by the last step. The purity of the purified product was more than 97%, the protein recovery was 72%, the endotoxin residue was less than 0.5 EU/mg, and the DNA residue was 3 ng/mg.
(2) Study the refolding process of scFv98H column chromatography (on-column) and optimize its conditions. Select Qxl strong anion exchange chromatography medium suitable for refolding of scFv98H. The refolding conditions of Qxl scFv98H were optimized. The refolding fluid flow rate, refolding pH, refolding load and the content of modifier in refolding fluid were investigated. The optimum flow rate is 1.5ml/min, the optimum pH9.0, the optimum loading is 100mg, the content of refolding liquid rheodenaturant is 0.1 mol/L, refolding program: ureaconcentration 8 mol/l 85940.1 mol/l, time 3 h, flow rate is 1.5ml/min; denaturfluid is 20 mmol/l, 8 mol/l urea, pH9.0, pH9.0, refoldfluid is 0.1 mol/L urea, 10% glyc, 1% glycglycglycglycglycglycine, 50 mmol/L, 50 mmol/L/L, 2 mmol/EDTA, 2 mmol/EDTA, pH9.2 mmol/L, pH9.0 mmol/EDTA, pH9.x9.0 column refs The protein recovery of cFv98H was 65%, and the protein specific activity was 700IU/mg.
(3) On the basis of the above studies, the purification and renaturation process of scFv98H was determined. The purity of the product was over 97% and the total recovery was 48.6%. The molecular weight of the product was 27681.4844Da determined by AB SCIEX TOF/TOF and Western blot. It had good antigenicity and passed the abnormal toxicity test in mice. The neutralization activity of RFFIT was 610IU/mg, the endotoxin content of bacterial endotoxin was less than 0.5EU/mg by gel-limulus test, and the DNA residue was 3 ng/mg by fluorescence staining.
A laboratory-scale separation, purification and renaturation process for scFv98H was established. The results will lay a foundation for future pilot-scale and industrial scale production of scFv98H.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.99

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