CN107344963B - The method and its application of in vitro refolding vimentin - Google Patents
The method and its application of in vitro refolding vimentin Download PDFInfo
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Abstract
This application provides the applications of in vitro refolding wild type or the method for saltant type vimentin, the wild type or saltant type vimentin that are prepared by this method, and the method for the vimentin of the correct refolding of identification.
Description
Technical field
The application relates generally to protein engineering field, more specifically, this application involves the preparation method of vimentin,
The especially method and its application of in vitro refolding vimentin.
Background technology
It has been found that vimentin contains two glycine to arginic mutation in rheumatoid arthritis people, and
The arginine deamination of protein surface is citrullinated.With the citrullinated saltant type vimentin (mutant of in-vitro recombination expression
Citrullinated vimentin, MCV) detect Serum Antibody content as antigen, it is that a kind of new rheumatoid closes
The scorching detection method of section, sensitivity and specificity are all than existing RA factor and cyclic citrullinated peptide clinical detection
Method is high.
Vimentin is one of cytoskeleton intermediate filament family member.The gross three-dimensional structure of vimentin is heretofore unknown,
Although PDB (Protein Data Bank) have it is multiple reported vimentin fragment structure (4YPC, 4YV3,3G1E,
3S4R, 3SSU, 3SWK, 3TRT, 3UF1,3KLT), but maximum segment also only covers more than 100 a amino acid.The people of heterogenous expression
Vimentin and saltant type vimentin poorly soluble, refolding is carried out after needing denaturation in vitro.However, people's vimentin and prominent
The method that the conditions in vitro of the refolding of modification vimentin is reported at present is indefinite.For example, having been reported using chromatography, use
Polyhistidine marks saltant type vimentin, and by it, affinity chromatography is incorporated on nickel column under Denaturing, then with without denaturation
The buffer solution elution containing imidazoles of agent.But the method is less reproducible, the saltant type vimentin after denaturation can not be washed
It is de-, or be in precipitated form after elution, hence it is evident that fail correctly to fold.In addition, vimentin fold after protein configurations there is no it is qualitative
Standard easily forms the intermediate filament of different molecular weight size after folding, can not accurate quantitative analysis for subsequent antibody content detect.
Thus, there is an urgent need to establish the correct method for folding vimentin and/or quality control, to meet easy and low consumption
Ground diagnoses the demand of rheumatoid arthritis.
Invention content
In a first aspect, the application provides in vitro refolding wild type or the method for saltant type vimentin, including following step
Suddenly：The wild type or saltant type vimentin prepared product for making recombinant expression acquisition include denaturant and reducing agent with pH for 6-10
Buffer fluid contacts to dissolve the prepared product, and obtain protein solution；And in the presence of redox mixture, by saturating
The stage concentration for reducing denaturant in protein solution of analysis method, with refolding wild type or saltant type vimentin.
In some embodiments, the stage concentration for reducing denaturant in protein solution will be described including passing through dialysis
Including being reduced to the concentration period of denaturant 50%, 25% and of initial concentration in the buffer solution of denaturant and reducing agent
0%.In some embodiments, by using three kinds reduced comprising the concentration gradient of the denaturant and the denaturant
Dialyzate, the concentration of denaturant is respectively to be denaturalized in the buffer solution comprising denaturant and reducing agent in three kinds of dialyzates
50%, 25% and the 0% of the concentration of agent.
In some embodiments, wild type or saltant type vimentin prepared product are obtained by following steps：Make host
Cell expresses wild type or saltant type vimentin；Host cell is cracked, the wild type or saltant type wave of inclusion bodies are obtained
Shape protein preparation.
In some embodiments, denaturant is urea and/or guanidine hydrochloride.In some embodiments, including denaturant
With urea in the buffer solution of reducing agent and/or a concentration of 1~10M of guanidine hydrochloride.In some embodiments, including denaturant and
A concentration of 4~8M of urea and/or guanidine hydrochloride in the buffer solution of reducing agent.In some embodiments, including denaturant and also
A concentration of 8M of urea and/or guanidine hydrochloride in the buffer solution of former agent.In the specific embodiment, stage reduction protein solution
The concentration of middle denaturant can be implemented to dialyse successively using three kinds of dialyzates, urea and/or guanidine hydrochloride in three kinds of dialyzates
Concentration be respectively 4M, 2M and 0M.
In some embodiments, reducing agent is dithiothreitol (DTT) (DTT) or β mercaptoethanols.In some embodiments,
Including a concentration of 1~20mM of dithiothreitol (DTT) or β mercaptoethanols in the buffer solution of denaturant and reducing agent.In some embodiment party
In case, including a concentration of 5~15mM of dithiothreitol (DTT) or β mercaptoethanols in the buffer solution of denaturant and reducing agent.At some
In embodiment, including a concentration of 10mM of dithiothreitol (DTT) or β mercaptoethanols in the buffer solution of denaturant and reducing agent.
In some embodiments, redox mixture is reduced glutathione/oxidized form of glutathione.At some
In embodiment, the ratio of reduced glutathione and oxidized form of glutathione is 1:10.
In some embodiments, the pH of buffer solution is 8, and includes：300mM NaCl, 10mM DTT, 1mM EDTA,
And 8M urea or guanidine hydrochloride.
In some embodiments, vimentin behaviour vimentin.
In some embodiments, host cell is Escherichia coli.In some embodiments, Bacillus coli expression by
The saltant type vimentin of DNA sequence encoding shown in SEQ ID NO.2.
Second aspect, the application provide the kit in vitro refolding wild type or saltant type vimentin, packet
It is 6-10 containing pH, includes the buffer solution of denaturant and reducing agent, and the concentration comprising the denaturant and the denaturant
A variety of dialyzates that gradient reduces.
In some embodiments, kit also includes redox mixture.
In some embodiments, denaturant is urea and/or guanidine hydrochloride.In some embodiments, described to include change
A concentration of 1~10M of urea and/or guanidine hydrochloride in the buffer solution of property agent and reducing agent.In some embodiments, described to include
A concentration of 4~8M of urea and/or guanidine hydrochloride in the buffer solution of denaturant and reducing agent.In some embodiments, the packet
A concentration of 8M of urea and/or guanidine hydrochloride in buffer solution containing denaturant and reducing agent.
In some embodiments, reducing agent is dithiothreitol (DTT) (DTT).In some embodiments, described to include change
A concentration of 1~20mM of dithiothreitol (DTT) in the buffer solution of property agent and reducing agent.In some embodiments, described to include denaturation
A concentration of 5~15mM of dithiothreitol (DTT) in the buffer solution of agent and reducing agent.In some embodiments, described includes denaturant
With a concentration of 10mM of dithiothreitol (DTT) in the buffer solution of reducing agent.
In some embodiments, redox mixture is reduced glutathione/oxidized form of glutathione.At some
In embodiment, the ratio of reduced glutathione and oxidized form of glutathione is 1:10.
In some embodiments, the pH of buffer solution is 8, including：300mM NaCl, 10mM DTT, 1mM EDTA, and
8M urea or guanidine hydrochloride.In the specific embodiment, concentration of the kit comprising urea or guanidine hydrochloride is respectively 4M, 2M and 0M
Three kinds of dialyzates.
The third aspect, the method that the application provides the vimentin for identifying correct refolding, including the use of circular dichroism detector
Detection waveform protein sample, if detecting α helical configurations and β-pleated sheet configuration being not present again, it indicates that vimentin is correctly rolled over
It is folded.
In some embodiments, circular dichroism detector detection includes following condition：At room temperature, to vimentin sample into
The Scanning Detction of row 180-600nm linearly polarized lights, the absorption coefficient for detecting left-right rotary circularly polarized light are poor.
Fourth aspect, the application provide the mutant human waveform egg of the refolding prepared by the method described in first aspect
In vain.In some embodiments, the mutant human vimentin is relative to people's wild type vimentin, in 16 and 59 bit aminos
Sour residue mutations are arginine.In some embodiments, SEQ ID NO are utilized:Coded sequence shown in 2 generates the saltant type
People's vimentin.Present invention also provides SEQ ID NO:The nucleic acid molecules of the people's vimentin of encoding mutant type shown in 2.
5th aspect, the application provide the wild type or saltant type of the refolding prepared by the method described in first aspect
Purposes of the vimentin in the reagent for preparing the biological sample moderate resistance vimentin antibodies for detecting individual.
The application also provides the reagent of the biological sample moderate resistance vimentin antibodies for detecting individual, and it includes pass through the
The wild type or saltant type vimentin of refolding prepared by the method described in one side.
As described above, saltant type vimentin is contained in the blood of patient with rheumatoid arthritis, it includes essences at three
Histidine mutations, and the arginine deamination of protein surface is citrullinated.Therefore, pair that will be obtained by first aspect the method
Saltant type vimentin is answered after the processing of polypeptide arginine deaminase is citrullinated, to can be used for detecting blood or serum in vitro
The citrullinated vimentin antibodies content of anti-saltant type of people in equal tissue fluid, and then diagnose rheumatoid arthritis.
Therefore, the application provides the mutant human vimentin of the refolding prepared by the method described in first aspect
(for example, sporting arginine at 16,59 relative to people's wild type vimentin) and optional arginine deaminase are being made
It is ready for use on the reagent of the citrullinated vimentin antibodies of biological sample moderate resistance saltant type of detection individual and is used for diagnostics classes wind
Purposes in the reagent of wet arthritis.
The application also provides the examination of the citrullinated vimentin antibodies of biological sample moderate resistance saltant type for detecting individual
Agent or reagent for diagnosing rheumatoid arthritis, it includes the prominent of the refolding prepared by the method described in first aspect
Modification people vimentin (for example, sporting arginine at 16,59 relative to people's wild type vimentin) and optional essence
Propylhomoserin deaminase.
Brief Description Of Drawings
Fig. 1 shows the plasmid map of constructed expression saltant type vimentin.
Fig. 2 shows the circular dichroism spectra spectrograms of the saltant type vimentin of refolding.
Fig. 3 shows electrophoresis result of the saltant type vimentin of refolding through polypeptide arginine deaminase before and after the processing,
In, swimming lane 1 identifies for molecular weight of albumen；Swimming lane 2 is+0.2 μ g polypeptide arginine of 1 μ g people's saltant type vimentins (G16/59R)
Deaminase (Peptidyl arginine deaminase, PAD), mixes loading with SDS sample-loading buffers immediately；Swimming lane 3 is 1 μ
+ 0.2 μ g PAD of g people's saltant type vimentin (G16/59R), mix with SDS sample-loading buffers again after 55 DEG C of 3 hours of reaction
Sample；Swimming lane 4 and 5 is respectively 2 and 3 double albumen loadings.
Fig. 4 is shown to be suffered from using the exemplary citrullinated saltant type vimentin detection rheumatoid arthritis of the application
The result of person and the citrullinated saltant type vimentin antibodies of healthy individuals serum moderate resistance.
Sequence briefly explains
SEQ ID NO:1 shows the amino acid sequence of the wild type human vimentin recorded in ncbi database.
SEQ ID NO:2 show the coded sequence of the exemplary mutations vimentin of the application, wherein people's wild type wave
16 and 59 codon glycines of shape albumen are sported arginine codon, have in addition also carried out the optimizations such as codon optimization
Design.
SEQ ID NO:3 show the amino acid sequence of the exemplary mutations vimentin of the application.
Detailed description of the Invention
Unless otherwise mentioned, the term involved by the application understands according to the routine of person of ordinary skill in the relevant.
In a first aspect, the application provides in vitro refolding wild type or the method for saltant type vimentin, including following step
Suddenly：The wild type or saltant type vimentin prepared product for making recombinant expression acquisition include denaturant and reducing agent with pH for 6-10
Buffer fluid contacts to dissolve the prepared product, and obtain protein solution；And in the presence of redox mixture, by saturating
The stage concentration for reducing denaturant and reducing agent in protein solution of analysis method, with refolding wild type or saltant type vimentin.
This method is suitable for the body of wild type/natural vimentin or any saltant type vimentin known in the art
Outer refolding.
In some embodiments, saltant type vimentin includes the 16th and the 59th in naive vimentin sweet ammonia
Acid mutation is arginine.The mutant is a kind of blood markers object of rheumatoid arthritis, therefore passes through the application method system
The mutant of standby refolding can be used for detecting the antibody that is caused by the mutant in the biological sample of individual, so diagnose or
Auxiliary diagnosis rheumatoid arthritis.
In some embodiments, wild type or saltant type vimentin prepared product are obtained by following steps：Make host
Cell expresses wild type or saltant type vimentin；Host cell is cracked, the wild type or saltant type wave of inclusion bodies are obtained
Shape protein preparation.
In some embodiments, host cell is Escherichia coli.In some embodiments, Bacillus coli expression by
The saltant type vimentin of DNA sequence encoding shown in SEQ ID NO.2.In a particular embodiment, can by wild type or
The coded sequence of saltant type vimentin is placed in strong promoter (such as T7 or PBADDeng) in the lower plasmid of control, importing Escherichia coli,
After induced expression, wild type or saltant type vimentin are present in the form of insoluble in inclusion body.
Cell cracking is carried out before or while the dissolving step that the step of cracking host cell can be described below.Cell is split
Solution can be for example, by mechanical shearing such as French compressing cell (French pressure cell), enzymic digestion, supersound process, homogenate
Change, bead vortex, detergent processing, organic solvent, freeze thawing, aluminium oxide or husky grinding, denaturant processing etc. are completed.Or
Person, can in the presence of disulfide bond reducing agent or cysteine blocking agent lytic cell.
The above method include make wild type or saltant type vimentin prepared product (for example, insoluble inclusion bodies) with
The Buffer fluid contacts for including denaturant and reducing agent, to make it dissolve.Can be used as centrifuged, filtering (including ultrafiltration), precipitate,
A variety of methods such as flocculation or fixation make insoluble or aggregation substance be detached with soluble protein, to obtain protein solution.
In some embodiments, including the pH of the buffer solution of denaturant and reducing agent is 6~10.In some embodiments
In, including the pH of the buffer solution of denaturant and reducing agent is 6~8.In some embodiments, including denaturant and reducing agent
The pH of buffer solution is about 8.
The purpose of refolding is to obtain the native conformation and natural disulphide bonds of albumen.In this application, vimentin
Refolding is to be enough to allow the protein renaturation be by making the concentration of denaturant in protein solution pass through stage be reduced to of dialysis
The level of solvable biologically active form.
In some embodiments, the stage concentration for reducing denaturant in protein solution will be described including passing through dialysis
Including decreasing below to the concentration period of denaturant several stages of initial concentration in the buffer solution of denaturant and reducing agent
Until 0.As unrestricted example, the stage concentration for reducing denaturant in protein solution may include that will become in buffer solution
It is reduced to 50%, 25% and the 0% of initial concentration to the concentration period of property agent.As unrestricted example, stage drop
In low protein solution the concentration of denaturant can by using include corresponding to desired each stage concentration denaturant carry out
Dialysis.In some embodiments, the three kinds of dialyzates reduced comprising the concentration gradient of denaturant and denaturant can be used,
The concentration of denaturant is respectively the concentration of denaturant in the buffer solution comprising denaturant and reducing agent in three kinds of dialyzates
50%, 25% and 0%.
Oxidant or redox mixture are added in dialyzate to allow natural disulphide bonds to rebuild, to promote to roll over again
It is folded.The example of oxidant includes but not limited to oxygen, cystine, oxidized form of glutathione, cystamine and ethylene dithiol alkyd.Redox
The example of mixture includes but not limited to cysteine/oxygen, cysteine/cystine, cysteine/cystamine, cysteamine/Guang
Amine, reduced glutathione/oxidized form of glutathione etc..
In some embodiments, denaturant is urea and/or guanidine hydrochloride.In some embodiments, including denaturant
With urea in the buffer solution of reducing agent and/or a concentration of 1~10M of guanidine hydrochloride.In some embodiments, including denaturant and
A concentration of 4~8M of urea and/or guanidine hydrochloride in the buffer solution of reducing agent.In some embodiments, including denaturant and also
A concentration of 8M of urea and/or guanidine hydrochloride in the buffer solution of former agent.In the specific embodiment, stage reduction protein solution
The concentration of middle denaturant can be implemented to dialyse successively using three kinds of dialyzates, urea and/or guanidine hydrochloride in three kinds of dialyzates
Concentration be respectively 4M, 2M and 0M.
In some embodiments, reducing agent is dithiothreitol (DTT) (DTT) or β mercaptoethanols.In some embodiments,
Including a concentration of 1~20mM of dithiothreitol (DTT) or β mercaptoethanols in the buffer solution of denaturant and reducing agent.In some embodiment party
In case, including a concentration of 5~15mM of dithiothreitol (DTT) or β mercaptoethanols in the buffer solution of denaturant and reducing agent.At some
In embodiment, including a concentration of 10mM of dithiothreitol (DTT) or β mercaptoethanols in the buffer solution of denaturant and reducing agent.
In some embodiments, redox mixture is reduced glutathione/oxidized form of glutathione.At some
In embodiment, the ratio of reduced glutathione and oxidized form of glutathione is 1:10.
In some embodiments, the pH of buffer solution is 8, and includes：300mM NaCl, 10mM DTT, 1mM EDTA,
And 8M urea or guanidine hydrochloride.
Second aspect, the application provide the kit in vitro refolding wild type or saltant type vimentin, packet
It is 6-10 containing pH, includes the buffer solution of denaturant and reducing agent, and the concentration comprising the denaturant and the denaturant
A variety of dialyzates that gradient reduces.
In some embodiments, kit also includes redox mixture.
In some embodiments, denaturant is urea and/or guanidine hydrochloride.In some embodiments, described to include change
A concentration of 1~10M of urea and/or guanidine hydrochloride in the buffer solution of property agent and reducing agent.In some embodiments, described to include
A concentration of 4~8M of urea and/or guanidine hydrochloride in the buffer solution of denaturant and reducing agent.In some embodiments, the packet
A concentration of 8M of urea and/or guanidine hydrochloride in buffer solution containing denaturant and reducing agent.
In some embodiments, reducing agent is dithiothreitol (DTT) (DTT).In some embodiments, described to include change
A concentration of 1~20mM of dithiothreitol (DTT) in the buffer solution of property agent and reducing agent.In some embodiments, described to include denaturation
A concentration of 5~15mM of dithiothreitol (DTT) in the buffer solution of agent and reducing agent.In some embodiments, described includes denaturant
With a concentration of 10mM of dithiothreitol (DTT) in the buffer solution of reducing agent.
In some embodiments, redox mixture is reduced glutathione/oxidized form of glutathione.At some
In embodiment, the ratio of reduced glutathione and oxidized form of glutathione is 1:10.
In some embodiments, the pH of buffer solution is 8, including：300mM NaCl, 10mM DTT, 1mM EDTA, and
8M urea or guanidine hydrochloride.In the specific embodiment, concentration of the kit comprising urea or guanidine hydrochloride is respectively 4M, 2M and 0M
Three kinds of dialyzates.
The third aspect, the method that the application provides the vimentin for identifying correct refolding, including the use of circular dichroism detector
Detection waveform protein sample, if detecting α helical configurations and β-pleated sheet configuration being not present again, it indicates that vimentin is correctly rolled over
It is folded.
Present inventor find, under the conditions of salinity appropriate and pH, vimentin formed oligomer form without
The intermediate filament that molecular size range differs can be formed, and finds that the secondary structure of the vimentin of correct refolding is more than for the first time in turn
90% is α spirals, such as shown in Figure 2.Therefore, in some embodiments, if detecting the circle with configuration shown in Fig. 2
Two chromatographic results, it indicates that the correct refolding of vimentin.
In some embodiments, circular dichroism detector detection includes following condition：At room temperature, to vimentin sample into
The Scanning Detction of row 180-600nm linearly polarized lights, the absorption coefficient for detecting left-right rotary circularly polarized light are poor.As unrestricted
Example, circular dichroism detector detection includes following condition：At room temperature, to the waveform egg of 0.08mg/mL in the cuvette of 2mm optical paths
White sample carries out the Scanning Detction of 180-600nm linearly polarized lights, and the absorption coefficient for detecting left-right rotary circularly polarized light is poor.
Fourth aspect, the application provide the mutant human waveform egg of the refolding prepared by the method described in first aspect
In vain.In some embodiments, the mutant human vimentin is relative to people's wild type vimentin, in 16 and 59 bit aminos
Sour residue mutations are arginine.In some embodiments, SEQ ID NO are utilized:Coded sequence shown in 2 generates the saltant type
People's vimentin.Present invention also provides SEQ ID NO:The nucleic acid molecules of the people's vimentin of encoding mutant type shown in 2.SEQ ID
Coded sequence shown in NO.2 passes through the optimization of present inventor, thus particularly suitable for the present processes.
5th aspect, the application provide the wild type or saltant type of the refolding prepared by the method described in first aspect
Purposes of the vimentin in the reagent for preparing the biological sample moderate resistance vimentin antibodies for detecting individual.
The application also provides the reagent of the biological sample moderate resistance vimentin antibodies for detecting individual, and it includes pass through the
The wild type or saltant type vimentin of refolding prepared by the method described in one side.
As described above, saltant type vimentin is contained in the blood of patient with rheumatoid arthritis, it includes essences at three
Histidine mutations, and the arginine deamination of protein surface is citrullinated.Therefore, saltant type vimentin the application obtained
In vitro after the processing of polypeptide arginine deaminase is citrullinated, it can be used in the tissue fluid such as easy detection blood or serum
The citrullinated vimentin antibodies content of anti-saltant type of people, and then diagnose rheumatoid arthritis.
As unrestricted example, ELISA enzyme linked immunoassay can be used (for example, see embodiment).But it should
Understand, citrullinated saltant type vimentin provided by the present application is also applied for other immunologic detection methods to detect blood or blood
The citrullinated vimentin antibodies content of anti-saltant type of people in clear equal tissue fluid, including it is turbidimetry, colloidal gold method, POCT, glimmering
Light, chemiluminescent labeling etc..
Therefore, this application provides a kind of methods and its phase of new in vitro refolding wild type or saltant type vimentin
The kit answered.The external correct refolding of the saltant type vimentin of heterogenous expression may be implemented by this method so that base
The diagnosis of the rheumatoid arthritis of citrullinated saltant type vimentin antibodies is resisted to become easy and low consumption in detection.
In some embodiments, refolding provided by the present application can be handled using polypeptide arginine deaminase PAD
Saltant type vimentin.In some embodiments, the mass ratio 1 of PAD and saltant type vimentin:5, it is small that 3 are reacted at 55 DEG C
When.Thus obtained citrullinated saltant type vimentin carries out Quality Control detection through sds gel electrophoresis and shows protein arginine quilt
Citrullinated, migration rate slows down in gel electrophoresis.
In some embodiments, micro drop can be carried out with citrullinated saltant type vimentin provided herein
The coating of fixed board plate.In some specific embodiments, the tablet after coating is made a definite diagnosis with clinic through CCP (cyclic citrulline polypeptide)
Rheumatoid arthritis people and 100 times of dilute serums (100 μ L, actually use 1 μ L serum) of Healthy People be incubated 20 points altogether
Clock three times after board-washing, is incubated 20 minutes altogether with HRP (horseradish peroxidase) the anti-human serum secondary antibodies being coupled, three times after board-washing,
Substrate, which is done, with 3,3 ', 5,5 '-tetramethyl benzidines (TMB) carries out immune integrated enzyme reaction.
Embodiment
Following embodiment is merely to illustrate and the purpose of unrestricted the application range.Unless otherwise specified, embodiment is pressed
More solito experiment condition, such as Sambrook《Molecular cloning experiment handbook》(Sambrook J&Russell DW,
Molecular cloning:A laboratory manual, 2001), or according to the condition of manufacturer's specification suggestion.
The structure of the synthesis and plasmid of 1 saltant type vimentin dna of embodiment
Saltant type vimentin, schematic diagram such as Fig. 1 of constructed plasmid map are synthesized in the way of full genome synthesis
It is shown.SEQ ID NO:1 and 3 show wild type human vimentin and the exemplary mutations vimentin amino acid sequence of the application
Row, wherein SEQ ID NO:3 and SEQ ID NO:1 compares, 16 and 59 codon glycine quilts of people's wild type vimentin
Sport arginine.
SEQ ID NO:Sequence shown in 1 comes from vimentin (homo sapiens, chromosome 10, GRCh38.p2；NC_000010,
Gene NCBI accession number GI:578818565, Protein Accession number：P08670).It is introducing except Mutated codons, also directed to expression
Codon optimization is carried out to coded sequence with host cell E. coli BL21 (DE3), while considering following factor：Evade
EagI、EcoRI、HindIII, KpnI, NdeI, NcoI, NotI, SacI, XbaI, XhoI restriction enzyme site, avoid having an impact base
Because of the secondary structure of expression, the sequence such as SEQ ID NO after optimization:Shown in 2.In addition, for the ease of purifying, in mutation waveform egg
The coded sequence of six histidine tags is added to after white coded sequence.By the good sequence of specialized company's compounding design and it is loaded into
On pET28a (+) carrier, expression unit is set up：T7 promoter-lac operon-MCV-His label-T7 terminators.
The refolding of 2 saltant type vimentin of embodiment
Plasmid constructed in embodiment 1 is transformed into e. coli bl21 (DE3).
After induced expression, after Bacillus coli cells are crushed with the methods of high-pressure homogeneous instrument or ultrasonic method freeze-thaw method, low speed
It centrifuges (3000g) and removes unbroken cell and cell fragment, then through high speed centrifugation (14000g), harvest precipitation is as forgiven
Body --- insoluble saltant type vimentin.
By solubilization of inclusion bodies in pH be 8.0, comprising 300mM NaCl, 10mM DTT, 1mM EDTA and 8M urea it is slow
It in fliud flushing, is incubated 10 minutes at room temperature, you can dissolving inclusion body.14000g centrifugation removal precipitations, obtain supernatant.
Using dialysis, (basic dialyzate is 100mM phosphate buffers, pH 8.0, the paddy Guang containing oxidized form and reduced form
Sweet peptide mixer (ratio 1：10)) the interim concentration for reducing urea in supernatant.Dialysis procedure divides three phases to carry out, point
The dialyzate containing 4M, 2M and 0M is not used to carry out.The folding again of saltant type vimentin is finally completed in the solution of the urea containing 0M
It is folded, obtain the phosphate buffer of the saltant type vimentin containing refolding.
The identification of 3 saltant type vimentin refolding configuration of embodiment
The concentration for the saltant type vimentin through refolding that embodiment 2 is obtained is adjusted to about 0.2mg/ml, with circle
Two chromatographies are detected (at room temperature, carries out 180- to the vimentin sample of 0.08mg/mL in the cuvette of 2mm optical paths
The Scanning Detction of 600nm linearly polarized lights, the absorption coefficient for detecting left-right rotary circularly polarized light are poor), the results are shown in Figure 2.
From Figure 2 it can be seen that saltant type vimentin is in apparent negative value in 208 and 222nm absorption coefficient differences, absorbed in 192nm
Coefficient is in positive value, shows the secondary structure based on the saltant type vimentin spiral containing α through refolding, is in correct after protein folding
Configuration.
The effect of embodiment 4 saltant type vimentin and polypeptide arginine deaminase
This example demonstrates the citrullinated waveforms of the correct saltant type of the configuration after refolding obtained by embodiment 2
Albumen can react with polypeptide arginine deaminase.
The saltant type vimentin obtained with embodiment 2 is reacted with polypeptide arginine deaminase (PAD), PAD and mutation
The mass ratio 1 of type vimentin:5,3 hours (pH7.4 5mM CaCl are reacted at 55 DEG C2In solution).Product is then subjected to electricity
It swims (12% polyacrylamide gel electrophoresis, 200V80 minutes, coomassie brilliant blue staining, decoloration).
Citrullinated saltant type vimentin after deamination is citrullinated due to the change with SDS binding forces, in gel electricity
Different migration rates is shown in swimming, it is specific as shown in Figure 3.Thus obtained citrullinated saltant type vimentin is solidifying through SDS
Gel electrophoresis carries out Quality Control detection and shows that protein arginine is citrullinated, and migration rate slows down in gel electrophoresis.
The detection of 5 citrullinated saltant type vimentin of embodiment resists citrullinated saltant type vimentin antibodies
The citrullinated saltant type vimentin obtained with embodiment 4 carries out the coatings of 96 orifice plates, tablet after coating and
100 times of dilute serum (100 μ for the rheumatoid arthritis people and Healthy People that clinic is made a definite diagnosis through CCP (cyclic citrulline polypeptide)
L actually uses 1 μ L serum) it is incubated 20 minutes altogether, three times after board-washing, with the anti-human serum of HRP (horseradish peroxidase) couplings
Secondary antibody is incubated 20 minutes altogether.Three times after board-washing, with 3,3 ', 5,5 '-Tetramethylbenzidine (TMB) do substrate and are exempted from
Epidemic disease integrated enzyme reaction.As shown in figure 4,18 patients serum's positive rates are 100%, negative findings are also all presented in 6 Healthy Peoples.Thus
It confirms, the citrullinated saltant type vimentin prepared according to the application method can be effective in detection rheumatoid arthritis
Resist citrullinated saltant type vimentin antibodies.
Sequence table
<110>The bio tech ltd Bei Nuobo of Shenzhen
<120>The method and its application of in vitro refolding vimentin
<130> 16C12641CN
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 466
<212> PRT
<213>Homo sapiens
<400> 1
Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly
1 5 10 15
Gly Pro Gly Thr Ala Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr
20 25 30
Thr Ser Thr Arg Thr Tyr Ser Leu Gly Ser Ala Leu Arg Pro Ser Thr
35 40 45
Ser Arg Ser Leu Tyr Ala Ser Ser Pro Gly Gly Val Tyr Ala Thr Arg
50 55 60
Ser Ser Ala Val Arg Leu Arg Ser Ser Val Pro Gly Val Arg Leu Leu
65 70 75 80
Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe
85 90 95
Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp
100 105 110
Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn
115 120 125
Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser
130 135 140
Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln
145 150 155 160
Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp
165 170 175
Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu
180 185 190
Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln
195 200 205
Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val
210 215 220
Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu
225 230 235 240
Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile
245 250 255
Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val
260 265 270
Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu
275 280 285
Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg
290 295 300
Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg
305 310 315 320
Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr
325 330 335
Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala
340 345 350
Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu
355 360 365
Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln
370 375 380
Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr
385 390 395 400
Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro
405 410 415
Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro
420 425 430
Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu
435 440 445
Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp
450 455 460
Leu Glu
465
<210> 2
<211> 1398
<212> DNA
<213>Artificial sequence
<220>
<223>Arginine mutation is introduced in wild type vimentin 16 and 59 amino acids residues, and it is excellent to carry out codon
Change
<400> 2
atgtctaccc gctctgtgtc ttcttcttct tatcgtcgta tgtttcgtgg tccgggtaca 60
gcctcacgcc cgagcagctc tcgtagctat gttaccacct ctacccgcac ctattcactg 120
ggttcagcac tgcgtccgag tacctcacgt agcctgtatg ctagctctcc gggccgtgtt 180
tatgccaccc gctcttcagc cgtgcgcctg cgttctagcg tgccgggtgt tcgtctgctg 240
caggattcag ttgattttag tctggcagat gcgattaata ccgaatttaa aaatacccgt 300
accaatgaaa aagtggaact gcaggaactg aatgatcgtt ttgctaatta tattgataaa 360
gtgcgttttc tggaacagca gaataaaatt ctgctggctg aactggaaca gctgaaaggt 420
cagggcaaaa gtcgcctggg cgatctgtat gaagaagaaa tgcgcgaact gcgtcgccag 480
gtggatcagc tgaccaatga taaagcacgt gttgaagttg aacgcgataa tctggcggaa 540
gatattatgc gcctgcgcga aaaactgcag gaagaaatgc tgcagcgcga agaagcagaa 600
aataccctgc agagttttcg ccaggatgtg gataatgcta gcctggctcg cctggatctg 660
gaacgcaaag ttgaaagcct gcaggaagaa attgcgtttc tgaaaaaact gcatgaagaa 720
gaaattcagg aactgcaggc tcagattcag gaacagcatg ttcagattga tgtggatgtt 780
tcaaaaccgg atctgaccgc tgccctgcgt gatgttcgtc agcagtatga aagcgttgca 840
gcaaaaaatc tgcaggaagc tgaagaatgg tataaatcaa aatttgccga tctgtcagaa 900
gcagcgaatc gcaataatga tgcgctgcgc caggcaaaac aggaaagcac cgaatatcgc 960
cgtcaggtgc agagtctgac ctgtgaagtt gatgcgctga aaggcaccaa tgaaagtctg
1020
gaacgccaga tgcgcgaaat ggaagaaaat tttgccgtgg aagcggcgaa ttatcaggat
1080
accattggcc gtctgcagga tgaaattcag aatatgaaag aagaaatggc acgccatctg
1140
cgcgaatatc aggatctgct gaatgttaaa atggccctgg atattgaaat tgccacctat
1200
cgtaaactgc tggaaggtga agaatcacgt atttcactgc cgctgccgaa ttttagcagc
1260
ctgaatctgc gtgaaaccaa tctggatagt ctgccgctgg tggataccca tagtaaacgt
1320
accctgctga ttaaaaccgt tgaaacccgt gatggccagg tgattaatga aaccagtcag
1380
catcatgatg atctcgag 1398
<210> 3
<211> 466
<212> PRT
<213>Homo sapiens
<400> 3
Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Arg
1 5 10 15
Gly Pro Gly Thr Ala Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr
20 25 30
Thr Ser Thr Arg Thr Tyr Ser Leu Gly Ser Ala Leu Arg Pro Ser Thr
35 40 45
Ser Arg Ser Leu Tyr Ala Ser Ser Pro Gly Arg Val Tyr Ala Thr Arg
50 55 60
Ser Ser Ala Val Arg Leu Arg Ser Ser Val Pro Gly Val Arg Leu Leu
65 70 75 80
Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe
85 90 95
Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp
100 105 110
Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn
115 120 125
Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser
130 135 140
Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln
145 150 155 160
Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp
165 170 175
Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu
180 185 190
Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln
195 200 205
Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val
210 215 220
Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu
225 230 235 240
Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile
245 250 255
Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val
260 265 270
Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu
275 280 285
Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg
290 295 300
Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg
305 310 315 320
Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr
325 330 335
Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala
340 345 350
Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu
355 360 365
Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln
370 375 380
Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr
385 390 395 400
Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro
405 410 415
Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro
420 425 430
Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu
435 440 445
Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp
450 455 460
Leu Glu
465
Claims (9)
1. the method for in vitro refolding wild type or saltant type vimentin, includes the following steps：
The wild type or saltant type vimentin prepared product for making recombinant expression acquisition include urea and dithiothreitol (DTT) with pH for 8
Buffer fluid contacts to dissolve the prepared product, and obtain protein solution, wherein in the buffer solution urea a concentration of 8M, two
A concentration of 10mM of sulphur threitol；And
In the presence of redox mixture, by the stage concentration for reducing urea in protein solution of dialysis, with refolding
Wild type or saltant type vimentin, wherein the stage concentration for reducing urea in protein solution includes will be described by dialysis
Including being reduced to the concentration period of urea 50%, 25% and of initial concentration in the buffer solution of urea and dithiothreitol (DTT)
0%, wherein the three kinds of dialyzates reduced comprising the concentration gradient of urea and urea are used, urea in three kinds of dialyzates
Concentration is respectively 50%, 25% and 0% of the concentration of urea in the buffer solution comprising urea and dithiothreitol (DTT)；
The wherein described saltant type vimentin be relative to people's wild type vimentin, be in 16 and 59 amino acids residue mutations
Arginine.
2. the method as described in claim 1, wherein the wild type or saltant type vimentin prepared product pass through following steps
It obtains：
Make host cell expression wild type or saltant type vimentin；
Host cell is cracked, the wild type or saltant type vimentin prepared product of inclusion bodies are obtained.
3. the method as described in any one of claim 1-2, wherein the redox mixture be reduced glutathione/
Oxidized form of glutathione.
4. the ratio of method as claimed in claim 3, wherein reduced glutathione and oxidized form of glutathione is 10:1.
5. including the purposes in kit below in vitro refolding wild type or saltant type vimentin：PH is 8, comprising dense
The buffer solution for the dithiothreitol (DTT) of 10mM and the urea of a concentration of 8M is spent, and is dropped comprising the concentration gradient of urea and urea
Three kinds of low dialyzates, the concentration of urea is respectively the buffering for including dithiothreitol (DTT) and urea in three kinds of dialyzates
50%, 25% and the 0% of the concentration of urea in liquid.
6. purposes as claimed in claim 5, wherein the kit also includes redox mixture.
7. purposes as claimed in claim 6, wherein the redox mixture is reduced glutathione/oxidized form paddy Guang
Sweet peptide.
8. the ratio of purposes as claimed in claim 7, wherein reduced glutathione and oxidized form of glutathione is 10:1.
9. kit as claimed in claim 5, wherein the buffer solution comprising dithiothreitol (DTT) and urea also includes 300mM
NaCl and 1mM EDTA.
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| CN117164691B (en) * | 2022-05-26 | 2024-08-30 | 深圳市睿盟创新生物科技有限公司 | Vimentin variants and preparation and use of citrullinated products thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104321650A (en) * | 2012-04-16 | 2015-01-28 | 生物辐射实验室股份有限公司 | Multiplex immunoassay for rheumatoid arthritis and other autoimmune diseases |
| CN105693841A (en) * | 2016-01-20 | 2016-06-22 | 武汉轻工大学 | Method for extracting and purifying vimentin |
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2017
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104321650A (en) * | 2012-04-16 | 2015-01-28 | 生物辐射实验室股份有限公司 | Multiplex immunoassay for rheumatoid arthritis and other autoimmune diseases |
| CN105693841A (en) * | 2016-01-20 | 2016-06-22 | 武汉轻工大学 | Method for extracting and purifying vimentin |
Non-Patent Citations (2)
| Title |
|---|
| Mutation and citrullination modifies vimentin to a novel autoantigen for rheumatoid arthritis;Holger Bang等;《Arthritis & Rheumatism 》;20070730;第56卷(第8期);摘要，第2505页左栏第3段，第2508页左栏第3段-第2509页左栏第3段，表1-2，图3 * |
| Reconstitution of intermediate-sized filaments from denatured monomeric vimentin;Winfried Renner等;《Journal of Molecular Biology》;19810625;第149卷(第2期);第288-289页第d-f节，第287页第2段 * |
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Effective date of registration: 20190820 Address after: 518033 Unite Plaza A, 5022 Riverside Avenue, Futian Street, Futian District, Shenzhen City, Guangdong Province, 4009-A4010L Patentee after: Shenzhen Jinshun New Materials Technology Co., Ltd. Address before: Room 1-210, No. 10 Bio-incubator Building, High-tech Middle Road, Nanshan District, Shenzhen City, Guangdong Province, 518057 Patentee before: SHENZHEN BEINUOBO BIOTECHNOLOGY CO., LTD. |