CN103254294A - CD34-SG17 polypeptide fragment as well as preparation method and application thereof - Google Patents
CD34-SG17 polypeptide fragment as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN103254294A CN103254294A CN201310145927XA CN201310145927A CN103254294A CN 103254294 A CN103254294 A CN 103254294A CN 201310145927X A CN201310145927X A CN 201310145927XA CN 201310145927 A CN201310145927 A CN 201310145927A CN 103254294 A CN103254294 A CN 103254294A
- Authority
- CN
- China
- Prior art keywords
- polypeptide fragment
- resin
- polypeptide
- amino acid
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a CD34-SG17 polypeptide fragment as well as a preparation method and application thereof. The nucleotide sequence of the polypeptide fragment is represented by SEQ ID No.1. The CD34-SG17 polypeptide fragment disclosed by the embodiment of the invention is high in hydrophilicity, simple in structure, convenient to synthesize and purify and relatively high in immunogenicity; and an antibody prepared by the CD34-SG17 polypeptide fragment is high in specificity, high in sensitivity and accurate in an expression part of a tissue cell.
Description
Technical field
The present invention relates to chemosynthesis technical field, more specifically, relate to a kind of CD34-SG17 polypeptide fragment and its preparation method and application.
Background technology
At present, cell streaming antibody commonly used carries out immunity with cell mostly, or this is the acquisition of cell extract immune animal, though can identify this cell, but epitope is unclear, experimental data theoretical inaccurate, poor specificity, preparation method's complexity, and poor repeatability, endlessly artificially control, so the antibody that produces has error, can produce detrimentally affect to the experiment in later stage is nonspecific reaction.
Summary of the invention
The present invention is intended to one of solve the problems of the technologies described above at least to a certain extent.
For this reason, one object of the present invention is to propose that a kind of wetting ability is strong, immunogenicity strong and be convenient to the CD34-SG17 polypeptide fragment of synthetic and purifying.
According to the CD34-SG17 polypeptide fragment of the embodiment of the invention, the nucleotide sequence of described polypeptide fragment is shown in SEQ ID N0.1.
According to the CD34-SG17 polypeptide fragment of the embodiment of the invention, wetting ability is strong, and is simple in structure, be convenient to synthetic and purifying, and this polypeptide fragment have stronger immunogenicity, good with the antibodies specific of its preparation, highly sensitive, the expressive site in histocyte is accurate.
Another object of the present invention is to propose a kind of preparation method of CD34-SG17 polypeptide fragment, and its concrete operations step can may further comprise the steps:
A) provide the amino acid (Fmoc-AA) of 9-fluorenyl methoxy carbonyl acyl group protection and make its activation, obtain activated amino acid;
B) with described activated amino acid and P-hydroxymethyl phenoxy methyl poly ethenoid resin (HMP resin) reaction, obtain being connected with amino acid whose resin;
C) remove the described Fmoc protecting group that is connected with amino acid whose resin;
D) will remove the amino acid whose resin of being connected with of Fmoc protecting group and activate, with the resin after the activation and excessive activated carboxyl component coupling, and slough the amino protecting group of the postactivated amino component of coupling;
E) repeating step a) to step d) 17 times, obtains peptide resin;
F) described peptide resin is separated, obtain CD34-SG17 polypeptide compound crude product.
According to the preparation method of the CD34-SG17 polypeptide fragment of the embodiment of the invention, its building-up process is to finish at the Peptide synthesizer of automatization, and preparation technology is easy, and structure is accurate, coupling rate height, and the cycle is short, the level of automation height.
In addition, preparation method according to the above embodiment of the present invention can also have following additional technical characterictic:
According to one embodiment of present invention, in step a), described Fmoc-AA is by activating with people's Dopamine HCL decarboxylase (DCC) and hydroxybenzotriazole (HOBT) reaction.
According to one embodiment of present invention, described activated carboxyl component from left to right is followed successively by: Serine, Methionin, leucine, glutamine, leucine, methionine(Met), L-glutamic acid, Methionin, Histidine, glutamine, Serine, asparagine, leucine, arginine, Methionin, glycine and halfcystine.
According to one embodiment of present invention, step f) comprises:
F-1) react in conjunction with scavenging agent and described peptide resin with trifluoroacetic acid (TFA), so that described CD34-SG17 polypeptide fragment is separated from peptide resin;
F-2) the reaction after-filtration falls resin, and goes out described scavenging agent by underpressure distillation;
F-3) with described CD34-SG17 polypeptide fragment with water dissolution after, with extraction liquid extraction, obtain CD34-SG17 polypeptide compound crude product.
According to one embodiment of present invention, described scavenging agent is the mixing solutions of 1 (EDT), thio phenyl methyl ether and water, step f-1) reaction times be 3h.
According to one embodiment of present invention, described extraction liquid is ether.
According to one embodiment of present invention, also comprise step: g) adopt high performance liquid chromatography with the separation and purification of described CD34-SG17 polypeptide compound crude product, obtain the CD34-SG17 polypeptide fragment.
Another purpose of the present invention is to propose application and CD34-SG17 polypeptide fragment the application in preparation detection CD34 streaming antibody kit of CD34-SG17 polypeptide fragment in preparation CD34 streaming antibody.
Additional aspect of the present invention and advantage part in the following description provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Description of drawings
Above-mentioned and/or additional aspect of the present invention and advantage are from obviously and easily understanding becoming the description of embodiment in conjunction with following accompanying drawing, wherein:
Fig. 1 is the efficient liquid phase chromatographic analysis synoptic diagram according to the CD34-SG17 polypeptide of the embodiment of the invention;
Fig. 2 is that CD34 antibody according to the embodiment of the invention is at human breast carcinoma tissue immunohistochemical staining synoptic diagram;
Fig. 3 is that CD34 antibody according to the embodiment of the invention is at people's stomach organization immunohistochemical staining synoptic diagram;
Fig. 4 a, Fig. 4 b and Fig. 4 c are respectively according to the antibody of the embodiment of the invention expression synoptic diagram at anti-people, rat CD34, mouse CD34.
Embodiment
Below further describe the present invention by specific embodiment, the following example is used for illustration purpose, but not is used for limiting the scope of the invention.The test method of unreceipted actual conditions in the following example can be operated according to the common described condition of molecular cloning handbook.Used test materials in the following example if no special instructions, is and buys gained from routine biochemistry reagent shop.
CD34-SG17 polypeptide fragment according to first aspect present invention embodiment is at first described below.
According to the CD34-SG17 polypeptide fragment of first aspect present invention embodiment, its nucleotide sequence is shown in SEQ ID N0.1.By sequence table as can be seen, this polypeptide fragment is made up of 17 amino-acid residues, be Serine, Methionin, leucine, glutamine, leucine, methionine(Met), L-glutamic acid, Methionin, Histidine, glutamine, Serine, asparagine, leucine, arginine, Methionin, glycine and halfcystine, and increased a halfcystine at carboxyl terminal, can be so that coupling carrier albumen.According to the CD34-SG17 polypeptide fragment of the embodiment of the invention, wetting ability is strong, and is simple in structure, be convenient to synthetic and purifying, and this polypeptide fragment have stronger immunogenicity, good with the antibodies specific of its preparation, highly sensitive, the expressive site in histocyte is accurate.
Specifically describe the preparation method according to the CD34-SG17 polypeptide fragment of second aspect present invention embodiment below, its concrete operations step can may further comprise the steps:
A) provide the amino acid (Fmoc-AA) of 9-fluorenyl methoxy carbonyl acyl group protection and make its activation, obtain activated amino acid;
B) with described activated amino acid and P-hydroxymethyl phenoxy methyl poly ethenoid resin (HMP resin) reaction, obtain being connected with amino acid whose resin;
C) remove the described Fmoc protecting group that is connected with amino acid whose resin;
D) will remove the amino acid whose resin of being connected with of Fmoc protecting group and activate, with the resin after the activation and excessive activated carboxyl component coupling, and slough the amino protecting group of the postactivated amino component of coupling;
E) repeating step a) to step d) 17 times, obtains peptide resin;
F) described peptide resin is separated, obtain CD34-SG17 polypeptide compound crude product.
According to the preparation method of the CD34-SG17 polypeptide fragment of the embodiment of the invention, its building-up process is to finish at the Peptide synthesizer of automatization, and preparation technology is easy, and structure is accurate, coupling rate height, and the cycle is short, the level of automation height.
Particularly, in step a), described Fmoc-AA is by inferior to (DCC) and hydroxybenzotriazole (HOBT) reaction activation with dicyclohexyl carbon two.
In order to access the polypeptide fragment of CD34-SG17 structure, when carrying out coupling, described activated carboxyl component is followed successively by: Serine, Methionin, leucine, glutamine, leucine, methionine(Met), L-glutamic acid, Methionin, Histidine, glutamine, Serine, asparagine, leucine, arginine, Methionin, glycine and halfcystine.
The concrete grammar that the CD34-SG17 polypeptide fragment is separated can comprise:
F-1) react in conjunction with scavenging agent and described peptide resin with trifluoroacetic acid (TFA), so that described CD34-SG17 polypeptide fragment is separated from peptide resin;
F-2) the reaction after-filtration falls resin, and goes out described scavenging agent by underpressure distillation;
F-3) with described CD34-SG17 polypeptide fragment with water dissolution after, with extraction liquid extraction, obtain CD34-SG17 polypeptide compound crude product.
Selection about scavenging agent does not have particular restriction, as long as described CD34-SG17 polypeptide fragment can be separated from peptide resin, preferably, described scavenging agent is the mixing solutions of 1 (EDT), thio phenyl methyl ether and water.Fully carry out step f-1 for what guarantee to react) preferred 3h of reaction times.
The selection of described extraction liquid does not have particular restriction yet, considers the cost problem, and according to one embodiment of present invention, described extraction liquid is ether.
In order to obtain the higher CD34-SG17 polypeptide fragment of purity, according to one embodiment of present invention, its preparation method also comprises step: g) adopt high performance liquid chromatography with the separation and purification of described CD34-SG17 polypeptide compound crude product, obtain the CD34-SG17 polypeptide fragment.
According to the CD34-SG17 polypeptide of the embodiment of the invention can be in preparation CD34 streaming antibody application, can identify the active somatic cell surface protein by the antibody of CD34-SG17 polypeptide preparation, thereby can be used for cell streaming detection technique.Further, then the CD34-SG17 polypeptide fragment can be used in preparation detection CD34 streaming antibody kit.
Below in conjunction with preparation method and the application of specific embodiment description according to the CD34-SG17 polypeptide fragment of the embodiment of the invention.
The preparation of embodiment 1CD34-SG17 polypeptide fragment
One, experiment material
HMP resin(P-hydroxymethyl phenoxy methyl poly ethenoid resin, Wang resin) purchases the CSBio company in the U.S.
Fmoc-AA (amino acid of 9-fluorenyl methoxy carbonyl acyl group protection) purchases the company in Merck
NMP n-formyl sarcolysine base pyrrolidone is purchased the company in Merck
The DCM methylene dichloride is purchased the company in Merck
MeoH methyl alcohol is purchased the company in Merck
The Piperidine piperidines is purchased the company in Merck
The DMAP dimethyl aminopyridine is purchased the company in Sigma
The HOBT hydroxybenzotriazole is purchased the company in Sigma
The DCC dicyclohexylcarbodiimide is purchased the company in Sigma
The TFA trifluoroacetic acid is purchased the company in Sigma
EDT1,2-dithioglycol purchase the company in Sigma
The thio phenyl methyl ether is purchased the company in Sigma
Crystallization phenol is purchased in Beijing chemical reagents corporation
Acetonitrile is purchased the company in Merck
Two, laboratory apparatus
Polypeptide automatic DNA synthesizer DNA: U.S. CSBio 336 type hyperchannel polypeptide automatic DNA synthesizer DNAs
Rotary Evaporators: Japanese YAMATO CE50 type Rotary Evaporators
High performance liquid chromatograph: Waters 600 E types
Freeze drier: U.S. LABCANCO
Vacuum cycle water pump: Great Wall, the Zhengzhou SH-B of science and trade company type
Whizzer: U.S. SIGMA company
Three, experimental procedure
1, the activation of amino acid (Fmoc-AA)
The structural formula of described Fmoc-AA is as follows:
Also can be expressed as:
Described Fmoc-AA is by activating with dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBT) reaction, and its reaction formula is as follows:
2, activated amino acid is connected on the resin.
Described activated amino acid and P-hydroxymethyl phenoxy methyl poly ethenoid resin (HMP resin) are reacted under the DMAP condition, obtain being connected with amino acid whose resin, its reaction formula is:
3, remove the Fmoc protecting group that is connected with amino acid whose resin.
Under the effect of Piperidine piperidines, remove the Fmoc protecting group that is connected with amino acid whose resin, concrete reaction formula is as follows:
4, amino acid whose activation
By activating with people's dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBT) reaction, its reaction formula is as follows:
5, with step 4 gained resin and excessive activated carboxyl component coupling, and slough the amino protecting group of the postactivated amino component of coupling.
6, repeat above-mentioned steps 1 to step 5, repeat 17 times, and the activated carboxyl component of each coupling from left to right is followed successively by: halfcystine, glycine, Methionin, arginine, leucine, asparagine, Serine, glutamine, Histidine, Methionin, L-glutamic acid, methionine(Met), leucine, glutamine, leucine, Methionin and Serine just can obtain peptide resin.
7, the separation of CD34-SG17 polypeptide
React in conjunction with the scavenging agent that mixes with 1 (EDT), thio phenyl methyl ether and water and described peptide resin with trifluoroacetic acid (TFA), so that described CD34-SG17 polypeptide fragment is separated from peptide resin; The reaction after-filtration falls resin, and goes out described scavenging agent by underpressure distillation; With described CD34-SG17 polypeptide fragment with water dissolution after, use extracted with diethyl ether, obtain CD34-SG17 polypeptide compound crude product.
8, the separation and purification of CD34-SG17 polypeptide and analysis
Adopt high performance liquid chromatography with the separation and purification of described CD34-SG17 polypeptide compound crude product.
Particularly, its operational condition is:
Chromatographic instrument Waters600 U.S. Waters company
Mobile phase A-0.1%TFA(trifluoroacetic acid) H
2O
The B-0.1%TFA(trifluoroacetic acid) in 60% acetonitrile
Detect wavelength 214nm
Flow velocity 10ml/ minute
Gradient from 20-60%B in 30 minutes
Thus, can obtain purity up to 98% CD34-SG17 polypeptide.
Further, also can carry out efficient liquid phase chromatographic analysis to the CD34-SG17 polypeptide that separates after purifying, its analysis condition is:
Chromatographic column: C
184.6 * 150mm
Moving phase: the H A-0.1%TFA(trifluoroacetic acid)
2O
The B-0.1%TFA(trifluoroacetic acid) in acetonitrile
Detect wavelength: 214nm
Flow velocity: 1ml/min
Gradient from 0-60%B liquid in 30 minutes
Analytical results is seen shown in Figure 1, and analytical results shows that CD34-SG17 polypeptide purity reaches more than 98%.
Preparation and the detection of embodiment 2 CD34-SG17 antibody
One, test materials
According to embodiment 1 preparation gained CD34-SG17 polypeptide;
Experiment New Zealand's large ear rabbit.
Two, testing sequence
Because the CD34-SG17 polypeptide does not have complete immunogenicity, needs coupling carrier albumen just can be prepared into complete immunogen.Therefore, at first with CD34-SG17 polypeptide and carrier proteins (KLH, hemocyanin or keyhole limpet hemocyanin, keyhole limpet hemocyanin) coupling, coupling agent is MBS.
To test with the New Zealand large ear rabbit through fundamental immunity (Freund's complete adjuvant+polypeptide antigen is fully emulsified) with repeatedly after booster immunization (Freund+polypeptide antigen is fully emulsified) the back multiple spot immunity, the row ear vein was got blood when IgG antibody concentration peaked in serum, separation of serum obtains the antibody of CD34-SG17.
By the sensitivity of ELISA method detection antibody, namely tire.
Detect the specificity of antibody by Western blot method.
Detect the expressive site of this antibody in cell by immunohistochemical method.Its operating process is: paraffin section de-waxing is to water, through 0.01M(mol/L) after the multiple and 3% hydrogen peroxide digestion of Citric Acid repair liquid (pH6.0) hot repair, with normal lowlenthal serum sealing, hatch with having diluted primary antibodie (Anti-CD34), 4 ℃ are spent the night, the Streptavidin that adds biotin labeled goat anti-rabbit igg and HRP mark successively is at last with DAB colour developing, neutral gum mounting.Estimate to express the position: mainly be expressed in the little vascular endothelial cell in the tissue
With fluorescein PE and FITC on this IgG antibody mark, detect through flow cytometer then.Fluidic cell detects operating process: extract endothelial progenitor cells from tissue, add the mouse CD34 of the Chinese People's Anti-Japanese Military and Political College of FITC mark respectively, each 2 μ L of the mouse CD34 of the Chinese People's Anti-Japanese Military and Political College of PE mark.Blank adds the homotype control antibodies of FITC and PE mark.Hatch by flow cytometer and detect.
Three, test-results
Detect the sensitivity of antibody by the ELISA method: detected result shows that the antibody titer of CD34-SG17 reaches 1:128000, and the sensitivity of antibody is very high.
Detect the specificity of antibody by Western blot method: the molecular weight of CD34 albumen is 39kDa, do Western blot experiment with the antibody of the present invention's preparation, the result shows that molecular weight is 39kDa, identical with theoretical molecular, and do not have assorted band, prove that the specificity of antibody of the present invention is fabulous.
Detect the expressive site of this antibody in tumor tissues by immunohistochemical method, its result as shown in Figures 2 and 3, Fig. 2 represents CD34 antibody in human breast carcinoma tissue immunohistochemical staining result, and Fig. 3 represents that CD34 antibody is in people's stomach organization immunohistochemical staining result.
Its operating process is: paraffin section de-waxing is to water, after 0.01M Citric Acid repair liquid (pH6.0) hot repair digests with 3% hydrogen peroxide again, with normal lowlenthal serum sealing, hatch with having diluted primary antibodie (Anti-CD34), 4 ℃ are spent the night, the Streptavidin that adds biotin labeled goat anti-rabbit igg and HRP mark successively is at last with DAB colour developing, neutral gum mounting.
Express the position: mainly be expressed in the little vascular endothelial cell in the tissue, expressive site is accurate, and the antibody of identity basis embodiment of the invention expressive site in human breast carcinoma and people's stomach organization cell is accurate.
Detect the specificity of streaming antibody by flow cytometer.Its result is shown in Fig. 4 a, Fig. 4 b and Fig. 4 c.
Wherein, Fig. 4 a represents control group streaming result:
Homotype contrast: the normal rabbit IgG(bs-0295P-PE of PE mark)
The fluorescence intensity of X coordinate axis: PE is done cross quadrant door according to the cell imaging of contrast, negative on the vertical line left side.
Y coordinate axis: FSC-H is that forward angle detects, and is used for choosing the cell scope.
Fig. 4 b represents test group (positive group) streaming result:
Primary antibodie: the anti-CD34 antibody of the rabbit of PE mark (bs-2038R-PE)
The X coordinate axis: have the part cell to combine CD34-PE, cell mass obviously moves to right, and the cell on vertical line the right is considered to the CD34 positive.
In Fig. 4 c, the E line is represented homotype contrast (negative control), and the F line is represented the CD34-PE positive, and the Y coordinate axis is represented cell counting, and the X coordinate axis is represented the fluorescence intensity of PE, visible CD34-PE positive cell showed increased, and peak value moves to right.
Detect operating process: extract mouse boosting cell, add the anti-CD34(bs-2038R-PE of rabbit of PE mark respectively), blank adds the homotype control antibodies of PE mark.Hatch by flow cytometer and detect, a certain amount of CD34 positive cell is arranged in the mouse boosting cell.
According to the above embodiment of the present invention as can be seen, the present invention has set up a kind of very desirable immunogen polypeptide, and successfully prepare the novel method that detects antibody for the cell streaming, that is: according to structure and the function of CD34 albumen, by the albumen database analysis software, design a plurality of epitopes, adopt solid-phase peptide synthesis to synthesize the preparation that polypeptide and coupling carrier albumen are finished antigen respectively, immune animal, filter out best epitope by experiment, successfully prepare high specificity, highly sensitive, the polyclonal cell streaming of the anti-CD34 of the rabbit of good stability detects antibody.For numerous scientific research personnel provide high-quality antibody, new approaches have been opened up for exploitation streaming antibody simultaneously.
In the description of this specification sheets, concrete feature, structure, material or characteristics that the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means in conjunction with this embodiment or example description are contained at least one embodiment of the present invention or the example.In this manual, the schematic statement to above-mentioned term not necessarily refers to identical embodiment or example.And concrete feature, structure, material or the characteristics of description can be with the suitable manner combination in any one or more embodiment or example.
Although illustrated and described embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment under the situation that does not break away from principle of the present invention and aim within the scope of the invention, modification, replacement and modification.
Sequence table
SEQUENCE LISTING
<110>
＜120〉a kind of CD34-SG17 polypeptide fragment and its preparation method and application
<130> /
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> PRT
＜213〉artificial sequence
<400> 1
Ser Lys Leu Gln Leu Met Glu Lys His Gln Ser Asp Lue Arg Lys Leu Gly Cys
Claims (10)
1. a CD34-SG17 polypeptide fragment is characterized in that, the nucleotide sequence of described polypeptide fragment is shown in SEQ ID N0.1.
2. a method for preparing according to the described CD34-SG17 polypeptide fragment of claim is characterized in that, may further comprise the steps:
A) provide the amino acid (Fmoc-AA) of 9-fluorenyl methoxy carbonyl acyl group protection and make its activation, obtain activated amino acid;
B) with described activated amino acid and P-hydroxymethyl phenoxy methyl poly ethenoid resin (HMP resin) reaction, obtain being connected with amino acid whose resin;
C) remove the described Fmoc protecting group that is connected with amino acid whose resin;
D) will remove the amino acid whose resin of being connected with of Fmoc protecting group and activate, with the resin after the activation and excessive activated carboxyl component coupling, and slough the amino protecting group of the postactivated amino component of coupling;
E) repeating step a) to step d) 17 times, obtains peptide resin;
F) described peptide resin is separated, obtain CD34-SG17 polypeptide compound crude product.
3. method according to claim 2 is characterized in that, in step a), described Fmoc-AA is by activating with people's Dopamine HCL decarboxylase (DCC) and hydroxybenzotriazole (HOBT) reaction.
4. method according to claim 2, it is characterized in that described activated carboxyl component from left to right is followed successively by: Serine, Methionin, leucine, glutamine, leucine, methionine(Met), L-glutamic acid, Methionin, Histidine, glutamine, Serine, asparagine, leucine, arginine, Methionin, glycine and halfcystine.
5. method according to claim 2 is characterized in that, step f) comprises:
F-1) react in conjunction with scavenging agent and described peptide resin with trifluoroacetic acid (TFA), so that described CD34-SG17 polypeptide fragment is separated from peptide resin;
F-2) the reaction after-filtration falls resin, and goes out described scavenging agent by underpressure distillation;
F-3) with described CD34-SG17 polypeptide fragment with water dissolution after, with extraction liquid extraction, obtain CD34-SG17 polypeptide compound crude product.
6. method according to claim 5 is characterized in that, described scavenging agent is the mixing solutions of 1 (EDT), thio phenyl methyl ether and water, step f-1) reaction times be 3h.
7. method according to claim 5 is characterized in that, described extraction liquid is ether.
8. method according to claim 2 is characterized in that, also comprises step:
G) adopt high performance liquid chromatography with the separation and purification of described CD34-SG17 polypeptide compound crude product, obtain the CD34-SG17 polypeptide fragment.
9. the application of CD34-SG17 polypeptide fragment according to claim 1 in preparation CD34 streaming antibody.
10. the application of CD34-SG17 polypeptide fragment according to claim 1 in preparation detection CD34 streaming antibody kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310145927.XA CN103254294B (en) | 2013-04-24 | 2013-04-24 | CD34-SG17 polypeptide fragment as well as preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310145927.XA CN103254294B (en) | 2013-04-24 | 2013-04-24 | CD34-SG17 polypeptide fragment as well as preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103254294A true CN103254294A (en) | 2013-08-21 |
| CN103254294B CN103254294B (en) | 2015-04-01 |
Family
ID=48958547
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310145927.XA Active CN103254294B (en) | 2013-04-24 | 2013-04-24 | CD34-SG17 polypeptide fragment as well as preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103254294B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105254717A (en) * | 2015-08-18 | 2016-01-20 | 广州一代医药科技有限公司 | Polypeptide specifically binding to CD34 molecule and application thereof |
| CN115894665A (en) * | 2022-07-14 | 2023-04-04 | 北京博奥森生物技术有限公司 | THRB-CVD20 polypeptide fragment, polyclonal antibody prepared by utilizing polypeptide fragment and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1691964A (en) * | 2002-09-06 | 2005-11-02 | 曼康公司 | Epitope sequences |
| CN102021242A (en) * | 2010-10-25 | 2011-04-20 | 清华大学 | Method for analyzing epitope of protein |
-
2013
- 2013-04-24 CN CN201310145927.XA patent/CN103254294B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1691964A (en) * | 2002-09-06 | 2005-11-02 | 曼康公司 | Epitope sequences |
| CN102021242A (en) * | 2010-10-25 | 2011-04-20 | 清华大学 | Method for analyzing epitope of protein |
Non-Patent Citations (2)
| Title |
|---|
| 柏树令等: "CD34抗原的生物学特性及其临床应用", 《解剖科学进展》 * |
| 韩晓红等: "应用流式细胞仪检测CD34+细胞方法学评价", 《中国免疫学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105254717A (en) * | 2015-08-18 | 2016-01-20 | 广州一代医药科技有限公司 | Polypeptide specifically binding to CD34 molecule and application thereof |
| CN105254717B (en) * | 2015-08-18 | 2018-08-24 | 中山大学 | The polypeptide combined with CD34 molecular specificities and its application |
| CN115894665A (en) * | 2022-07-14 | 2023-04-04 | 北京博奥森生物技术有限公司 | THRB-CVD20 polypeptide fragment, polyclonal antibody prepared by utilizing polypeptide fragment and application thereof |
| CN115894665B (en) * | 2022-07-14 | 2024-01-05 | 北京博奥森生物技术有限公司 | THRB-CVD20 polypeptide fragment, polyclonal antibody prepared by using polypeptide fragment and application of polyclonal antibody |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103254294B (en) | 2015-04-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4229704B2 (en) | Fc region polypeptide binding molecule | |
| EP2578681B1 (en) | Iga-binding peptide and iga purification using same | |
| AU2002254683A1 (en) | Binding molecules for Fc-region polypeptides | |
| CN109425662A (en) | A kind of method and system for identifying albumen | |
| CA2542104A1 (en) | Method for the identification of epitopes related to immunogenicity in biopharmaceuticals | |
| CA2179562C (en) | Peptide useful in the separation and purification of immunoglobulins | |
| CN103254294B (en) | CD34-SG17 polypeptide fragment as well as preparation method and application thereof | |
| EP2614371B1 (en) | Method for labeling of compounds | |
| CN115850448B (en) | Lactoferrin polypeptide fragment, preparation method thereof, prepared antibody and application | |
| CN108084047A (en) | A kind of propionyl for preparing specificity methylates lysine ubiquitin antibody method | |
| Verdoliva et al. | Affinity puricication of polyclonal antibodies using immobilized multimeric peptides | |
| RU2014102721A (en) | METHOD FOR OBTAINING AN AGENT CONNECTING TO PREPRO-VAZOPRESSIN OR ITS FRAGMENTS | |
| EP1094080A3 (en) | Anti-dnase-gamma antibody, and production and use thereof | |
| CN108148124A (en) | A kind of Human HNRPA 0 polypeptide and its preparation method for antibody | |
| CN102993276B (en) | Polypeptide derivative with immunocompetence as well as preparation method and application of internal reference antibody of polypeptide derivative with immunocompetence | |
| CN101928332A (en) | Preparation method of Human HNRPA0 polypeptide and antibody thereof | |
| CN107091928A (en) | A kind of affine filler of cymbidium mosaic virus antibody and preparation method thereof | |
| KR20120093151A (en) | Improved screening of biopolymers | |
| CN103111093B (en) | The preparation of flag peptide tag recombinant protein immunoaffinity purification enriching column and application thereof | |
| RU2356576C1 (en) | SYNTHETIC ANTIGEN ABILITY TO BIND β1-ADRENORECEPTOR AUTOANTIBODIES | |
| EP0779297A1 (en) | Antigenic peptides with glycine substitutions | |
| CN114437176B (en) | Novel light labeling reagent and application thereof in cell surface proteome and N-glycosylation enrichment analysis | |
| CN101955510A (en) | Affinity peptide specific to herceptin and segment thereof | |
| CN102206254B (en) | Lysine decarboxylase epitope, anti-decarboxylase epitope antibody and application thereof | |
| CN101928334A (en) | Preparation method of mouse Ehf polypeptide and antibody thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |