CN108117590A - The preparation method of one species specificity, 61 threonine phosphorylation antibody of anti-Kif4A protein 11s and application - Google Patents
The preparation method of one species specificity, 61 threonine phosphorylation antibody of anti-Kif4A protein 11s and application Download PDFInfo
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- CN108117590A CN108117590A CN201611079249.1A CN201611079249A CN108117590A CN 108117590 A CN108117590 A CN 108117590A CN 201611079249 A CN201611079249 A CN 201611079249A CN 108117590 A CN108117590 A CN 108117590A
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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Abstract
Preparation method and application the invention discloses a species specificity 61 threonine phosphorylation antibody of anti-Kif4A protein 11s, the anti-Kif4A albumen T1161 phospho-ABs of specificity are of great significance to furtheing investigate influence of its phosphorylation state to the adjusting of Kif4 itself Functions and to entire cell mitogen process, the Antibody preparation and application are basic regulation mechanism of the phosphorylation state of announcement Kif4 molecules during cell cycle mitosis, its relation with cancer generation of preliminary analysis, to the basic law of profound understanding cell mitogen, and the design of the treatment and prevention of tumour strategy based on Kif4 molecular targets provides Research foundation.
Description
Technical field
The present invention relates to biological medicine technology application more particularly to a species specificity 61 threonines of anti-Kif4A protein 11s
The preparation method of phospho-AB and application.
Background technology
The research of driving protein molecular is the another hot spot of life science in recent years.Not only have to the announcement of its function
Help deep understand normal cell growth, tissue development and organ dysfunction or many disease development fundamental mechanisms
It discloses and new approaches is provided.Chromosome drives protein molecular Kif4A to be positioned at chromosome and spindle in the cell mitogen phase,
Participate in chromatic agglutination, chromosome separation, spindle is formed and cytokinesis process.Therefore for the anti-Kif4A albumen of specificity
T1161 phospho-ABs are to furtheing investigate its phosphorylation state to the adjusting of Kif4 itself Functions and having to entire cell
The influence of silk fission process is of great significance.
The content of the invention
The present invention overcomes in the prior art the shortcomings that, provide a species specificity 61 threonines of anti-Kif4A protein 11s
The preparation method of phospho-AB and application.
In order to solve the above-mentioned technical problem, the present invention is achieved by the following technical solutions：
A kind of Kif4A-T1161 phosphorylated polypeptide has the amino acid sequence Ser-Phe- as described in SEQ ID No.1
Phe-Asn-Pro-Val-Cys-Ala-Thr-Pro-Asn-Ser-Lys-Ile-Leu-Lys- Flu-Met-Cys, wherein ammonia of reviving
Acid carries out phosphorylation, i.e. one phosphate radical of tyre.
A kind of Kif4A-T1161 phosphorylations antigen, by the Kif4A-T1161 phosphorylated polypeptide and hemocyanin KLH
Prepared by crosslinking forms, and is carried out according to following specific steps：
(1) G-25 cross-link dextrans 800rpm, 5min are centrifuged, abandons supernatant, added in and buffered with the isometric PBS of glucan
Liquid mixing；
(2) 134 microlitres of KLH are added in into 25ml vials, the PBS that 1866 microlitres of pH are 6.0, final KLH are final concentration of
10mg/ml；
(3) claim 0.006g MBS, be dissolved in 200 microlitres of DMSO；
(4) under agitation, the MBS dispersions of step (3) are slowly added into the KLH dispersions of step (2)
In, it is slowly added in a manner that liquid-transfering gun pipette tips point is extended below liquid level, persistently stirs 30min；
(5) a 25ml plastic suction pipet (as chromatographic column) is taken, is cut off with scissors suitable for reading, glass fibre blocks Xia Kou,
The abundant mixing of G25 cross-link dextrans in step (1) is poured into pipe, pure glucan about 5-6ml is 7.4 using 50ml pH
PBS cleaning albumen affinity column；
(6) agitated finely dispersed KLH-MBS mixed liquors in step (4) are added in into the pillar that step (5) prepares；
(7) after KLH-MBS mixed liquors fully enter pillar, washed 1 time with pH 7.4PBS, start simultaneously at the EP with 1.5ml
Pipe connects liquid below, often pipe 1ml, using BCA determination of protein concentration kit measurement protein concentrations, selects highest 3 pipe of concentration
Liquid is mixed, and is crosslinked for following polypeptides；
(8) by the 10mg polypeptides (amino acid sequence described in SEQ ID No.1：Ser-Phe-Phe-Asn-Pro-Val-
Cys-Ala-Thr-Pro-Asn-Ser-Lys-Ile-Leu-Lys-Flu-Met-Cys 200 microlitres of pH 7.4PBS dissolvings) are added in,
The KLH mixtures that the polypeptide addition 1.5ml of dissolving is obtained by step (7), stir 1h；
(9) will pass through step (8) crosslinked substance pH 7.4PBS dialysis 12 it is small when, dialyzate (pH is replaced per 12h
7.4PBS), each 800ml；The liquid after dialysis is taken out, adds in pH 7.4PBS to total volume 10ml, often pipe 1ml is dispensed, and -80
Degree freezes.
The preparation method of the anti-61 threonine phosphorylation antibody of Kif4A protein 11s of one species specificity, according to the following steps into
Row：
Step 1 prepares polypeptide and the crosslinked antigens of KLH：
(1) G-25 cross-link dextrans 800rpm, 5min are centrifuged, abandons supernatant, added in and buffered with the isometric PBS of glucan
Liquid mixing；
(2) 134 microlitres of KLH are added in into 25ml vials, the PBS that 1866 microlitres of pH are 6.0, final KLH are final concentration of
10mg/ml；
(3) claim 0.006g MBS, be dissolved in 200 microlitres of DMSO；
(4) under agitation, the MBS dispersions of step (3) are slowly added into the KLH dispersions of step (2)
In, it is slowly added in a manner that liquid-transfering gun pipette tips point is extended below liquid level, persistently stirs 30min；
(5) a 25ml plastic suction pipet (as chromatographic column) is taken, is cut off with scissors suitable for reading, glass fibre blocks Xia Kou,
The abundant mixing of G25 cross-link dextrans in step (1) is poured into pipe, pure glucan about 5-6ml is 7.4 using 50ml pH
PBS cleaning albumen affinity column；
(6) agitated finely dispersed KLH-MBS mixed liquors in step (4) are added in into the pillar that step (5) prepares；
(7) after KLH-MBS mixed liquors fully enter pillar, washed 1 time with pH 7.4PBS, start simultaneously at the EP with 1.5ml
Pipe connects liquid below, often pipe 1ml, using BCA determination of protein concentration kit measurement protein concentrations, selects highest 3 pipe of concentration
Liquid is mixed, and is crosslinked for following polypeptides；
(8) by the 10mg polypeptides (amino acid sequence described in SEQ ID No.1：Ser-Phe-Phe-Asn-Pro-Val-
Cys-Ala-Thr-Pro-Asn-Ser-Lys-Ile-Leu-Lys-Flu-Met-Cys 200 microlitres of pH 7.4PBS dissolvings) are added in,
The KLH mixtures that the polypeptide addition 1.5ml of dissolving is obtained by step (7), stir 1h；
(9) will pass through step (8) crosslinked substance pH 7.4PBS dialysis 12 it is small when, dialyzate (pH is replaced per 12h
7.4PBS), each 800ml；The liquid after dialysis is taken out, adds in pH 7.4PBS to total volume 10ml, often pipe 1ml is dispensed, and -80
Degree freezes.
Step 2：Antigen emulsifies
The antigen dialysed prepared by step 1 and Freund's complete adjuvant and incomplete Freund's adjuvant, according to 1：1 volume ratio
Uniformly mixing is made antigen and Freund's complete adjuvant mixed liquor, antigen and incomplete Freund's adjuvant mixed liquor, is operating respectively
In, Freund's adjuvant is added drop-wise in antigen, 4 DEG C shake up 2 it is small when, vibration, ultrasound, to formation Water-In-Oil droplet.
Step 3：Animal immune
The male new zealand white rabbit of 6 weeks or so of health is selected, animal house, which is raised one week, makes its suitable environment.
Before experiment, 1.5ml blood is taken as preimmune serum in the auricular vein of rabbit.
It is first week, (i.e. real at the back of rabbit and groin injection 200-300ug antigens and Freund's complete adjuvant mixed liquor
Apply the antigen emulsified in example 2).
3rd week, inject 200ug antigens and incomplete Freund's adjuvant mixed liquor.
6th week, inject 200ug antigens and incomplete Freund's adjuvant mixed liquor.It is drawn blood, prepared with White Rabbit auricular vein
Serum (containing special anti-Kif4A-T1161 phospho-ABs).
Period, the 5th week, ear edge took blood.Take the potency and specificity of Virus monitory antibody.7th week, ear edge took blood.It takes
The potency and specificity of Virus monitory antibody.
Compared with prior art, the beneficial effects of the invention are as follows：The anti-Kif4A albumen T1161 phospho-ABs pair of specificity
Further investigate influence of its phosphorylation state to the adjusting of Kif4 itself Functions and to entire cell mitogen process
It is of great significance, the Antibody preparation and application are to disclose the phosphorylation state of Kif4 molecules in cell cycle mitosis process
In basic regulation mechanism, its relation for occurring with cancer of preliminary analysis, to the basic law of profound understanding cell mitogen,
And the design of the treatment and prevention of tumour strategy based on Kif4 molecular targets provides Research foundation.
Description of the drawings
Fig. 1 is the anti-Kif4A albumen T1161 phospho-ABs specific recognition mitotic cell of polypeptide competitiveness experimental verification
The Kif4A of middle T1161 phosphorylations.
Fig. 2 handles the anti-Kif4A albumen T1161 phospho-ABs spy of experimental verification for protein kinase C DK micromolecular inhibitors
The Kif4A of T1161 phosphorylations in different identification mitotic cell.
Fig. 3 is that the T1161 point mutation Protein Detection of exogenous expression verifies that anti-Kif4A albumen T1161 phospho-ABs are special
The Kif4A of different identification specific recognition T1161 phosphorylations.
Specific embodiment
The present invention is described in further detail with specific embodiment below in conjunction with the accompanying drawings：
Used drug and instrument are as follows in following embodiment：
G-25 cross-link dextrans are purchased from Hefei Lan Xu Bioisystech Co., Ltd,
KLH (hemocyanin), MBS (3- maleimide yl benzoic acid succinimide esters crosslinking agent), DMSO (dimethyl
Sulfoxide), Freund's complete adjuvant, incomplete Freund's adjuvant be purchased from Sigma-Aldrich Co., Ltd,
Albumen affinity column is purchased from Shenzhen comma Bioisystech Co., Ltd,
BCA determination of protein concentration kit is purchased from Beijing Suo Laibao Science and Technology Ltd,
Embodiment 1-prepare polypeptide and the crosslinked antigens of KLH
(1) G-25 cross-link dextrans 800rpm, 5min are centrifuged, abandons supernatant, added in and buffered with the isometric PBS of glucan
Liquid mixing；
(2) 134 microlitres of KLH are added in into 25ml vials, the PBS that 1866 microlitres of pH are 6.0, final KLH are final concentration of
10mg/ml；
(3) claim 0.006g MBS, be dissolved in 200 microlitres of DMSO；
(4) under agitation, the MBS dispersions of step (3) are slowly added into the KLH dispersions of step (2)
In, it is slowly added in a manner that liquid-transfering gun pipette tips point is extended below liquid level, persistently stirs 30min；
(5) a 25ml plastic suction pipet (as chromatographic column) is taken, is cut off with scissors suitable for reading, glass fibre blocks Xia Kou,
The abundant mixing of G25 cross-link dextrans in step (1) is poured into pipe, pure glucan about 5-6ml is 7.4 using 50ml pH
PBS cleaning albumen affinity column；
(6) agitated finely dispersed KLH-MBS mixed liquors in step (4) are added in into the pillar that step (5) prepares；
(7) after KLH-MBS mixed liquors fully enter pillar, washed 1 time with pH 7.4PBS, start simultaneously at the EP with 1.5ml
Pipe connects liquid below, often pipe 1ml, using BCA determination of protein concentration kit measurement protein concentrations, selects highest 3 pipe of concentration
Liquid is mixed, and is crosslinked for following polypeptides；
(8) 10mg polypeptides (amino acid sequence described in SEQ ID No.1) are added in into 200 microlitres of pH 7.4PBS dissolvings, it will
The polypeptide of dissolving adds in 1.5ml and passes through the KLH mixtures that step (7) obtains, and stirs 1h；
(9) will pass through step (8) crosslinked substance pH 7.4PBS dialysis 12 it is small when, dialyzate (pH is replaced per 12h
7.4PBS), each 800ml；The liquid after dialysis is taken out, adds in pH 7.4PBS to total volume 10ml, often pipe 1ml is dispensed, and -80
Degree freezes.
2-antigen of embodiment emulsifies
The antigen dialysed prepared by embodiment 1 and Freund's complete adjuvant and incomplete Freund's adjuvant, according to 1：1 volume
Than uniformly mixing, antigen and Freund's complete adjuvant mixed liquor, antigen and incomplete Freund's adjuvant mixed liquor are made respectively, is operating
In, Freund's adjuvant is added drop-wise in antigen, 4 DEG C shake up 2 it is small when, vibration, ultrasound, to formation Water-In-Oil droplet.
3-animal immune of embodiment
A. the male new zealand white rabbit of 6 weeks or so of health is selected, animal house, which is raised one week, makes its suitable environment.
B. before testing, 1.5ml blood is taken as preimmune serum in the auricular vein of rabbit.
It is first week, (i.e. real at the back of rabbit and groin injection 200-300ug antigens and Freund's complete adjuvant mixed liquor
Apply the antigen emulsified in example 2).
3rd week, inject 200ug antigens and incomplete Freund's adjuvant mixed liquor.
5th week, ear edge took blood.Take the potency and specificity of Virus monitory antibody.
6th week, inject 200ug antigens and incomplete Freund's adjuvant mixed liquor.
7th week, ear edge took blood.Take the potency and specificity of Virus monitory antibody.
It is drawn blood with White Rabbit auricular vein, prepares serum (containing special anti-Kif4A-T1161 phospho-ABs)
Embodiment 4-using serum progress molecular biology experiment detection
1. as shown in Figure 1, (10- is stayed overnight using 30nM taxols (Taxol) drug culture human cervical carcinoma cell HeLa
12h), suspension cell (synchronizing in the cell mitogen phase) is collected, protein electrophoresis (SDS-PAGE) and albumen are carried out after cracking
Blot hybridization (Westernblot) is tested.Application specific microtubulin-resisting (Tubulin) and anti-Kif4A antibody conduct respectively
Control shows that tubulin and Kif4A protein contents are identical in sample.In the anti-Kif4A-T1161 phospho-ABs of application specific
(pT1161) and using phosphorylated polypeptide and non-esterified polypeptide the anti-Kif4A-T1161 phospho-ABs (pT1161) neutralized
Carry out hybridization check.The anti-Kif4A-T1161 phospho-ABs (pT1161) of the results show and non-esterified polypeptide neutralize anti-
Kif4A-T1161 phospho-ABs (pT1161) can identify the Kif4A in m period cell, but be phosphorylated in polypeptide
The antibody of sum cannot identify the Kif4A albumen in m period cell.
2. as shown in Figure 2, using 30nM taxols (Taxol) and differential protein kinase c DK micromolecular inhibitors
Roscovitine drug culture human cervical carcinoma cells HeLa is stayed overnight, and is collected suspension cell and (is synchronized in cell mitogen
Phase), protein electrophoresis (SDS-PAGE) and western blot hybridization (Westernblot) experiment are carried out after cracking.Application specific resists
Kif4A-T1161 phospho-ABs (pT1161) carry out hybridization check.The results show that after handling cell using Roscovitine,
CDK kinase activities are suppressed, and Kif4A cannot be by CDK phosphorylations, thus pT1161 antibody cannot be detected in cell pyrolysis liquid
Kif4A, opposite common anti-Kif4A antibody can detect the Kif4A albumen in cell pyrolysis liquid.
3. as shown in Figure 3, Kif4A-T1161 albumen (wild-type protein WT) is mutated by point of application mutating technology
The expression matter of Kif4A-A1161 (non-phosphorylating mutant protein TA) or Kif4A-E1161 (intending phosphorylation mutant albumen TE)
Grain, when cell transfecting expresses exogenous GFP fusion proteins (WT, TA, TE) and small application taxol culture cell 24.Collect cell
Cracking carries out protein electrophoresis and western blot hybrid experiment.Application specific microtubulin-resisting (Tubulin) and anti-Kif4A respectively
Antibody is as endogenous tubulin in control test sample and Kif4A albumen and exogenous GFP fusion proteins.Reapply spy
The anti-Kif4A-T1161 phospho-ABs (pT1161) of the opposite sex carry out hybridization check.The results show that after taxol treatment cell, outside
The wild type GFP-Kif4AWT albumen and plan phosphorylation mutant Protein G FP-Kif4ATE of source property expression can be by pT1161
Antibody identifies that the non-phosphorylating mutant protein GFP-Kif4ATA of opposite exogenous expression cannot be identified by the antibody.
The present invention is described in detail above, but the content is only presently preferred embodiments of the present invention, it is impossible to be recognized
To be used to limit the practical range of the present invention.Any changes and modifications in accordance with the scope of the present application should all still return
Belong within the patent covering scope of the present invention.
Sequence table
<110>Tianjin Normal University
<120>The preparation method of one species specificity, 61 threonine phosphorylation antibody of anti-Kif4A protein 11s and application
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> PRT
<213>Artificial sequence
<400> 1
Ser Phe Phe Asn Pro Val Cys Ala Thr Pro Asn Ser Lys Ile Leu Lys
1 5 10 15
Flu Met Cys
Claims (10)
1. a kind of Kif4A-T1161 phosphorylated polypeptide, it is characterised in that：With the amino acid sequence as described in SEQ ID No.1,
Wherein threonine carries out phosphorylation.
2. a kind of Kif4A-T1161 phosphorylations antigen, it is characterised in that：By Kif4A-T1161 phosphorylated polypeptide and hemocyanin
Prepared by KLH crosslinkings forms, and Kif4A-T1161 phosphorylated polypeptide has the amino acid sequence as described in SEQ ID No.1, wherein reviving
Propylhomoserin carries out phosphorylation.
3. a kind of Kif4A-T1161 phosphorylations antigen according to claim 2 is carried out according to following specific steps：
(1) G-25 cross-link dextrans 800rpm, 5min are centrifuged, abandons supernatant, added in the PBS buffer solution isometric with glucan and mix
It is even；
(2) 134 microlitres of KLH, the PBS, the final final concentration of 10mg/ of KLH that 1866 microlitres of pH are 6.0 are added in into 25ml vials
ml；
(3) claim 0.006g MBS, be dissolved in 200 microlitres of DMSO；
(4) under agitation, the MBS dispersions of step (3) are slowly added into the KLH dispersions of step (2), adopted
It is slowly added to the mode that liquid-transfering gun pipette tips point is extended below liquid level, persistently stirs 30min；
(5) a 25ml plastic suction pipet (as chromatographic column) is taken, is cut off with scissors suitable for reading, glass fibre blocks Xia Kou, will walk
Suddenly the abundant mixing of G25 cross-link dextrans pours into pipe in (1), and pure glucan about 5-6ml is clear using the PBS that 50ml pH are 7.4
Washing protein affinity column；
(6) agitated finely dispersed KLH-MBS mixed liquors in step (4) are added in into the pillar that step (5) prepares；
(7) after KLH-MBS mixed liquors fully enter pillar, washed 1 time with pH 7.4PBS, start simultaneously at and existed with the EP pipes of 1.5ml
Liquid is connect below, often pipe 1ml, using BCA determination of protein concentration kit measurement protein concentrations, select the highest 3 pipe liquid of concentration
It is mixed, is crosslinked for following polypeptides；
(8) by the 10mg polypeptides (amino acid sequence described in SEQ ID No.1：Ser-Phe-Phe-Asn-Pro-Val-Cys-
Ala-Thr-Pro-Asn-Ser-Lys-Ile-Leu-Lys-Flu-Met-Cys, wherein threonine carry out phosphorylation) add in 200
The polypeptide of dissolving is added in the KLH mixtures that 1.5ml passes through step (7) and obtain, stirs 1h by microlitre pH 7.4PBS dissolving；
(9) will pass through step (8) crosslinked substance pH 7.4PBS dialysis 12 it is small when, dialyzate (pH is replaced per 12h
7.4PBS), each 800ml；The liquid after dialysis is taken out, adds in pH 7.4PBS to total volume 10ml, often pipe 1ml is dispensed, and -80
Degree freezes.
4. a kind of preparation method of Kif4A-T1161 phosphorylations antigen, it is characterised in that：It is carried out according to following specific steps：
(1) G-25 cross-link dextrans 800rpm, 5min are centrifuged, abandons supernatant, added in the PBS buffer solution isometric with glucan and mix
It is even；
(2) 134 microlitres of KLH, the PBS, the final final concentration of 10mg/ of KLH that 1866 microlitres of pH are 6.0 are added in into 25ml vials
ml；
(3) claim 0.006g MBS, be dissolved in 200 microlitres of DMSO；
(4) under agitation, the MBS dispersions of step (3) are slowly added into the KLH dispersions of step (2), adopted
It is slowly added to the mode that liquid-transfering gun pipette tips point is extended below liquid level, persistently stirs 30min；
(5) a 25ml plastic suction pipet (as chromatographic column) is taken, is cut off with scissors suitable for reading, glass fibre blocks Xia Kou, will walk
Suddenly the abundant mixing of G25 cross-link dextrans pours into pipe in (1), and pure glucan about 5-6ml is clear using the PBS that 50ml pH are 7.4
Washing protein affinity column；
(6) agitated finely dispersed KLH-MBS mixed liquors in step (4) are added in into the pillar that step (5) prepares；
(7) after KLH-MBS mixed liquors fully enter pillar, washed 1 time with pH 7.4PBS, start simultaneously at and existed with the EP pipes of 1.5ml
Liquid is connect below, often pipe 1ml, using BCA determination of protein concentration kit measurement protein concentrations, select the highest 3 pipe liquid of concentration
It is mixed, is crosslinked for following polypeptides；
(8) by the 10mg polypeptides (amino acid sequence described in SEQ ID No.1：Ser-Phe-Phe-Asn-Pro-Val-Cys-
Ala-Thr-Pro-Asn-Ser-Lys-Ile-Leu-Lys-Flu-Met-Cys, wherein threonine carry out phosphorylation) add in 200
The polypeptide of dissolving is added in the KLH mixtures that 1.5ml passes through step (7) and obtain, stirs 1h by microlitre pH 7.4PBS dissolving；
(9) will pass through step (8) crosslinked substance pH 7.4PBS dialysis 12 it is small when, dialyzate (pH is replaced per 12h
7.4PBS), each 800ml；The liquid after dialysis is taken out, adds in pH 7.4PBS to total volume 10ml, often pipe 1ml is dispensed, and -80
Degree freezes.
A 5. species specificity 61 threonine phosphorylation antibody of anti-Kif4A protein 11s, which is characterized in that with Kif4A-T1161 phosphorus
After acidifying antigen is emulsified, it is immunized and prepares in animal, Kif4A-T1161 phosphorylations antigen is by Kif4A-T1161 phosphorylations
Polypeptide is prepared with hemocyanin KLH crosslinkings and formed, and Kif4A-T1161 phosphorylated polypeptide has the ammonia as described in SEQ ID No.1
Base acid sequence, wherein threonine carry out phosphorylation.
6. anti-61 threonine phosphorylation antibody of Kif4A protein 11s of a species specificity according to claim 5, feature exist
In carrying out as steps described below：
Step 1：Antigen emulsifies
Kif4A-T1161 phosphorylations antigen is uniformly mixed with Freund's complete adjuvant according to isometric ratio, by Kif4A-T1161 phosphorus
Antigen is acidified with incomplete Freund's adjuvant according to 1：1 volume ratio uniformly mixes, and antigen is made respectively and is mixed with Freund's complete adjuvant
Liquid, antigen and incomplete Freund's adjuvant mixed liquor,
Step 2, animal immune
New zealand white rabbit is selected to carry out animal immune, 1.5ml blood is taken as preimmune serum in the auricular vein of rabbit；First
Week, at the back of rabbit and groin injection 200-300ug antigens and Freund's complete adjuvant mixed liquor；3rd week, inject 200ug
Antigen and incomplete Freund's adjuvant mixed liquor；6th week, 200ug antigens and incomplete Freund's adjuvant mixed liquor are injected, in animal
Immunologic process middle ear edge vein haemospasia prepares serum, contains special anti-Kif4A-T1161 phospho-ABs in serum.
7. the preparation method of the anti-61 threonine phosphorylation antibody of Kif4A protein 11s of a species specificity, which is characterized in that under
State step progress：
Step 1：Antigen emulsifies
Kif4A-T1161 phosphorylations antigen is uniformly mixed with Freund's complete adjuvant according to isometric ratio, by Kif4A-T1161 phosphorus
Antigen is acidified with incomplete Freund's adjuvant according to 1：1 volume ratio uniformly mixes, and antigen is made respectively and is mixed with Freund's complete adjuvant
Liquid, antigen and incomplete Freund's adjuvant mixed liquor,
Step 2, animal immune
New zealand white rabbit is selected to carry out animal immune, 1.5ml blood is taken as preimmune serum in the auricular vein of rabbit；First
Week, at the back of rabbit and groin injection 200-300ug antigens and Freund's complete adjuvant mixed liquor；3rd week, inject 200ug
Antigen and incomplete Freund's adjuvant mixed liquor；6th week, 200ug antigens and incomplete Freund's adjuvant mixed liquor are injected, in animal
Immunologic process middle ear edge vein haemospasia prepares serum, contains special anti-Kif4A-T1161 phospho-ABs in serum.
8. the preparation method according to right will go 7, which is characterized in that in step 1, Freund's adjuvant or Freund is endless
Full adjuvant is added drop-wise in antigen, 4 DEG C shake up 2 it is small when, vibration, ultrasound, to the droplet of formation Water-In-Oil.
9. a kind of Kif4A- described in a kind of Kif4A-T1161 phosphorylated polypeptide described in claim 1 or claim 2
A species specificity 61 threonine phosphorylation antibody of anti-Kif4A protein 11s described in T1161 phosphorylations antigen or claim 5
Application in studying its phosphorylation state and being adjusted to Kif4 itself Functions, in phosphorylation Kif4 in cytofilament fission process
Application in middle regulation mechanism research.
10. a kind of Kif4A- described in a kind of Kif4A-T1161 phosphorylated polypeptide described in claim 1 or claim 2
A species specificity 61 threonine phosphorylation antibody of anti-Kif4A protein 11s described in T1161 phosphorylations antigen or claim 5
In the application that phosphorylation Kif4 is related in cell mitogen and cancer in research, in the tumour based on Kif4 molecular targets
Application in protective agents preparation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611079249.1A CN108117590A (en) | 2016-11-30 | 2016-11-30 | The preparation method of one species specificity, 61 threonine phosphorylation antibody of anti-Kif4A protein 11s and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611079249.1A CN108117590A (en) | 2016-11-30 | 2016-11-30 | The preparation method of one species specificity, 61 threonine phosphorylation antibody of anti-Kif4A protein 11s and application |
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Cited By (2)
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| CN108840920A (en) * | 2018-06-19 | 2018-11-20 | 中山大学孙逸仙纪念医院 | A kind of human CYR 61 Protein S er167 site phosphorylation antigen, antibody and its preparation method and application |
| CN110294806A (en) * | 2019-07-08 | 2019-10-01 | 徐州医科大学 | A kind of phospho-AB and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108840920A (en) * | 2018-06-19 | 2018-11-20 | 中山大学孙逸仙纪念医院 | A kind of human CYR 61 Protein S er167 site phosphorylation antigen, antibody and its preparation method and application |
| CN108840920B (en) * | 2018-06-19 | 2021-09-28 | 中山大学孙逸仙纪念医院 | Human CYR61 protein Ser167 site phosphorylation antigen and antibody, and preparation method and application thereof |
| CN110294806A (en) * | 2019-07-08 | 2019-10-01 | 徐州医科大学 | A kind of phospho-AB and its application |
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