CN108277214A - One kind stress phosphorylation antigen polypeptide, antibody, preparation method and application - Google Patents
One kind stress phosphorylation antigen polypeptide, antibody, preparation method and application Download PDFInfo
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- CN108277214A CN108277214A CN201810154814.9A CN201810154814A CN108277214A CN 108277214 A CN108277214 A CN 108277214A CN 201810154814 A CN201810154814 A CN 201810154814A CN 108277214 A CN108277214 A CN 108277214A
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- phosphorylation
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
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- C12N9/6489—Metalloendopeptidases (3.4.24)
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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Abstract
It stress phosphorylation antigen polypeptide, antibody, preparation method and application the present invention provides one kind, the phosphorylation antigen polypeptide, it is characterized in that the antigen polypeptide include the 458th threonine site of people MMP9 albumen near 15 peptides as candidate polypeptide, wherein the threonine in the sites T184 is phosphorylation state, and C-terminal connects the amino acid sequence of a cysteine.The present invention also provides the antibody and its preparation method and application prepared by above-mentioned antigen polypeptide, include the kit and method for detecting cancer of the antibody.Antibody prepared by the present invention can detect stress under environment MMP9 albumen phosphorylation modification, to inquire into MMP9 albumen T458 site phosphorylations under stressed condition potential utility is provided for the diagnosis and treatment of clinical tumor disease to the influence during tumor cell proliferation, migration etc..
Description
Technical field
The present invention relates to antibody production techniques fields, and in particular to it is a kind of for the sites people MMP9 albumen T458 stress phosphorus
It is acidified antigen polypeptide, antibody, preparation method and application.
Background technology
Metastases are considered as the process of a multi-step, need the collective effect of multiple genes and complete.Certain spies
Fixed gene can allow tumour cell to fall off from primary tumo(u)r, adhere to endothelial cell, invade extracellular matrix
(extracellular matrix, ECM), tumour cell pass through blood vessel, and mushroom out, finally orga- nogenesis one a long way off
A new tumour.And matrix metalloproteinase (Matrix metalloproteinases, MMPs) is the weight for participating in this process
Want gene.MMPs is the major physiological medium of ECM degradations.Matrix metalloproteinase is family's zinc dependence endopeptidase, main
Function is degradation ECM.MMPs participates in a variety of physiology and pathologic process, such as form generation, wound healing, tissue repair and remodeling].
In addition, MMPs increases the growth of cell by performance, migrate, intrusion, the ability of transfer and angiogenesis, therefore invading in tumour
It attacks and plays an important role with transfer.MMP9 is type Ⅳ collagenase, also known as gelatinase, is secreted with inactive zymogen forms,
Gelatin and multiple protein after hydrolysis activates in degradable ECM, play an important role in tumor-infiltrated and transfer.
Matrix Metallopeptidase 9 (MMP9) is also referred to as 92kDa IV Collagenase Types or Gelatinase B (GELB), is MMP families enzyme
Member, be responsible for degradation denaturation and basilar memebrane collagen and pass through process soluble protein promote inflammation, soluble protein
Including protease inhibitors, chemotactic factor (CF) and cell factor.MMP9 also by film binding molecule (such as growth factor precursor and by
Body, tyrosine kinase receptor (TKR), cell adhesion molecule proteolysis control the migration, invasion and transfer of tumour cell.
In disease, MMP9 is secreted by many cell types including leucocyte, such as neutrophil cell, monocyte/macrophage are thin
It is born of the same parents and lymphocyte and fibroblast, myofibroblast, epithelial cell, smooth muscle cell, endothelial cell, osteoclastic thin
Born of the same parents and tumour cell.
Clinical and experimental evidence shows that MMP9 levels increase and cancer development, transfer and the patient survival of shortening phase
Connection, because it is played a crucial role by digesting basilar memebrane and extracellular matrix components in tumor cell invasion and transfer.With people
The neutrophil gelatinase-associated lipocalin (NGAL) that MMP9 in neutrophil cell is covalently attached protects MMP9
From proteolytic degradation and increases the enzymatic activity of MMP9 and then enhance tumor invasion and diffusion.Serum middle and high concentration
MMP9/NGAL compounds are related to progression free survival phase shorter in clear cell renal cell carcinoma and poor Overall survival.
The effect of MMP-9 is related to colorectal cancer, cancer of pancreas, breast cancer, lung cancer, oophoroma, carcinoma of urinary bladder and gastric cancer.
Invention content
The invention discloses one kind stress phosphorylation antigen polypeptide, antibody, preparation method and application, for MMP9 albumen
The antigen polypeptide of T458 phosphorylation sites, can the expression of specific recognition human tumor cells MMP9 albumen T458 phosphorylation sites
Polyclonal antibody with and preparation method thereof, apply the specific recognition and cancer cell detection kit and cancer in cancer cell
The detection method of cell.
The first aspect of the present invention provides a kind of phosphorylation antigen polypeptide, it is characterised in that the antigen polypeptide is behaved
15 peptides screen to obtain as candidate polypeptide near the 458th threonine site of MMP9 albumen, wherein Soviet Union's ammonia in the sites T184
Acid is phosphorylation state, and C-terminal connects the amino acid sequence of a cysteine.
The second aspect of the present invention provide it is a kind of for the sites people MMP9 albumen T458 stress phospho-AB, it is special
Sign is that the antibody is the antibody for people's MMP9 albumen T458 site amino acids phosphorylations, wherein being adopted in the Antibody preparation
It is with the antigen polypeptide being made of following amino acid sequence, particular sequence：CEPRPPTTTT (p) PQPT, wherein T (p) tables
It lets others have a look at MMP9 albumen T458 site amino acids phosphorylation threonines, C-terminal connects a cysteine.
The third aspect of the present invention provide it is a kind of for the sites people MMP9 albumen T458 stress phosphorylation Anti-TNF-α
The preparation method of body, it is characterised in that including (1) to the secondary structure of the 458th location proximate amino acid sequence of MMP9 albumen, exempt from
Epidemic focus, hydrophilic and hydrophobic, surface accessibility etc. are analyzed, and it is artificial synthesized to determine that suitable one section of peptide sequence carries out；(2) it will close
At the carrier mcKLH of polypeptide and maleimide ammonia activation be coupled, this coupled product is carried out desalting column is immunized new west after purification
Blue rabbit；(3) antibody titer is detected with ELISA method by four immune rabbit anteserums, potency is collected and exempted from after reaching ideal value
The agarose affinity purification column purification antibody of the coated cyanogen bromide-activated of polypeptide is used in combination in epidemic disease rabbit anteserum；(4) to purified antibodies into
Row ELISA, western bot are identified.
The fourth aspect of the present invention provide it is a kind of for people MMP9 albumen T458 sites stress phospho-AB answer
With wherein described carry out specific recognition cancer cell expression using according to the antibody of claim 2 or claim 3 preparation
The sites people MMP9 albumen T458.Wherein the cancer cell is preferably human nasopharyngeal carcinoma, cervical carcinoma, gastric cancer, glioma cell.
A kind of cancer cell detection kit of fifth aspect present invention, including quotient it is above-mentioned be directed to the sites people MMP9 albumen T458
Stress phospho-AB, antigen retrieval buffers, PBS buffer solutions, enzyme blocking agent 3%H2O2, horseradish enzyme mark sheep anti-Mouse/rabbit
IgG polymer, DAB color developing agents, haematoxylin dye liquor, ethyl alcohol, environment friendly transparent agent, 0.5% ammonium hydroxide and ultra-pure water.
The present invention also provides a kind of methods of cancer cell detection, it is characterised in that uses above-mentioned cancer cell detection reagent
Box includes the following steps：
(1) by organizational routine paraffin section to be detected, by environment friendly transparent agent, ethyl alcohol distinguishes shaking table dewaxing, ultra-pure water drift
It washes；
(2) antigen retrieval buffers are heated to boiling, paraffin section is put into the antigen retrieval buffers of boiling, it is medium-to-high grade
Microwave treatment, room temperature cooling are placed in ultra-pure water, impregnate, shaken wash 3 times with PBS later；
(3) sample is placed in endogenous peroxydase blocking agent 3%H2O2, is protected from light incubation at room temperature, PBS buffer solution is shaken
It washes；
(4) take out slice, be added dropwise by it is diluted for the sites people MMP9 albumen T458 stress phospho-AB, be put into
It is incubated in box, 4 DEG C of refrigerator overnights are put into PBS buffer solution and fully wash；
(5) horseradish enzyme mark sheep anti-Mouse/rabbit igg polymer, incubation at room temperature is added dropwise after drying tissue surrounding liquid, PBS delays
Fliud flushing is washed, and is added dropwise and is now matched appropriate DAB color developing agents, color development at room temperature, tap water color development stopping, is dyed, is washed in haematoxylin dye liquor
Slice is placed in 0.5% ammonium hydroxide afterwards and is impregnated, continues to wash；
(6) slice is sequentially placed into ethyl alcohol, slice is placed in clarifier after taking-up, with neutral gum mounting, optics is aobvious
Micro- microscopic observation.
According to previous work as a result, inventor predicts that MMP9 the 458th threonine (T458) of albumen is a potential phosphorus
Polyadenylation sites, may be related to the stabilization of the albumen and activation function.The present invention contains the phosphoric acid using engineer and synthesis
One section of MMP9 polypeptide (pT458) for changing site is coupled, warp with the keyhole blood indigo plant carrier protein (KLH) of maleic amide activation
New zealand rabbit is immunized after desalting and purifying, after four times immune and ELISA bioactivities, acquisition rabbit anteserum is simultaneously coated through polypeptide
The agarose gel purification column purification of cyanogen bromide-activated.The polyclonal antibody, can by identifications such as ELISA, western blot
The sites MMP9 albumen pT458 of specific recognition human nasopharyngeal carcinoma CNE1 cells expression.
The present invention has selected MMP9 albumen comprising 15 peptides near the 458th threonine site (T458) as candidate more
Peptide, and the Peptide systhesis comprising pT458 and comlete antigen preparation are carried out by artificial means.To the 458th location proximate of MMP9 albumen
Secondary structure, immunogenicity, hydrophilic and hydrophobic, surface accessibility of amino acid sequence etc. are analyzed, and determine suitable one section of peptide
Sequence carries out artificial synthesized；The carrier mcKLH of the polypeptide of synthesis and the activation of maleimide ammonia is coupled, this coupled product is carried out
New zealand rabbit is immunized in desalting column after purification；Antibody titer is detected with ELISA method by four immune rabbit anteserums, potency
It is collected after up to ideal value and rabbit anteserum is immunized, the agarose (CNBr-activated of the coated cyanogen bromide-activated of polypeptide is used in combination
Sepharose) affinity purification column purification antibody；The identifications such as ELISA, western bot are carried out to purified antibodies.Qualification result
Show the polyclonal antibody can the sites specific recognition MMP9 albumen pT458, can be used for detecting the phosphoric acid in the tumour cell site
Change level, provides a kind of tool to explore tumor cell proliferation and metastasis research, also provide help, and energy for diagnosing tumor
Its clinical prognosis is instructed to judge.
Wherein, chemically synthesized polypeptide antigen is small molecule, itself is difficult the antigenicity having had, and animal can only be induced to produce
Raw very weak immune response, thus it is critically important to be crosslinked with carrier protein.Carrier protein contains many epitopes, can
T helper cell is stimulated, and then induces B cell reaction.For with the crosslinked carrier protein of polypeptide there are many, wherein most generally using
Carrier be keyhole limpet hemocyanin (keyhole limpet hemacyanin, KLH), bovine serum albumin(BSA) (bovine serum
Albumin, BSA), ovalbumin (ovalbumin, OVA) and bovine thyroglobulin (bovine thyroglobulin, THY).
KLH has higher antigenicity, is that the most commonly used polypeptide is crosslinked carrier.BSA is also often used as peptide carrier, but due to BSA
Often be used as detection experiment blocking agent and make this method production antibody application on there is certain limitations.
Advantageous effect of the invention is：
The antigen in the present invention one section of site MMP9 albumen pT458 containing the phosphorylation site using engineer and synthesis
Polypeptide, and prepare corresponding polyclonal antibody.The polyclonal antibody can specificity by identifications such as ELISA, western blot
Identify MMP9 albumen pT458 phosphorylation sites, and relative to its high expression in multiple cancerous tissues cell of cancer beside organism, it is poor
It is different that there is statistical significance.The present invention phosphorylation polyclonal antibody can specific recognition MMP9 albumen pT458 phosphorylation sites,
The phosphorylation level that can be used for detecting the tumour cell site provides one kind to explore tumor cell proliferation and metastasis research
Tool also provides help for diagnosing tumor, and its clinical prognosis can be instructed to judge.
Description of the drawings
Fig. 1 is to analyze people's MMP9 protein characteristics using DNAstar softwares.The sequence marked in frame is the polypeptide sequence selected
Row, this polypeptide sequence are located near the 458th threonine of MMP9 albumen, and antigenicity, hydrophily and surface accessibility are stronger.
Fig. 2 is the OD values of pMMP9-Thr458 antibody and non-phosphorylating MMP9-Thr458 antibody, and ELISA detects pMMP9-
Thr458 antibody is specific phosphorylation site antibody (* P ＜ 0.05).
Fig. 3 is the western-blot qualification results of moderate resistance MMP9 (pT458) polyclonal antibody of the present invention.Loading is CNE1
Cell (CNE cell line) lysate.The cell uses pcDNA6.0/myc-His-ATF1 wild types and pcDNA6.0/ respectively
Myc-His-ATF1 (T184A) mutant plasmid is transiently transfected.
Fig. 4 is the preparation flow of the present invention.
Fig. 5 is the cellular immunity group qualification result of anti-mm P9 (pT458) polyclonal antibody；Cell is nasopharyngeal carcinoma cell
CNE2, hepatocellular carcinoma H22, stomach cancer cell MGC803.
Fig. 6 is the immunohistochemistry qualification result of moderate resistance MMP9 (pT458) polyclonal antibody of the present invention.Used sample is
Cervical cancer cell pathological section.
Fig. 7 is the immunohistochemistry qualification result of moderate resistance MMP9 (pT458) polyclonal antibody of the present invention.Used sample is
Gastric cancer and glioma cell pathological section.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate this hair
It is bright, rather than limit the scope of the invention.Under the premise of without departing substantially from technical solution of the invention, the present invention is made
Those of ordinary skill in the art's any change easy to implement fall within scope of the presently claimed invention.
Embodiment 1
Step 1：The design and synthesis of MMP9 albumen pT458 polypeptides
1.1MMP9 amino acid sequence
People MMP9 protein amino acid sequences (NP_004985) are obtained according to GenBank：
As a result：People's MMP9 albumen contains 708 amino acid.
1.2 analyze people MMP9 protein characteristics (table 1) with DNAstar softwares：
Table 1
| Analysis project (Analysis) | Holoprotein (Whole protein) |
| Molecular weight (Molecular weight) | 784580.47m.w. |
| Length (Length) | 708 |
| Isoelectric point (Isoelectric Point) | 5.82 |
As a result：People's MMP9 molecular weight of albumen is 784580.47 dalton, and isoelectric point 5.82 is acidic protein.
1.3 analyze people MMP9 protein immunogenics, hydrophilic and hydrophobic and surface accessibility (Fig. 1) with DNAstar softwares：
As a result：The 449th to the 462nd, people MMP9 albumen totally 14 amino acid antigenicities, hydrophily and surface accessibility
It is relatively strong, and the 458th threonine includes wherein.
1.4 MMP9 synthetic peptide sequences：
By above-mentioned analysis, the polypeptide sequence of selection is CEPRPPTTTTPQPT (449aa-462aa, SEQ ID No：1).
Step 2：Peptide systhesis and and carrier protein couplet
2.1 Peptide systhesis
For ease of with carrier protein couplet, a cysteine, and the 458th threonine is added in C-terminal in synthesis polypeptide
For phosphorylation state：CEPRPPTTTT(p)PQPT(SEQ ID No：2).Simultaneously synthesizing one section the 458th containing non-phosphorylating
Threonine polypeptide sequence：CEPRPPTTTTPQPT.Polypeptide is synthesized by hundred strange biotechnology (Suzhou) Co., Ltds.
Peptide systhesis flow:
A. Peptide systhesis：It is synthesized using solid-phase synthesis, i.e., first by the hydroxyl of the hydroxyl end amino acid for the peptide chain of being synthesized
It is connected with the same insoluble macromolecule resin of the structure of covalent bond, being then incorporated in the amino acid on solid phase carrier with this makees
It is moiety by sloughing amino protecting group and with excessive activated carboxyl component reaction, spreading peptide chain, repetitive operation, until
Until reaching the peptide chain length to be synthesized, finally peptide chain is cleaved from resin, is handled by purifying etc. to get being wanted
Polypeptide.
B. it purifies：RP-HPLC is purified
1) HPLC conditions：
Mobile phase：A) 0.1%TFA aqueous solutions；B) 0.1%TFA acetonitrile solutions
Gradient：A/B (90/40) arrives A/B (40/90) 30min
Flow velocity：1ml/min
Temperature：Room temperature (23 DEG C)
Detection：214nm's is ultraviolet
Sample：The crude product of freeze-drying
2) step：
A. crude product is dissolved in mobile phase
B. 20-30mg (2-2.5ml) sample is injected
C. main peak is collected into 50ml pipes
D. it is lyophilized
3) it identifies：LC/MS conditions：
Mobile phase：A) 0.05%TFA aqueous solution B) 0.1%TFA acetonitrile solutions
Gradient：A/B (90/10) arrives A/B (40/60) 15min
Flow velocity：1ml/min
Temperature：Room temperature (23 DEG C)
Detection：214nm's is ultraviolet
MS API：ESI
The result of 2 Peptide systhesis of table
| Peptide systhesis number | Polypeptide sequence | Purity (%) |
| 150803001 | NH2-CEPRPPTTTp[Thr]PQPT-CONH2 | >90% |
| 150803002 | NH2-CEPRPPTTTTPQPT-CONH2 | >90% |
2.2 polypeptides and carrier protein couplet
Chemically synthesized polypeptide antigen is small molecule, itself is difficult the antigenicity having had, and animal can only be induced to generate very
Weak immune response, thus it is critically important to be crosslinked with carrier protein.Carrier protein contains many epitopes, can stimulate
T helper cell, and then induce B cell reaction.For with the crosslinked carrier protein of polypeptide there are many, wherein the load most generally used
Body is keyhole limpet hemocyanin (keyhole limpet hemacyanin, KLH), bovine serum albumin(BSA) (bovine serum
Albumin, BSA), ovalbumin (ovalbumin, OVA) and bovine thyroglobulin (bovine thyroglobulin, THY).
KLH has higher antigenicity, is that the most commonly used polypeptide is crosslinked carrier.BSA is also often used as peptide carrier, but due to BSA
Often be used as detection experiment blocking agent and make this method production antibody application on there is certain limitations.
Polypeptide coupling procedures：
A. solution prepares：Coupling buffer includes Na2HPO4, NaH2PO4, NaCl, EDTA, adjusts pH to 7.2；
B. experimental procedure：
1) column bed prepares：Pure water and coupling buffer column scrubber bed；
2) polypeptide prepares：A small amount of DMF dissolves polypeptide, stands half an hour, waits for grainless insoluble matter in solution, add appropriate AH
Liquid is configured to the polypeptide solution of 6mg/ml, and the amount for needing to be coupled is separated from polypeptide solution；
3) KLH, Sulfo-SMCC prepare：According to mass ratio, coupled peptide total amount：Pure KLH=1：1, calculate pure KLH's
Amount, according to mass ratio, pure KLH：Sulfo-SMCC=10：1, calculate the amount of Sulfo-SMCC；
4) KLH and Sulfo-SMCC reactions are collected with reactant：By the KLH weighed be dissolved in suitable AH liquid be configured to eventually it is dense
10mg/ml is spent, Sulfo-SMCC is dissolved into the solution of 100mg/ml with DMSO, and the two is mixed and is shaken up, between reacting at room temperature 4h simultaneously
Disconnected mixed shake makes it fully react, with chromatography post separation sample；
5) reactant of KLH and Sulfo-SMCC is coupled with polypeptide：It needs that corresponding amount KLH is added in coupled peptide to each pipe
With Sulfo-SMCC reactants, 2h or ambient temperature overnight are reacted at room temperature, vertical mixed instrument mixing is used in combination, extremely by the polypeptide being coupled
In -20 DEG C of preservations.
Note：Using KLH carrier protein couplet synthesis polypeptides, gained coupling peptide is used as immunizing antigen.
Step 3：It is prepared by anti-mm P9 polypeptide rabbit polyclonal antibodies
3.1 immune and blood samplings：
3.2 antibody titer ELISA detection methods
3.2.1 serum ELISA detections：
A. solution prepares：
Coating buffer：50mM Na2CO3 (pH9.6), 20mM Tris-HCl (pH8.5) or 10mM PBS (pH7.4)
Confining liquid：General closing BSA, skimmed milk power, casein, gelatin etc.
Cleaning solution：PBST or pure water
B. experimental procedure：
1) antigen is dissolved in by debita spissitudo in coating buffer；
2) 100ul antigens are added in corresponding hole, 4 DEG C overnight；
3) it empties liquid and pats dry residual liquid, cleaning solution rinses 3 times；
4) add 200ul confining liquids per hole, 37 DEG C are incubated 1 hour；
5) it empties liquid and pats dry residual liquid, cleaning solution rinses 3 times；
6) add 100ul primary antibodies per hole, 37 DEG C are incubated 1 hour；
7) it empties liquid and pats dry residual liquid, cleaning solution rinses 3 times；
8) add 100ul secondary antibodies per hole, 37 DEG C are incubated 1 hour；
9) it empties liquid and pats dry residual liquid, cleaning solution rinses 5 times；
10) residual liquid in hole is patted dry, 100ul developing solutions are added per hole, 37 DEG C are protected from light colour developing 10min；
11) add 50ul 2M H2SO4 color development stoppings per hole, and read 450nm OD values immediately.
C. serum ELISA testing results：
Note：The ELISA detections of RB55631-55632 serum turn sun (1:32000, P/N values>2.1), two steps will be arranged affine
It purifies and carries out ELISA Identification of the antibodies.
3.2.2 antibody affinity purification result：
3.2.3 antibody ELISA result
Conclusion：
The immune RB55631-55632 serum ELISA test positive of the project；Two steps are affine method antibody purification, is obtained altogether
Obtain phosphorylation antibody 3.89mg.
The identification of step 4 anti-mm P9 (pT458) polyclonal antibody
The ELISA of 4.1 anti-mm P9 (pT184) polyclonal antibodies is identified
A) ELISA experimental procedures
(1) by polypeptide MMP9-Thr458 and pMMP9-Thr458,0.2 μ g/100ul are dissolved into 1 × CBS coating buffers, are spread
To 100ul in the hole of each ELISA Plate, 4 DEG C are coated with overnight.
(2) take out the ELISA Plate being coated within second day, dry coating buffer, clappers.
(3) plus confining liquid (3%BSA), each hole add 200ul, 37 DEG C of incubation 2h.
Close after, take out ELISA Plate, board-washing three times, in each hole be added 300ul PBST stand 2min, after get rid of
Plate is clapped on clean gauze, the liquid in hole is patted dry and is preferred by dry (avoiding the pollution between Kong Yukong).
(5) next step experiment can be carried out directly, it is also possible to which valve bag packages, and 4 DEG C save backup.
(6) by MMP9 primary antibodies with 1:4000 diluted concentration is added per hole 100ul, 37 DEG C of incubation 1h.
(9) ELISA Plate is taken out after 1h, HRP secondary antibodies, 37 DEG C of incubation 30min are added in board-washing afterwards three times.
(10) ELISA Plate is taken out after 30min, board-washing is added 37 DEG C of TMB color developing agents and is protected from light incubation 15min afterwards three times.
(11) ELISA Plate to be taken out and (becomes blue) after 15min, terminate liquid is added immediately, microplate reader shakes 30s, and 450nm is read, point
Analyse result.
B) ELISA experiments detection pMMP9-458 antibody is specific phosphorylation site antibody
Phosphorylation in view of the sites MMP9-Thr458 is there is not yet relevant report, therefore inventor authorized company customizes
PMMP9-Thr458 antibody tests whether detection pMMP9-Thr458 antibody is specific phosphorylation site antibody through ELISA.Knot
Fruit shows that the OD values of pMMP9-Thr458 antibody and pMMP9-Thr458 antigen bindings are significantly greater than pMMP9-Thr458 antibody
With the OD values (see Fig. 2) of non-phosphorylating MMP9-Thr458 antigen bindings, and pMMP9-Thr458 antibody and pMMP9-Thr458
The OD values of antigen binding are reduced with the dilution of antibody concentration, and it is specificity as a result to prompt the pMMP9-Thr458 antibody
The sites pMMP9-Thr458 antibody.
The Western-Blot of 4.2 anti-mm P9 (pT458) polyclonal antibodies is identified
A it) is transiently transfected using the transfection reagent Jetpei of Polyplus companies, by taking 6cm culture dishes as an example：
(1) when cell is grown to 80%-90%, and cell state is good, cell count after cell dissociation, each 6cm are carried out
Culture dish spreads 5 × 105-1 × 106 cell, and cell suspension and culture medium total volume are 5ml
(2) 3 μ g DNA are taken, the plasmid volume and NaCl volume summations for calculating gained are 250ul, then take 6ul transfection examinations
The transfection reagent mixed is added in the DNA mixed, gently by agent jetpei, 244ul NaCl constant volumes to 250ul mixings
After mixing simply centrifugation, it is placed at room temperature for 15-30min.
(3) it by the above-mentioned rotaring redyeing system prepared, gently adds in the corresponding culture dish marked, after mixing, is put into 37
DEG C, 5%CO2 constant incubators carry out cell culture.
(4) cell can carry out Western-Blot identifications after 24-48h.
B) the Western-Blot qualification results of anti-mm P9 (pT458) polyclonal antibody
CNE1 cells are logical with pMMP9-Thr458 antibody after transiently transfecting cDNA, MMP9-WT, MMP9-Thr458Ala
Cross Western blot detection Thr458 whether phosphorylation, as a result show the MMP9-Thr458 phosphorylations of wild type MMP9 (WT)
Level is (see the Fig. 3) for the MMP9-Thr458 phosphorylation levels for being apparently higher than MMP9-Thr458 catastrophe points (T458A).As a result it carries
Show that MMP9Thr458Ala is the site of phosphorylation.
The cellular immunity groupization of 4.3 anti-mm P9 (pT458) polyclonal antibodies is identified
A) the cellular immunity group step of anti-mm P9 (pT458) polyclonal antibody：
(1) special cell climbing sheet is placed in 6 orifice plates culture dish, by the cell density of 2*104/ml by cell inoculation in training
It supports in ware and carries out cell climbing sheet, immunocyte histochemical stain identification is carried out after cell covers with 50%-70%.
(2) culture medium in six orifice plates is exhausted, PBS, which shakes, washes 3 each 1min of sample.
(3) 15min is fixed in fixer.
(4) PBS, which shakes, washes 1 5min of sample.
(5) 0.1%Triton X-100 (DPBS matches) are incubated 1 20min.
(6) PBS, which shakes, washes 3 each 3min of sample.
(7) 2%BSA closings are incubated 30min.
(8) PBS, which shakes, washes 1 5min of sample
(9) primary antibody (p-MMP9)) (PBS matches, titre 1 for incubation：200, wet box) 4OC is stayed overnight or 37OC 60min.It is negative right
According to most handy primary antibody derived sera, PBS liquid is otherwise used
(10) PBS cleans 4 each 5min of sample.
(11) secondary antibody working solution is incubated (wet box) 37OC 30min.
(12) PBS, which shakes, washes 4 each 5min of sample.15C liquid (wet box) 37OC 30min.
(13) PBS cleans 3 each 5min of sample.
(14) DAB colour developings (being protected from light, under the microscope to brown) about 1-5min.
(15) 2 1min of distillation washing.
(16) haematoxylin redyes 0.5~1min.
(17) it originally washes.
(18) 8% ammonium hydroxide 30s.
(19) glycerine or neutral gum rotor.
(20) under the microscope.
B) the cellular immunity group qualification result of anti-mm P9 (pT458) polyclonal antibody：
It chooses nasopharyngeal carcinoma cell CNE2, hepatocellular carcinoma H22, stomach cancer cell MGC803 and carries out the experiment of cellular immunity groupization,
As a result display anti-mm P9 (pT458) experimental groups (as shown in Figure 6) visible brown color dyeing in endochylema or karyon, be positive knot
Fruit, control group endochylema or karyon are shown in blue dyeing, and be negative result.Be repeated several times, as a result be consistent before.Prompt anti-mm P9
(pT458) polyclonal antibody can be used for tumor cell levels detection.
The histogenic immunity groupization of 4.4 anti-mm P9 (pT458) polyclonal antibodies is identified
A) experimental procedure：Cervical carcinoma, gastric cancer, each 50 progress immunohistochemistry of glioma patient are selected, P-MMP9 is verified
(T458) differential expression of the antibody in cancer, cancer beside organism.
1 organizational routine paraffin section thickness 3-5um, anti-flake dry after dragging for piece, are put into 65 DEG C of ovens and bake piece 2 hours.
2 slice dewaxing aquation programs
(1) environment friendly transparent agent I, II, III shaking tables dewax each 12 minutes；
(2) each 3 minutes of the ethyl alcohol of 100%-95% -80% -70% -50%, shaking table.
3 ultra-pure waters rinse 5 minutes, and washing is had to fully.
4 antigen hot repairs are multiple：Heat will extremely be boiled in EDTA (1X) antigen retrieval buffers and microwave box, paraffin section is put into
In the antigen retrieval buffers for entering boiling, medium-to-high grade microwave treatment 20-30 minutes.
5 stop heating, and room temperature cools down 20-30 minutes.
6 will set ultra-pure water by slice after antigen retrieval, impregnate 2 times it is 5 minutes each, shaken later with PBS wash 3 times it is 5 minutes each.
Sample is placed in endogenous peroxydase blocking agent 3%H2O2 by 7, is protected from light incubation at room temperature 15 minutes.PBS buffer solution
Shake wash 3 times it is 5 minutes each.
8 take out slice, and primary antibody (concentration proportioning, PBS dilutions 1 is added dropwise:200) 60ul is put into special incubation after primary antibody is added dropwise
In box, 4 DEG C of refrigerator overnights.
9 slice be put into PBS buffer solution clean 3 times it is 5 minutes each, fully wash, prevent because washing it is not clean caused by non-spy
Opposite sex dyeing.(preceding PBS twice is outwelled)
10 dry 70ul horseradish enzyme marks sheep anti-Mouse/rabbit igg polymer are added dropwise after tissue surrounding liquid (according to actual conditions
It is required that Covering samples), it is incubated at room temperature 30 minutes, PBS buffer solution is washed 3 minutes × 3 times.
11 colour developings are added dropwise and now match appropriate DAB color developing agents, color development at room temperature, 5-20 minutes.Tap water color development stopping.First pass
Tap water need to focus on, originally water washing 3 minutes.
12 redye, and are dyed 40 seconds in haematoxylin dye liquor, after washing 1 minute, slice are placed in 0.5% ammonium hydroxide and is impregnated 30 seconds
Washing 5-10 minutes is continued in left and right.
13 are sequentially placed into slice in -100% ethyl alcohol of -5% ethyl alcohol of -80% ethyl alcohol of 70% ethyl alcohol each 3 minutes.
Slice is sequentially placed into clarifier I, II, III each 5 minutes after 14 taking-ups
15 neutral gum mountings, optical microphotograph is under the microscope.
B) the histogenic immunity group qualification result of anti-mm P9 (pT458) polyclonal antibody
By cervical carcinoma, gastric cancer, glioma slice and the microscopic observation (as shown in Figure 7) after immunohistochemical experiment, compare
Expression in anti-mm P9 (pT458) polyclonal antibodies cancer, cancer beside organism, it is seen that cancerous tissue is visible pale brown in endochylema or karyon
Color is positive.Cancer beside organism's visible blue in endochylema or karyon, is negative.As a result anti-mm P9 (pT458) Anti-TNF-α is prompted
Body expresses in cancerous tissue, not expressing in cancer beside organism, and there are notable differences for the two.
The phosphorylation polyclonal antibody of the above-mentioned experiment results proved present invention can specific recognition MMP9 albumen pT458 phosphoric acid
Change site, can be used for detecting the phosphorylation level in tumour cell site such as gastric cancer cervical carcinoma rhinocarcinoma, it is thin to explore tumour
Born of the same parents are proliferated and metastasis research provides a kind of tool, also provide help for diagnosing tumor, and its clinical prognosis can be instructed to judge.
Sequence table
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<120>One kind stress phosphorylation antigen polypeptide, antibody, preparation method and application
<141> 2018-02-23
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Claims (7)
1. a kind of phosphorylation antigen polypeptide, it is characterised in that the 458th threonine site of the antigen polypeptide behaviour MMP9 albumen
Near 15 peptides screen to obtain as candidate polypeptide, wherein the threonine in the sites T458 be phosphorylation state, and C-terminal connection one
The amino acid sequence of a cysteine.
2. it is a kind of for the sites people MMP9 albumen T458 stress phospho-AB, it is characterised in that the antibody is to be directed to people
The antibody of MMP9 albumen T458 site amino acids phosphorylations, wherein being used in the Antibody preparation by following amino acid sequence group
At antigen polypeptide, particular sequence is：CEPRPPTTTT (p) PQPT, wherein T (p) indicate the sites people MMP9 albumen T458 ammonia
Base acid phosphoric acid threonine, C-terminal connect a cysteine.
3. it is a kind of for the sites people MMP9 albumen T458 stress phosphorylation polyclonal antibody preparation method, it is characterised in that packet
(1) is included to the secondary structure of the 458th location proximate amino acid sequence of MMP9 albumen, immunogenicity, hydrophilic and hydrophobic, surface accessibility
Etc. being analyzed, it is artificial synthesized to determine that suitable one section of peptide sequence carries out；(2) polypeptide of synthesis and maleimide ammonia are activated
This coupled product is carried out desalting column and new zealand rabbit is immunized after purification by carrier mcKLH couplings；(3) pass through four immune rabbit blood
Antibody titer is detected with ELISA method clearly, potency collects immune rabbit anteserum after reaching ideal value, and the coated bromination of polypeptide is used in combination
The agarose affinity purification column purification antibody of cyanogen activation；(4) ELISA, western bot is carried out to purified antibodies to identify.
4. it is a kind of for the sites people MMP9 albumen T458 stress phospho-AB application, wanted using right wherein described
Seek 2 people's MMP9 albumen described or that specific recognition cancer cell expression is carried out according to antibody prepared by claim 3 preparation method
The sites T458.
5. it is according to claim 4 stress phospho-AB application, wherein the cancer cell is human nasopharyngeal carcinoma, uterine neck
Cancer, gastric cancer, glioma cell.
6. a kind of cancer cell detection kit, including be directed to described in claim 2 or according to prepared by claim 3 preparation method
The sites people MMP9 albumen T458 stress phospho-AB, antigen retrieval buffers, PBS buffer solutions, enzyme blocking agent 3%H2O2, peppery
Root enzyme mark sheep anti-Mouse/rabbit igg polymer, DAB color developing agents, haematoxylin dye liquor, ethyl alcohol, environment friendly transparent agent, 0.5% ammonium hydroxide and super
Pure water.
7. a kind of method of cancer cell detection, it is characterised in that cancer cell detection kit described in claim 1 is used, including
Following steps：
(1) by organizational routine paraffin section to be detected, by environment friendly transparent agent, ethyl alcohol distinguishes shaking table dewaxing, ultra-pure water rinsing；
(2) antigen retrieval buffers are heated to boiling, paraffin section is put into the antigen retrieval buffers of boiling, medium-to-high grade microwave
Processing, room temperature cooling are placed in ultra-pure water, impregnate, shaken wash 3 times with PBS later；
(3) sample is placed in endogenous peroxydase blocking agent 3%H2O2, is protected from light incubation at room temperature, PBS buffer solution, which is shaken, washes；
(4) take out slice, be added dropwise by it is diluted for the sites people MMP9 albumen T458 stress phospho-AB, be put into incubation
In box, 4 DEG C of refrigerator overnights are put into PBS buffer solution and fully wash；
(5) horseradish enzyme mark sheep anti-Mouse/rabbit igg polymer, incubation at room temperature, PBS buffer solution is added dropwise after drying tissue surrounding liquid
Washing is added dropwise and now matches appropriate DAB color developing agents, color development at room temperature, tap water color development stopping, is dyed in haematoxylin dye liquor, will after washing
Slice, which is placed in 0.5% ammonium hydroxide, to be impregnated, and continues to wash；
(6) slice is sequentially placed into ethyl alcohol, slice is placed in clarifier after taking-up, with neutral gum mounting, light microscope
Lower observation.
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| CN116444677A (en) * | 2023-06-09 | 2023-07-18 | 中国人民解放军军事科学院军事医学研究院 | FoxM1 protein Y575 phosphorylated polyclonal antibody, preparation method and application thereof |
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