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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重组人组织因子的表达纯化及复性研究

药
物
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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

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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不同细胞因子组合对人脐血干细胞体外扩增作用研究

不同细胞因子组合对人脐血干细胞体外扩增作用研究黄体龙;杨跃煌【期刊名称】《齐齐哈尔医学院学报》【年(卷),期】2016(037)014【摘要】目的：研究不同的细胞因子组合对人脐血干细胞体外的扩增作用，优化筛选脐血干细胞扩增的细胞因子。

方法根据细胞因子不同，实验分五组：①对照组；②SF（ SCF＋FL）组；③SFT（ SCF＋FL＋TPO）组；④SFT6（SCF＋FL＋TPO＋IL-6）组；⑤SFT36（SCF＋FL＋TPO＋IL-3＋IL-6）。

采集人脐带血，分离纯化CD34＋细胞，接种于含有以上不同因子的培养体系中培养，分别于接种当天及第7天检测有核细胞数、集落形成单位（ CFU）数及CD34＋细胞数。

结果SFT36组有核细胞数、CD34＋细胞数及集落形成单位数分别增加（622±179）％，（784±213）％，（804±272）％，与其他4组有显著性差别。

结论 SFT36（ SCF＋FL＋TPO＋IL-3＋IL-6）组5种因子组合是体外扩增脐血合适的细胞因子组合。

%Objective To investigate the effect of different cytokine combinations on the expansion of umbilical cord blood stem cells invitro .Methods CD34 +cells from cord blood were expanded under different combinations of cytokines:①Control②SF(SCF+FL)③SFT(SCF+FL+TPO) ④SFT6(SCF+FL+TPO+IL-6) ⑤ SFT36(SCF+FL+TPO+IL-3 +IL-6).Results In group SFT36, the number of nucleated cells (MNC), CD34 +cell and colony forming units (CFU) were increased (622 ±179)%, (784 ±213)%, (804 ± 272)%, and the ot her 4 groups had significant differences .Conclusions Our experimental results show that thecombination of SCF +FL+TPO+IL-3 +IL-6 and 5 factors is an appropriate combination of cytokines for the expansion of umbilical cord blood .【总页数】3页(P1765-1767)【作者】黄体龙;杨跃煌【作者单位】650228云南，昆明医科大学附属儿童医院，昆明市儿童医院血液科;650228云南，昆明医科大学附属儿童医院，昆明市儿童医院血液科【正文语种】中文【相关文献】1.人骨髓间充质干细胞对脐血干细胞体外扩增支持作用的研究 [J], 费小明;陆化;吴雨洁;周迎峰;周小玉;唐宇鸿;沈文怡;汪承亚2.不同细胞因子组合对脐带血AC133+细胞体外扩增效率的影响 [J], 刘玉龙;戴宏;姜忠;周丽英;胡勤芳;郭晓葵;周剑影3.人骨髓间充质干细胞联合细胞因子的无血清培养体系体外扩增脐血干细胞研究[J], 费小明;吴雨洁;唐宇宏;沈文怡;陆化;李建勇;汪承亚4.不同细胞因子组合体外诱导人脐血干细胞向胰岛样细胞分化 [J], 侯萍;赵冬阳;李革飞;李晓丰;李剑平5.不同细胞因子组合对c-kit+Lin-细胞的体外扩增研究 [J], 张守华;廖彩仙;张春兴;苏俊;赖勇强;周杰因版权原因，仅展示原文概要，查看原文内容请购买。

造血细胞生长因子在脐血体外扩增中作用研究的进展

造血细胞生长因子在脐血体外扩增中作用研究的进展
赵兰滨;李梅君
【期刊名称】《数理医药学杂志》
【年(卷),期】2006(19)2
【摘要】人脐血中造血干/祖细胞增殖能力强,但单份脐血数量有限,因此目前脐血移植多用于低体重儿童.造血细胞生长因子具有促进脐血扩增的作用,在不同的造血细胞生长因子联合作用下,可以有目的地进行体外扩增,从而使单份脐血有望解决高体重儿童及成人脐血移植存在的造血干/祖细胞数量不足的问题,为脐血广泛应用于临床移植作出贡献.
【总页数】3页(P191-193)
【作者】赵兰滨;李梅君
【作者单位】锦州市中心医院血液科,锦州,121000;锦州医学院第一附属医院血液科
【正文语种】中文
【中图分类】R318.12
【相关文献】
1.脐血造血细胞体外扩增的研究进展 [J], 许力
2.脐血内皮细胞对脐血早期造血细胞的体外扩增作用 [J], 尹利明;程腊梅;王绮如;谭孟群
3.造血细胞生长因子对脐血CD34+细胞短期体外扩增的作用 [J], 叶韵斌;徐莉敏;陈志哲;林建银;陈强;周智锋;李洁羽
4.人脐血贴壁层细胞对脐血造血细胞体外扩增的作用 [J], 鞠晓红;赵珍谊;方芳;马爱新
5.脐血造血细胞的体外扩增:2.造血细胞生长因子和培养液更换的作用 [J], 应小飞;张元兴;谭文松
因版权原因，仅展示原文概要，查看原文内容请购买。

脐带血造血干细胞扩增技术研究进展

脐带血造血干细胞扩增技术研究进展摘要：自从1988年运用脐带血移植治愈了Fanconi患者以来，便开始了脐血的研究，脐带血已经广泛应用于临床，体外进行脐带血造血干细胞(CB HSCs)的扩增可以产生数量较多的细胞，解决了单份脐带血移植的缺陷。

本文介绍了脐带血造血干细胞扩增技术的研究进展。

关键词：脐带血；造血干细胞；扩增技术1脐带血造血干细胞(CB HSCs)可移植性研究表明CB中有丰富的HSCs，其含量与骨髓（BM）是一致的，但是HSCs亚群的比例不同。

CB的单个细胞中CD34+的细胞含量低于BM，CD34+/CD38-的细胞含量与BM相似，CD34+/CD38-/HLA-DR-的细胞含量高于BM，CB HSCs的扩增和增殖能力要高于BM HSCs，可以对体外扩增的HSCs造血能力进行重建。

如果将CB以及BM CD34+细胞输入体内，可以产生同样的造血能力，如果将它们移植入SCID-hu bone 小鼠体内，CB的植入效果要优于BM。

CB HSCs会分泌一些满足自身成长的因子，受到细胞因子的作用，CB HSCs会迅速的进行细胞增殖，且其细胞粒端长于BM细胞，表明CB HSCs具有可移植性。

2影响体外扩增的因素CB HSCs的体外扩期特性受到许多因素的影响，如培养条件、细胞因子等。

下面分别进行介绍。

2.1起始培养细胞进行造血干细胞扩增研究时，用分选CD34+做为起始培养细胞，进行体外扩增，CD34+细胞的纯度对其定向分化能力具有决定性的作用，因为这些非纯化细胞会消耗大量的营养物质，改变培养体系的PH值，通过细胞纯化可以减少脐带血标本中的非目标细胞的含量。

通过对CD34+的纯化效果与HSCs的扩增效果进行对比，可以看到CD34+细胞和总HSCs扩增比例要高于HSCs。

CD34+的纯度对细胞扩增程度也会产生影响，如果纯度为62%，则可以收到最好的细胞扩增效果，实验表明并非纯度越高扩增效果就越好，具体原因可能是CD34+纯化技术会丢失部分细胞。

人干细胞生长因子cDNA克隆、表达和纯化及其造血种属特异性

人干细胞生长因子cDNA克隆、表达和纯化及其造血种属特异性【摘要】人干细胞生长因子（human stem cell growth factor，hSCGF）是一种早期造血调控因子。

已知人干细胞生长因子具有两种形式，包括全长分子hSCGF以及在Ca2+依赖糖识别结构域（calcium-dependent carbohydrate recognition domain，CRD）内缺失78氨基酸的截短型分子hSCGFβ。

hSCGFβ的造血刺激活性具有严格的种属特异性。

本研究目的在于探讨hSCGF能否协同刺激小鼠粒/单系祖细胞增殖。

为克服hSCGF的cDNA GC含量较高的困难，本研究以两步PCR的方法从人胎肝cDNA文库（Clontech）中成功克隆hSCGF cDNA，进而将hSCGF成熟肽编码序列亚克隆于原核表达载体pGEX4T-2中，融合表达。

研究结果表明，通过低温诱导（28℃），重组表达产物主要以可溶蛋白的形式存在于裂解上清中，利用亲和层析纯化融合表达蛋白。

对重组蛋白的造血刺激活性分析表明，不同于截短型分子hSCGFβ，全长分子hSCGF能够刺激小鼠骨髓粒/单系造血祖细胞增殖。

结论： hSCGF CRD不直接结合受体，可能具有其他生物学功能。

【关键词】 cDNA克隆Cloning，Expression and Purification of Human Stem Cell Growth Factor cDNA and Its Species-specificity in HematopoiesisAbstract Stem cell growth factor (SCGF) is an early-acting hematopoitic cytokine that has two isoforms including hSCGF with full length molecules and hSCGFβ，78 amino acids of which lost in the conserved calcium-dependent carbohydrate-recognition domain (CRD).It has been demonstrated that hSCGFβ is strictly species-specific inregulating he-matopoiesis.This study was aimed to explore whether human SCGF can exert synergistic stimulatory effect on heterogenous murine CFU-GM progenitor.Firstly，hSCGF cDNA was amplified from human fetal liver cDNA library by using two-step PCR.The hSCGF mature peptide coding sequence was subsequently placed at downstream of glutathione S-transferase (GST) sequence in GST gene fusion expression vector. The results indicated that there existed an additional 60 kD protein compared with mock BL21 when the cells hosting recombinant plasmid were induced with IPTG at 37℃.SDS-PAGE analysis demonstrated that the GST-hSCGF fusion protein mainly existed in insoluble form.When induced at low temperature (28℃)，the recombinant protein was mostly soluble.The GST-fusion recombinant protein was subsequently purified by usingaffinity chromatography.The clonogenic assay revealed that，unlike hSCGFβ，hSCGF had the granulocyte/ macrophage promoting activity (GPA)for murine bone marrow GM progenitor. It is concluded that，in contrast to human SCGFβ，the intact molecular hSCGF may have no species specificity，implying that CRD domain in human SCGFβ does not directly bind to corresponding SCGF receptor，but may have certain biological function.Key words human stem cell growth factor; CRD region; cDNA clone; fusion expression; hematopoietic species specificity细胞因子在造血过程中起到十分重要的调控作用［1，2］。

重组人CRP的表达纯化及其内化进入HeLa细胞的观察

重组人CRP的表达纯化及其内化进入HeLa细胞的观察陈腾祥;李红梅;胡水旺;赵明哲;刘亚伟;刘靖华;姜勇【期刊名称】《中国病理生理杂志》【年(卷),期】2008(024)006【摘要】目的:构建人C-反应蛋白(CRP)原核表达载体,表达纯化his-EGFP-CRP蛋白,观察其能否内化进入HeLa肿瘤细胞.方法:利用特异性引物,从p91023/CRP载体上将编码人CRP的基因序列亚克隆到原核表达载体pET14b/MCS-EGFP-(N)36上;对阳性克隆进行PCR、酶切和测序鉴定,并将其转化到大肠杆菌BL21(DE3)中诱导表达,表达的重组蛋白通过亲和色谱纯化,梯度透析复性,并与HeLa细胞孵育,利用荧光显微镜观察其内化入胞.结果:PCR、双酶切和测序鉴定表明,pET14b/EGFP-hCRP原核表达质粒构建正确;转化实验发现,该质粒在BL21(DE3)中能够被大量诱导表达;蛋白纯化及荧光显微镜观察结果表明,复性后的表达产物可结合于HeLa细胞膜,孵育一定时间后,可定位于胞质及胞核.结论:成功地构建了带增强型绿色荧光蛋白(EGFP)标签的人CRP原核表达载体,该载体能够在大肠杆菌BL21(DE3)中被诱导表达重组蛋白his-EGFP-CRP,纯化复性后的重组人CRP能与HeLa肿瘤细胞结合,并能内化入胞,移位入核.【总页数】6页(P1155-1160)【作者】陈腾祥;李红梅;胡水旺;赵明哲;刘亚伟;刘靖华;姜勇【作者单位】南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室,广东,广州510515;南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室,广东,广州510515;南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室,广东,广州510515;南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室,广东,广州510515;南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室,广东,广州510515;南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室,广东,广州510515;南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室,广东,广州510515【正文语种】中文【中图分类】R730.23;Q344+.13【相关文献】1.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[J], 范洁;丁欣欣;蒋永平2.重组天花粉蛋白的原核表达、纯化及其对宫颈癌HeLa细胞增殖的影响 [J], 尤程程;黄利鸣;王艳林;韩钰;李雪梅;曹春雨3.大肠杆菌中表达的重组人粒细胞-巨噬细胞集落刺激因子/白细胞介素-3(rhGM-CSF/IL-3)融合蛋白的纯化 [J], 戴盛明;肖桂元;周少雄;郑兴武;饶颖竹;周甘平;吴茂莲4.重组质粒pLMP1-p53mt在人鼻咽癌HNE1和人宫颈癌HeLa细胞中的表达分析 [J], 何迎春;田道法;贺安意;江洁琼;刘宇勤5.重组人TGF┐β1诱导免疫耐受及其对Hela细胞MHC抗原表达的实验研究 [J], 李勇;韩本立;王会信因版权原因，仅展示原文概要，查看原文内容请购买。

重组人干细胞因子的研究

重组人干细胞因子的研究吴军;巩新;唱韶红;赵志虎;左从林;马清钧【期刊名称】《生物工程学报》【年(卷),期】2003(019)006【摘要】利用基因工程的方法制备了高纯度的人干细胞因子,并对制备的重组人干细胞因子的结构和生物活性进行了研究.结果表明rhSCF在磷酸盐缓冲液中以非共价的二聚体形式存在,其精确分子量、质量肽谱、氨基酸组成及N端、C段序列等均与理论值一致,二硫键位置正确.rhSCF单用或与rhG-CSF合用,对猴外周血造血祖细胞有明显的动员作用.【总页数】7页(P698-704)【作者】吴军;巩新;唱韶红;赵志虎;左从林;马清钧【作者单位】军事医学科学院生物工程研究所,北京,100071;军事医学科学院生物工程研究所,北京,100071;军事医学科学院生物工程研究所,北京,100071;军事医学科学院生物工程研究所,北京,100071;北京昭衍新药研究中心;军事医学科学院生物工程研究所,北京,100071【正文语种】中文【中图分类】Q786【相关文献】1.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[J], 范洁;丁欣欣;蒋永平2.重组人干细胞因子对猕猴外周血干细胞的动员作用 [J], 熊国林;郝静;柳晓兰;董波;赵振虎;邱丽玲;陈松森;雒蓬轶;王若竹;罗庆良3.重组人白细胞介素11联合重组人粒细胞集落刺激因子动员自体外周血干细胞移植治疗急性白血病 [J], 徐丹;孟凡义;易正山;孙竞4.重组人干细胞因子与重组人粒细胞集落刺激因子联合用药的猴毒性研究 [J], 宣尧仙;陈国灿;陈云祥;徐潘生;杨红忠;陈颖;陈浩;卢祺炯;李峰;陈名友;雒蓬轶;刘忠荣;王若卓;钱伯初5.重组人粒细胞刺激因子促进骨髓间充质干细胞修复急性肺损伤的机制研究 [J], 乔广明;王志敏;张雪欣;时亚娟;齐晓玉;贠美玲;岳智杰因版权原因，仅展示原文概要，查看原文内容请购买。

脐带血NKT细胞的体外扩增及其表型研究

脐带血NKT细胞的体外扩增及其表型研究【摘要】目的建立体外有效扩增脐带血NKT细胞的方法及其表型的研究。

方法分别采用单加IL-2，及同时加IL-2和α-半乳糖神经酰胺（α-Galcer）的方法，从人脐带血单个核细胞（UCB-MNCs）中扩增NKT细胞。

用流式细胞检测技术测定NKT细胞（TCR Vα24+ TCR Vβ11+）的比例及其他表型特征。

结果 UCB-MNCs在第7天时，其TCR Vαβ+ NKT细胞的扩增量占淋巴细胞的（24.48±4.19）%，是原来的（8.74±4.37）×102倍（P＜0.01），且大多不表达NK1.1（CD161）；TCR Vβ11+较TCR Vα24+高表达。

结论α-Galcer对脐血NKT细胞有特异地扩增功能。

以表型划分，脐血中的NKT细胞多为NK1.1-，一种新的NKT细胞亚型。

【关键词】脐带血；NKT细胞；α-Galcer；细胞表型The proliferation of natural killer T cells in umbilical cord blood and the different phenotype between them【Abstract】 Objective The first objective of this study was to establish an effective method to proliferate natural killer T （NKT）cells in umbilical cord blood （UCB）.The second one was to study the phenotype.Methods Mononuclear cells （MNCs） from UCB were cultured in the presence of IL-2 （100U/ml），with or without alpha-galcer.TCRValpha24Vbeta11 double positive NKT cells and its other phenotypes were determined by flow cytometry.Results After 7 days expansion，TCRVαβ+ NKT cells from UCB-MNCs increased（8.74±4.37）×102 times，but most of them did not express NK1.1 and its TCR Vβ11+ was higher than TCR Vα24+.Conclusion Human TCRValpha24Vbeta11 double positive NKT cells can expand following the administration of alpha-galcer.Most of the UCB-NKT was NK1.1-，a new subset of NKT cells.【Key words】 umbilical cord blood；natural killer T （NKT）cells；alpha-galcer；phenotype脐带血是一种来源广泛的新资源，其中的低免疫原性细胞正日益受到人们的关注。

不同细胞因子组合对体外培养人脐带血造血干细胞的扩增效果

不同细胞因子组合对体外培养人脐带血造血干细胞的扩增效果刘云霞;孟杰;刘立新;郭亚春;王庆林;徐大为【期刊名称】《中国组织工程研究》【年(卷),期】2007(011)003【摘要】目的:观察细胞因子对培养的人脐带血造血干细胞的扩增效果,寻求体外最佳细胞因子组合.方法:实验于2002-06/2004-04在承德医学院基础研究所及承德医学院附属医院中心实验室完成.①以足月顺产健康新生儿的脐带血(由承德医学院附属医院妇产科提供,新生儿家属均签署实验知情同意书)作为实验标本.无菌条件下平均采血量30～60 mL,梯度离心分离脐带血单个核细胞.②细胞体外扩增DMEM 培养体系(含体积分数0.2的胎牛血清)为0.25 μg/L干细胞因子,0 25 μg/L白细胞介素3,0.5 μg/L白细胞介索6,0.25 μg/L粒-巨噬细胞集落刺激因子,0.8 μg/L粒细胞集落刺激因子,0.05 μg/L血小板生成素,0.8μg/L FLt-3配基.此7种细胞因子及剂量按不同组合分为6组:干细胞因子+白细胞介素3+白细胞介素6组、干细胞因子+白细胞介素3+白细胞介素6+粒-巨噬细胞集落刺激因子组、干细胞因子+白细胞介素3+白细胞介素6+粒细胞集落刺激因子组、干细胞因子+白细胞介素3+白细胞介素6+粒-巨噬细胞集落刺激因子+粒细胞集落刺激因子组、干细胞因子+白细胞介素3+白细胞介素6+血小板生成素+FLt-3配基组、干细胞因子+血小板生成素+FLt-3配基组.各组单个核细胞终浓度为1×108个L-1.③培养第7,14,21天进行细胞形态学观察;培养第0,5,10,14,18,21天观察不同细胞因子组合对脐带血干细胞扩增的效果;应用流式细胞仪对培养前后细胞表面标志CD34+进行检测.结果:①脐带血干细胞形态学观察结果:体外培养第14天,细胞胞体小,胞浆量少,胞核体积大,多不规则,为早期造血干细胞.②细胞体外扩增培养情况:应用干细胞因子+白细胞介素3+白细胞介素6+血小板生成素+FLt-3配基这一细胞因子组合,脐带血干细胞可保持＞98%的细胞存活率;培养第7天出现典型早期造血干细胞,第21天细胞总数达到高峰(F=60.228,P＜0.01).③细胞表面标志CD34+的动态变化:干细胞因子+白细胞介素3+白细胞介素6+血小板生成素+FLt-3配基这一细胞因子组合为生长刺激剂,应用后体外培养的造血干细胞于第14天达高峰,与培养前比较差异有显著性意义(F=20.782,P均＜0.01),之后逐渐下降.结论:应用干细胞因子+白细胞介素3+白细胞介素6+血小板生成素+FLt-3配基细胞因子组合可长期维持脐带血干细胞的体外生长,且体外培养第14天是临床移植的最佳时机.【总页数】4页(P401-404)【作者】刘云霞;孟杰;刘立新;郭亚春;王庆林;徐大为【作者单位】承德医学院基础部,河北省承德市,067000;承德医学院附属医院神经外科,河北省承德市,067000;承德医学院基础部,河北省承德市,067000;承德医学院基础部,河北省承德市,067000;湖南师范大学医学部免疫教研室,湖南省长沙市,410006;承德医学院基础部,河北省承德市,067000【正文语种】中文【中图分类】R3【相关文献】1.人胎盘提取物对脐带血造血干细胞的体外扩增实验研究 [J], 刘禄斌;刘畅;张华;刘菊莲;陈凤娴2.细胞因子FL、TPO对脐带血造血干细胞的扩增作用 [J], 许倩;刘云霞;周晓春;王庆林;颜勇3.不同细胞因子组合对脐带血AC133+细胞体外扩增效率的影响 [J], 刘玉龙;戴宏;姜忠;周丽英;胡勤芳;郭晓葵;周剑影4.人脐带血来源造血干细胞体外扩增的研究进展 [J], 董忱;赵龙;张斌;陈虎5.不同培养体系对脐带血造血干细胞扩增的影响 [J], 郝牧;邱录贵;吴瞳;李斯丹;孟恒星因版权原因，仅展示原文概要，查看原文内容请购买。

脐血干细胞体外程序扩增的实验研究

脐血干细胞体外程序扩增的实验研究张霞;项莺松;张新;金志军;杨平;蔡建明;杨云纺【期刊名称】《上海医学》【年(卷),期】2001(24)2【摘要】目的利用一种新的体外扩增脐带血原始细胞的方法 ,使得干细胞在数量增加的同时 ,又能尽量保持原始性。

方法在相同细胞因子组合下 ,用体外液体培养比较脐血有核细胞程序扩增组与一次扩增组及有无基质层条件下的优劣。

结果培养 2 8d后 ,虽然程序扩增组MNC细胞绝对数低于一次扩增组 ,但其CD34 +细胞数明显高于一次扩增组,且有基质层组细胞总数,原始细胞数均高于无基质层组。

结论体外程序扩增脐血干细胞是一种有效的扩增干细胞的方法 ,从而使得脐血的应用更为广泛。

【总页数】4页(P95-98)【关键词】造血干细胞;免疫表型;脐血;基因扩增【作者】张霞;项莺松;张新;金志军;杨平;蔡建明;杨云纺【作者单位】第二军医大学长征医院妇产科;第二军医大学海医系放射医学研究所;山东省新泰市第一人民医院【正文语种】中文【中图分类】R457【相关文献】1.人脐血间充质干细胞对脐血CD+34细胞体外扩增作用的研究 [J], 周敦华;黄绍良;张绪超;魏菁;吴燕峰;黄科;黎阳;方建培2.人骨髓间充质干细胞对脐血干细胞体外扩增支持作用的研究 [J], 费小明;陆化;吴雨洁;周迎峰;周小玉;唐宇鸿;沈文怡;汪承亚3.人骨髓间充质干细胞联合细胞因子的无血清培养体系体外扩增脐血干细胞研究[J], 费小明;吴雨洁;唐宇宏;沈文怡;陆化;李建勇;汪承亚4.脐血间充质干细胞的体外扩增及向类肝细胞分化的实验研究 [J], 韩翠萍;刘吉勇;高蕾;裴庆山;孙欣欣;文婷玉5.人骨髓基质干细胞克隆对脐血造血干细胞体外扩增作用的研究 [J], 林竞韧;郭坤元;严定安因版权原因，仅展示原文概要，查看原文内容请购买。

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
因版权原因，仅展示原文概要，查看原文内容请购买。