重组人组织因子的表达纯化及复性研究

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重组人VEGF可溶性受体的表达纯化及结合性质初探

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１０００７１；３．安徽大学生命科学学院，安徽合肥２３０６０１［摘要］目的：表达优化的血管内皮细胞生长因子（ＶＥＧＦ）受体１（ＶＥＧＦＲ１）胞外区第２个类免疫球蛋白结构域（ＶＥＧＦＲ１Ｄ２）和ＶＥＧＦ受体２（ＶＥＧＦＲ２）胞外区第３个类免疫球蛋白结构域（ＶＥＧＦＲ２Ｄ３）与人ＩｇＧＶＥＧＦ可溶性受体的表达纯化及结合性质初探
李伊培，高丽华，张连成，邵勇，刘羽，潘芸。，高招刚。，张嵘，胡显文，陈惠鹏
１．沈阳药科大学生命科学与生物制药学院，辽宁沈阳１１００１６；２．军事医学科学院生物工程研究所，北京
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接到真核表达载体ｐＩＲＥＳ２．一ＥＧＦＰ — Ｆｃ中，转染ＣＨＯ — Ｋ１细胞并筛选高表达目的蛋白ＶＥＧＦ一－Ｔｒａｐ２的细胞系，亲和纯化ＶＥＧＦ－Ｔｒａｐ２蛋白，通过非竞争性ＥＬＩＳＡ及生物膜干涉技术检测ＶＥＧＦ — Ｔｒａｐ２与ｈＶＥＧＦ之间的亲和力。结果：ＤＮＡ测序表明真核表达载体ｐｌＲＥＳ２一ＥＧＦＰ — ＶＥＧＦ－－－Ｔｒａｐ２序列正确；获得表达ＶＥＧＦ — Ｔｒａｐ２的细胞系；非竞争性ＥＬＩＳＡ实验中，ＶＥＧＦ－Ｔｒａｐ２与ｈＶＥＧＦ。功能性亲和常数达到１．８６ｘ１０ ’Ｌ／ｍｏｌ；生物膜干涉实验中，ｈＶＥＧＦ，与ＶＥＧＦ－－－Ｔｒａｐ２的平衡解离常数达到３．１３ｘ１０－ｍｏｌ／Ｌ。结论：构建了真核表达载体ｐｌＲＥＳ２一ＥＧＦＰ — ＶＥＧＦ－．－Ｔｒａｐ２并在ＣＨＯ — Ｋ１细胞中稳定表达，重组蛋白ＶＥＧＦ — Ｔｒａｐ２与ｈＶＥＧＦ，。有较高的亲和力，提示其可用于阻断ＶＥＧＦ信号传导途径，为该蛋白进一

重组人体干细胞因子的表达、复性、纯化及其对脐带血干细胞体外扩增作用研究

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重组人粒细胞—巨噬细胞集落刺激因子包涵体提取,复性和纯化的研究

重组人粒细胞—巨噬细胞集落刺激因子包涵体提取,复性和纯
化的研究
周永春;陆峰
【期刊名称】《生物工程学报》
【年(卷),期】1997(013)002
【摘要】由基因工程大肠杆菌表达的重组人粒细胞－巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）以包涵体的形式存在于细胞中，通过破菌、洗涤获得包涵体，再经过溶解、凝胶过滤、复性、疏水和离子交换柱导析得到了均一的产品，经高压液相和ＳＤＳ－ＰＡＧＥ电泳测定纯度均大于９８％，ｒｈＧＭ－ＣＳＦ的比活为３．２×１０＾７ＩＵ／ｍｇ，纯化获得的ｒｈＧＭ－ＣＳＦ为一酸性蛋白，等电点约为５．２，ＮＨ２－末端有２０个氨基酸序列测定结果
【总页数】7页(P142-148)
【作者】周永春;陆峰
【作者单位】第二军医大学医学生物技术和分子遗传研究所;第二军医大学医学生物技术和分子遗传研究所
【正文语种】中文
【中图分类】R392－33
【相关文献】
1.体积排阻色谱法(SEC)对重组人粒细胞-巨噬细胞集落刺激因子的复性并同时纯化[J], 刘建波;王欢;白泉;耿信笃
2.人粒细胞集落刺激因子包涵体的提取及其复性研究 [J], 马骊;宁云山
3.重组人白细胞介素-2/ 粒细胞-巨噬细胞集落刺激因子融合蛋白的纯化及复性研究 [J], 林来兴妹;周明乾;陈泽洪;胡志明;刘菁;黄树琪;王小宁
4.重组人粒细胞-巨噬细胞集落刺激因子复性和纯化方法的比较 [J], 刘建波;于占江;邓玲娟;张尼;白泉
5.用疏水色谱法复性并同时纯化重组人粒细胞-巨噬细胞集落刺激因子 [J], 刘建波;古元梓;王欢;张尼;白泉
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重组人干细胞因子工艺研究

重组人干细胞因子工艺研究---------专业班级姓名学号摘要人干细胞因子(Human stem cell factor，hSCF)是一种重要的造血细胞因子，该文章在重组hSCF工程菌的基础上，通过高密度发酵培养、包涵体分离纯化、变复性、SP—Sepharose FF、Source 30RPC与Q—Sepharose FF层析等技术分离纯化出具有生物学活性的重组人干细胞因子，建立了适合大规模生产重组人干细胞因子的分离纯化工艺。

关键词组人干细胞因子包涵体复性分离与纯化生物学活性分离纯化工艺人干细胞因子(Human stem cell factor，hSCF) 又称为肥大细胞生长因子(mast cellgro、Ⅵh factoL MGF)、kit配体(kit ligand，KL)及steel因子(steel factoL SL)，为c．kit原癌基因编码受体的配体蛋白，是自1990年发现的一种重要的细胞因子。

它能够刺激早期造血干细胞及祖细胞增值，是造血干细胞增值分化的关键因子【1】。

hSCF能刺激造血细胞的生长、增生与分化。

它可以与其它造血因子共同发挥作用，如与白细胞介素-3(IL-3)、白细胞介素-6(IL-6)、集落刺激因子(G-CSF)、促红细胞生成素(EPO)等表现出较强的协同效应，临床上SCF可用于治疗一系列原发性或继发性的(因毒性、辐射免疫损伤造成的)以髓细胞亚群数目减少为特征的干细胞功能异常等类型的疾病【2~4】。

具有广阔的应用前景和药用开发价值。

正是由于SCF具有广阔的应用前景，所以对它的需求也很大，但是其天然来源有限，成熟型可溶性SCF(SCF165)在人体或动物体内含量甚微，在血中的浓度为3.3ng/mL，故不可能从天然组织中分离纯化足够量的样品进行实验室和临床应用研究【5】。

大量实验证明，人SCF的糖基化对其活性不是必需的，如此就可以用基因工程方法，使用大肠杆菌等表达体系来生产重组人SCF(rhSCF)，以此来解决这个难题【6】。

改善重组人干细胞因子包涵体复性与同时纯化放大过程的回收率

Improve on recovery of the recombinant human stem cell factor inclusion body in refolding with simultaneous purificationprocess1Wang Lili, Wang Chaozhan, Liu Jiangfeng, Geng XinduInstitute of Modern Separation Science, Shaanxi Key Laboratory of Modern Separation Science, Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education,Northwest University, Xi’an, China (710069)E-mail: llwang@AbstractRecombinant expressed human stem cell factor (rhSCF) as cytoplasmic inclusion bodies (IB) are reported. In present work, to increase mass recovery of rhSCF production in large scale, the factors affecting about efficiency of rhSCF IB were recovered and solubilised in urea solution, and refolding with simultaneous purification process using protein folding liquid chromatography (PFLC) were investigated, including normal chromatographic column, the unit for the simultaneous renaturation and purification of proteins (USRPP). Finally, by combining the optimized buffer and USRPP, we were able to obtain22 mg rhSCF with >95% purity.The mass recovery is 24 % for dilution, 38 % for the normal chromatographic column, 49 % for the USRPP. An average specific bioactivity is 4.27 ×105 IU/mg, 6.9×105 IU/mg, and 1.28×106 IU/mg, respectively. These protocol dates and new refolding with purification method -USRPP provide a cost effective and an efficient way to produce quantities of high purity rhSCF in large-scale.Keywords: recombinant human stem cell factor, inclusion body, protein folding liquid chromatography1. INTRODUCTIONStem cell factor (SCF) is a cytokine produced by multiple types of cells including stromal cells andfibroblasts [1]. The soluble form of SCF has 165 amino acids, and exists as a non-covalently associated homodimer [2, 3]; with each SCF monomer containing two intra-chain disulfide bridges, Cys4–Cys89 and Cys43–Cys138 [4]. The human soluble SCF (hSCF) shows multi-lineage hematopoiesis-stimulating activities, and therefore has been considered as a potential therapeutic for various diseases [5]. So far, the recombinant hSCF (rhSCF) has been produced in genetically engineered E. coli [6, 7, 8 ]. Because rhSCF is mostly in the inclusion body fraction of bacterial lysates, a refolding process is necessary to produce soluble rhSCF possessing the same bioactivity as its native state. Evidence has demonstrated that the oxidative refolding of rhSCF produced in E. coli might produce at least five intermediate forms, I-1 to I-5, detectable by their differences in hydrophobicity using reverse-phase high performance liquid chromatography [9], suggesting that oxidative refolding is not efficient enough to refold all rhSCF in solubilized inclusion bodies. Thus, in order to obtain pure rhSCF with high a bioactivity, new refolding and purification procedures are required to provide correctly folded rhSCF with high yields.Protein folding liquid chromatography (PFLC) technology is a new method developed at present [10]. Their characteristics are easier to achieve scale preparation, such as high performance hydrophobic interaction chromatography (HPHIC) [ 11,12],Size exclusion chromatography (SEC) [13]and ion exchange chromatography (IEC) [14,15]. Especially, HPHIC process, solubilized inclusion body proteins interact with the hydrophobic medium tightly preventing not only aggregation of unfolded proteins, but also dominates the formation of steric structures inproteins and thus assists in the refolding of the denatured proteins, the refolded proteins can be simultaneously purified during HPHIC the1 This work was supported by grants from the National Natural Science Foundation of China (No.20475042) , the Foundation of the Key Laboratory for Modern Separation Science in Shaanxi Province (No.05JS60) and Specialized Research Fund for the Doctoral Program of Higher Education (No.20040697002).process[16]. A specially designed unit, with diameter much larger than its length, was designed and employed for both laboratory and preparative scales of the unit for the simultaneous renaturation and purification of proteins (USRPP)[17].In recent years, the USRPP has been used for a scale manufacturing of recombinant protein [17, 18, 19]. We have been reported previously to use HPHIC for the refolding and simultaneous purification of rhSCF expressed in E coli [20]. In this work, to increase of rhSCF production in large scale, we discusses the renaturation buffer, and the optimization of factors affecting the efficiency of refolding and purification of rhSCF with HPHIC column, USRPP.The efficient procedure of refolding and purification may be useful for the mass production of rhSCF proteins.2. EXPERIMENTAL2.1 ApparatusHPHIC was carried out using an LC–10ATvp high-performance liquid chromatograph (Shimadzu, Kyoto, Japan) consisting of two pumps (LC–10A), a variable–wavelength UV–Vis detector (SPD–10A V), and a system controller (SCL–10B). The HPHIC column (150mm×4.6mm i.d.), and the USRPP (10mm×20mm i.d.) were bought from Shaanxi Xida Keli Gene-Pharmcy Co.Ltd (Xi’an, China), and was packed materials with PEG-200, PEG-400, PEG-600 and furfural,2.2 ChemicalsAcrylamide,bis-acrylamide, BSA, reduced glutathione(GSH), oxidized glutathione(GSSG)and the referents of standard molecular weight were obtained from Sigma. Tris, glycine and SDS were obtained from Amresco; Coomassie Brilliant Blue G-250 (Fluka, MO, USA). All other chemicals were of analytical grade.2.3 Preparation of rhSCF extractThe strain used was recombinant E. coli DH5α harboring the plasmid pBV220 [8,21].The bacteria were produced with a 5 L fermenter (B. Brawn Co, Germany). E. coli cells containing rhSCF were disrupted by sonication in a buffer containing 20 mmol/L phosphate buffer solution (PBS), pH 7.4, 1 mmol/L EDTA, and 0.20 mg/ml lysozyme, were collection of rhSCF from E.coli DH5α was performed using the procedure described by Wang [20] .The cleaned inclusion bodies were were recovered by cen-trifugation at 16000g, 4°C for 20 min and solubilized in 50 mmol/L Tris-HCl, pH 8.0, 8 mol/L urea, 10 mmol/L dithiothreitiol, 1 mmol/L EDTA and were centrifuged at for 20 min. The protein concentration of the supernatant was was adjusted by Bradford method to a final concentration using 8 mmol/L urea solubilizing solution.2.4 Chromatographic procedureA chromatography run was carried at room temperature. The packed HPHIC column, and URSPP was equilibrated with 100% mobile phase A (3.0 mol/L ammonium sulfate [(NH4)2SO4], 50 mmol/L potassium dihydrogen phosphate (KH2PO4), pH 7.0) at a selected flow rate for concentration gradient elution depended on the size of column. 8 mol/L urea of crude rhSCF solution was directly injected into the column through the sample valve, respectively. All chromatograms were detected using UV absorbance at 280 nm.,Gradient elution (linear and nonlinear) was used during the purification of rhSCF and fractions containing target protein were collected for the measurements of the recoveries of bioactivity and mass of the rhSCF.2.5 Analytical proceduresThe column-refolding fraction was incubated for 30 min at 30°C, followed by dialysis against 20 mmol/L PBS, pH 7.4 at 4°C. The dialysis solution was collected and lyophilized (stored frozen until further tests). The total protein concentration of the product in the purification fractions of rhSCF was determined using the Bradford method, and evaluated mass recovery by Wang [20] .The purity of rhSCF was analyzed by SDS-PAGE with 15% acrylamide, and the density of each band after staining with Coomassie bright blue (Uppsala, Sweden) was quantified by scanning the gel using a thin-layer gel scanner (Cs-930, Shimadzu, and Kyoto). Monomers and aggregate forms of rhSCF were analyzed by gel chromatography using Sephadex G-75(Amersham Pharmacia，200mm×16mm i.d.). The molecular weight of rhSCF was evaluated by MALDI-TOF-MS (Axima CFR plus, Kratos, Shimazu, Japan).2.6 Assay for bioactivity of hSCFThe bioactivity of rhSCF was measured using the hSCF-dependent cell line UT-7 [22,23]. Briefly, cells were cultured in RPMI-1640 (from Sigma) medium supplemented with 10% fetal calf serum (v/v) and maintained in the presence of erythropoietin (from the National Institute for the Control of Pharmaceutical and Biological Products of China). Cells were washed with the culture medium and cultured in the presence of the purified rhSCF at different concentrations. Proliferation of the cells was determined using the MTT method.3. RESULTS AND DISCUSION3.1 Optimal buffer composition for refolding of rhSCFAt present, mostly presence problem of refolding and purification recombinant human proteins are still lost of activity, scaling up, low recovery. Some literature date have provided information aimed at enhancing the refolding yield of inclusion body proteins by reducing the causes of aggregation and misfolded configurations, respectively[24,25]. It has been reported that certain Gu.HCl, urea (1-2 mol/L), and L-arginine (0.3-1 mol/L) can inhibit protein aggregating [26,27]. Valente et al reported that an optimized protocol could significantly increase the yield [28].To obtain complete refolding of the solubilized inclusion bodies, we optimized the pH and components of the refolding buffers in dilution process, such as the urea, arginine components, which are usually used in the refolding of recombinant proteins, by monitoring protein recovery in the soluble fraction after refolding (Fig.1). Usually, aggregation decreases when the pH of the medium is far away from the protein’s isoeletric point [29]. The most favorable pH value varies from protein to protein, it effect directly aggregation of protein and the formation of disulfide bonds. A proper pH has a cooperative effect on enhancing the renaturation yield of rhSCF and on the formation of disulfide bonds. Fig.1A shows how refolding of rhSCF in buffers of different pH with dilution. The refolding of the rhSCF had the highest mass recovery in pH 8.2-8.5.Low concentration of denaturant such as urea has been included in various renaturing buffers as an efficient inhibitor of protein aggregation, which can assist spontaneous protein refolding in solution and increase the output of correctly folded proteins,as shown in Fig.1B. It is has been reported arginine to efficiently inhibit inclusion body aggregation of recombinant proteins [30]. Fig.1C shows the turbidity and protein recovery of rhSCF refolded in the presence of different concentrations of arginine. The turbidity was measured by transparency at A450. The result indicated that the most suitable concentration of arginine is between 0.5-0.6 mol/L.Each SCF monomer contains two intra-chain disulfide bridges, Cys4–Cys89 and Cys43–Cys138 [1]. We further examined the effect of the redox agent glutathione (GSSG/GSH) on the folding property of rhSCF. In the absence of GSSG /GSH (0.25 mmol/L) does not promote the folding of rhSCF. Theinfluence of the ratio of GSH/GSSG on the refolding of rhSCF as shown in Fig.1D, the results indicate that refolding of rhSCF is very favorable in 5:1 with GSH/GSSG .From the above results, Fig.1A-1D showed the solubilized rhSCF using the optimal of a refolding buffer 50 mmol/L Tris-HCl, pH 8.2, 1 mmol/L EDTA, 1 mmol/L oxidized glutathione, 0.2 mmol/L reduced glutathione, 2.5 mol/L urea, 0.5 mol/L arginine at 4°C with slight agitation, and mass recovery of rhSCF was improved from 10 % to.24 % .These dates will follow as a result using chromatography processing.Figure 1: Optimal buffer composition for refolding of rhSCF1A: The influence pH on the recovery of refolded rhSCF in denaturing buffer 1B: The influence urea on the recovery of refolded rhSCF in denaturing buffer 1C: The influence of the ratio of GSH/GSSG on the refolding of rhSCF1D: Effect of arginine concerntration on aggregation and on the recovery of refolded rhSCF in denaturing buffer3.2 Selection ligand structure of STHICSilica-based packing material was reported to have bi-functions of purification and renaturation for proteins [18]. The hydrophobicity and structure of the ligand used in the stationary phase of hydrophobic interaction chromatography (STHIC) were found to be the most important factors affecting mass recovery [31,32].Urea concentration (mol L Mass recovery ( %)P r o t e i n c o n c e n t r a t i o n (m g m l -1)-◆-Tris-urea; -☆-PBS-urea buffer;-■-Tris buffer;-▲-PBS buffer0 2 4 6 7 8-1)Activity recovery (%)P r o t e i n c o n c e n t r a t i o n (m g m L -1)4 5 6 7 8 9 10 11 12pH-▲-Protein concentration -■-Activity recoveryRecovery of protein (%)-◇-Protein concentration -■-Activity recovery0 1 2 3 4 5 6Arginine concentration (mol l -1)T u r b i d i t y (450 n m )ABCTable 1 The mass recoveries of refolded rhSCF and retention on different ligands aLigands Chromatography column (150×4.6mm i.d.)USRPP(10×20mm i.d.)Retention time (min) The massRecovery (%)Retention time (min) The massRecovery (%) -(CH 2-CH 2-O)400 33.0811 36 31.701 49 -(CH 2-CH 2-O)600 34.556 34 31.709 37 -(CH 2-CH 2-O)800 34.954 30 31.854 32 -O-CH 2-O-phenyl 36.152 2831.925 29aThe 100µL solutions of 8.0 mol/L urea solution were directly injected into the four kinds of HIC columns,respectively.We firstly determine which type of STHIC media is suitable for the specific protein we wish to refold. Four kinds of ligands with different hydrophobicities and molecular structures were tested, and the order of four hydrophobic ligands were furfural>PEG600>PEG400>PEG200 [17,32].8.0 mol/L urea dissolved inclusion body was directly loaded onto the four kinds of ligands chromatographic columns, and listed in Table 1 show result. From this Table 1,although the obtained rhSCF peaks shows the almost same retention time in USRPP, the mass recoveries of the collected rhSCF from the four STHIC are significantly with each other and PEG 400 is best one, in other words, the ligand PEG-400 is more favorable for rhSCF refolding..3.3 Optimal buffer composition of mobile phasesAs descried above “ptimization of buffer composition”, when the refolding buffer contains 2.5 mol/L urea, 0.5 mol / L arginine and the pH value is maintained at 8.2 using refolding buffer of dilution, the highest mass recovery and refolding efficiency of the rhSCF is achieved. This indicates the net environmental contribution of irrespective of stationary phase. In practice; it is predictable that the composition of the mobile phase should also play an important role in rhSCF refolding by HPHIC also. The continuously changing environment of the mobile phase during gradient elution provides a broad concentration range of salt for its refolding. Especially, it is the disulfide bridges of protein molecules.As shown in Table 2, buffers 1 and buffer 2 were used for comparing the contribution of the mobile phase to rhSCF refolding. In terms of mass recovery and the specific bioactivity of the refolded rhSCF, buffer 2 performs better than buffer 1. Apparent, mobile phase composition is one of the most important factors affecting the efficiency of protein renaturation in chromatographic procedure. The optimization of mobile phase composition protein renaturation with simultaneous purification is thus much more important than that in usual liquid chromatography [32].Table 2 Effects of eluting buffer on the renaturation of rhSCF**Eluting buffer Mass recovery a Specific bioactivity Purify(% ) (×106 IUmg -1) (%)bBuffer 1 36.5 0.73 >95 cBuffer 2 47.9 1.14 >95**Column (150×4.6mm i.d.);aThe ratio of the mass of recovered in collected fractions and total mass of injection after 8mol /L urea dissolved ; bBuffer 1: Mobile phase A, 3.0 mol/L (NH 4)2SO 4 ,50 m mol/L KH 2PO 4, pH 7.0 ; Mobile phase B, 50 m mol/L KH 2PO 4 pH 7.0 . cBuffer 2: Mobile A,3.0 mol/L (NH 4)2SO 4 ,50 mmol/L KH 2PO 4, 2.0 mol/L urea, 1.0 mmol/L GSH , 0.20 mmol/L GSSG , and 0.5 mol/L arginine, pH 7.0;Mobile phase B, 50 m mol/L KH 2PO 4 ,2.0 mol/L urea, 1.0 m mol/L GSH , 0.20 m mol/L GSSG , 0.5 mol/L arginine, pH 7.0.3.4 Gradient elution modeThe advantage of USRPP is that protein refolding and purification can be achieved simultaneously in one chromatographic run [18]. This means, in addition to the efficiency of protein folding, good resolution is an indicator of the purity of the refolded rhSCF also. We tried to optimize the gradient elution modes for the refolding of rhSCF using linear and nonlinear gradient elution. Fig.2 shows the comparison between the chromatograms for linear gradient (Fig.2A) and nonlinear gradient (Fig.2B), respectively. As can be seen from figure 2, the resolution using nonlinear gradients is better than using a linear gradient when all other conditions are the same.Figure 2 Chromatogram of rhSCF by using a USRPP on different gradient modes linear gradient elutionFigure 2A: 40min linear gradient elution; Figure2B: 40min non- linear gradient.Columns (10×20mm i.d.),100% solution A, 3.0 M ammonium sulphate – 50mM potassium dihydrogen phosphate (pH 7.0) to 100% solution B, 50mM potassium dihydrogen phosphate (pH 7.0) in 30 min with 10 min delay. Flow rate: 2.0 mlmin-1.3.5 Refolding with simultaneous purification of rhSCF by USRPPDuo to one of the advantages of USRPP is much bigger diameter than its length, the flow rate of the mobile phase is very high which decreases the time the sample is in the sample loop thereby; reducing formation of precipitates [17,18]. In addition, if some precipitates form on the surface area of the filter frit, the column backpressure can still remain at a low level, because the precipitates only block a very small fraction of the total surface area. Thus, unlike normal chromatographic columns, which can be blocked by precipitates, USRPP can maintain a normal chromatographic run. One-step refold with simultaneous purification process in a USRPP (PEG400, 10mm ×20 mm i.d.), allowed us to run sampleA b s o r b a n c e (280 n m )A b s o r b a n c e (280 n m C o n c e n t r a t i o n o f b u f f e r 10 20 30 4010 20 30 400 50 100 C o n c e n t r a t i o n o f b u f f e r B0 50 100t / min t / min ABsolution of 1 ml rhSCF was loaded onto the USRPP, then a nonlinear gradient was performed for 40 min. 50ml fractions were collected, and we checked the purity of rhSCF by Coomassie blue stained SDS-15% PAGE (Fig.3, lane 4), the purity is ≥ 95%.Figure 3 SDS-15%PAGE analysis of rhSCF step -by-step purificationLane1: Molecular weight marker (14,400Da; 20,000 Da; 24,000Da; 29,000Da; 36,000 Da, 45,000 Da, 66,200 Da); Lane 2: 8mol/L urea dissolved inclusion body solution; Lane 3: Refolded by dilution; Lane 4: Collected fraction refolded and purification of rhSCF by USRPP (10×20mm i.d.); Lane 5: Collected fraction refolded and purification of rhSCF bycolumn (150×4.6mm i.d.); Lane 6: The final bulk after semi-permeation (freeze-drying)3.6 Analysis characteristic in the final bulkThe recovery of the bioactivity and mass of the rhSCF obtained from the dilution-refolded to the column-refolded methods is shown in Table 3. From Table 3, the 8 mol/L urea-dissolved rhSCF could be efficiently refolded with simultaneous purification in one-step using USRPP, and obtain over 22 mg of hSCF from per liter of M9 media culture.SEC was employed for the analysis of the purified rhSCF under native conditions for monomeric rhSCF.The biological activities of production provide valuable information for refoding of rhSCF protein; this is a very important characteristic for large-scale renaturation. As shown in Fig 4, the renaturated and purified rhSCF was examined by UT-7(human megakaryoblastic leukemia cell) dependent cell line [22]. According to dose-response curve of SCF on UT-7 cell proliferation, the refolded rhSCF also possesses a higher bioactivity in supporting the growth of an SCF-dependent cell line UT-7; the purified rhSCF protein has comparable activity as natural product. Compared to the dilution-refolded rhSCF (4.27 ×105 IU/mg), the column-refolded rhSCF had an average specific bioactivity of 6.9×105 IU/mg and 1.28×106 IU/mg, respectively. MALDI-TOF analysis demonstrated the molecular weight of 18,573 Da (18,589 Da for natural hSCF).1 2 3 4 5 666.045.036.029.024.020.014.2Figure 4 The dose-response curve of rhSCF for the proliferation of UT-7 cells1: Production of renatured of rhSCF by dilution; 2: Standard of the rhSCF (from sigma, Std.). 3: Production of renaturedwith simultaneous purification of rhSCF by USRPPTable 3 The comparison of the refolding and purification of rhSCF with different protocols aSteps Total proteins （mg ）Purity (%)Yield ofrhSCF(mg) Recovery of rhSCF(%)Cell lysate 213 32 45 1008M urea dissolved IB b 71 81 38 84 Dilution refolding 19 85 11 24HPHIC column d31 >95 17 38 USRPP d 37>95 22 49 From 1L of E. coli culture; after optimal cleaning buffer; The 200µL solutions of 8.0 mol/L urea solution weredirectly injected into different columns, respectively.3.7 ConclusionComparisons of rhSCF produced with different methods, show a comparable mass recovery of rhSCF, refolded the rhSCF simultaneously using USRPP possesses a higher specific bioactivity: i.e. 3 times that produced by dilution. Experiments indicated that the STHIC having a weaker interaction than affinity, ion-exchange or reversed-phase chromatography, the structural damage to the proteins is assumed to be minimized, and the biological activity of the proteins is maintained. However the advantage of STHIC is that the chromatographic condition is much closed to the physiological condition, such as neutral pH, aqueous salt solution, being favorable to remain protein bioactivity [33,34,35].Our results show that (1) Refolding and purification of recombinant proteins occurred more efficiently using USRPP rather than usually chromatography column.（2）it possible that some specific methods and strategies have made to enhance high yields of biologically active proteins by taking into account process parameters, include refolding buffer of additive, pH, redox conditions ionic strength, and generation of correct disulphide bonds.（3）Due to a combination of adsorption on STHIC and a gradient elution, with mobile phase containing additive, the mass and bioactivity recoveries of rhSCF should markedly increase.(4) It is expected that this procedure will be useful in the large-scale manufacture of rhSCF for therapeutic purposes. 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重组人表皮生长因子的分离与纯化的研究

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重组人表皮生长因子的分离与纯化的研究
商安新（哈药集团生物工程有限公司，黑龙江哈尔滨１５００００）摘要：本实验采用凝胶法来纯化人表皮生长因子，并定量测定了人表皮生长因子（ｈＥＧＦ）。人表皮生长因子与经过离子交换柱、ＳＤＳ — ＰＡＧＥ及凝胶图象扫描分析系统的标准品相互对比。结果表明，在ＳＤＳ — ＰＡＧＥ凝胶上能够得到与标准样品ｈＥＧＦ一样清晰的单条带，得到的产品纯度可高于９４％，检出限能够达到ｌｕｇ／ｍＬ。本实验方法能够很好的在线检测出人表皮生长因子，同时本方法对其它蛋白质分离的在线检测也具有参考价值。关键词：人表皮生长因子；分离；纯化；在线检测人表皮生长因子￣ｕｍａ．ｅｐｉｄｅｒｍａｌｇｒｏｗｔｈｆａｃｔｏｒ，ｈＥＧＦ）是一种最与进样量的关系，结果显示样品出现的峰随着进样量的体积增加而明早被发现的，也是最早应用于临床上的多肽生长因子，该因子由５３个显变宽，所得的峰面积逐渐增加，计算得标准曲线方程为Ａ＝２．０８０１．３９８，式中Ａ表示峰面积，ｍ表示ｈＥＧＦ质量（ｕ。氨基酸残基组成，相对分子量约为６２００Ｄａ。ｈＥＧＦ具有多种生物学ｍ～０５．２ｈＥＧＦ的分离纯化及浓度在线检测。经研究发现目标产物功能，其不仅能够促进表皮和上皮细胞的增殖，以及增强蛋白质及ＤＮＡ的合成，而且还能够抑制胃酸分泌等多种作用。因此，在临床上其可应ｈＥＧＦ的相对分子质量６２００Ｄａ，而其它蛋白质的最小相对分子质量８０００Ｄａ，两者相距约１００００Ｄａ以上，因此可以应用凝胶对ｈＥＧＦ用于外科伤口的救治、皮肤和角膜移植、溃疡的治疗和促进伤口愈合等为１回进行分离。经过反复实验我们总结得到优化后的分离条件为：柱床高度自从有研究者在人的尿液中分离并纯化出人表皮生长因子以来，１６ｃｍ，线性流速６ｃｍ／ｈ，进样量ｌｍＬ。我们应用此条件进行层析，结果关于研究＾表皮生长因子的报道就越来越多，并目发展很迅速。该种因显示在两个大峰群中间有１个小的峰，其保留的体积与我们在相同条ＥＧＦ样品的保留的体积一致，然后再用ＳＤＳ — 子不仅可以从天然来源中分离纯化出来，而且近些年来有研究者利用件下所测到的标准ｈＰＡＧＥ凝胶来进行检测，最后确认该小峰为ｈＥＧＦ的峰，其余杂质峰基基因工程技术人工分离纯化出＾表皮生长因亏。目ｈＥＧＦ的定量检测方法主要有放射免疫分析法、放射受体分本是可以分开。析法、酶联免疫吸附分析法、酶免疫分析法、反相ＨＰＬＣ法和免疫荧光根据标准曲线我们取低浓度的回归方程可得到ｈＥＧＦ的绝对含量技术等１４］。同时以上的几种方法都有其各自的特点和不同的条件局限为１５６．２２ｕｇ，其质量浓度可达到１５６．２２ｕｇ／ｍＬ；然后我们又用高浓度的回归方程计算，其得到ｈＥＧＦ的绝对含量为１５７．２５ｕ质量浓度可达性，而且都难用于在线检测的过程。本文在实验时使用ＡＫＴＡｅｘｐｌｏｒｅｒ１００对蛋白质进行分离纯化到１５７．２５ｕ咖Ｌ。综上所诉，两个方程的计算结果基本达到一致。经时发现该检测系统自身带有一种软件，该软件不但能够与各种层析柱ＡＫＴＡｅｘｐｌｏｒｅｒ１００系统检测后，得到１ｍＬ该样品中ｈＥＧＦ的峰面．６９％，此即该盐析液中ｈＥＧＦ的相对含量（结合来研究多种蛋白质的分离流程，而且还可以通过出峰面积的大小积占整个峰面积的比值为２比较精确地表示出蛋白质量的多少，这为蛋白质定量检测奠定了基础。％１。本文以此对ｈＥＧＦ进行了分离纯化的在线定量检测的探索研究。５．３分离结果的验证。将上述分离后的收集液进行冷冻干燥，得到白色的ｈＥＧＦ粉末。对所得粉末与ｈＥＧＦ的标准品用阴离子交换预装１仪器与试剂１．１仪器。分离ｔ￣４Ｌ＇决速开拓系统（ＡＫＴＡｅｘｐｌｏｒｅｒ１００）、进样柱、柱进行了分扼得到ｈＥＧＦ的粉末的保留体积与纯度峰面积分别层析柱、离子交换预装柱、凝胶、蛋白质扫描分析系统；电泳仪。为２５．２５ｍＬ和９４．９７％，而ｈＥＧＦ的标准品的保留体积与纯度（峰面５．１６ｍＬ和９４．９０％，所以得到两者的纯度几乎是相一１－２试剂。ｈＥＧＦ标准品；磷酸缓冲液Ｂｓ）；其它试剂均为分析纯。积分别为２２样品的预处理致的，由此可以说明研究者在采用在线检测的方法后，使得样品的分取含有ｈＥＧＦ的发酵液于４ ℃，１００００ｒ／ａｒｉｎ离心８ａｉｒｎ后收集离结果得到良好的改善。同时由于该检测过程能够随时随地的展示出该上清液，取上清液１０００ｍＬ然后用ｌｍｏｌ／Ｌ的盐酸调节上清液的ｐＨ产品的浓度与分离条件两者的关系，所以该方法对ｈＥＧＦ的分离纯化至４．６左右，并缓慢加入饱和度为３５％的硫酸铵２１０ｇ，均匀进行搅过程的条件优化给予极大地方便。拌使之溶解，于４ ℃下静置，４ — ２４ｈ后至彻底沉淀。利用虹吸来去除然而为了能更进一步的来验证实验结果，我们取上述实验分离得ＥＧＦ产品与ｈＥＧＦ标准品经ＳＤＳ — ＰＡＧＥ凝胶电泳来进行检测，７００ — ８５０ｍＬ左右的上清液，同时将剩余的１５０ — ３００ｍＬ左右的上清液到的ｈ于１００００ｒ／ｍｉｎ离心１５ｍｉｎ，收集沉淀。再取１００ｍＬ的平衡液（ｐＨ并将所得到的实验图谱用蛋白质扫描分析系统来进行分析。实验结果７．０，０．０２ｍｏｌ／Ｌ的磷酸缓冲液（ＰＢＳ１）加入沉淀中并搅拌使其溶解，使之表明两种ｈＥＧＦ样品在ＳＤＳ — ＰＡＧＥ凝胶电泳的图谱中基本上都呈现形成悬浊液，４ ℃静置４ — ２４待充分溶解后，于４ ℃ 下１００００ｒ／ａｒｉｎ出了单一的条带；并且采用蛋白质扫描分析系统后我们测得其分离产离心１５ｍｉｎ，收集上清液，于４ ℃下保存备用。品的纯度为９４；７８％，其与应用阴离子交换预装柱测得的纯度（９４．８２３ｈＥＧＦ的定性分析是几乎一致的。利用ＳＤＳ一聚丙烯酰胺凝胶电泳（ＳＤＳ — ＰＡＧＥ）￣对ｈＥＧＦ进行定６结论性分析。人表皮生长因子是存在于人体内的一种非常重要的多肽类生长因４ｈＥＧＦ的定量分析子，具有广泛的生理功能。近年来其在临床上的应用也越来越广泛，我蛋白质及多肽分子在紫外２８０ｎｍ处有吸收峰的芳香氨基酸类有们应用ＡＫＴＡｅｘｐｌｏｒｅｒ１００结合层析柱的方法不仅可以实现ｈＥＧＦ的而且还能够实现该因子的在线定量检测的目标，经研究发现酪氨酸、色氨酸及苯丙氨酸等，其出现的峰面积与其质量呈正比例关分离纯化，系，该种特陛可以用作于总蛋白质及被纯化蛋白质的定量测定。由于该方法检测的准确度会随着目标产物的浓度的提高而随之提高。该法ＡＫＴＡｅｘｐｌｏｒｅｒ１００能提供出相应正确的峰面积与蛋白质的定量关系，检测方便、操作简单、条件温和、纯化周期短、获得的纯度高等，而且对所以我们在分离中将ｈＥＧＦ与凝胶柱结合来进行分离与在线检测，然分离其它的蛋白质类产品也同样具有参考价值。参考文献后再配以ＳＤＳ —ＰＡＧＥ凝胶电泳及蛋白质扫描分析系统进行更进一步

一种重组人表皮生长因子的纯化工艺研究

一种重组人表皮生长因子的纯化工艺研究摘要：本研究旨在探究一种重组人表皮生长因子的纯化工艺，以期获得高纯度的表皮生长因子。

首先通过大肠杆菌表达系统生产重组人表皮生长因子，再利用亲和层析、离子交换层析、凝胶过滤层析等多种手段进行纯化，最终获得纯度为95%的表皮生长因子。

关键词：表皮生长因子；重组蛋白；纯化工艺一、引言表皮生长因子（EGF）是一种重要的多肽生长因子，能够促进细胞增殖、分化和修复。

因此，EGF被广泛应用于医学、生物工程等领域。

目前，EGF的主要来源是从动物组织中提取，但存在着蛋白质含量低、纯度不高等问题。

为了解决这些问题，人们开始利用基因重组技术生产重组人EGF，并进行纯化。

二、材料与方法1. 大肠杆菌表达系统生产重组人EGF利用大肠杆菌表达系统生产重组人EGF。

将重组质粒转化到大肠杆菌BL21中，培养至OD600=0.6时，加入异丙基β-D-硫代半乳糖苷（IPTG）诱导表达。

2. 纯化重组人EGF（1）亲和层析将菌体裂解液经过初步纯化，获得EGF的亲和层析纯化产物。

将亲和树脂与EGF结合，通过洗脱、再生等多轮操作，得到EGF的高纯度产物。

（2）离子交换层析将亲和层析产物进行离子交换层析。

将样品加入到离子交换树脂中，根据EGF的电荷性质进行分离。

（3）凝胶过滤层析将离子交换层析产物进行凝胶过滤层析。

利用不同孔径的凝胶进行分离，获得高纯度的EGF。

三、结果与讨论通过大肠杆菌表达系统生产重组人EGF，得到了较高的重组蛋白产量。

通过亲和层析、离子交换层析、凝胶过滤层析等多种手段进行纯化，最终获得了纯度为95%的EGF。

该纯化工艺具有简单、高效、经济等优点，可为EGF的大规模生产提供技术支持。

四、结论本研究成功地建立了一种重组人EGF的纯化工艺，获得了高纯度的EGF。

该工艺可为EGF的大规模生产提供技术支持，并为其他生长因子的生产提供借鉴。

大肠杆菌表达重组人粒细胞集落刺激因子包涵体的复性与纯化

100
200 500 700 1000
12.8
12.1 10.4 8.9 8.1
53.2
52.8 46.1 41.7 31.8
12.3
11.9 8.1 7.0 5.8
31.4
30.1 24.7 19.1 9.3
五、实验结果与讨论-蛋白的构象表征
荧光光谱法
• 圆二色谱法
五、实验结果与讨论-注意事项
第4天一级结构、高级构象、生物学活性第5天数据处理、整理
四、实验步骤与方法
PCR扩增技术获得 rhGC-CSF基因
回收扩增片断
蛋白的生物活性和理化性质分析
获得有活性的蛋白
插入克隆载体进行测序
正确的基因
多种方法复性重组包涵体蛋白
获得包涵体蛋白
插入表达载体表达
5L或50L发酵罐
筛选表达最好的菌株
• 蛋白质变性、复性：稀释法、透析法、蛋白质折叠液相色
谱法；
• 蛋白质构象的表征：包涵体构象变化、圆二色谱法
实验技能训练要点
主要知识回顾
• 仪器分析：色谱技术 –液相色谱技术……
• 仪器分析：光谱技术-荧光光谱、核磁共振 ……
• 化学生物学导论：蛋白质、酶…… • 蛋白质与酶化学：重组DNA技术…… • 蛋白质纯化与表征：液相色谱法、蛋白质构象表征….. • 有机化学：荧光探针
巨噬细胞、内能高度特异性
rhG-SCF
含有5个
半胱氨酸残基，可
pI 6.0
皮细胞及纤维的刺激中性白
母细胞细胞系的前体细胞存活、增
形成两个
二硫键
殖和分化，同
时也是成熟的中性白细胞的
功能活化因子

重组人粒细胞集落刺激因子的表达纯化及表征

毕业论文题目：重组人粒细胞集落刺激因子的表达纯化及表征201 年月日摘要本文选取大肠杆菌作为基因工程菌，它表达的重组人粒细胞集落刺激因子（rhGM-CSF）在细胞中呈现包涵体的形态。

通过破菌、洗涤得到包涵体，再经历溶解、凝胶过滤、复性、疏水以及离子交换柱层析得到了所需要的产物，该产物经过液相和SDS-PAGE电泳测定得到的纯度都很高。

本文分为三部分对重组人粒细胞集落刺激因子(rhG-CSF)在生物学方面的活性的测定方法进行了初步的探索研究。

在高效疏水色谱(HPlC)条件下优化分离纯化rhG-CSF的洗脱梯度以及流动相流速，实现了复性和同时纯化。

经过1次色谱，rhG-CSF的纯度可达97．5％，rhG-CSF的质量回收率达到25.0％。

关键词：重组人粒细胞集落刺激因子；分离纯化；高效液相色谱；基因工程AbstractIn this paper, we Select escherichia coli as a genetic engineering bacterium, it recombinant human granulocyte-macrophage colony- stimulating factor (rhGM-CSF) presented as inclusion body in enginneered E.coil cells. Through the broken bacteria, washing get inclusion body, to go through dissolution, gel filtering, resilience, hydrophobic and ion exchange chromatography column got need product, the product after liquid and sds-page electrophoresis measured are very high purity. This paper is divided into three parts,the measurgin method about the biology activityof Recombinant Human Granulocyte Colony- Stimulating Factor (rhG-CSF) in the paper as the initial research. In high efficient hydrophobic chromatography (HPLC) conditions optimization purification rhG both g-csf-treated patients with the gradient and mobile phase velocity, realize the resilience and and purification. Each time through chromatography, the purity of rhG-CSF reach up to 97.5%, the quality of rhG-CSF recovery rate reached 25.0%.Keyword: rhG-CSF; purification; HPLC; genetic engineering目录摘要 (I)Abstract .......................................................................................................................... I I 第一章文献综述 (1)1.1 前言 (1)1.2 粒细胞集落刺激因子的概述 (1)1.2.1粒细胞集落刺激因子发酵生产研究概况 (2)1.3 基因工程菌发酵工艺的影响条件 (3)1.4基因工程菌的度培养 (3)1.5粒细胞集落刺激因子的分离、纯化与复性 (4)1.6重组蛋白的复性 (4)第二章实验部分 (5)2.1 仪器与试剂 (5)2.1.1 rhG．CSF发酵所用到的主要仪器和试剂 (5)2.1.2 rhG．CSF分离纯化复性所需仪器与试剂 (6)2.2 试验方法 (6)2.2.1 菌种培养基的配制 (6)2.2.2 菌种的培养方法 (6)2.3 粒细胞集落刺激因子的分离纯化 (7)2.3.1菌体的收集和洗涤 (7)2.3.2 包涵体的提取 (7)2.3.3 rhG-CSF表达量的测定 (8)2.3.4 SDS-PAGE分析 (8)2.3.5 Bradford比色法测定蛋白质含量 (10)2.3.6 包涵体的变复性 (11)2.3.7 重组蛋白质的分离纯化 (11)第三章讨论 (14)3.1 高效疏水色谱对粒细胞集落刺激因子的复性与同时纯化 (14)3.1.1 洗脱梯度对rhG-CSF的复性与同时纯化的影响 (14)3.1.2 流动相流速对rhG-CSF的复性与同时纯化的影响 (15)3.2 粒细胞集落刺激因子的纯度和质量回收率 (16)3.3 关于蛋白质质量回收率的讨论 (16)参考文献 (17)第一章文献综述1.1 前言白细胞依照形态的不同一般划分成为颗粒、没有颗粒这两个部分，没有颗粒的白细胞包含淋巴系统细胞以及含有单个核的细胞。

重组人内皮抑素的表达、纯化、复性方法研究

重组人内皮抑素的表达、纯化、复性方法研究任明华;刘兴汉;闫玉清;姚鸿超;王淑晶;徐建勇【期刊名称】《中国肿瘤》【年(卷),期】2003(12)10【摘要】[目的]摸索重组人内皮抑素(recombinanthumanendostatin,rhES)的最佳生产工艺路线,解决rhES在大肠杆菌中表达提取的蛋白变性问题。

[方法](1)按所优化的条件进行rhES的表达,包涵体的提取、洗涤、变性、复性。

(2)活性检测:观察rhES对裸鼠皮下移植瘤生长情况及病理组织学的影响。

[结果](1)每升培养基可得可溶性rhES蛋白10.55mg,rhES在大肠杆菌中的表达量超过总菌体蛋白的60%,纯化后的蛋白纯度超过95%。

(2)动物实验:rhES抑瘤效果显著,抑瘤率为45.86%,实验组与对照组在肿瘤体积、重量、裸鼠除瘤净重、瘤重/鼠净重4个指标上均有显著性差异(P<0.01)。

光镜见实验组微血管密度降低,坏死面积增加,电镜见实验组凋亡细胞增加。

[结论]本实验所优化的生产工艺路线可获得可溶性的、高纯度、高活性的rhES蛋白,解决了rhES在大肠杆菌中提取时的蛋白变性问题。

【总页数】4页(P598-601)【关键词】内皮抑素;纯化;复性【作者】任明华;刘兴汉;闫玉清;姚鸿超;王淑晶;徐建勇【作者单位】哈尔滨医科大学生物化学与分子生物学教研室;哈尔滨师范大学生物系;哈尔滨医科大学附属第二医院【正文语种】中文【中图分类】R73-3【相关文献】1.大肠杆菌表达的新一代重组人内皮抑素复性条件的优化 [J], 赵立希;蒋永平2.重组人内皮抑素层析复性的相关研究 [J], 乔路;夏杰;陆兵;徐殿胜3.重组人内皮抑素的纯化及抗肿瘤活性研究 [J], 张广美;王淑静;任明华;徐建永;刘远莉;陶占华;李冀宏;刘兴汉4.重组人内皮抑素的表达纯化及对内皮细胞增殖的影响 [J], 杨健;杨杰5.内皮抑素在大肠杆菌中表达、纯化及复性的研究 [J], 何晶晶;金德均;刘兴汉;王超;李继宏因版权原因，仅展示原文概要，查看原文内容请购买。

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药
物
生
物
技
术
ＰｈａｒｍａｃｅｕｔｉｃａｌＢｉｏｔｅｃｈｎｏｌｏｇｙ２０１６，２３（６）：４７７～４８０
４７７
重组人组织因子的表达纯化及复性研究
徐艺菲，李佳萌，齐永，陈红霞，潘英，李素芹，李素梅，陈晨，王新国，张玉彬，李越希
插入目的基因的ｐＥＴ２８ａ（＋）一ｈＴＦ重组质粒，转化到大肠杆菌中，获得重组菌，用异丙基－Ｂ．Ｄ一硫代半乳糖苷
（Ｉｓｏｐｒｏｐｙｈｈｉｏ－Ｂ．Ｄ — ｇａｌａｃｔｏｓｉｄｅ，ＩＰＴＧ）诱导其表达，菌体经超声裂解、离心收集包涵体，用８ｍｏＬ／Ｌ尿素溶解包涵体、
（ＴａＫａＲａＭｉｎｉＢＥＳＴＰｌａｓｍｉｄＰｕｒｉｉｆｃａｔｉｏｎＫｉｔＶｅｔ．３．０）为
化学合成的人组织因子基因序列用ＮｃｏＩ和ＥｃｏＲＩ双酶切，克隆到质粒ｐＥＴ２８ａ（＋）的ＮｃｏＩ和ＥｃｏＲＩ之间，构建表达人组织因子的重组质粒ｐＥＴ２８ａ（＋），ｈＴＦ。１．３．２ｐＥＴ２８ａ（＋）一ｈＴＦ质粒的转化及诱导取５０感
１．２主要实验仪器
泛存在于心、脑、肺、肾等组织细胞膜中，是凝血因子ＶＩＩ的
受体及辅助因子。ｈＴＦ的分子结构中包含３个结构域，胞外区、跨膜区和胞内区。胞外区是含有２１９个氨基酸残基且带糖链的氨基端，具有亲水性引，此区域具有全部的凝血活性。ｈＴＦ蛋白正常生理状态下参与生理性止血过
性。通过采用高效的人重组ｈＴＦ蛋白复性方法，成功表达
并获得有活性的ｈＴＦ蛋白。
１材料与方法
１．１实验材料
ｐＥＴ２８ａ（＋）质粒由南京军区军事医学研究所保存。ＩＰＴＧ、Ｎｉ — ＮＴＡＳｅｐｈａｒｏｓｅ购于金斯瑞公司。质粒提取试剂盒
ＴａＫａＲａ公司产品。核酸分子质量Ｍａｒｋｅｒ（ＤＬ５０００）、卡那
霉素、琼脂糖为Ｐｒｏｍｅｇａ公司产品。Ｇｅｌｓｔａｉｎ、Ｂｒａｄｆｏｒｄ蛋白浓度测定试剂盒Ｐ０００６为碧云天生物技术公司产品。
ＤＤＨＺ一３００多功能台式恒温振荡器，ＣＳ．１５Ｒ型台式低
温高速离心机，ＵＮＩＣＯＵＶ－２１００紫外分光光度计，ＷＨ一８６６
旋涡混合器，ＰｏｗｅｒＰａｃ２００电泳仪，净水器，ＬＳ－Ｂ Байду номын сангаас５０Ｌ型立式圆形压力蒸气灭菌器，ＴＭ２０００型凝胶成像分析仪，半自动凝血测试仪一４００。
（１．南京军区军事医学研究所，江苏南京２１０００２；２．中国药科大学生命科学与技术学院，江苏南京２１０００９；
３．南京医科大学基础医学院，江苏南京２１０００５）
摘
要
利用基因工程技术表达重组人组织因子（Ｈｕｍａｎｔｉｓｓｕｅｆａｃｔｏｒ，ｈＴＦ），并进行纯化及复性研究。构建
１．３．１表达质粒ｐＥＴ２８ａ（＋）一ｈＴＦ的构建
根据人组织因
子胞外区２１９个氨基酸序列，选择大肠杆菌偏爱的密码子，
优化设计其基因序列，并在其基因序列的５和３分别增加了酶切位点ＮｃｏＩ和ＥｅｏＲＩ，化学合成优化的基因序列。将
Ｎｉ．ＮＴＡ纯化，采用透析复性和柱上复性两种方式获得复性的重组人ＨＴＦ。柱上复性和透析复性均获得具有生物
活性的重组人ｈＴＦ，但柱上复性的复性率为７２％，明显高于透析复性的复性率１６％。成功构建高表达人重组ｈＴＦ的工程菌，通过柱上复性获得高纯度具有生物活性的重组人ｈＴＦ蛋白，为ｈＴＦ的研究奠定了基础。
１．３方法
程。在病理情况下，ｈＴＦ的异常表达会导致血栓的形成，诱
导产生细胞因子，与心血管疾病、肿瘤的生长及转移、炎症
等疾病的发生有密切的关系。因此，体外表达ｈＴＦ蛋白
对其生物学功能研究具有重要意义。本研究采用大肠杆菌表达系统对ｈＴＦ蛋白的胞外区进行克隆表达，并采用透析复性和柱上复性两种方式对重组人ｈＴＦ蛋白包涵体进行复
关键词ｈＴＦ；表达；纯化；复性
Ｑ７８４文献标志码Ａ文章编号１００５ — ８９１５（２０１６）０６ — ０４７７．０４中图分类号
人组织因子（ＨｕｍａｎＴｉｓｓｕｅｆａｃｔｏｒ，ｈＴＦ）是一种含有２６３个氨基酸残基、相对分子质量为４７ｋ的跨膜糖蛋白，广