WO2019075892A1 - 一种胎盘样硫酸软骨素a或其衍生物的亲和层析纯化方法 - Google Patents

一种胎盘样硫酸软骨素a或其衍生物的亲和层析纯化方法 Download PDF

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WO2019075892A1
WO2019075892A1 PCT/CN2017/116624 CN2017116624W WO2019075892A1 WO 2019075892 A1 WO2019075892 A1 WO 2019075892A1 CN 2017116624 W CN2017116624 W CN 2017116624W WO 2019075892 A1 WO2019075892 A1 WO 2019075892A1
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chondroitin sulfate
affinity chromatography
derivative
placenta
surface antigen
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French (fr)
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张居作
范秀军
张键
程国钢
陈指龙
张保珍
肖仲琳
韩金雨
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Shenzhen Institute of Advanced Technology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to the field of biomedicine, in particular to a method for affinity chromatography purification of a placenta-like chondroitin sulfate A or a derivative thereof.
  • the principle of affinity chromatography is to utilize the specificity and reversibility of the affinity between biomolecules.
  • the coupled affinity is based on the stationary phase adsorption medium, and the target in the mobile phase is affinity-adsorbed through the stationary phase, while in the mobile phase.
  • the impurities are washed out, and finally by changing the mobile phase conditions, such as pH or ion concentration and species, the affinity adsorption between the ligand and the target is dissociated, and the target is released into the mobile phase, thereby obtaining a high-purity target. Since the advent of self-affinity chromatography technology, this technology has developed very rapidly and has achieved remarkable results in the field of biotechnology.
  • Immunoaffinity Chromatography which uses the specific binding characteristics of antigen-antibodies, one of which is a ligand to adsorb the other, this method can obtain a high purification factor and maintain high natural activity; 3.
  • Metal ion pro And chromatography which uses metal ions to form a chelate with a certain protein, with immobilized metal ions as a ligand, such as the use of histidine imidazole groups to specifically bind to nickel ions to purify the group. a fusion protein of a tyrosine tag; 4.
  • biotin affinity chromatography which utilizes a part of the intermolecular or intramolecular interaction force to artificially cure a molecule or a part of a molecule as a ligand. Purify the target protein.
  • polysaccharide affinity purification methods There are still few establishment and application of polysaccharide affinity purification methods. The key difficulty is to find ligands that can bind specifically to polysaccharides.
  • the protein was mixed with chloroform and n-butanol to form a gel insoluble matter, and the polysaccharide was dissolved in the aqueous phase, and repeated operations were repeated to remove impurities such as protein, but this
  • the polysaccharide obtained by the method is crude and contains various polysaccharide components.
  • the invention utilizes the principle that the cell surface polysaccharide is specifically recognized by the ligand, and the placental-like chondroitin sulfate A (pl-CSA) on the surface of the trophoblast cell can be specifically recognized and combined by the erythrocyte surface antigen VAR2CSA infected by the malaria parasite, and is synthesized and synthesized.
  • VAR2CSA rVAR2
  • the pl-CSA minimal binding moiety is a ligand
  • the affinity chromatography column is prepared by coupling with NHS-activated agarose to establish a pl-CSA affinity chromatography purification method.
  • One aspect of the present invention provides a method for purifying a placenta-like chondroitin sulfate A or a derivative thereof, which comprises chromatographic purification of placenta-like chondroitin sulfate A or a derivative thereof by affinity chromatography.
  • the affinity chromatography column is a conjugate of a recombinant Plasmodium infection erythrocyte surface antigen protein and an affinity chromatography matrix.
  • the chromatographic purification method is to apply a crude product of placenta-like chondroitin sulfate A or a derivative thereof to an affinity chromatography column, and wash with a washing liquid until no impurities flow out, and then The eluate was eluted and the pure product of placenta-like chondroitin sulfate A or its derivative was collected.
  • the volume ratio of the crude plate loading of the placenta-like chondroitin sulfate A or its derivative to the chromatography column is 1:0.5-2, preferably 1:0.8-1.2, more preferably 1:1. .
  • the elution volume of the wash liquor is more than 3 times the column volume, more preferably more than 5 times the column volume, more preferably 5-10 times the column volume.
  • the eluent has an elution volume of more than 5 times the column volume, more preferably 8 More than the column volume, more preferably 10 column volumes.
  • the detergent used for affinity chromatography has a pH of 7-9, more preferably a pH of 7-8, more preferably a pH of 7.2, and an eluent pH of 2-4, more preferably.
  • the pH is selected from 2.2 to 3.6, and the pH is preferably 3.0.
  • the eluent comprises a protective agent.
  • the protective agent is glycine, vitamin C, vitamin E, sodium sulfite, sodium hydrogen sulfite, sodium metabisulfite, sodium thiosulfate.
  • the detergent comprises a salt solution.
  • the detergent is 0.1 M NaCl, 0.02 M Na 2 HPO 4 , with a pH of 7.2.
  • the eluent is 0.1 M glycine and has a pH of 3.
  • the pH of the effluent is detected after elution with the eluent, and the pure placenta-like chondroitin sulfate A is collected at a pH of 6.8, and the peak of collection is determined by an ultraviolet monitor.
  • the genetic information of the recombinant Plasmodium-infected erythrocyte surface antigen protein in the conjugate of the recombinant Plasmodium-infected erythrocyte surface antigen protein and the affinity chromatography matrix is ID: GU249598, and the code includes ID1-ID2.
  • a cysteine-rich region (1273-3897) selected from one or more of agarose, cellulose, cross-linked dextran, polyacrylamide, and porous glass beads a conjugate of a recombinant Plasmodium-infected erythrocyte surface antigen protein and an affinity chromatography matrix, wherein the recombinant Plasmodium-infected erythrocyte surface antigen protein is recombinantly synthesized by a minimally binding portion of Plasmodium-infected erythrocyte surface antigen VAR2CSA and pl-CSA;
  • the recombinant Plasmodium is infected with the erythrocyte surface antigen protein, and the recombinant Plasmodium infection erythrocyte surface antigen protein is coupled with the affinity chromatography matrix through a chemical bond.
  • the sequence of the recombinant Plasmodium infection erythrocyte surface antigen protein is shown in SEQ ID No. 2.
  • SEQ ID No. 1 is as follows
  • the conjugate of the recombinant Plasmodium-infected erythrocyte surface antigen protein and the affinity chromatography matrix is prepared by coupling the recombinant Plasmodium-infected erythrocyte surface antigen protein and the affinity chromatography substrate through a condensation crosslinking agent.
  • the crosslinking agent is selected from one or more of DCC, DIC, EDC, NHS, Hobt.
  • Another aspect of the present invention provides a method for preparing a placenta-like chondroitin sulfate A or a derivative thereof, comprising the steps of:
  • the purification method in the step 2) is a crude purification method of the above-described placenta-like chondroitin sulfate A or a derivative thereof.
  • the method for preparing the crude placenta-like chondroitin sulfate A is:
  • the enzyme is removed after the lysed tissue cell slurry is deactivated to obtain a crude product of placenta-like chondroitin sulfate A or a derivative thereof.
  • the preparation method of the recombinant Plasmodium infection erythrocyte surface antigen protein is:
  • the gene synthesis ligand encodes a gene fragment, which synthesizes a 2760 bp gene fragment by gene synthesis, and encodes a cysteine-rich region including ID1-ID2 with reference to ID:GU249598 (1273-3897, That is, the nucleotide sequence of SEQ ID No. 2) is SEQ ID No. 1, and a coding sequence of 6 ⁇ His tag is provided at the end thereof;
  • Another aspect of the present invention provides a material for purifying placenta-like chondroitin sulfate A or a derivative thereof, which is a recombinant Plasmodium infection erythrocyte surface antigen protein.
  • Another aspect of the present invention provides a chromatography column for purifying placenta-like chondroitin sulfate A or a derivative thereof, which is a conjugate of a recombinant Plasmodium-infected erythrocyte surface antigen protein and an affinity chromatography vector.
  • Another aspect of the invention provides the use of a recombinant Plasmodium-infected erythrocyte surface antigen protein for the preparation of a chromatography column for the purification of placenta-like chondroitin sulfate A or a derivative thereof.
  • the placental-like chondroitin sulfate A derivative is selected from the group consisting of a protein having chondroitin sulfate A, chondroitin sulfate A proteoglycan, and one or more placenta-derived exosomes modified with chondroitin sulfate A.
  • the derivative Since the derivative has a placental-like chondroitin sulfate A structure, it can be purified using the specific affinity chromatography column of the present invention.
  • Another aspect of the invention provides the use of a recombinant Plasmodium-infected erythrocyte surface antigen protein for purifying placental-like chondroitin sulfate A or a derivative thereof.
  • an affinity chromatography purification system for placenta-like chondroitin sulfate A which includes a sample introduction device, an electrode tee, a constant current pump, and an affinity chromatography from upstream to downstream.
  • the sample introduction device comprises three injection containers which are respectively connected to the electrode tee through three channels, and the electrode three-way is a liquid level induction tee.
  • Three injection containers were used to prime the pl-CSA crude, wash solution and eluent.
  • the flow rate of the injection constant current pump and the discharge constant current pump are identical.
  • a chromatography column for purifying placenta-like chondroitin sulfate A or a derivative thereof according to the present invention is provided in an affinity chromatography column.
  • the electrode two-way is a pH-sensing two-pass
  • the collecting device comprises two collecting capacities, which are respectively connected to the electrode two-pass through two channels, and two collecting containers are respectively used for pick-and-place Waste liquid and product.
  • the ultraviolet detecting device for detecting the effluent is respectively connected with the electrode tee and the electrode two-pass, and the ultraviolet monitor controls the change of the light absorption value of the mobile phase and the change of the pH value.
  • the electrode tee and the electrode are two-way to open or close the injection valve and the collector valve.
  • the invention utilizes the principle that the cell surface polysaccharide is specifically recognized by the ligand, and the placental-like chondroitin sulfate A (pl-CSA) on the surface of the trophoblast cell can be specifically recognized and combined by the erythrocyte surface antigen VAR2CSA infected by the malaria parasite, and is synthesized and synthesized.
  • the VAR2CSA and pl-CSA minimal binding moiety (rVAR2) are ligands, and the affinity chromatography column is prepared by coupling with NHS activated agarose to establish a pl-CSA affinity chromatography purification method.
  • Difficulties in purification are not adsorbed by rVAR2, and are easily removed by washing to obtain high-purity pl-CSA; while the washing condition pl-CSA is matched by rVAR2
  • the base is firmly bound, the eluent conditions are easily released, and a high recovery rate is obtained.
  • A prokaryotic expression plasmid
  • B prokaryotic expression plasmid double restriction enzyme digestion (1: BamHI single digestion; 2: SalI single digestion; 3: BamHI, SalI double digestion; M: DNA marker DL15000);
  • C prokaryotic expression PCR identification (1-16: PCR identification of different bacterial cells; M: DNA marker DL2000).
  • A Process product for separation and purification of pl-CSA from pig placenta (1 and 2: lysis, enzymatic hydrolysis products; 3 and 4: deproteinized, 0.45 ⁇ m filter filtered product; 5. pure after chromatographic system) pl-CSA);
  • B process product for separation and purification of pl-CSA from cells (1: enzymatic, deproteinized, 0.45 ⁇ m filter filtered product; 2: pure pl-CSA after chromatography system).
  • Slurry Slurry
  • VAR2CSA gene sequence FCR3 strain, GenBank accession no. GU249598
  • GU249598 the gene fragment SEQ ID No.1 with a strong affinity for pl-CSA, with ID : GU249598 is a reference, the length is 2760 bp, which contains a coding sequence of 6 ⁇ His tag, encoding a cysteine-rich region (1273-3897) containing ID1-ID2, and its coding sequence is SEQ ID No. .1 is shown.
  • SEQ ID No. 1 is as follows
  • the BamH I restriction site was added at the 5' end, and the Sal I restriction site was added to the 3'end; the gene fragment SEQ ID No. 1 was ligated with the pET28a(+) prokaryotic expression vector to transform E. coli BL21 competent cells. Prokaryotic expression was carried out, and Ni 2+ affinity chromatography was carried out using a rVAR2 terminal 6 ⁇ His tag to obtain high purity rVAR2 (684.1 ⁇ g/mL).
  • the successfully coupled agarose gel was stored as a rVAR2 affinity chromatography column of pl-CSA at 4 ° C; Pipe, electrode tee, constant current pump, chromatography column, pipeline, constant current pump, electrode two-way, sample or waste liquid collection bottle, complete the establishment of affinity chromatography purification system.
  • the crude pl-CSA obtained in the step (3) is passed through the electrode three through the constant flow pump into the column at 1 ml/(1 ml column), when the crude liquid level of the pl-CSA is lower than the volume of the first column.
  • the electrode tee is closed and allowed to stand for 30 min, so that the crude pl-CSA is fully combined with the affinity ligand on the column;
  • the electrode tee opens the washing solution (0.1 M NaCl, 0.02 M Na 2 HPO 4 , pH 7.2 ), remove the impurities by 10 times the column volume washing liquid, the liquid level is lower than the first 10 times the volume of the column volume, the electrode tee is closed;
  • the electrode tee turns on the eluent (0.1M Glycine glycine, pH 3.0) , elute pl-CSA with 5 times column volume eluent, electrode dip collects waste liquid at pH ⁇ 6.8, starts at pH ⁇ 6.8, and collects pl when UV detector shows UV absorption -CSA

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Abstract

一种胎盘样硫酸软骨素A或其衍生物的纯化方法，所述纯化方法为以亲和层析的方法对胎盘样硫酸软骨素A或其衍生物进行层析纯化，其中亲和层析柱材为重组疟原虫感染红细胞表面抗原蛋白与亲和层析基质的偶联物，所述层析纯化的方法为将胎盘样硫酸软骨素A或其衍生物粗品上样于亲和层析柱上，并以洗涤液进行洗涤至没有杂质流出，再以洗脱液进行洗脱并收集胎盘样硫酸软骨素A或其衍生物纯品。所述纯化方法能够克服多糖纯化的困难，分子大小和结构相近的其他多糖不被rVAR2吸附，容易被洗涤去除，获得高纯度的pl-CSA；而洗涤液条件pl-CSA被rVAR2配基牢固结合，洗脱液条件容易释放，获得了较高的回收率。

Description

一种胎盘样硫酸软骨素A或其衍生物的亲和层析纯化方法 技术领域
本发明涉及生物医药领域，具体涉及一种胎盘样硫酸软骨素A或其衍生物的亲和层析纯化方法。
背景技术
亲和层析的原理是利用生物分子间亲和力的特异性和可逆性，偶联亲和配基于固定相吸附介质，流动相中的目标物通过固定相时被亲和吸附，而流动相中的杂质被洗出，最后通过改变流动相条件，如pH值或者离子浓度和种类，解离配基与目标物之间的亲和吸附，使目标物释放进入流动相，从而获得高纯度目标物，自亲和层析技术出现至今，这一项技术发展非常迅速，在生物技术领域取得了瞩目的成绩，现已经被广泛用于蛋白质、肽类、酶制剂、抗原抗体、激素、核酸等。亲和层析柱中被固定的配基选择是亲和纯化的关键因素，根据配基与生物大分子之间相互作用体系的不同，可分为四种不同的类型，包括：1.生物亲和层析，这种方法是利用特异性相互作用的配对物质来纯化其中的一种物质，典型的配对物质包括酶-底物、酶-抑制剂、激素-受体等；2.免疫亲和层析，这种方法是利用抗原抗体的特异性结合特性，以其中一方为配基吸附另一方，这种方法能获得高纯化倍数的物质，且能保持高的天然活性；3.金属离子亲和层析，这种方法是利用金属离子能与某种蛋白形成螯合物，以固定化的金属离子为配基，如利用组氨酸咪唑基团能与镍离子特异性结合来纯化具有组氨酸标签的融合蛋白；4.拟生物素亲和层析，这种方法是利用部分分子间或者分子内部的相互作用力，以人工合成固化的一种分子或者分子的一部分为配基，来纯化目的蛋白。但是目前还鲜有多糖亲和纯化方法的建立和应用，关键的难点在于发现能够与多糖特异性亲和结合的配体。
多糖的提取方案通常采用醇提取、水提取、或者有机溶剂提取等方法分离总的多糖或者其中某一类性质的多糖，如利用Sevag液(V氯仿:V正丁醇＝4:1)加入含多糖的样品中，剧烈振摇5min后4000rpm离心10min，利用蛋白质与氯仿和正丁醇相互作用形成凝胶不溶物，而多糖溶解在水相，多次反复操作，以达到去除蛋白等杂质，但此种方法所获得的多糖为粗品，含有多种多糖组分，后续的研 究、开发和应用等方面的质量不可控，多糖种类不可控，很难区分不同多糖间的性质、作用类型和作用方式，如肝素中的其他多糖的残存严重阻碍了其临床应用。为了从粗多糖中纯化出某一种单体，比较成熟的方法是利用分子筛层析柱，如DEAE-纤维素-52层析柱、Sephadex G-100层析柱和AB树脂等，这种方法已经广发应用多糖的纯化，如板蓝根多糖纯化、肝素纯化、螺旋藻多糖纯化等，这种方法在没有更好的方法出现之前具有广泛的优越性，但其缺陷在于纯度还是较低，回收率低，为了提高纯度，往往要牺牲超过50％的回收率，而对于分子大小和结构相近的物质更是无计可施。
发明内容
本发明利用细胞表面多糖被配基特异性识别的原理，既滋养层细胞表面的胎盘样硫酸软骨素A(pl-CSA)能被疟原虫感染红细胞表面抗原VAR2CSA特异性的识别结合，以重组合成的VAR2CSA(rVAR2)，既pl-CSA最小结合部分为配基，通过与NHS活化的琼脂糖偶联制备亲和层析柱材，建立pl-CSA亲和层析纯化方法，这一方法能够克服多糖纯化的困难(回收率低、纯度低)，包括分子大小和结构相近的其他多糖不被rVAR2吸附，很容易被洗涤去除，获得高纯度的pl-CSA；而在洗涤液条件，pl-CSA被rVAR2配基牢固结合，洗脱液条件容易释放，从而获得了较高的回收率。
本发明一个方面提供了一种胎盘样硫酸软骨素A或其衍生物的纯化方法，所述纯化方法为以亲和层析的方法对胎盘样硫酸软骨素A或其衍生物进行层析纯化，其中亲和层析柱材为重组疟原虫感染红细胞表面抗原蛋白与亲和层析基质的偶联物。
在本发明的实施方案中，层析纯化的方法为将胎盘样硫酸软骨素A或其衍生物的粗品上样于亲和层析柱上，并以洗涤液进行洗涤至没有杂质流出，再以洗脱液进行洗脱并收集胎盘样硫酸软骨素A或其衍生物的纯品。
在本发明的实施方案中，胎盘样硫酸软骨素A或其衍生物的粗品上样与层析柱材体积比为1：0.5-2，优选为1：0.8-1.2，更优选为1：1。
在本发明的实施方案中，洗涤液的洗脱体积为3倍以上柱体积，更优选为5倍以上柱体积，更优选为5-10倍柱体积。
在本发明的实施方案中，洗脱液的洗脱体积为5倍以上柱体积，更优选为8 倍以上柱体积，更优选为10倍柱体积。
在本发明的实施方案中，亲和层析所用的洗涤剂pH值7-9，更优选择pH值7-8，更优选择pH值7.2，洗脱剂pH值为2-4，更优选择pH值2.2-3.6，更优选择pH值3.0。
在本发明一个具体的实施方案中，所述洗脱剂中包含保护剂，优选地，所述保护剂为甘氨酸、维生素C、维生素E、亚硫酸钠、亚硫酸氢钠、焦亚硫酸钠、硫代硫酸钠、甲硫氨酸、硫脲、乙二胺四醋酸二钠、枸橼酸中的一种或几种的组合物。
在本发明一个具体的实施方案中，所述洗涤剂中包含盐溶液。
在本发明的一个具体的实施方案中，所述洗涤剂为0.1M NaCl，0.02M Na2HPO4，选择pH值为7.2。
在本发明的一个具体的实施方案中，所述洗脱剂为0.1M甘氨酸，pH值为3。
在本发明的实施方案中，当以洗脱液进行洗脱后，检测流出物的pH值，当pH值为6.8时开始收集胎盘样硫酸软骨素A纯品，以紫外监测仪确定收集峰值。
在本发明中，重组疟原虫感染红细胞表面抗原蛋白与亲和层析基质的偶联物中所述重组疟原虫感染红细胞表面抗原蛋白的基因信息为ID：GU249598，编码包含ID1-ID2在内的一段富含半胱氨酸的区域(1273-3897)，所述亲和层析载体选自琼脂糖、纤维素、交联葡聚糖、聚丙烯酰胺、多孔玻璃珠中的一种或多种；重组疟原虫感染红细胞表面抗原蛋白与亲和层析基质的偶联物中，所述的重组疟原虫感染红细胞表面抗原蛋白为疟原虫感染红细胞表面抗原VAR2CSA与pl-CSA最小结合部分重组合成的重组疟原虫感染红细胞表面抗原蛋白，重组疟原虫感染红细胞表面抗原蛋白与亲和层析基质通过化学键进行偶联。所述重组疟原虫感染红细胞表面抗原蛋白的序列如SEQ ID No.2所示。
SEQ ID No.1如下所示
Figure PCTCN2017116624-appb-000001
Figure PCTCN2017116624-appb-000002
Figure PCTCN2017116624-appb-000003
Figure PCTCN2017116624-appb-000004
在本发明中，重组疟原虫感染红细胞表面抗原蛋白与亲和层析基质的偶联物的制备方法为将重组疟原虫感染红细胞表面抗原蛋白与亲和层析基质通过缩合交联剂进行偶和，所述的交联剂选自DCC、DIC、EDC、NHS、Hobt中的一种或多种偶联。
本发明另一个方面提供了一种胎盘样硫酸软骨素A或其衍生物的制备方法，其包括如下步骤：
1)制备胎盘样硫酸软骨素A或其衍生物的粗品；
2)纯化胎盘样硫酸软骨素A或其衍生物的粗品，得到胎盘样硫酸软骨素A或其衍生物的纯品。
其中步骤2)中的纯化方法采用本发明上述的胎盘样硫酸软骨素A或其衍生物的粗品纯化方法。
在本发明的技术方案中，制备胎盘样硫酸软骨素A粗品的方法为：
1-1)将猪胎盘破碎或者将连续培养的滋养层细胞半机械分离，得到组织细胞浆液；
1-2)将组织细胞浆液以裂解液进行裂解，得到裂解的组织细胞浆液；
1-3)将裂解的组织细胞浆液灭酶后除去蛋白，得到胎盘样硫酸软骨素A或其衍生物的粗品。
在本发明中，所述重组疟原虫感染红细胞表面抗原蛋白的制备方法为：
1)基因合成配基编码基因片段，既通过基因合成手段合成2760bp的基因片段，以ID：GU249598为参照，编码包含ID1-ID2在内的一段富含半胱氨酸的区域(1273-3897，即SEQ ID No.2)的核苷酸序列即SEQ ID No.1，并在其末端设置6×His标签的编码序列；
2)配基rVAR2的重组表达，既将基因片段通过5端BamH I、3端Sal I双酶切位点连接pET28a(+)载体，转化大肠杆菌BL21感受态细胞，通过PCR和测序验证阳性克隆；将阳性克隆接种卡那霉素LB液体培养基，37℃摇床220rpm培养16个小时后，加入0.5mM的IPTG进行诱导，4～6小时后收集菌体，超声波破碎收集重组蛋白混合液；
3)配基rVAR2的分离纯化，既通过Ni2+亲和层析纯化方法，获得rVAR2纯品。
本发明另一个方面提供了一种用于纯化胎盘样硫酸软骨素A或其衍生物的材料，其为重组疟原虫感染红细胞表面抗原蛋白。
本发明另一个方面提供了一种用于纯化胎盘样硫酸软骨素A或其衍生物的层析柱材，其为重组疟原虫感染红细胞表面抗原蛋白与亲和层析载体的偶联物。
本发明另一个方面提供了重组疟原虫感染红细胞表面抗原蛋白在制备纯化胎盘样硫酸软骨素A或其衍生物的层析柱材中的用途。
在本发明中，所述胎盘样硫酸软骨素A衍生物选自具有硫酸软骨素A的蛋白、硫酸软骨素A蛋白聚糖、被硫酸软骨素A修饰的胎盘源外泌体中一种或多种。由于其衍生物中具有胎盘样硫酸软骨素A结构，因此可以采用本发明的特异性亲和层析柱材对其进行纯化。
本发明另一个方面提供了重组疟原虫感染红细胞表面抗原蛋白在纯化胎盘样硫酸软骨素A或其衍生物中的用途。
本发明再一个方面提供了一种用于胎盘样硫酸软骨素A的亲和层析纯化系统，其从上游至下游依次包括进样装置、电极三通、进样恒流泵、亲和层析柱、排液恒流泵、电极二通、收集装置和紫外检测装置。
在本发明的亲和层析纯化系统中，进样装置包含3个进样容器，其通过三条通道分别与电极三通相连，电极三通为液位感应三通。3个进样容器分别用于乘放pl-CSA粗品、洗涤液和洗脱液。
在本发明的亲和层析纯化系统中，进样恒流泵与排液恒流泵的流速一致。
在本发明的亲和层析纯化系统中，亲和层析柱中设置有本发明所述的用于纯化胎盘样硫酸软骨素A或其衍生物的层析柱材。
在本发明的亲和层析纯化系统中，电极二通为酸碱度感应二通，收集装置包含2个收集容，其通过两条通道分别与电极二通相连，2个收集容器分别用于乘放废液和产物。
在本发明亲和层析纯化系统中，用于检测流出物的紫外检测装置与电极三通和电极二通分别连接，紫外监控仪通过检测流动相的光吸收值的改变和pH值的改变控制电极三通和电极二通，从而开启或关闭进样阀门和收集器阀门。
有益效果
本发明利用细胞表面多糖被配体特异性识别的原理，既滋养层细胞表面的胎盘样硫酸软骨素A(pl-CSA)能被疟原虫感染红细胞表面抗原VAR2CSA特异性的识别结合，以重组合成的VAR2CSA与pl-CSA最小结合部分(rVAR2)为配基，通过与NHS活化的琼脂糖偶联制备亲和层析柱材，建立pl-CSA亲和层析纯化方法，这一方法能够克服多糖纯化的困难(回收率低、纯度低)，包括分子大小和结构相近的其他多糖不被rVAR2吸附，很容易被洗涤去除，获得高纯度的pl-CSA；而洗涤液条件pl-CSA被rVAR2配基牢固结合，洗脱液条件容易释放，获得了较高的回收率。
附图说明
图1.本发明的技术路线图。
图2.本发明的工艺流程图。
图3.pET28a(+)-rVAR2原核表达。
A：原核表达质粒；B：原核表达质粒双酶切鉴定(1：BamHI单酶切；2：SalI单酶切；3：BamHI、SalI双酶切；M：DNA marker DL15000)；C：原核表达菌PCR鉴定(1-16：不同菌液PCR鉴定；M：DNA marker DL2000)。
图4.rVAR2的制备和纯化。
A：rVAR2蛋白在不同感受态中表达(1：TransBL21；2：BL21(DE3)；3：BL21；BL21(DE3)阴性菌)；B：重组菌抗6×His抗体WB验证(1：TransBL21；2：BL21(DE3)；3：BL21；BL21(DE3)阴性菌)；C：纯化过程中各组分(1：rVAR2粗样品；2：8M尿素裂解过滤后的样品；3：洗涤液样品；4：纯化后的rVAR2；5：BL21(DE3)阴性菌液；M：protein plus 250kD)D：纯化过程中各组分抗6×His抗体WB验证(1：rVAR2粗样品；2：8M尿素裂解过滤后的样品；3：洗涤液样品；4：纯化后的rVAR2；5：BL21(DE3)阴性菌液；M：protein plus 250kD)。
图5.Pl-CSA得纯化过程产物。
A：从猪胎盘中分离纯化pl-CSA的过程产物(1和2：裂解、酶解产物；3和4：去蛋白、0.45μm滤膜过滤后的产物；5.过层析系统后的纯pl-CSA)；B：从细胞中分离纯化pl-CSA的过程产物(1：酶解、去蛋白、0.45μm滤膜过滤后的产物；2：过层析系统后的纯pl-CSA)。
具体实施方式
实施例1制备并纯化胎盘样硫酸软骨素A
包括如下实验步骤：
(1)猪胎盘破碎(4℃破碎仪4M破碎60sec)或者连续培养的滋养层细胞系半机械分离(0.05％的胰酶加离心力甩：胰酶室温作用3min，离心力560g作用3min)，得到组织细胞浆液；
(2)将步骤(1)获得的组织细胞浆液与裂解液混合(V细胞浆液:V裂解液＝1:10)，裂解液中含RNase、DNase I、胰酶和protease K(各酶液终浓度为1U/ml或1μg/ml)，pH值7.0～9.0，优选条件是pH值8.0，超声波破碎仪50％的功率冰浴条件超声2sec、间隔2sec、时长30sec/ml，得到裂解的组织细胞浆液；
(3)将步骤(2)获得的裂解的组织细胞浆液4℃放置12～16h，然后85℃水浴2min灭活裂解液中的酶；加入1/4体积的Sevag液(V氯仿:V正丁醇＝4:1)，剧烈振摇5min后4100g离心10min，取上清；0.45μm的滤膜过滤既得到pl-CSA粗品，苯酚硫酸法检测多糖含量，一周内置于4℃保存，长期置于-20℃或-80℃。
(4)对疟原虫感染红细胞表面抗原VAR2CSA基因序列(FCR3虫株，GenBank accession no.GU249598)进行分析，合成与pl-CSA亲和能力较强肽段的基因片段SEQ ID No.1，以ID：GU249598为参照，长度2760bp，其中包含有6×His标签的编码序列，编码包含ID1-ID2在内的一段富含半胱氨酸的区域(1273-3897)蛋白，其编码序列如SEQ ID No.1所示。
SEQ ID No.1如下所示
Figure PCTCN2017116624-appb-000005
Figure PCTCN2017116624-appb-000006
Figure PCTCN2017116624-appb-000007
并在其5’端加入BamH I酶切位点，3’端加入Sal I酶切位点；将基因片段SEQ ID No.1与pET28a(+)原核表达载体相连，转化大肠杆菌BL21感受态细胞，进行原核表达，利用rVAR2末端6×His标签进行Ni2+亲和层析，获得高纯度的rVAR2(684.1μg/mL)。
(5)将步骤(4)中获得的高纯度的rVAR2与NHS活化琼脂糖(浓度4％)偶联：取琼脂糖溶于100mM pH5.5的MES溶液中，加入EDC，轻轻混匀，再加入NHS，轻轻混匀后室温避光反应2h；随后加入以1mg/g琼脂糖的用量加入rVAR2，轻轻充分混匀，室温避光反应24h，装柱，用pH值7.2的PBS洗出反应液，收集流出液，并进行浓缩，以SDS-PAGE检测脱落的rVAR2，4℃储存成功偶联的琼脂糖凝胶既为pl-CSA的rVAR2亲和层析柱；连接储液瓶、管道、电极三通、恒流泵、层析柱、管道、恒流泵、电极二通、样品或废液收集瓶，完成亲和层析纯化系统的组建。
(6)将步骤(3)中获得的pl-CSA粗品以1ml/(1ml柱材)经电极三通过恒流泵进入层析柱，当pl-CSA粗品液面低于第一个柱材体积液面时电极三通关闭，静置30min，使pl-CSA粗品与柱材上的亲和配基充分结合；电极三通开启洗涤液(0.1M NaCl，0.02M Na2HPO4，pH值7.2)，以10倍柱材体积洗涤液去除杂质，液面低于第一个10倍柱材体积液面，电极三通关闭；电极三通开启洗脱液(0.1M Glycine甘氨酸，pH值3.0)，以5倍柱材体积洗脱液洗脱pl-CSA，电极二通在pH值≥6.8时收集废液，在pH值＜6.8时开始，并且有紫外检测仪显示有紫外吸收时， 收集pl-CSA纯样品，第一个纯化流程完成；进出口恒流泵流速一致，电极三通为液位感应三通，电极二通为酸碱度感应二通，紫外监控仪通过检测流动相的光吸收值的改变和pH值的改变控制电极三通和电极二通，从而开启或关闭进样阀门和收集器阀门；收集高纯度pl-CSA，以苯酚硫酸法检测pl-CSA浓度。10倍柱材体积的洗涤缓冲液清洗柱材，完成纯化后将柱材置于含0.01％叠氮钠的洗涤缓冲液中，4℃储存。

Claims (10)

  1. 一种胎盘样硫酸软骨素A或其衍生物的纯化方法，所述纯化方法为以亲和层析的方法对胎盘样硫酸软骨素A或其衍生物进行层析纯化，其中亲和层析柱材为重组疟原虫感染红细胞表面抗原蛋白与亲和层析基质的偶联物，所述层析纯化的方法为将胎盘样硫酸软骨素A或其衍生物粗品上样于亲和层析柱上，并以洗涤液进行洗涤至没有杂质流出，再以洗脱液进行洗脱并收集胎盘样硫酸软骨素A纯品或其衍生物纯品。
  2. 根据权利要求1所述的纯化方法，重组疟原虫感染红细胞表面抗原蛋白与亲和层析基质的偶联物中，所述的重组疟原虫感染红细胞表面抗原蛋白为疟原虫感染红细胞表面抗原VAR2CSA与pl-CSA最小结合部分重组合成的重组疟原虫感染红细胞表面抗原蛋白，且重组疟原虫感染红细胞表面抗原蛋白末端链接His标签，此重组蛋白与亲和层析基质通过化学键进行偶联；优选地，所述重组疟原虫感染红细胞表面抗原蛋白的序列如SEQ ID No.2所示。
  3. 根据权利要求1-2任一项所述的纯化方法，胎盘样硫酸软骨素A或其衍生物粗品上样与层析柱材体积比为1：0.5-2，优选为1：0.8-1.2，更优选为1：1；
    亲和层析所用的洗涤剂pH值7-9，更优选为pH值7-8，更优选为pH值7.2；
    洗脱剂pH值为2-4，更优选为pH值2.2-3.6，更优选为pH值3.0。
  4. 根据权利要求1-2任一项所述的纯化方法，所述洗脱剂中包含保护剂，优选地，所述保护剂为甘氨酸、维生素C、维生素E、亚硫酸钠、亚硫酸氢钠、焦亚硫酸钠、硫代硫酸钠、甲硫氨酸、硫脲、乙二胺四醋酸二钠、枸橼酸中的一种或几种的组合物。
  5. 根据权利要求1-2任一项所述的纯化方法，当以洗脱液进行洗脱后，检测流出物的pH值，当pH＜6.8且紫外检测仪有紫外吸收时开始收集胎盘样硫酸软骨素A纯品或其衍生物纯品。
  6. 根据权利要求1-2任一项所述的纯化方法，洗涤液的洗脱体积为3倍以 上柱体积，洗脱液的洗脱体积为5倍以上柱体积；更优选洗涤液的洗脱体积为5倍以上柱体积，洗脱液的洗脱体积为8倍以上柱体积；最优选洗涤液的洗脱体积为5-10倍柱体积，洗脱液的洗脱体积为10倍柱体积。
  7. 一种纯化的胎盘样硫酸软骨素A或其衍生物，纯化的胎盘样硫酸软骨素A或其衍生物通过权利要求1-6任一项所示的纯化方法制备而得。
  8. 一种用于纯化胎盘样硫酸软骨素A或其衍生物的层析柱材，其为重组疟原虫感染红细胞表面抗原蛋白与亲和层析载体的偶联物，所述的重组疟原虫感染红细胞表面抗原蛋白为疟原虫感染红细胞表面抗原VAR2CSA与pl-CSA最小结合部分重组合成的重组疟原虫感染红细胞表面抗原蛋白，且重组疟原虫感染红细胞表面抗原蛋白与亲和层析基质通过化学键进行偶联；优选地，所述重组疟原虫感染红细胞表面抗原蛋白的序列如SEQ ID No.2所示；更优选地，所述亲和层析载体选自琼脂糖、纤维素、交联葡聚糖、聚丙烯酰胺、多孔玻璃珠中的一种或多种。
  9. 如权利要求8所述的重组疟原虫感染红细胞表面抗原蛋白与亲和层析载体的偶联物作为亲和层析纯化柱材中的用途，优选地，作为用于纯化胎盘样硫酸软骨素A或其衍生物的亲和层析纯化柱材中的用途，更优选地，所述胎盘样硫酸软骨素A衍生物选自具有硫酸软骨素A的蛋白、硫酸软骨素A蛋白聚糖、被硫酸软骨素A修饰的胎盘源外泌体中一种或多种。
  10. 一种用于胎盘样硫酸软骨素A或其衍生物的亲和层析纯化装置，其以权利要求8所述的重组疟原虫感染红细胞表面抗原蛋白与亲和层析载体的偶联物作为纯化胎盘样硫酸软骨素A或其衍生物的层析柱材，优选地，该装置从上游至下游依次包括进样装置、电极三通、进样恒流泵、亲和层析柱、排液恒流泵、电极二通、收集装置和紫外检测装置，其中，进样装置包含3个进样容器，其通过三条通道分别与电极三通相连，电极三通为液位感应三通；电极二通为酸碱度感应二通，收集装置包含2个收集容，其通过两条通道分别与电极二通相连；用于检测流出物的紫外检测装置与电极三通和电极二通分别连接。
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