CN106755032A - A kind of high-cutting slope along road albumen vivoexpression and the method for isolating and purifying - Google Patents
A kind of high-cutting slope along road albumen vivoexpression and the method for isolating and purifying Download PDFInfo
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- CN106755032A CN106755032A CN201611010470.1A CN201611010470A CN106755032A CN 106755032 A CN106755032 A CN 106755032A CN 201611010470 A CN201611010470 A CN 201611010470A CN 106755032 A CN106755032 A CN 106755032A
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- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000005520 cutting process Methods 0.000 title claims abstract description 16
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000000746 purification Methods 0.000 claims abstract description 14
- 238000005227 gel permeation chromatography Methods 0.000 claims abstract description 12
- 241000588724 Escherichia coli Species 0.000 claims abstract description 11
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims abstract description 8
- 239000002299 complementary DNA Substances 0.000 claims abstract description 7
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 7
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 5
- 239000012634 fragment Substances 0.000 claims abstract description 5
- 230000002068 genetic effect Effects 0.000 claims abstract description 5
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 43
- 239000011780 sodium chloride Substances 0.000 claims description 21
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 20
- 239000012266 salt solution Substances 0.000 claims description 11
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 150000002460 imidazoles Chemical class 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 102000009438 IgE Receptors Human genes 0.000 claims description 4
- 108010073816 IgE Receptors Proteins 0.000 claims description 4
- 238000005342 ion exchange Methods 0.000 claims description 4
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 238000006073 displacement reaction Methods 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 229910001453 nickel ion Inorganic materials 0.000 claims description 3
- 238000002525 ultrasonication Methods 0.000 claims description 3
- 239000011534 wash buffer Substances 0.000 claims description 3
- 238000002086 displacement chromatography Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000012408 PCR amplification Methods 0.000 abstract description 2
- 238000002955 isolation Methods 0.000 abstract description 2
- 238000005215 recombination Methods 0.000 abstract description 2
- 230000006798 recombination Effects 0.000 abstract description 2
- 102000005962 receptors Human genes 0.000 description 11
- 108020003175 receptors Proteins 0.000 description 11
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 4
- 208000024376 chronic urticaria Diseases 0.000 description 3
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- 206010052568 Urticaria chronic Diseases 0.000 description 2
- 239000000370 acceptor Substances 0.000 description 2
- 230000002804 anti-anaphylactic effect Effects 0.000 description 2
- 230000001363 autoimmune Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
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- 230000000694 effects Effects 0.000 description 2
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- 238000005516 engineering process Methods 0.000 description 2
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- 239000013612 plasmid Substances 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- 208000020446 Cardiac disease Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000024780 Urticaria Diseases 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 229940124623 antihistamine drug Drugs 0.000 description 1
- 239000000739 antihistaminic agent Substances 0.000 description 1
- 208000029407 autoimmune urticaria Diseases 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 210000000245 forearm Anatomy 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 238000002650 immunosuppressive therapy Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
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- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
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Abstract
The present invention discloses a kind of high-cutting slope along road albumen vivoexpression and the method for isolating and purifying, and is first SEQ ID NO by cDNA sequence:By after PCR amplifications, being integrated into escherichia coli vector PET32M, the carrier that will be built into is converted to e. coli bl21 so as to obtain thalline 1 genetic fragment；Cultivated to OD during thalline is placed in into 37 DEG C, the shaking table of 200rad/min600Untill 0.6 0.8, being subsequently adding IPTG makes the concentration of IPTG in bacterium solution reach 0.2mol/L, and continues at 37 DEG C, and 16h is cultivated under the conditions of the shaking table of 200rad/min；High-cutting slope along road albumen is obtained after thalline after culture is isolated and purified through preliminary purification, affinity chromatography, ion-exchange chromatography, gel chromatography successively.Isolation and purification method of the invention realizes solubility expression by vitro recombination by Escherichia coli, greatly reduces IgE high-affinity receptors albumen into product production cost and production difficulty.
Description
Technical field
The present invention relates to biology field, more particularly to a kind of high-cutting slope along road albumen vivoexpression and point
From the method for purifying.
Background technology
Chronic urticaria is one of dept. of dermatology's common disease, complicated with the cause of disease, symptom repeatedly, the characteristics of the course of disease is long, greatly
Some patientss can not find the clear and definite cause of disease, and be considered as " idiopathic ", and about 40% course of disease can be more than 10 years, although do not threaten life
Life, but its painful and worried and to work and social life influence for bringing even is no less than cardiac disorder.Shenzhen is every
Year, number of the infected was more than 10,000 people.The antianaphylactic treatment (such as antihistamine drug) conventional for quite a few patient is tended not to
Prove effective, this kind of patient belongs to Autoimmune Chronic Urticaria, exist in serum anti-Fc ε R I α (IgE acceptors) autoantibodies or
(and) anti-IgE autoantibodies, fall ill based on autoimmune mechanism.These autoantibodies can be with mast cell and basophilla grain
The α chain combinations of the IgE acceptor Fc ε R of cell surface, and lasting inflammatory stimulus effect is played, cause complement activation, patient loose
Cell and basophilic granulocyte degranulation release histamine.Such patient is invalid after rule was using antianaphylactic treatment 3 months to use instead
Immunosuppressive therapy can usually obtain certain curative effect, but have the shortcomings that time-consuming length, patients symptomatic relief are slow, painful big.
Existing Autologous Serum Skin Test (autologous serum skin test, ASST) is referred to as in the world
Greaves is tested, and is the clinical practice detection method for screening autoimmune urticaria.Peripheral blood in patients 3ml is generally extracted, often
The lower centrifugation serum of temperature, injects this by the forearm intradermal of bleeder, meanwhile, inject offside intradermal with normal saline
As control.Wheal size is observed at injection after 30 minutes being judged.But the method is got up with one in clinical implementation
Fixed difficulty, it is difficult to obtain the cooperation of patient.
Using antigen-antibody reaction, being a kind of good new method detects internal IgE receptor proteins autoantibody quantity
Method, but the premise of the method is that have the substantial amounts of IgE receptor proteins of high-purity.
The content of the invention
The purpose of the present invention is to overcome deficiency of the prior art, there is provided a kind of high-cutting slope along road proteosome appearance
Up to and the method that isolates and purifies.
To achieve the above object, high-cutting slope along road albumen vivoexpression proposed by the present invention and the side for isolating and purifying
Method, comprises the following steps：It is SEQ ID NO by cDNA sequence:1 genetic fragment is by after PCR amplifications, being integrated into Escherichia coli
In carrier PET32M, the carrier that will be built into is converted to e. coli bl21 so as to obtain thalline；
Cultivated to OD during thalline is placed in into 37 DEG C, the shaking table of 200rad/min600Untill 0.6-0.8, IPTG is subsequently adding
The concentration of IPTG in bacterium solution is reached 0.2mol/L, and continue at 37 DEG C, 16h is cultivated under the conditions of the shaking table of 200rad/min；Will
Thalline after culture obtains high affine after being isolated and purified through preliminary purification, affinity chromatography, ion-exchange chromatography, gel chromatography successively
Power IgE receptor proteins.
Further, the preliminary purification is to collect thalline after the thalline after cultivation is centrifuged through 4000g × 20min, and
20mL Tris-HCl containing 50mM are suspended in, in the solution of 20mM NaCl；Ultrasonication thalline, is centrifuged through 18000g × 20min
After collect supernatant.
Further, the method for the affinitive layer purification albumen is by the supernatant carry nickel ion affinity chromatograph
Post, and 5 column volumes are cleaned with cleaning fluid, 2 column volumes of elution；Wherein, cleaning fluid is 50mM Tris-HCl,
The solution of 20mM NaCl, 20mM imidazoles；Eluent is the solution of 50mM Tris-HCl, 20mM NaCl, 200mM imidazoles.
Further, the method for the ion-exchange purification albumen is for low by albumen displacement buffer solution in the eluent
Salting liquid, carry ion exchange column is eluted lower prop using the method high level salt solution of gradient elution, and low salt solutions are
50mM Tris-HCl, 20mM NaCl, high level salt solution is 50mM Tris-HCl, 1M NaCl.
Further, the method for the gel chromatography albumen is that above-mentioned ion-exchange chromatography is eluted into gained albumen to hang
Carry in gel chromatography column, chromatographic column is departed to it using wash buffer, buffer solution is Tris-HCl containing 50mM, 20mM NaCl
Solution.
Further, EK enzyme digestion albumen was used before by gel chromatography albumen, digestion solution is 50mM
The solution of Tris-HCl, 20mM NaCl.
Isolation and purification method of the invention passes through vitro recombination, by Bacillus coli expression IgE high-affinity receptor albumen,
IgE high-affinity receptor albumen then is isolated and purified using the method for affinity chromatography, gel chromatography and ion-exchange chromatography, and then
Obtain pure IgE high-affinity receptors albumen.Solubility expression is realized by Escherichia coli, IgE high-affinities is greatly reduced and is received
Body protein into produce production cost and production difficulty.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Structure according to these accompanying drawings obtains other accompanying drawings.
Fig. 1 is the electrophoresis detection figure of IgE high-affinity receptor PCR primers, and wherein fl is IgE high-affinity receptor albumen
The PCR primer of cDNA, M is marker；
Fig. 2 is carrier cleavage map, and wherein fl is the plasmid enzyme restriction result for loading IgE high-affinity receptor albumen cDNA, and M is
marker；
Fig. 3 is the final purification result figure of IgE high-affinity receptor albumen, and wherein fl is IgE high-affinity receptor albumen, M
It is marker.
The realization of the object of the invention, functional characteristics and advantage will be described further referring to the drawings in conjunction with the embodiments.
Specific embodiment
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein from start to finish
Same or similar label represents same or similar element or the element with same or like function.Below with reference to attached
It is exemplary to scheme the embodiment of description, it is intended to for explaining the present invention, and be not considered as limiting the invention, based on this
Embodiment in invention, the every other reality that those of ordinary skill in the art are obtained under the premise of creative work is not made
Example is applied, the scope of protection of the invention is belonged to.
High-cutting slope along road albumen vivoexpression of the present invention and the method for isolating and purifying, comprise the following steps：
(1) thalline is built
It is SEQ ID NO by the corresponding cDNA sequence of known high-cutting slope along road albumen:1 genetic fragment is by PCR
After amplification, be integrated into escherichia coli vector PET32M, by sequence verification vector construction it is correct after, by plasmid according to alkaline lysis
Simultaneously translation table reaches e. coli bl21 for method extracting, so as to obtain the thalline of conversion.As shown in figure 1, after genetic fragment is expanded through PCR
Electrophoretic analysis, fl is the PCR primer of IgE high-affinity receptor albumen cDNA, and M is marker.
(2) cultivate
Cultivated to OD during the thalline of above-mentioned acquisition is placed in into 37 DEG C, the shaking table of 200rad/min600Untill 0.6-0.8, so
Add IPTG to be induced afterwards, the concentration of IPTG in bacterium solution is reached 0.2mol/L, and continue at 37 DEG C, 200rad/min's shakes
16h is cultivated under the conditions of bed.
(3) preliminary purification
Thalline is collected after thalline after cultivation is centrifuged through 4000g × 20min, and is suspended in 20mL Tris- containing 50mM
In the solution of HCl, 20mM NaCl；Ultrasonication thalline, supernatant is collected after being centrifuged through 18000g × 20min.
(4) affinity chromatography
By the supernatant carry on nickel ion affinity chromatograph post, and 5 column volumes are cleaned with cleaning fluid, then with wash-out
Liquid elutes 2 column volumes；Wherein, cleaning fluid is the solution of 50mM Tris-HCl, 20mM NaCl, 20mM imidazoles；Eluent is
The solution of 50mM Tris-HCl, 20mM NaCl, 200mM imidazoles.
(5) ion-exchange chromatography
Using the Source 15Q fillers of GE, chromatographic column is carried out using low salt solutions as albumen displacement buffer solution first
Then balance, loading carry ion exchange column uses the method for gradient elution to use high level salt solution as elution buffer
Eluted down chromatographic column；Wherein, low salt solutions are 50mM Tris-HCl, 20mM NaCl, and high level salt solution is 50mM Tris-
HCl, 1M NaCl.
(6) gel chromatography
First use EK enzyme digestion albumen, digestion solution for 50mM Tris-HCl, 20mM NaCl solution, then by it is above-mentioned from
In sub- displacement chromatography wash-out gained albumen carry gel chromatography column, chromatographic column is departed to it using wash buffer, finally obtained
High-cutting slope along road albumen.As shown in figure 3, the final purification result figure of IgE high-affinity receptor albumen, wherein fl is that IgE is high
Affinity receptor protein, M is marker.The buffer solution is Tris-HCl containing 50mM, the solution of 20mM NaCl.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means to combine specific features, structure, material or spy that the embodiment or example are described
Point is contained at least one embodiment of the invention or example.In this manual, to the schematic representation of above-mentioned term not
Identical embodiment or example must be directed to.And, the specific features of description, structure, material or feature can be with office
Combined in an appropriate manner in one or more embodiments or example.Additionally, in the case of not conflicting, the skill of this area
Art personnel can be tied the feature of the different embodiments or example described in this specification and different embodiments or example
Close and combine.
The preferred embodiments of the present invention are the foregoing is only, the scope of the claims of the invention is not thereby limited, it is every at this
Under the inventive concept of invention, the equivalent structure transformation made using description of the invention and accompanying drawing content, or directly/use indirectly
It is included in scope of patent protection of the invention in other related technical fields.
Claims (6)
1. a kind of high-cutting slope along road albumen vivoexpression and the method for isolating and purifying, it is characterised in that including following step
Suddenly：It is SEQ ID NO by cDNA sequence:1 genetic fragment is integrated into escherichia coli vector PET32M after PCR is expanded,
The carrier that will be built into is converted to e. coli bl21 so as to obtain thalline；
Cultivated to OD during thalline is placed in into 37 DEG C, the shaking table of 200rad/min600Untill 0.6-0.8, being subsequently adding IPTG makes bacterium
The concentration of IPTG reaches 0.2mol/L in liquid, and continues at 37 DEG C, and 16h is cultivated under the conditions of the shaking table of 200rad/min；Will culture
Thalline afterwards obtains high-affinity after being isolated and purified through preliminary purification, affinity chromatography, ion-exchange chromatography, gel chromatography successively
IgE receptor proteins.
2. high-cutting slope along road albumen vivoexpression as claimed in claim 1 and the method for isolating and purifying, its feature exist
It is to collect thalline after the thalline after cultivation is centrifuged through 4000g × 20min in, the preliminary purification, and is suspended in 20mL to contain
In the solution of 50mM Tris-HCl, 20mM NaCl；Ultrasonication thalline, supernatant is collected after being centrifuged through 18000g × 20min.
3. high-cutting slope along road albumen vivoexpression as claimed in claim 2 and the method for isolating and purifying, its feature exist
In the method for the affinitive layer purification albumen is by the supernatant carry nickel ion affinity chromatograph post and clear with cleaning fluid
Wash 5 column volumes, 2 column volumes of elution；
Wherein,
Cleaning fluid is the solution of 50mM Tris-HCl, 20mM NaCl, 20mM imidazoles；
Eluent is the solution of 50mM Tris-HCl, 20mM NaCl, 200mM imidazoles.
4. high-cutting slope along road albumen vivoexpression as claimed in claim 3 and the method for isolating and purifying, its feature exist
It by albumen displacement buffer solution in the eluent is low salt solutions to be in the method for, the ion-exchange purification albumen, carry from
Sub- displacement chromatography post, lower prop is eluted using the method high level salt solution of gradient elution,
Low salt solutions are 50mM Tris-HCl, 20mM NaCl,
High level salt solution is 50mM Tris-HCl, 1M NaCl.
5. high-cutting slope along road albumen vivoexpression as claimed in claim 4 and the method for isolating and purifying, its feature exist
In the method for the gel chromatography albumen is that above-mentioned ion-exchange chromatography is eluted into gained albumen carry gel chromatography column
In, depart from chromatographic column to it using wash buffer,
Buffer solution is Tris-HCl containing 50mM, the solution of 20mM NaCl.
6. high-cutting slope along road albumen vivoexpression as claimed in claim 5 and the method for isolating and purifying, its feature exist
In, EK enzyme digestion albumen was used before by gel chromatography albumen, digestion solution is 50mM Tris-HCl, 20mM
The solution of NaCl.
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| CN201611010470.1A CN106755032A (en) | 2016-11-17 | 2016-11-17 | A kind of high-cutting slope along road albumen vivoexpression and the method for isolating and purifying |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660569A (en) * | 2012-04-21 | 2012-09-12 | 大连理工大学 | Method for preparing recombinant human IgE receptor protein and application of recombinant human IgE receptor protein |
| CN104212860A (en) * | 2014-08-21 | 2014-12-17 | 深圳北京大学香港科技大学医学中心 | Method for expressing human IgE high affinity receptor protein in vitro |
-
2016
- 2016-11-17 CN CN201611010470.1A patent/CN106755032A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660569A (en) * | 2012-04-21 | 2012-09-12 | 大连理工大学 | Method for preparing recombinant human IgE receptor protein and application of recombinant human IgE receptor protein |
| CN104212860A (en) * | 2014-08-21 | 2014-12-17 | 深圳北京大学香港科技大学医学中心 | Method for expressing human IgE high affinity receptor protein in vitro |
Non-Patent Citations (2)
| Title |
|---|
| NCBI: "GenBank登录号：BC005912.1", 《NCBI GENBANK》 * |
| NCBI: "GenBank登录号：BC015195.1", 《NCBI GENBANK》 * |
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