抗狂犬病毒G蛋白單鏈抗體scFv98H的純化和復(fù)性工藝研究
發(fā)布時(shí)間：2018-09-05 05:43
【摘要】：狂犬病是致死率最高的急性傳染病，是由狂犬病毒感染中樞神經(jīng)系統(tǒng)導(dǎo)致的一種人畜共患病，具有急性、接觸性、高致死性等特點(diǎn)。WHO建議采用暴露后預(yù)防（PEP）獲得快速的免疫保護(hù)，防治狂犬病的發(fā)生�？箍袢《狙迤鹬匾淖饔�，然而，抗狂犬病人免疫球蛋白（HRIG）來(lái)源有限，存在潛在的血液疾病污染，抗狂犬病馬免疫球蛋白（ERIG）容易引起過(guò)敏反應(yīng)，存在批間差異等缺點(diǎn)，因此，單克隆抗體由于無(wú)血液污染和可連續(xù)生產(chǎn)等優(yōu)點(diǎn)，被認(rèn)為可能成為上述兩種抗血清的理想替代品。但是，鼠源單抗容易引起人體極大副反應(yīng)，基因工程抗體分子量大，需要真核系統(tǒng)表達(dá)，價(jià)格昂貴，因此限制單抗廣泛應(yīng)用。單鏈抗體具有原核表達(dá)、分子小、穿透力強(qiáng)、免疫原性低，易于基因操作等特點(diǎn)，比較適合大批量生產(chǎn)和具有很好的應(yīng)用前景。本課題組谷鐵軍等人曾根據(jù)已進(jìn)入臨床的單抗雞尾酒混合抗體CR57/CR4098，設(shè)計(jì)并制備得到相應(yīng)的單鏈抗體FV57和FV4098，且這兩株抗體在體外實(shí)驗(yàn)中都表現(xiàn)出對(duì)狂犬病毒較好的結(jié)合活性和中和活性，尤其是FV4098在小鼠攻毒實(shí)驗(yàn)中提供保護(hù)率相似于市售的HRIG和ERIG。因?yàn)镕V4098基因序列的C端有His-tag，本論文稱其為scFv98H。所以本論文將在前期表達(dá)載體構(gòu)建，原核表達(dá)及大規(guī)模發(fā)酵的基礎(chǔ)上，嘗試建立一套適合scFv98H純化和包涵體復(fù)性工藝，為其將來(lái)中試規(guī)模和產(chǎn)業(yè)化規(guī)模生產(chǎn)scFv98H奠定基礎(chǔ)。 本論文研究?jī)?nèi)容主要包括以下幾個(gè)部分： （1）研究scFv98H分離純化工藝并優(yōu)化其條件。選擇適合scFv98H純化的Nuvia Q強(qiáng)陰離子交換層析介質(zhì)。對(duì)Nuvia Q純化scFv98H條件進(jìn)行優(yōu)化，主要考察起始緩沖液pH，上樣流速，單位體積層析介質(zhì)的載量。優(yōu)化后的條件為：采用流穿模式純化，20ml柱體積的最適pH7.2，最適上樣流速5ml/min，最適載量80mg。流動(dòng)相A為20mmol/LTris、8mol/L脲，pH7.2，流動(dòng)相B為20mmol/LTris、8mol/L脲、1mol/L NaCl，pH9.0。 復(fù)性后的scFv98H經(jīng)過(guò)凝膠過(guò)濾進(jìn)行精純，通過(guò)scFv98H分子量選擇合適的凝膠過(guò)濾層析介質(zhì)Superdex75。scFv98H純化條件為：流速為1.5ml/min，上樣量不超過(guò)柱體積的5%，采用一步洗脫方式，流動(dòng)相為0.1mol/L脲、10%甘油、1%甘氨酸、50mmol/L Tris、2mmol/L EDTA，pH9.0。通過(guò)最后一步精純得到scFv98H的單體。經(jīng)過(guò)純化得到產(chǎn)品的純度＞97%，蛋白回收率72%，內(nèi)毒素殘留量＜0.5EU/mg，DNA殘留量3ng/mg。 （2）研究scFv98H柱層析（柱上）復(fù)性工藝并優(yōu)化其條件。選擇適合scFv98H復(fù)性的Qxl強(qiáng)陰離子交換層析介質(zhì)。對(duì)Qxl復(fù)性scFv98H條件進(jìn)行優(yōu)化，主要考察復(fù)性液流速，復(fù)性pH，復(fù)性載量，復(fù)性液中變性劑含量。優(yōu)化后的條件為：20ml柱體積的Qxl柱上復(fù)性的最適流速1.5ml/min，最適pH9.0，最適載量100mg，復(fù)性液里變性劑含量0.1mol/L，復(fù)性方案：脲濃度8mol/l→0.1mol/l，時(shí)間3h，流速1.5ml/min；變性液為20mmol/l，8mol/l脲，pH9.0，復(fù)性液為0.1mol/L脲、10%甘油、1%甘氨酸、50mmol/L Tris、2mmol/L EDTA，pH9.0。通過(guò)Qxl柱上復(fù)性，scFv98H的蛋白回收率為65%，蛋白比活力為700IU/mg。 （3）在上述研究基礎(chǔ)上，確定了scFv98H純化和復(fù)性工藝路線，進(jìn)行了實(shí)驗(yàn)室規(guī)模scFv98H分離純化和復(fù)性，得到的產(chǎn)品經(jīng)SDS-PAGE和HPLC測(cè)定，純度達(dá)到97%以上，總回收率為48.6%。對(duì)其進(jìn)行鑒定分析，AB SCIEX TOF/TOF測(cè)得分子量為27681.4844Da，，Western blot顯示具有良好的抗原性，小鼠異常毒性試驗(yàn)合格，快速熒光灶抑制試驗(yàn)（RFFIT）檢測(cè)中和活性為610IU/mg，細(xì)菌內(nèi)毒素殘留量檢測(cè)法（凝膠法-鱟試劑）檢測(cè)內(nèi)毒素含量＜0.5EU/mg，外源性DNA殘留量測(cè)定法（熒光染色法）檢測(cè)DNA殘留量為3ng/mg。 通過(guò)本論文的研究，建立了實(shí)驗(yàn)室規(guī)模的scFv98H分離純化和復(fù)性工藝，研究結(jié)果為將來(lái)的中試規(guī)模和產(chǎn)業(yè)化規(guī)模生產(chǎn)scFv98H奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.99
本文編號(hào)：2223371
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.99
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