WO2019075892A1

WO2019075892A1 - Method for purifying placenta-like chondroitin sulfate a or derivative thereof by affinity chromatography

Info

Publication number: WO2019075892A1
Application number: PCT/CN2017/116624
Authority: WO
Inventors: 张居作; 范秀军; 张键; 程国钢; 陈指龙; 张保珍; 肖仲琳; 韩金雨
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2017-10-17
Filing date: 2017-12-15
Publication date: 2019-04-25
Also published as: CN108570118A; CN108570118B

Abstract

Disclosed is a method for purifying a placenta-like chondroitin sulfate A or a derivative thereof. The purification method performs the chromatographic purification of the placenta-like chondroitin sulfate A or derivative thereof by affinity chromatography, wherein the material of the affinity chromatographic column is a conjugate of a recombinant plasmodium-infected erythrocyte surface antigen protein with an affinity chromatographic matrix. The chromatographic purification method comprises loading crude placenta-like chondroitin sulfate A or derivative thereof onto the affinity chromatographic column, washing with a washing liquid until no impurities flow out, and then eluting with the eluent and collecting the purified placenta-like chondroitin sulfate A or derivative thereof. The purification method can overcome the difficulty in polysaccharide purification, making other polysaccharides with a similar molecular size and structure not be adsorbed by rVAR2 and easily removed by washing so as to obtain the highly purified pl-CSA; and making the washing condition pl-CSA firmly bonded by the rVAR2 ligand and the eluent condition easily released to obtain a higher recovery rate.

Description

Affinity chromatography purification method for placenta-like chondroitin sulfate A or its derivative

Technical field

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.

Background technique

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. It has been widely used in proteins, peptides, enzyme preparations, antigens and antibodies, hormones, nucleic acids and the like. The choice of ligands immobilized in affinity chromatography columns is a key factor in affinity purification. According to the interaction system between ligands and biomacromolecules, it can be divided into four different types, including: 1. Biological pro And chromatography, which uses a pair of interacting substances to purify one of the substances. Typical counterparts include enzyme-substrate, enzyme-inhibitor, hormone-receptor, etc. 2. 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. a 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. However, 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 polysaccharide extraction scheme usually uses alcohol extraction, water extraction, or organic solvent extraction to separate the total polysaccharide or a polysaccharide of a certain nature, such as using Sevag solution (V _chloroform : V _n-butanol = 4:1). In the sample of polysaccharide, it was shaken vigorously for 5 min, centrifuged at 4000 rpm for 10 min, and 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 quality of subsequent research, development and application is uncontrollable, the polysaccharide type is uncontrollable, and it is difficult to distinguish the nature, type of action and mode of action between different polysaccharides. The residual of other polysaccharides such as heparin severely hampers its clinical application. In order to purify a monomer from a crude polysaccharide, a more mature method is to use a molecular sieve column such as DEAE-cellulose-52 column, Sephadex G-100 column and AB resin. The purification of polysaccharides, such as the purification of Radix isatidis, the purification of heparin, the purification of spirulina polysaccharides, etc., has been widely used. This method has broad advantages before the emergence of a better method, but its drawback is that the purity is still low and the recovery rate is low. In order to improve the purity, it is often necessary to sacrifice more than 50% recovery rate, and for substances with similar molecular size and structure, there is nothing to do.

Summary of the invention

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, 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 of polysaccharides (low recovery, low purity), including other polysaccharides with similar molecular size and structure, are not adsorbed by rVAR2, and are easily removed by washing to obtain high-purity pl-CSA; while in washing conditions, pl-CSA It is firmly bound by the rVAR2 ligand, and the eluent conditions are easily released, resulting in higher recovery.

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.

In an embodiment of the present invention, 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.

In an embodiment of the present invention, 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. .

In an embodiment of the invention, 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.

In an embodiment of the invention, 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.

In an embodiment of the present invention, 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.

In a specific embodiment of the present invention, the eluent comprises a protective agent. Preferably, the protective agent is glycine, vitamin C, vitamin E, sodium sulfite, sodium hydrogen sulfite, sodium metabisulfite, sodium thiosulfate. A composition of one or more of methionine, thiourea, disodium edetate, and citric acid.

In a particular embodiment of the invention, the detergent comprises a salt solution.

In a particular embodiment of the invention, the detergent is 0.1 M NaCl, 0.02 M Na ₂ HPO ₄ , with a pH of 7.2.

In a particular embodiment of the invention, the eluent is 0.1 M glycine and has a pH of 3.

In an embodiment of the invention, 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.

In the present invention, 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

In the present invention, 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:

1) preparing a crude product of placenta-like chondroitin sulfate A or a derivative thereof;

2) Purification of a crude placenta-like chondroitin sulfate A or a derivative thereof to obtain a pure product of placenta-like chondroitin sulfate A or a derivative thereof.

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.

In the technical solution of the present invention, the method for preparing the crude placenta-like chondroitin sulfate A is:

1-1) breaking the pig placenta or semi-mechanically separating the continuously cultured trophoblast cells to obtain a tissue cell slurry;

1-2) lysing the tissue cell slurry with a lysate to obtain a lysed tissue cell slurry;

1-3) 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.

In the present invention, the preparation method of the recombinant Plasmodium infection erythrocyte surface antigen protein is:

1) 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;

2) Recombinant expression of ligand rVAR2, the gene fragment was ligated into pET28a(+) vector through 5-terminal BamH I, 3 terminal Sal I double-cleavage site, transformed into E. coli BL21 competent cells, and positive clones were verified by PCR and sequencing. The positive clones were inoculated into kanamycin LB liquid medium, cultured at 37 ° C for 220 hours at 220 rpm, and then induced by adding 0.5 mM IPTG. After 4 to 6 hours, the cells were collected, and the recombinant protein mixture was collected by ultrasonic disruption;

3) Separation and purification of ligand rVAR2, and obtaining pure rVAR2 by Ni ²⁺ affinity chromatography purification method.

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.

In the present invention, 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. Kind. 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.

According to still another aspect of the present invention, an affinity chromatography purification system for placenta-like chondroitin sulfate A is provided, which includes a sample introduction device, an electrode tee, a constant current pump, and an affinity chromatography from upstream to downstream. Column, drain constant current pump, electrode two-way, collecting device and ultraviolet detecting device.

In the affinity chromatography purification system of the present invention, 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.

In the affinity chromatography purification system of the present invention, the flow rate of the injection constant current pump and the discharge constant current pump are identical.

In the affinity chromatography purification system of the present invention, 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.

In the affinity chromatography purification system of the present invention, the electrode two-way is a pH-sensing two-pass, and 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.

In the affinity chromatography purification system of the present invention, 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.

Beneficial effect

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 (low recovery, low purity), including other polysaccharides with similar molecular size and structure, 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.

DRAWINGS

Figure 1. Technical roadmap of the present invention.

Figure 2. Process flow diagram of the present invention.

Figure 3. Prokaryotic expression of pET28a(+)-rVAR2.

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).

Figure 4. Preparation and purification of rVAR2.

A: rVAR2 protein is expressed in different competent states (1: TransBL21; 2: BL21 (DE3); 3: BL21; BL21 (DE3) negative bacteria); B: recombinant bacteria against 6 × His antibody WB verification (1: TransBL21; 2: BL21 (DE3); 3: BL21; BL21 (DE3) negative bacteria); C: each component in the purification process (1: rVAR2 crude sample; 2: 8 M urea cracked filtered sample; 3: washing liquid sample; 4: purified rVAR2; 5: BL21 (DE3) negative bacterial solution; M: protein plus 250kD) D: anti-6×His antibody WB verification of each component during purification (1: rVAR2 crude sample; 2:8M urea lysis Filtered sample; 3: wash solution sample; 4: purified rVAR2; 5: BL21 (DE3) negative bacterial solution; M: protein plus 250 kD).

Figure 5. Pl-CSA obtained the purification process product.

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 μm 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 μm filter filtered product; 2: pure pl-CSA after chromatography system).

Detailed ways

Example 1 Preparation and Purification of Placenta-like Chondroitin Sulfate A

Including the following experimental steps:

(1) Porcine placenta fragmentation (4°C crusher 4M broken 60 sec) or semi-mechanical separation of continuous cultured trophoblast cell lines (0.05% trypsin plus centrifugal force 胰: trypsin room temperature for 3 min, centrifugal force 560 g for 3 min), tissue Cell serum

(2) mixing the tissue cell slurry obtained in step (1) with the lysate (V _{cell slurry} : V _lysate = 1:10), and the lysate contains RNase, DNase I, trypsin and protease K (each enzyme solution The concentration is 1U/ml or 1μg/ml), the pH value is 7.0-9.0, preferably the pH value is 8.0, and the ultrasonic disruptor 50% power ice bath condition is ultrasonic 2 sec, interval 2 sec, duration 30 sec/ml, and the lysed tissue cells are obtained. Slurry

(3) The lysed tissue cell slurry obtained in the step (2) is placed at 4 ° C for 12 to 16 hours, and then the enzyme in the lysate is inactivated by a water bath at 85 ° C for 2 minutes; a 1/4 volume of Sevag solution is added (V _chloroform : V- _{n-butyl) Alcohol} = 4:1), shaken vigorously for 5 min, centrifuged at 4100 g for 10 min, and the supernatant was taken; the filter of 0.45 μm was filtered to obtain crude pl-CSA, and the content of polysaccharide was detected by phenol sulfuric acid method, which was stored at 4 ° C for one week and was placed for a long time. -20 ° C or -80 ° C.

(4) Analysis of the VAR2CSA gene sequence (FCR3 strain, GenBank accession no. GU249598) of the erythrocyte surface antigen infected by Plasmodium, and synthesize 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 μg/mL).

(5) Coupling the high-purity rVAR2 obtained in the step (4) with the NHS-activated agarose (concentration: 4%): agarose was dissolved in 100 mM MES solution of pH 5.5, EDC was added, and the mixture was gently mixed. Add NHS, gently mix and shake at room temperature for 2 h; then add rVAR2 at a dose of 1 mg/g agarose, mix thoroughly, dilute at room temperature for 24 h, install the column, wash with PBS pH 7.2. The reaction solution was taken out, the effluent was collected, and concentrated, and the detached rVAR2 was detected by SDS-PAGE. 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.

(6) 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. When the liquid level is closed, 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 pure sample, the first purification process is completed; the constant flow rate of the inlet and outlet constant flow pump is the same, the electrode tee is the liquid level induction tee, the electrode two pass is the pH induction two-way, and the ultraviolet monitor detects the light absorption value of the mobile phase. Change and pH change control electrode tee and electrode two-way, thereby opening or closing the injection valve and collector valve; collecting Purity pl-CSA, phenol sulfuric acid assay concentration pl-CSA. The column was washed with 10 column volumes of wash buffer. After purification, the column was placed in a wash buffer containing 0.01% sodium azide and stored at 4 °C.

Claims

A method for purifying a placenta-like chondroitin sulfate A or a derivative thereof, which is a chromatographic purification method for placental-like chondroitin sulfate A or a derivative thereof by affinity chromatography, wherein the affinity chromatography column The material is a conjugate of the recombinant Plasmodium erythrocyte surface antigen protein and the affinity chromatography matrix, and the chromatographic purification method is to load the crude placenta-like chondroitin sulfate A or its derivative onto the affinity chromatography column. And washing with washing liquid until no impurities flow out, and then eluting with the eluent and collecting pure chondroitin sulfate A or a pure derivative thereof.
The purification method according to claim 1, wherein the recombinant Plasmodium infection erythrocyte surface antigen protein is conjugated with an affinity chromatography matrix, and the recombinant Plasmodium infection erythrocyte surface antigen protein is Plasmodium erythrocyte surface antigen VAR2CSA and The pl-CSA minimal binding portion recombinantly synthesized recombinant Plasmodium is infected with the erythrocyte surface antigen protein, and the recombinant Plasmodium infected erythrocyte surface antigen protein end-linked His tag, and the recombinant protein is coupled to the affinity chromatography substrate by a chemical bond; preferably, The sequence of the recombinant Plasmodium infection erythrocyte surface antigen protein is shown in SEQ ID No. 2.
The purification method according to any one of claims 1 to 2, wherein the volume ratio of the placenta-like chondroitin sulfate A or its derivative to the chromatographic column is 1:0.5-2, preferably 1:0.8-1.2. More preferably 1:1;

The pH of the detergent used in affinity chromatography is 7-9, more preferably pH 7-8, more preferably pH 7.2;

The eluent has a pH of 2-4, more preferably a pH of 2.2-3.6, more preferably a pH of 3.0.
The purification method according to any one of claims 1 to 2, wherein the eluent contains a protective agent. Preferably, the protective agent is glycine, vitamin C, vitamin E, sodium sulfite, sodium hydrogen sulfite, sodium metabisulfite. A composition of one or more of sodium thiosulfate, methionine, thiourea, disodium edetate, and citric acid.
The purification method according to any one of claims 1 to 2, wherein after elution with an eluent, the pH of the effluent is detected, and when the pH is 6.8 and the ultraviolet detector has ultraviolet absorption, collection of placenta-like sulfate cartilage is started. A pure product of A or its pure product.
The purification method according to any one of claims 1 to 2, wherein the elution volume of the washing liquid is 3 times The volume of the upper column, the elution volume of the eluent is more than 5 times the column volume; more preferably, the elution volume of the washing solution is more than 5 times the column volume, and the elution volume of the eluent is 8 times or more the column volume; most preferably washing The elution volume of the liquid was 5-10 column volumes, and the elution volume of the eluate was 10 column volumes.
A purified placenta-like chondroitin sulfate A or a derivative thereof, which is prepared by the purification method shown in any one of claims 1 to 6 by using purified placenta-like chondroitin sulfate A or a derivative thereof.
A chromatography column for purifying placenta-like chondroitin sulfate A or a derivative thereof, which is a conjugate of a recombinant Plasmodium infection erythrocyte surface antigen protein and an affinity chromatography vector, the recombinant Plasmodium infected red blood cells The surface antigen protein is a recombinant Plasmodium erythrocyte surface antigen protein recombinantly synthesized by the minimally binding part of Plasmodium erythrocyte surface antigen VAR2CSA and pl-CSA, and the recombinant Plasmodium erythrocyte surface antigen protein and the affinity chromatography matrix are coupled by chemical bonds. Preferably, the sequence of the recombinant Plasmodium infection erythrocyte surface antigen protein is as shown in SEQ ID No. 2; more preferably, the affinity chromatography carrier is selected from the group consisting of agarose, cellulose, cross-linked dextran, One or more of polyacrylamide, porous glass beads.
The use of the conjugate of a recombinant Plasmodium-infected erythrocyte surface antigen protein and an affinity chromatography vector according to claim 8 as an affinity chromatography purification column, preferably as a purified placenta-like chondroitin sulfate A Or the use of an affinity chromatography purification column of a derivative thereof, more preferably, 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 sulfuric acid. One or more of chondroitin A modified placental derived exosomes.
An affinity chromatography purification device for placenta-like chondroitin sulfate A or a derivative thereof, which comprises the conjugate of the recombinant Plasmodium-infected erythrocyte surface antigen protein and the affinity chromatography carrier according to claim 8 a chromatography column of placenta-like chondroitin sulfate A or a derivative thereof, preferably, the device includes a sample introduction device, an electrode tee, an injection constant current pump, an affinity chromatography column, and a liquid discharge from the upstream to the downstream. a flow pump, an electrode two-way, a collecting device and an ultraviolet detecting device, wherein the sampling device comprises three injection containers, which are respectively connected to the electrode tee through three channels, the electrode tee is a liquid level sensing tee; the electrode two-way For the pH sensing two-way, the collecting device comprises two collecting capacities, which are respectively connected to the electrode two-pass through two channels; the ultraviolet detecting device for detecting the effluent is connected with the electrode tee and the electrode two-pass respectively.