CN106222222A - Preparation method for human recombinant leukemia inhibitory factor - Google Patents

Preparation method for human recombinant leukemia inhibitory factor Download PDF

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CN106222222A
CN106222222A CN201610642110.7A CN201610642110A CN106222222A CN 106222222 A CN106222222 A CN 106222222A CN 201610642110 A CN201610642110 A CN 201610642110A CN 106222222 A CN106222222 A CN 106222222A
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leukemia inhibitory
inhibitory factor
buffer
exchange chromatography
filler
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CN201610642110.7A
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杜伯雨
宋伟
关欣
李小璐
吴帆
郗雪艳
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湖北医药学院
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/54Interleukins [IL]
    • C07K14/5415Leukaemia inhibitory factor [LIF]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • C12P21/02Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Abstract

The invention relates to the field of medicines and particularly provides a method of preparation method for a human recombinant leukemia inhibitory factor by applying cell expression products of escherichia coli. In particular, the method includes the steps of: expressing leukemia inhibitory factor protein by applying the escherichia coli; performing low-temperature induction to achieve soluble expression of the leukemia inhibitory factor protein; and performing three-step purification, comprising cationic exchange chromatography, anionic exchange chromatography, and cationic exchange chromatography, to purify the leukemia inhibitory factor protein which has high purity and high activity. The method is easy to carry out, is low in cost and can be used for preparing the recombinant leukemia inhibitory factor in large scale.

Description

一种重组人白血病抑制因子的制备方法 A method of preparing a recombinant human leukemia factor inhibition

技术领域 FIELD

[0001]本发明涉及医药领域,具体为一种应用从大肠杆菌细胞表达产物中制备具有高纯度重组人白血病抑制因子的方法。 [0001] The present invention relates to the field of medicine, particularly high-purity recombinant human leukemia inhibitory factor having as a product prepared from E. coli cells expressing application.

背景技术 Background technique

[0002] 白血病抑制因子(Leukemia inhibitory factor,LIF)由180个氨基酸构成,属IL-6家族。 [0002] LIF (Leukemia inhibitory factor, LIF) consists of 180 amino acids, is the IL-6 family. 其分子内部含有6个半胱氨酸,可形成3对二硫键(07812-。78134,07818-。78131,cys60-cysl63)。 Intramolecular which contains 6 cysteines can form three disulfide bonds (07812-.78134,07818-.78131, cys60-cysl63). 其受体包括两个亚基,分别为LIFR和gpl30,均具有胞内区和跨膜区,已知可被LIF激活的下游信号通路包括JAK/STAT3、PI3K/AKT、ERKl/2以及mTOR信号通路;目前已知LIF可促进干细胞的增殖并防止其分化,同时对胚胎发育、神经系统、免疫和内分泌系统的功能特别是肿瘤细胞的增殖和发生都有显著的影响,在科研领域,LIF在胚胎干细胞等的培养上应用的较为广泛,主要是用于防止干细胞发生自发分化而维持其增殖状态。 Receptors which comprises two subunits, respectively gpl30 and LIFR, each having intracellular and transmembrane domains, known to be activated by LIF downstream signaling pathways including JAK / STAT3, PI3K / AKT, ERKl / 2 and mTOR signaling passage; known LIF stimulates proliferation of stem cells and prevent their differentiation, and function of embryonic development, the nervous system, immune and endocrine systems, particularly tumor cell proliferation and occurs have a significant influence in the field of scientific research, LIF in embryonic stem cells cultured more widely applied, is mainly used to prevent and maintain its proliferation of stem cells spontaneously differentiate.

[0003] Hilton DJ等人应用DEAE sepharose、Lentil Lectin sepharose、CM sepharose以及Pheny I娃胶反向层析方法从经内毒素刺激的Krebs腹水细胞培养上清中纯化LIF蛋白(Hilton DJ et al.Anal B1chem, 1988,173:359-367,W08807548),但由该方法所得的LIF蛋白其表达量较低,并且纯化步骤较为繁琐,并不适合LIF蛋白的大量制备。 [0003] Hilton DJ et al application DEAE sepharose, Lentil Lectin sepharose, CM sepharose, and reverse phase chromatography Pheny I baby gel method ascites cell culture supernatant of LIF protein was purified from toxin-stimulated Krebs (Hilton DJ et al.Anal B1chem, 1988,173: 359-367, W08807548), but obtained by a method of the LIF protein expression level is low, and the purification step is more complicated, a large number of proteins are not suitable for the preparation of LIF.

[0004]由于原核细胞表达的无糖基化修饰的LIF蛋白同真核细胞表达的LIF蛋白相比活性相当,因此也有研究者尝试应用原核细胞如大肠杆菌来表达LIF蛋白。 [0004] Since the expression of LIF with sugarless eukaryotic expression prokaryotic cell group of modified protein compared to LIF activity comparable to, and therefore some researchers tried to apply prokaryotic cells such as E. coli to express the LIF protein. Samal BB等人在大肠杆菌中表达的LIF蛋白以包涵体形式存在,在变复性之后参考上述Hilton DJ等人的方法对LIF蛋白进行了纯化(Samal BB et al.B1chim B1phys Acta,1995,1260: 27-34),但是由于包涵体在变性复性过程中需要大量的变性剂,并且复性的效率较低,因此该方法也不适合LIF蛋白的大量制备。 Samal BB et al LIF expressed protein in E. coli as inclusion bodies, refolding after Hilton DJ et al reference above method of LIF protein was purified (Samal BB et al.B1chim B1phys Acta, 1995,1260 : 27-34), but due to the inclusion bodies requires a large amount of a denaturing agent during the refolding denaturation, renaturation and less efficient, this method is not suitable for mass production of LIF protein. 此外还有研究者尝试以融合蛋白的形式在大肠杆菌表达LIF蛋白,同LIF融合的蛋白通常可促进LIF蛋白的可溶性表达(CN103981205,Jung AS etal.PLoS 0NE,2013,8:e83781),但由于以融合形式表达的蛋白需要经过后续的酶切步骤才可得到无标签的目的蛋白,其过程较为繁琐,并且容易引入其他蛋白(如用于酶切的酶类以及融合表达的蛋白标签)的污染,同时在纯化过程中因为需要应用亲和层析,其成本往往较尚O There are also researchers try to form a fusion protein expression LIF protein in E. coli, with LIF fusion protein normally promotes expression of soluble LIF protein (CN103981205, Jung AS etal.PLoS 0NE, 2013,8: e83781), but because in the form of a fusion protein to go through before the subsequent digestion step the unlabeled protein was obtained, the process is more cumbersome and easy to introduce contamination of other proteins (e.g., proteins and enzymes for cleavage of the tag of the expressed fusion) of also because of the need during purification by affinity chromatography, which is often more cost yet O

[0005]因此,发明一种能够制备高纯度、高活性且易操作、成本低的重组人白血病抑制因子的制备方法成为当务之急。 [0005] Accordingly, the invention is capable of preparing high-purity, high activity and easy to operate, low cost method for preparing recombinant factor human leukemia inhibitory become imperative.

发明内容 SUMMARY

[0006]本发明的目的在于提供一种从大肠杆菌细胞中制备具有高纯度的白血病抑制因子的方法。 [0006] The object of the present invention is to provide a method for high-purity leukemia inhibitory factor prepared from E. coli cells having. 其包括以下步骤: Comprising the steps of:

[0007] (I)将1^8-!1(^、恥(:^0了4混合,混合液中的三者的浓度分别达到201111、30-150111]\1和ImM,然后加入十二烷基硫酸钠、Triton X-100或者Tween-20中的一种,使其浓度达到 [0007] (I) the 1 ^ 8-1 (^, shame (:! ^ 0 4 mixing, the concentration of the mixture of three respectively 201111,30-150111] \ 1 and ImM, followed by addition of twelve sodium alkyl sulfate, or one of Tween-20 Triton X-100, to a concentration of

0.5%-0.05%,得到裂解液; 0.5% -0.05%, resulting lysate;

[0008] (2)对包含有人白血病抑制因子表达载体的大肠杆菌放入LB培养基、SOB培养基、SOC培养基、2 X YT培养基或TB培养基中,在25-37°C进行培养,在0D600nm达到0.5-0.8时加Λ0.5-1.0mM的异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导,调整大肠杆菌的培养温度至16-25°C继续培养16-30小时后收获细菌,加入裂解液后对菌体进行破碎,分离菌体沉淀和上清液,使用NaH2PO4溶液调整上清液的pH至6.0; [0008] (2) the E. coli vector was placed in an LB medium containing leukemia inhibitory factor, SOB medium, the SOC medium, 2 X YT medium or TB medium and cultured at 25-37 ° C , 0D600nm reached 0.5-0.8 when added Λ0.5-1.0mM isopropyl -β-D- thiogalactoside (IPTG) induction, the culture temperature is adjusted to 16-25 ° C E. coli culture was continued at 16 bacteria were harvested after 30 hours after addition of the cell lysate was crushed, and the supernatant isolated bacterial pellet using NaH2PO4 pH of the supernatant solution was adjusted to 6.0;

[0009] (3)初步纯化:使用阳离子交换色谱填料的色谱柱,以磷酸、NaCl、EDTA按照25mM、50mM和ImM配制缓冲液A,以磷酸、NaCl、EDTA按照25mM、500mM和ImM配制缓冲液B,以缓冲液A平衡色谱柱,将获得的上清液进行上样,然后使用缓冲液A平衡色谱柱,再以磷酸、NaCl、EDTA按照三者浓度25mM、10mM和ImM配制溶液,加入非离子表面活性剂,得到含有0.01%- [0009] (3) Preliminary purification: cation exchange chromatography column packing, phosphoric acid, NaCl, EDTA accordance with 25mM, 50mM and ImM formulation buffer A, phosphoric acid, NaCl, EDTA according to 25mM, 500mM formulation buffer and ImM B, supernatant column equilibrated with buffer a, the samples were obtained, and then using the column equilibrated with buffer a, and then phosphoric acid, NaCl, EDTA solution was prepared in accordance with the three concentrations of 25mM, 10mM and ImM, non-added ionic surfactant, containing 0.01% to give -

0.1 %非离子表面活性剂的清洗缓冲液,对结合在填料上的蛋白进行清洗,在基线稳定后应用以缓冲液A和B体积比1:4配制的洗脱缓冲液对结合在层析填料上的蛋白样品进行洗脱,收集洗脱峰,对收集的洗脱峰进行超滤换液处理,将洗脱组分的缓冲液置换为PH 7.0的25mM磷酸缓冲液; Washing buffer with 0.1% non-ionic surfactants, binding to proteins on the filler cleaning applications in a stable baseline with buffer A and B volume ratio of 1: 4 elution buffer formulation on binding chromatographic packing protein elution was carried out on samples collected elution peak, collected elution peak was changed ultrafiltration process, the elution buffer component is replaced with 7.0 PH 25mM phosphate buffer;

[0010] (4)中度纯化:使用阴离子交换色谱填料的色谱柱,以25mM磷酸缓冲液(pH7.0)平衡色谱柱后,将合并后换液的洗脱液组分进行上样,使用以25mM磷酸缓冲液(pH 7.0)平衡色谱柱,在紫外吸收升高以及回落至20mAU-200mAU收集流穿的含有样品的组分; [0010] (4) intermediate purification: anion exchange chromatography using a column packing, in the 25mM phosphate buffer (pH7.0) equilibrate the column, the liquid component of the combined transducer eluate is loaded onto a, using in 25mM phosphate buffer (pH 7.0) balance column, and raising the UV absorption dropped to the sample containing the component 20mAU-200mAU collected in the flow-through;

[0011 ] (5)精细纯化:使用阳离子交换色谱填料的色谱柱,以pH 7.0的25mM磷酸缓冲液平衡色谱柱后,将上述收集的流穿组分上样,再次使用PH 7.0的25mM磷酸缓冲液平衡色谱柱,以磷酸、NaCl按照25mM、500mM配制pH 7.0的缓冲液C,之后根据样品纯化的规模线性变换pH7.0的25mM磷酸缓冲液和缓冲液C的相对比例,应用线性梯度洗脱的方式对结合在填料上的样品进行洗脱,在紫外吸收升高以及回落至20mAU-200mAU收集洗脱峰组分,得到最终的重组人白血病抑制因子洗脱液。 [0011] (5) Fine purification: cation exchange chromatography using a column packing, in the 25mM phosphate buffer, pH 7.0 equilibrium column flow-through was loaded on the above-described components collected using a 25mM phosphate buffer PH 7.0 again liquid equilibrium column, phosphoric acid, NaCl 25mM accordance, 500 mM formulation buffer C pH 7.0, followed by purification according to the relative proportions of the size of the sample linear transformation of 25mM phosphate buffer pH7.0 buffer C and the use of the linear gradient elution manner on the filler bound samples were eluted down and increased absorption in the ultraviolet to 20mAU-200mAU component elution peak is collected to give the final recombinant human leukemia inhibitory factor eluent.

[0012]初步纯化所用的阳离子交换色谱的填料配基可为羧基型的弱阳离子类填料、膦酸基型的中强阳离子类交换填料或者磺酸基型的强阳离子类交换色谱填料,使用的非离子表面活性剂为Triton X-1OO^Triton X-114或者Tween 20。 [0012] Preliminary purified by cation exchange chromatography packing ligands may filler carboxyl group-type weakly cationic, phosphonic acid type in the strong cationic exchange packing or strongly cationic sulfonic acid group-exchange chromatography filler, using a non-ionic surfactant is Triton X-1OO ^ Triton X-114 or Tween 20.

[0013]中度纯化所用的阴离子交换色谱填料为季胺基型的强碱型阴离子交换色谱填料,或者为伯胺基、仲胺基以及叔胺基型的弱碱型阴离子交换色谱填料。 [0013] The intermediate purification by anion exchange chromatography with filler-type quaternary amine strong base anion exchange chromatography filler, or a primary amine, secondary amine and tertiary amine-type weak base anion exchange chromatography packing.

[0014]精细纯化所用的强阳离子色谱的填料配基为具有磷酸根基团的阳离子交换色谱填料,优先选择轻基磷灰石(Hydroxyapatite)。 [0014] Strong cation chromatography ligands fine filler used was purified with a cation exchange chromatography phosphate groups of the filler, preferred light HA (Hydroxyapatite).

附图说明 BRIEF DESCRIPTION

[0015]图1为对包含有人白血病抑制因子表达载体的大肠杆菌进行诱导后鉴定人白血病抑制因子表达的凝胶电泳扫描结果。 [0015] Figure 1 is the E. coli containing the expression vector was leukemia inhibitory factor induction of gel electrophoresis to identify human leukemia inhibitory factor expression scan results.

[0016] 图2为应用充填阳离子交换填料SP Sepharose Fast Flow的色谱柱对前期处理过的大肠杆菌上清液进行纯化后所得的洗脱曲线图。 [0016] FIG 2 is filled with cation exchange packing applications SP Sepharose Fast Flow column pre-treatment of E. coli is a graph of the elution supernatant obtained after purification.

[0017] 图3为应用充填阴离子交换填料Q Sepharose Fast Flow的色谱柱对从SPSepharose Fast Flow色谱柱收集的洗脱液进行纯化后所得的洗脱曲线图。 [0017] FIG. 3 is filled with anion exchange packing applications Q Sepharose Fast Flow column to the column were collected from the eluate eluted SPSepharose Fast Flow graph obtained after purification.

[0018] 图4为应用充填阳离子交换填料Hydoxyapatite的色谱柱对从Q Sepharose FastFlow色谱柱收集的洗脱液进行纯化后所得的洗脱曲线图。 [0018] FIG. 4 is a cation exchange chromatography column filled with a filler applied Hydoxyapatite collected from the column of Q Sepharose FastFlow eluent elution profile obtained after purification FIG.

[0019]图5为应用SDS-PAGE对各纯化步骤所得的人白血病抑制因子蛋白样品的纯度检测结果。 [0019] FIG. 5 is applied to each of the SDS-PAGE of purification steps resulting purity of the detection result of human leukemia inhibitory factor protein samples.

[0020]图6为对所得的人白血病抑制因子蛋白样品进行体外实验的活性测定结果。 [0020] FIG. 6 is a resulting human leukemia inhibitory factor protein samples were measured in vitro data activity.

具体实施例 Specific Example

[0021 ] 1.大肠杆菌裂解液的制备 1. Preparation of the E. coli lysate [0021]

[0022] 将1^8-!1(^、恥(:140了4混合,混合液中的三者的浓度分别达到20111]\1、601111和1111]\1,然后加入Triton X-100,使其浓度达到0.5%,得到裂解液备用。 ! [0022] The 1 ^ 8-1 (^, shame (: 4 mixture 140, the mixture of the three concentrations, respectively 20111] \ and 1,601111 1111] \ 1, followed by addition of Triton X-100, to a concentration of 0.5%, resulting lysate standby.

[0023]接种含有人白血病抑制因子表达载体的大肠杆菌至LB培养基中,在37°C、200转过夜培养,之后以1:50的体积比接种于新鲜配制的LB培养基中,在370C度、200转振摇培养至细菌0D600nm检测结果达到0.6时,加入终浓度为0.5mM的IPTG进行诱导,然后降低细菌培养液的温度至16°C,并同时改变细菌的培养温度至16°C,继续200转振摇培养20小时后收获细菌。 [0023] inoculate a human leukemia inhibitory factor expression vector to E. coli in LB medium at 37 ° C, 200 rpm overnight culture, then inoculated in a volume ratio of 1:50 in freshly prepared LB medium at 370C degrees to 200 rpm shaking culture bacteria detection result 0D600nm reached 0.6, IPTG 0.5mM final concentration is induced, and then reducing the temperature of the liquid bacterial culture to 16 ° C, while changing the culture temperature to 16 ° C the bacteria and continue to 200 rpm shaking culture the bacteria were harvested after 20 hours.

[0024]将所收获细菌称重后,根据细菌湿重按照1:10的体积比加入裂解液(如细菌湿重为Ig,则加入的裂解液为1mL)进行破碎,破碎细菌后分离上清,使用0.2M的NaH2PO4溶液调整裂解液上清的PH至6.0备用。 [0024] The bacteria were harvested after weighing, bacterium according wet weight ratio of 1:10 by volume lysis buffer was added (e.g., an Ig wet weight bacteria, the lysis buffer was added 1 mL) is crushed, the supernatant was separated after crushing the bacteria using 0.2M NaH2PO4 solution lysate supernatant was adjusted to 6.0 backup PH. 对裂解液沉淀和上清中人白血病抑制因子的表达情况应用SDS-PAGE进行检测,结果如图1所示,图中箭头所示为表达的人白血病抑制因子蛋白。 Lysates and supernatants precipitated on human leukemia inhibitory factor (VEGF) application for SDS-PAGE, and the results shown in FIG. 1 by an arrow shown in FIG expressed human leukemia inhibitory factor protein.

[0025] 2.应用阳离子交换色谱对裂解液中的白血病抑制因子进行捕获 [0025] 2. The application of cation exchange chromatography lysates capture leukemia inhibitory factor

[0026] 以磷酸、似(:140了4按照25111]\1、50111]\1和1111]\1配制缓冲液4,以磷酸、似(:140了4按照25mM、500mM和ImM配制缓冲液B,然后以缓冲液A平衡装填有阳离子交换色谱填料SPsepharose fast flow的色谱柱,在基线稳定后将裂解液上清进行上样,然后使用缓冲液A平衡色谱柱,再以磷酸、NaCl、EDTA按照三者浓度25mM、10mM和ImM配制溶液,加入TritonX-114,得到含有0.1 % Triton X_114的清洗缓冲液,对结合在填料上的蛋白进行清洗,在基线稳定后应用以缓冲液A和B体积比1:4配制的洗脱缓冲液对结合在层析填料上的蛋白样品进行洗脱,收集洗脱峰。应用阳离子交换色谱对裂解液中的白血病抑制因子进行捕获阶段纯化的色谱结果如图2所示,其中UV1_280为280nm的紫外吸收曲线,Cond为电导率曲线,阴影所示为收集的洗脱液组分。 [0026] In phosphate, like (: 4 140 25111 according to] \ 1,50111] \ 1, and 1111] \ 1 4 formulation buffer, phosphoric acid, like (: 140 4 according to 25mM, 500mM formulation buffer and ImM B, and then equilibrated with buffer a cation exchange chromatography column packed with chromatography packing SPsepharose fast flow of the sample after the lysate supernatant was stable baseline, and then using the column equilibrated with buffer a, and then phosphoric acid, NaCl, EDTA concentration according to the three 25mM, 10mM and ImM solution was prepared, added TritonX-114, to give wash buffer containing 0.1% Triton X_114 of binding proteins on the filler cleaned, applied in buffer a and B in a stable baseline volume ratio of 1: chromatographic results of binding proteins on chromatography media samples were prepared 4 elution buffer elution, elution peak was collected on a cation-exchange chromatography lysates leukemia inhibitory factor purified in FIG capture phase Figure 2, wherein UV1_280 280nm UV absorption curve, Cond is the conductivity curve, as indicated by hatching in the eluent fractions collected.

[0027]对收集的洗脱峰进行超滤换液处理,将洗脱组分的缓冲液置换为pH 7.0的25mM磷酸缓冲液。 [0027] The eluted peaks were collected to ultrafiltration process was changed, the elution buffer component is replaced with 25mM phosphate buffer, pH 7.0.

[0028] 3.应用阴离子交换色谱对捕获的白血病抑制因子进行中度纯化 [0028] 3. Application of the anion exchange chromatography capture intermediate purification leukemia inhibitory factor

[0029] 以25mM磷酸缓冲液(pH 7.0)平衡装填有阴离子交换色谱填料Q sepharose fastflow的色谱柱后,在基线稳定后将上述合并后换液的洗脱液组分进行上样,使用25mM磷酸缓冲液(pH 7.0)平衡色谱柱,在紫外吸收升高以及回落至20mAU-200mAU收集流穿的含有样品的组分。 After [0029] In 25mM phosphate buffer (pH 7.0) balance the anion exchange chromatography column packed with chromatography packing Q sepharose fastflow, in other words after the baseline was stabilized after the above-described components were combined eluate sample, the use of phosphoric acid 25mM buffer (pH 7.0) balance column, and raising the UV absorption dropped to the sample containing the component 20mAU-200mAU collected in the flow-through. 应用阴离子交换色谱对白血病抑制因子进行中度纯化的色谱结果如图3所示,其中UV1_280为280nm的紫外吸收曲线,Cond为电导率曲线,Conc B为设定的洗脱液比例曲线;阴影所示为收集的含有人白血病抑制因子的组分。 Application of chromatographic results of anion exchange chromatography leukemia inhibitory factor purified intermediate 3, wherein UV1_280 UV absorption profile at 280nm, is the conductivity curve Cond, Conc B eluent set scale curve; shading component factor is shown containing human leukemia inhibitory collected.

[0030] 4.应用阳离子交换色谱对白血病抑制因子进行精细纯化 [0030] 4. The application of cation exchange chromatography purification fine leukemia inhibitory factor

[0031 ] 以pH 7.0的25mM磷酸缓冲液平衡装填有轻基磷灰石(Hydroxyapatite)的色谱柱后,在基线稳定后将上述步骤收集的组分进行上样,上样后应用25mM磷酸缓冲液(pH 7.0)平衡色谱柱,以磷酸、NaCl按照25mM、500mM配制pH 7.0的缓冲液C,之后按照三倍色谱柱体积的洗脱溶液体积,从100%的pH 7.0的25mM磷酸缓冲液变换至100 %的缓冲液C,应用线性梯度洗脱的方式对结合在填料上的样品进行洗脱,在紫外吸收升高以及回落至20mAU-200mAU时收集洗脱峰组分,并进行鉴定。 [0031] In 25mM phosphate buffer, pH 7.0 equilibrium HA loaded with light (The Hydroxyapatite) column, the component will be loaded on the above-described step of collecting baseline stabilization, the application of 25mM phosphate buffer after loading (pH 7.0) balance column, phosphoric acid, NaCl 25mM follow, C formulation buffer 500 mM, pH 7.0, then three times the column volume of elution solution volume, from 25mM phosphate buffer pH 7.0 100% conversion to 100% buffer C, using linear gradient elution manner bound sample was eluted on the filler, and increased when the UV absorption dropped to 20mAU-200mAU component elution peak was collected and identified. 应用阳离子交换色谱对白血病抑制因子进行精细纯化的色谱结果如图4所示,其中UV1_280为280nm的紫外吸收曲线,Cond为电导率曲线,Conc B为设定的洗脱液比例曲线;阴影所示为收集的洗脱液的组分。 Application of chromatographic results of cation exchange chromatography purified fine leukemia inhibitory factor shown in Figure 4, wherein UV1_280 280nm UV absorption curve, Cond the conductivity curve, Conc B eluent set scale curve; hatching component of the eluent collected.

[0032]将各纯化步骤所得的人白血病抑制因子蛋白样品的纯度进行检测,结果如图5所示,图中箭头所指示的为表达的人白血病抑制因子蛋白。 [0032] Each of the resulting purification step of human leukemia inhibitory factor protein purity of the samples were detected, the results shown in Figure 5, indicated by an arrow in FIG expression of human leukemia inhibitory factor protein.

[0033] 5.人白血病抑制因子的体外活性测定 [0033] The human leukemia inhibitory factor activity in vitro assay

[0034] 将Ml细胞接种至96孔板,40000个/孔,加入50yL培养基(DMEM+10%Fetal calfserum),然后加入50yL按照比例稀释的经纯化得到的人白血病抑制因子蛋白样品;应用市售的人白血病抑制因子作为对照,按照相同的稀释方法进行稀释,并且保持终体积亦为50μL,加入已经接种Ml细胞的96孔板中。 [0034] The Ml cells were seeded into 96-well plates, 40,000 / well, was added 50yL medium (DMEM + 10% Fetal calfserum), followed by addition of purified human 50yL according to the ratio obtained diluted leukemia inhibitory factor protein samples; application City human leukemia inhibitory factor sold as a control, were diluted in the same dilution method, and also to maintain a final volume of 50 L, have been added to cells seeded in 96 well plates Ml. 在37°C培养不同时间后应用CCK-8检测试剂按照厂家推荐的方法测定对Ml细胞增殖的抑制情况,同市售商品化人白血病抑制因子的活性比较,结果如图6所示,其中Pur1-LIF代表应用本发明所阐述方法所制备的人白血病抑制因子蛋白所获得结果,Mill-LIF为应用市售的人白血病抑制因子蛋白所获得结果。 After incubation at 37 ° C at different time application CCK-8 assay reagent detection Ml inhibition of cell proliferation in accordance with the method recommended by the manufacturer, with the commercialization of a commercially available human leukemia inhibitory factor activity of comparison, the results shown in Figure 6, wherein Pur1- Representative applications of the present invention LIF production method as set forth in human leukemia inhibitory factor protein results obtained, Mill-LIF for the application of commercially available human leukemia inhibitory factor protein results obtained.

[0035]现通过以上实施方式并且结合附图来进一步描述本发明的有益效果,应理解为实施方式仅用于例证的目的,不限制本发明的范围,同时本领域普通技术人员根据本发明所做的显而易见的改变和修饰也包含在本发明范围之内。 [0035] By the above embodiments and now in conjunction with the accompanying drawings be further described advantageous effects of the present invention, it should be understood that for the purpose of illustration of the embodiments only, not limiting the scope of the present invention, while those of ordinary skill in the art in accordance with the present invention make obvious changes and modifications are also included within the scope of the present invention.

Claims (7)

1.一种重组人白血病抑制因子的制备方法,包括以下步骤: (1)将TriS-HCUNaCl、EDTA混合,混合液中的三者的浓度分别达到20mM、30-150mM和ImM,然后加入十二烷基硫酸钠、Triton X-100或者Tween-20中的一种,使其浓度达到0.5%-0.05%,得到裂解液; (2)对包含有人白血病抑制因子表达载体的大肠杆菌放入培养基中在25-37°C进行培养,在0D600nm达到0.5-0.8时加入0.5-1.0mM的异丙基-β-D-硫代半乳糖苷进行诱导,调整大肠杆菌的培养温度至16-25°C继续培养16-30小时后收获细菌,加入裂解液后对菌体进行破碎,分离菌体沉淀和上清液,使用NaH2PO4溶液调整上清液的pH至6.0; (3)初步纯化:使用阳离子交换色谱填料的色谱柱,以磷酸、NaCl、EDTA按照25mM、50mM和ImM配制缓冲液A,以磷酸、NaCl、EDTA按照25mM、500mM和ImM配制缓冲液B,然后以缓冲液A平衡色谱柱,将获得的上清液进行上样,然后继续使用 1. A method of preparing recombinant human Factor leukemia inhibitory, comprising the steps of: (1) the concentration of the TriS-HCUNaCl, EDTA mixture, a mixture of three respectively 20mM, 30-150mM and ImM, followed by addition of twelve sodium alkyl sulfate, Triton or one of Tween-20 X-100, to a concentration of 0.5% -0.05%, resulting lysate; (2) comprising a leukemia inhibitory factor was placed in culture medium of E. coli vector were cultured at 25-37 ° C, in the induction of 0.5-1.0mM 0D600nm reached 0.5-0.8 when added isopropyl -β-D- thiogalactoside, the culture temperature was adjusted to 16-25 ° E. coli C 16-30 hours after cultured bacteria were harvested, lysates of the cells after addition of crushed cells separated precipitate and supernatant, using a pH adjustment of the supernatant to 6.0 NaH2PO4 solution; and (3) preliminary purification: cationic exchange chromatography column packing, phosphoric acid, NaCl, EDTA accordance with 25mM, 50mM and ImM a formulation buffer, phosphoric acid, NaCl, EDTA according to 25mM, 500mM and ImM formulation buffer B, and then column equilibrated with buffer a, the supernatant obtained was loaded, and then continue to use 冲液A平衡色谱柱,再以磷酸、NaCl、EDTA按照三者浓度25mM、10mM和ImM配制溶液,加入非离子表面活性剂,得到含有0.01%-0.1%非离子表面活性剂的清洗缓冲液,对结合在填料上的蛋白进行清洗,在基线稳定后应用以缓冲液A和B体积比1:4配制的洗脱缓冲液对结合在层析填料上的蛋白样品进行洗脱,收集洗脱峰,对收集的洗脱峰进行超滤换液处理,将洗脱组分的缓冲液置换为PH7.0的2511^磷酸缓冲液; (4)中度纯化:使用阴离子交换色谱填料的色谱柱,以pH 7.0的25!1^磷酸缓冲液平衡色谱柱后,将合并后换液的洗脱液组分进行上样,继续使用以PH 7.0的25!1^磷酸缓冲液平衡色谱柱,在紫外吸收升高以及回落至20mAU-200mAU收集流穿的含有样品的组分; (5)精细纯化:使用阳离子交换色谱填料的色谱柱,以pH 7.0的25mM磷酸缓冲液平衡色谱柱后,将上述收集的流穿组分上样,再次使用 A flushing equilibrium column, and then phosphoric acid, NaCl, EDTA solution was prepared in accordance with the three concentrations of 25mM, 10mM and ImM, non-ionic surfactant is added to give a wash buffer containing 0.01% -0.1% non-ionic surfactant, binding protein on the filler cleaning applications in a stable baseline with buffer a and B volume ratio of 1: protein binding in the samples eluted on a chromatographic packing 4 prepared elution buffer, eluted peaks were collected , elution peak collected was changed ultrafiltration process, the elution buffer component is replaced PH7.0 ^ phosphate buffer 2511; (4) intermediate purification: anion exchange chromatography column packing, pH 7.0 at 25! ^ 1 phosphate buffer for equilibrium column, the eluant was changed after the combined fractions were loaded on, to continue to use the 25 PH 7.0! ^ 1 column balanced phosphate buffer, ultraviolet absorbent component comprising raised and dropped to the sample 20mAU-200mAU collected flowthrough; and (5) fine purification: cation exchange chromatography using a column packing to the column balance buffer 25mM phosphate, pH 7.0, collecting the above the flow-through component of the sample, again using PH 7.0的25mM磷酸缓冲平衡色谱柱,以磷酸、NaCl按照25mM、500mM配制pH 7.0的缓冲液C,之后根据样品纯化的规模线性变换pH 7.0的25mM磷酸缓冲液和缓冲液C的相对比例,应用线性梯度洗脱的方式对结合在填料上的样品进行洗脱,在紫外吸收升高以及回落至20mAU-200mAU收集洗脱峰组分,得到最终的重组人白血病抑制因子洗脱液。 The PH 7.0 25mM phosphate buffer balanced column with phosphate, NaCl 25mM follow, C formulation buffer 500 mM pH 7.0, after the sample was purified according to the size of the relative proportions of linear transformation 25mM phosphate pH 7.0 buffer and buffer C, the application linear gradient elution manner bound sample was eluted on the filler, and the increased absorption dropped to 20mAU-200mAU component elution peak is collected to give the final recombinant human leukemia inhibitory factor in the ultraviolet eluent.
2.根据权利要求1所述的重组人白血病抑制因子制备方法,其特征在于使用的大肠杆菌培养基为LB培养基、SOB培养基、SOC培养基、2 X YT培养基、TB培养基。 2. The recombinant E. coli culture medium according to claim 1 leukemia inhibitory factor production method, which is characterized by using LB medium, SOB medium, the SOC medium, 2 X YT medium, TB medium.
3.根据权利要求1所述的重组人白血病抑制因子制备方法,其特征在于初步纯化所用的阳离子交换色谱的填料配基可为羧基型的弱阳离子类填料、膦酸基型的中强阳离子类交换填料或者磺酸基型的强阳离子类交换色谱填料。 The recombinant according to claim 1 leukemia inhibitory factor production method, wherein a filler used in the group preliminary purification by cation exchange chromatography may be a weak carboxyl cationic type filler, the strong acid cationic group type type strong cation exchange packing or a sulfonic acid group-exchange chromatography filler.
4.根据权利要求1中所述的重组人白血病抑制因子制备方法,其特征在于中度纯化所用的阴离子交换色谱填料为季胺基型的强碱型阴离子交换色谱填料,或者为伯胺基、仲胺基以及叔胺基型的弱碱型阴离子交换色谱填料。 The recombinant of claim 1 in preparation of leukemia inhibitory factor, characterized in that the intermediate purification by anion exchange chromatography with filler quaternary amine strong base type anion-exchange chromatography filler, or a primary amine, secondary and tertiary amino group-type weak base anion exchange chromatography packing.
5.根据权利要求1所述的重组人白血病抑制因子制备方法,其特征在于精细纯化所用的强阳离子色谱的填料配基为具有磷酸根基团的阳离子交换色谱填料。 The recombinant according to claim 1 prepared leukemia inhibitory factor, wherein the strong cation chromatography ligands fine filler used was purified with a cation exchange chromatography phosphate groups filler.
6.根据权利要求1所述的重组人白血病抑制因子制备方法,其特征在于使用的非离子表面活性剂为Triton X-1OO^Triton X-114或者Tween 20。 6. The recombinant of claim 1 prepared according to claim leukemia inhibitory factor, wherein the nonionic surfactant used is Triton X-1OO ^ Triton X-114 or Tween 20.
7.根据权利要求5所述的重组人白血病抑制因子制备方法,其特征在于使用的磷酸根基团阳离子交换色谱填料为羟基磷灰石。 7. The recombinant human 5 production method according to claim leukemia inhibitory factor, characterized in that the phosphate groups of the cation exchange chromatography using a filler is hydroxyapatite.
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CN1616489A (en) * 2004-09-30 2005-05-18 中国科学技术大学 Method for purifying and recombining human iterleukin-12
WO2009058812A1 (en) * 2007-10-30 2009-05-07 Genentech, Inc. Antibody purification by cation exchange chromatography
CN102477098A (en) * 2010-11-29 2012-05-30 清华大学深圳研究生院 Fusion protein and application thereof in preparation of human leukemia inhibitory factor
CN103981205A (en) * 2014-05-23 2014-08-13 上海同科生物科技有限公司 Method for recombinant expression of human leukemia inhibitory factor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1119018A (en) * 1993-03-05 1996-03-20 先灵公司 Purification of human interleukin-10
CN1616489A (en) * 2004-09-30 2005-05-18 中国科学技术大学 Method for purifying and recombining human iterleukin-12
WO2009058812A1 (en) * 2007-10-30 2009-05-07 Genentech, Inc. Antibody purification by cation exchange chromatography
CN102477098A (en) * 2010-11-29 2012-05-30 清华大学深圳研究生院 Fusion protein and application thereof in preparation of human leukemia inhibitory factor
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