CN2727741Y - A chromatography column for separation and extraction - Google Patents

Abstract

The utility model discloses a separating and extracting chromatographic column which is composed of a chromatographic column and an adenosine diphosphate resin which is packed in the chromatographic column. The two portions are assembled. The chromatographic column is a hollow tubular object. The interior of the bottom at one end of the chromatographic column is provided with a sieve plate that allows the liquor to get through but prevents the resin particles from losing. The chromatographic column is filled with the functional filler adenosine diphosphate resin inside. The utility model can abstract the protein in the heat shock protein family and the heat shock protein-the polypeptide complex formed by the polypeptide molecular groupings in non-covalent bonding to the HSP, from the biological tissue, the cell homogenate and other hybrid protein miscible liquids rapidly, easily and effectively.

Description

A kind of separation and Extraction chromatographic column

Technical field

The utility model relates to the equipment that biomedicine field is used, and particularly relates to a kind of separation and Extraction chromatographic column.

Background technology

With single albumen purifying from the solution that tissue extract or albumen mix a series of very complicated and loaded down with trivial details separation and Extraction processes of needs experience of coming out, expend time in and grow and ultimate yield is very low.Along with the development of biochemical technology, a kind of easy and simple to handle, method---affinity chromatography of separating the high separation and purification biomacromolecule of specificity appears.Its principle is to utilize the characteristic of specific adsorption between the biomacromolecule or combination to reach the purpose of fast purifying target protein.

Summary of the invention

The objective of the invention is to provide for the personnel that are engaged in biomedical research and preparation biopreparate a kind of can be quickly and easily from foreign protein solution separation and purification go out heat shock protein (HSP), particularly be purified into the heat-shock protein-polypeptide complex chromatographic column of (Heat shock protein peptidecomplexes is called for short HSPPC).

The very important biological characteristics of HSP is to catch polypeptide and the albumen that morphs in body tissue and the cell, or the entrained exogenous molecules group of invasion pathogen forms HSPPC as antigen, HSPPC gives the T lymphocyte with the polypeptide antigen molecular group submission that they capture, the immune system recognition of excitating organism and the oneself protein of removing variation and the molecule and the cell of external source.

Because the N of HSP end has the function of ATP enzyme, so its decapacitation and ATP (Adenosinetriphosphate, abbreviation ATP, Chinese title atriphos) outside the effect, can also be specifically with solid-phase resin on the ADP (Adenosine 3 ' of coupling, 5 ' the diphosphate ADP that abridges, Chinese claims adenosine diphosphate (ADP)) the phase affinity.HSPPC as with the ATP effect, the peptide molecule group among the HSPPC will be from HSPPC be dissociated out, the antigen presentation function of HSP will be lost.Test discovery HSPPC combines then with ADP and does not dissociate.Therefore according to these characteristics and actual demand, employing is coupled to ADP on the solid phase Agarose resin, when the ADP-Agarose resin of the foreign protein flow of solution that contains HSP or HSPPC in chromatographic column, affinity interaction takes place in the ADP on HSP in the foreign protein solution or HSPPC and the solid-phase resin, therefore HSP and HSPPC are adsorbed and are trapped on the solid-phase resin, and other foreign protein that affinity interaction does not take place with ADP then flows through resin and can not be attracted on the resin.The ADP-Agarose chromatographic column can not only be quickly and easily from foreign protein solution separation and purification go out HSP, and can also separation and purification go out HSPPC.

What the present invention relates to is preparation a kind of " separation and Extraction chromatographic column ", its feature is made up of chromatographic column and ADP-Agarose resin two parts, wherein, chromatographic column is to be made by the built-in sieve plate 2 of the tube 1 of hollow type and lower end, and the purpose of sieve plate is to allow flow of solution to cross but prevent that resin runs off.Sieve plate also can be reached other material substitution of identical function by nylon mesh screen, ceramic wafer, glass fibre etc.The lower end of chromatographic column is flow of solution outlet 3, but the socket flexible rubber hose.With Adenosine3 ', 5 '-diphosphate-Agasoe resin, abbreviation ADP-Agarose resin 4 is packed into promptly forms the separation and Extraction chromatographic column in the chromatographic column.

What of the size of chromatographic column and potting resin can prepare commodity-type ADP-Agarose separation and Extraction chromatographic column in enormous quantities, all size according to.The application that this separation extract layer is analysed post can alleviate the labour intensity of biology and purifying HSP of medical laboratory or HSPPC significantly and shorten purge process.

Heat shock protein is the intracellular family protein that is present in that a class homology is high, sequence is very conservative, can be divided into a plurality of members according to its molecular size and architectural characteristic, mainly be present in the endochylema as albumen such as HSP-27, HSP-40, HSP-60, HSP-70, HSP-90, HSP-110, gp96 albumen mainly is present in the endoplasmic.They are in low-level expression status at ordinary times, in case the expression of HSP just can be increased sharply when sudden change of physiological, pathologic or environment appears in cell, so heat shock protein be otherwise known as " stress protein ".Growth, differentiation, genetic transcription and the self-protection of assembling, transmembrane transport and the antigen presentation pair cell of HSP by participating in combination of proteins, folding, subunit play a significant role.Another key property of HSP is that it has in conjunction with the foreign pathogens of invasion internal body and the ability of body endogenous cell variant protein matter and polypeptide.They combine with foreign protein and polypeptide, endogenous variant protein and peptide molecule group, form that HSPPC participates in antigen presentation and its HSP itself does not produce antigenicity, so HSP be otherwise known as " molecular chaperones ".Prepare and a kind ofly can make biology laboratory and the clinical medicine laboratory is quick, convenient and separation and purification efficiently goes out HSP or HSPPC chromatographic column has urgent demand and crucial meaning in actual applications.

Application person can adjust extraction conditions and needn't arrest and be limited to the experiment condition that the present invention narrates according to breadboard condition, extraction experience separately.

Description of drawings

Fig. 1 is the utility model structural representation.

Embodiment

Embodiment 1

1. assembling chromatographic column:

(1), Adenosine 3 ', 5 ' diphosphate-Agarose resin (ADP-Agarose resin) can be ordered from Sigma company.

(2), select the chromatography void column of plastics or glass manufacturing for use.The specification of post is not limit, and commonly used have 1 * 5cm, 1.5 * 10cm, a 2 * 20cm etc., can select for use according to the actual requirements and prepared in batches, as preparing the ADP-Agarose resin chromatographic column of different capabilities such as being filled with 5ml, 10ml, 15ml.

(3), equilibrium liquid (20mM Tris-Acetate, 20mM NaCl,, 3mM MgCl ₂, 0.5mMPMSF, 15mM β-mercaptoethanol, pH7.5).

Use is rinsed well and dry chromatography void column.The ADP-Agarose resin that equilibrium liquid was fully soaked is filled into resin in the chromatographic column with equilibrium liquid with suction pipe.Make and resin natural subsidence in void column, must not leave bubble in the post bed until required volume.

The packing of post: the chromatographic column two ends that can is finished seal to prevent resin drying and pollution with sealing film.The chromatographic column that sealing is good is packed into and is sealed the back in the aluminium plastic packaging bag and put into packing box in the lump together with operation instructions.

2, the preparation of tissue fluid:

Take out about 1cm on one's body from rat ³Tumor mass, remove blood stains with normal saline flushing, repair normal structure, the tumor tissues that cuts into fine grained chippings is put into-20 ℃ of refrigerator and cooled freezes and it is inserted homogenizer after 2 hours.Grinding buffer solution (the 10mM NaHCO that in homogenizer, adds an amount of precooling ₃, 0.5mMPMSM pH7.5) grinds.Tissue homogenate after the grinding is got its supernatant through refrigerated centrifuge 8000rpm 10 minutes after centrifugal and is crossed ADP-Agarose separation and Extraction chromatographic column.

The chromatography reaction: remove special suggestion, the whole operation process is recommended in 4 ℃ of environment carries out.Do balance with sealing off the finished product ADP-Agarose separation and Extraction chromatographic column that takes out the back slightly with equilibrium liquid, get rat tumor tissue homogenate supernatant after centrifugal and mix with isopyknic balance liquid phase and directly be added on the ADP-Agarose chromatographic column.Fully affine for the ADP that makes HSPPC in the homogenate supernatant and resin coupling, the post current control all entered the post bed at 3ml/ hour until the homogenate supernatant.

Washing: the equilibrium liquid flushing chromatographic column that contains 0.5M NaCl that uses 20 times of bed volumes, the post current control was at approximately 1ml/ minute, use then equilibrium liquid fully wash chromatographic column until the ultraviolet monitoring value stabilization of effluent in OD280＜0.02, reach the purpose that all foreign proteins in the post are fully removed.

Wash-out: chromatographic column is moved under the conventional room temperature, soaked in the post resin 30 minutes, then the HSPPC that separates with ADP with the eluent wash-out of 5 times of bed volumes with eluent (equilibrium liquid that contains 3mM ADP).Under ultraviolet monitoring, collect the effluent in the peak.

Separate once more: the excessive needs of HSPPC under the wash-out such as fruit volume concentrate.Use high pressure liquid chromatograph (HPLC) to separate once more, HSPPC is separated with free ADP molecule and a spot of other not removed foreign protein.Collect the effluent at peak, HSPPC place, can obtain the HSPPC of purity more than 90% through the separation of HPLC several times.

The evaluation of separator: the purity of utilizing HSPPC albumen usable highly effective liquid chromatography (HPLC) that the method for affinity chromatography is purified into or polyacrylamide gel electrophoresis (SDS-PAGE) to analyze eluted product.Available ultraviolet spectrophotometry is done the mensuration of protein content.Adopt SDS-PAGE electrophoresis and western blot test to reach the purpose of protein urine.

3, the pre-service of cell liquid:

People's tumor cell line Hella cell is scraped from collect the centrifuge tube centrifugal 10 minutes of 5000rpm/min together with nutrient solution from double dish.Abandoning supernatant is leniently broken up the cell of precipitation with equilibrium liquid and flushing and then centrifuging and taking precipitation lightly.Collect the cell precipitation about about weight in wet base 1 gram.

Precipitation cell down moves to the equilibrium liquid that plastic test tube adds the 5ml precooling again and breaks up also suspension cell, does the cell ultrasonication then.The centrifuging and taking supernatant is crossed chromatographic column.

The chromatography reaction: in 4 ℃ of environment the clasmatosis supernatant is added on the good ADP-Agarose separation and Extraction chromatographic column of balance, the flow speed control of post was at approximately 2ml/ hour.After supernatant entered post bed and resin and fully reacts, earlier with the equilibrium liquid 100ml flushing chromatographic column that contains 0.5M NaCl, and then the ultraviolet monitoring absorption value of washing until effluent with a large amount of equilibrium liquids was stabilized in OD ₂₈₀＜0.02.

Wash-out: the HSPPC that the mode wash-out that the employing competition substitutes and the ADP of resin coupling have affinity.At room temperature use eluent (equilibrium liquid that contains 3mM ADP) to soak the interior resin of chromatographic column 30 minutes, make HSPPC separate with the ADP of solid-phase resin coupling.Use the eluent wash-out of 5 times of bed volumes again, under ultraviolet monitoring, collect effluent in the peak.

Separate once more: effluent places in the bag filter in the peak that wash-out is collected, under 4 ℃ of environment to the HPLC sample buffer dialysis of precooling 3 times.Will be after the solution concentration of dialysing separate HSPPC and free compositions such as ADP, separate and collect effluent in the HSPPC peak and do the quantitative and qualitative evaluation of albumen with HPLC.

Preserve: the HSPPC albumen of separating can be placed on to preserve in-20 ℃ of refrigerators after freeze drying did not influence its biologic activity in 1 year.

The evaluation of separator: the HSPPC that separation and purification goes out can adopt methods such as SDS-PAGE gel electrophoresis, high performance liquid chromatography (HPLC) and ultraviolet spectrometry to do purity of protein and quantitative detection.Adopt Western blot (Western Blot) to do qualitative test of protein.

Claims (2)

1, a kind of separation and Extraction chromatographic column is characterized in that, it is to be combined by the chromatographic column and the adenosine diphosphate (ADP)-agarose resin two parts of filling in post.

2, separation and Extraction chromatographic column according to claim 1 is characterized in that, described chromatographic column is a kind of hollow type tube, and one bottom portion is built-in with the sieve plate that allows solution to pass through but stop resin particle to run off.

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