CN109293762A - DRC3f antigen polypeptide, the polyclonal antibody of anti-DRC3f and application - Google Patents
DRC3f antigen polypeptide, the polyclonal antibody of anti-DRC3f and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
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- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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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
The invention belongs to the pharmaceutical product technical fields containing antigen or antibody, disclose a kind of DRC3f antigen polypeptide, the polyclonal antibody of anti-DRC3f and application；The secondary structure of DRC3f protein amino acid sequence, immunogenicity, hydrophilic and hydrophobic, surface accessibility etc. are analyzed, it is artificial synthesized to determine that suitable one section of peptide sequence carries out；Carrier KLH, BSA of the polypeptide of synthesis and the activation of maleimide ammonia are coupled, this coupled product is subjected to desalting column, bal/c mouse is immunized after purification；Antibody titer is detected with ELISA method by repeatedly immune mice serum, potency, which reaches, collects immune serum after ideal value, and with Protein G-protein purification column antibody purification；The identification such as ELISA, western bot is carried out to purified antibodies.Qualification result shows that the polyclonal antibody can specific recognition DRC3f albumen.
Description
Technical field
The invention belongs to the pharmaceutical product technical field containing antigen or antibody more particularly to a kind of DRC3f antigen are more
Peptide, the polyclonal antibody of anti-DRC3f and application.
Background technique
Complement C_3 is the highest molecule of content in human body endocomplement system, while being also the key molecule of complement system.?
During complement activation, three complement activation pathways require to form C5 activating enzymes by activation C3.The activation of C3 forms C3b,
The iC3b and 17 Peptide C 3f of inactivation are decomposed under can then helping by CR1 the or H factor by i factor.The latter acts in carboxypeptidase-N
De- arginine down is quickly generated 16 Peptide D RC3f.More and more researchs think that DRC3f has important physiological function: Wo Men
Find that DRC3f content in SSc patients serum obviously increases in early-stage study, and related to SSc disease activity；Li Man etc.
Research thinks that DRC3f may be the biomarker of Severe degree hepatitis；It is penetrating that C3f and DRC3f can also improve vascular endothelial cell
Property, the core element HWESAS of the two also has class growth hormone sample effect.Since DRC3f molecular weight is small, detection method is limited,
There is not antibody sale on the market at this stage, currently, mainly applying Matrix-assisted laser desorption ionization time of flight mass spectrometry yet
(matrix-assisted laser desorption/ionization time-of flight mass
Spectrometry, MALDI-TOF MS) detection serum in DRC3f content, specific method is: first separate complete examination
Agent is purchased from Bruker Daltonics company of Germany, specifically: magnetic bead is added in magnetic bead combination buffer and is mixed, is added later
Serum is mixed and is stood.Sample cell, which is put into Beads enrichment device, keeps magnetic bead adherent, abandons suspension, is cleaned with magnetic bead cleaning buffer solution
Twice.Magnetic bead elution buffer is added in sample cell, the supernatant after elution moves into new sample cell, with magnetic bead stabilizing buffer
Piping and druming can be analyzed by mass spectrometry after mixing.The standard items of 1u L take 1u L when point target after mixing with 10u L matrix, and point exists
On 600um Anchorchip standard items position, Anchorchip target is put into mass spectrum after drying at room temperature, selection mode: LP-
Clinprot, acquisition range: 1000~12000Da of relative molecular weight.
In conclusion problem of the existing technology is:Matrix-assisted laser desorption ionization (MALDI-
TOF MS) most of laboratories are not purchased, and instrument price is expensive, secondly expensive using magnetic bead consumptive material expense in experiment, experimental implementation
Complexity, time-consuming and requires sample purity high for pretreatment.
Solve the difficulty and meaning of above-mentioned technical problem:The small only 16 polypeptide compositions of DRC3f molecular weight, due to not having needle
To the specific antibody of the polypeptide, can only be examined at this stage by Matrix-assisted laser desorption ionization time of flight mass spectrometry
It looks into, limits the functional study to DRC3f.Therefore, new high sensitivity is actively found, the high detection method of specificity is for grinding
Study carefully the function of DRC3f polypeptide and its important, but since DRC3f molecular weight is small, immunogenicity is general, due to DRC3f molecular weight
Small, immunogenicity is general, and therefore, we are by being immunized mouse after coupling it with KLH, using DRC3f as 96 orifice plate of antigen coat,
Detection discovery, produces the antibody of the anti-DRC3f of specificity.Meanwhile we are checked by WB and are found, which being capable of specificity
The DRC3f that couples of identification KLH, but cannot check the expression of Complement C_3 in rat liver tissue, the antibody is prompted to have
Stronger specificity can be used for DRC3f functional study.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of DRC3f antigen polypeptides, and anti-DRC3f's is polyclonal
Antibody and application.
The invention is realized in this way a kind of DRC3f antigen polypeptide, the amino acid sequence of the DRC3f antigen polypeptide are
SEQ ID NO:1.
It is immunogene by the DRC3f antigen polypeptide another object of the present invention is to provide one kind is antigen immune
The polyclonal antibody that balb/c mouse obtains.
Another object of the present invention is to provide a kind of preparation method of polyclonal antibody, the polyclonal antibody
Animal will be immunized after the mouse DRC3f polypeptide and carrier protein couplet in preparation method, collects immune animal blood and prepares anti-blood
Clearly, antiserum is isolated and purified to obtain polyclonal antibody.
Further, the carrier protein is KLH.
Further, the potency of the polyclonal antibody is 1: 12000.
In conclusion advantages of the present invention and good effect are: second level knot of the present invention to DRC3f protein amino acid sequence
Structure, immunogenicity, hydrophilic and hydrophobic, surface accessibility etc. are analyzed, and it is artificial synthesized to determine that suitable one section of peptide sequence carries out；It will
Carrier KLH, BSA of polypeptide and maleimide the ammonia activation of synthesis are coupled, this coupled product progress desalting column is immunized after purification
Balb/c mouse；Antibody titer is detected with ELISA method by repeatedly immune mice serum, potency is received after reaching ideal value
Collect immune serum, and with Protein G-protein purification column antibody purification；ELISA, western are carried out to purified antibodies
The identification such as bot.Qualification result show the polyclonal antibody can specific recognition DRC3f albumen, carry out grinding for its function for the later period
Study carefully and effective research means are provided.
Detailed description of the invention
Fig. 1 is DRC3f antigen polypeptide provided in an embodiment of the present invention, the polyclonal antibody of anti-DRC3f and application
Fig. 2 is provided in an embodiment of the present invention through DNAstar software analysis DRC3f immunogenicity, hydrophilic and hydrophobic and surface
The polypeptide sequence segment for selecting synthesis is shown in box for accessibility schematic diagram.
Fig. 3 is provided in an embodiment of the present invention for anti-rabbit C3 complement antibody and mice serum schematic diagram；
In figure: 1BSA；2DRC3f-KLH；3 rat livers；4 rat livers；5Marker；A primary antibody is anti-for anti-rabbit C3 complement
Body；B is mice serum.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
DRC3f antigen polypeptide provided in an embodiment of the present invention, amino acid sequence SEQ ID NO:1
(SSKITHRIHWESASLL) DNA sequence dna is agctccaa gatcacccac cgtatccact gggaatctgc
cagcctcctg。
The polyclonal antibody of anti-DRC3f provided in an embodiment of the present invention is with aforementioned KLH-DRC3f, BSA-DRC3f antigen
Polypeptide is immunogene, and immune animal is prepared.
As shown in Figure 1, the preparation method of the polyclonal antibody of anti-DRC3f provided in an embodiment of the present invention includes following step
It is rapid:
The keyhole blood indigo plant carrier protein KLH of S101:DRC3f antigen polypeptide and maleic amide activation, through desalting and purifying
Afterwards as immunogene and adjuvant mixed immunity bal/c mouse, during which it is immunized more than twice；
S102: after ELISA detection serum antibody titer up to after 1:8000, acquiring mice serum, by purifying, ELISA and
After western blot identification, the polyclonal antibody of resisting GPC 3 is obtained.
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 for being crosslinked with carrier protein.Carrier protein contains many epitopes, can stimulate
T helper cell, and then induce B cell reaction.There are many carrier proteins for being crosslinked with polypeptide, 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, most strong with immunogenicity after DRC3f antigen polypeptide crosslinking of the invention, therefore the present invention
Select KLH as coupling carrier albumen.BSA is also often used as peptide carrier, but since BSA is often used as detection test
Blocking agent and make this method produce antibody application on there is certain limitations.
Application principle of the invention is further described combined with specific embodiments below.
Embodiment 1 prepares DRC3f antigen polypeptide
The design and synthesis of 1.DRC3f polypeptide: people DRC3f is obtained according to the DRC3f sequence (accession number :) on GenBank
Protein sequence contains 16 amino acid.
2. analyzing DRC3f protein characteristic with DNAstar software, the molecular weight of the albumen is 1865.11 dalton, isoelectric point
It is 9.85, is basic protein.
3. dividing DRC3f protein immunogenic, hydrophilic and hydrophobic and surface accessibility with DNAstar software, the results showed that immune
Originality is lower, and hydrophily is general.
The preparation of 2 DRC3f antigen polypeptide of embodiment and and carrier protein couplet
1. Peptide systhesis
To be convenient for and carrier protein couplet, increase a cysteine in DRC3f antigen polypeptide C-terminal, it may be assumed that
VDDAPGNSQQATPKDC.Polypeptide is synthesized by Shanghai Xin Hao Biotechnology Co., Ltd.
2. polypeptide and carrier protein couplet
3. coupled product purifies
The preparation of the anti-DRC3f polypeptide mouse polyclonal antibody of embodiment 3 and purifying
1. animal immune
14 bal/c female mices, weight 15-20g is immunized in coupled product.Coupled product and Freund's complete adjuvant or Freund are endless
Full adjuvant is purchased from Sigma company, separately has two bal/c female mices to do negative control not immune.
Immune programme is shown in Table 3, first dilutes 100 μ g antigens with PBS, until 1.00mL, is mixed with isometric adjuvant, vortex oscillation
100min, detection emulsification is as a result, take drop antigen drop in waterborne, insoluble diffusion in one minute.
4. antibody titer ELISA is detected
(1) experiment reagent
A. phosphate buffer (PBS) (10 × concentration): sodium chloride (NaCl) 50g, potassium chloride (KCl) 1.25g, di(2-ethylhexyl)phosphate
Hydrogen potassium (KH2PO4) 1.25g, disodium hydrogen phosphate (Na2HPO4·12H2O) 18.1g, distilled water add to 1000mL, with 1M HCl tune pH
To 7.2.
B. confining liquid: Normal Goat Serum 5mL, 1 × PBS (being free of tween) 95mL.
C. washing buffer: Tween-20 (Tween 20) 0.2mL, 1 × PBS 1000mL.
D. substrate develop the color A liquid: sodium acetate (CH3COONa) 13.6g, citric acid (C6H8O7·H2O) 1.6g, hydrogen peroxide
(H2O230%) 0.3mL, distilled water add to 500mL.
E. substrate develop the color B liquid: disodium ethylene diamine tetraacetate (EDTANa2) 0.2g, citric acid (C6H8O7·H2O) 0.95g is sweet
Oil (C3H8O3) 50mL, tetramethyl benzidine (TMB) 0.2g, distilled water adds to 500mL.
F. terminate liquid (2M H2SO4): take dense H2SO427.62mL is added slowly in the distilled water of 473mL, and mixing is
It can.
G. antigen coat liquid (0.1M carbonate buffer solution): pH 9.6, sodium carbonate (Na2CO3) 1.59g, sodium bicarbonate
(NaHCO3) 2.93g, Sodium azide (NaN3) 0.2g, distilled water adds to 1000mL.
H. the goat anti-mouse igg (commercialization) of horseradish peroxidase-labeled.
(2) experimental procedure
A. antigen coat: pure 12.5 μ g/mL of DRC3f polypeptide antigen final concentration takes 100 μ L to be added poly- after being diluted with coating buffer
In each hole of the enzyme-linked detection plate of styrene, after 4 DEG C are stayed overnight, washing lotion is washed 3 times.
B. close: every hole adds 200 μ L confining liquids, and 37 DEG C after two hours, are washed 3 times, patted dry.4 DEG C of refrigerators are set to save backup.
C.ELISA detection
1. plus sample to be tested: separating serum after mouse orbit blood sampling, with PBS doubling dilution, 100 holes μ L/ are loaded onto coating
In good ELISA Plate, meanwhile, choosing non-immune serum is negative control, with PBS doubling dilution, 4 DEG C of overnight incubations, washing
It 3 times, pats dry.
2. plus secondary antibody: according to the potency of ELIAS secondary antibody select extension rate 1:8000,100 holes μ L/, 37 DEG C of incubation 30min,
Washing 3 times, pats dry.
3. colour developing: substrate colour developing each hole 80 μ L/ of A, B liquid, 37 DEG C of colour developing 15min are added.
4. terminating: 80 hole μ L/ of terminate liquid is added.
5. reading: each hole OD value is measured with 450nm Single wavelength, to be greater than 2.5 with the ratio (P/N) of negative control hole OD value
It is limited, as critical point that is positive or determining potency is judged as, the results are shown in Table 1, and 3 immune serum potency reach
1:102400 or more.
5. antibody purification
(1) saturated ammonium sulphate method
A. saturated ammonium sulfate solution (SAS) is prepared: by 767g (NH4)2SO4It is slowly added in 1L distilled water while stirring.With
Ammonium hydroxide or sulfuric acid are transferred to sulfuric acid pH7.0.This i.e. saturation degree be 100% ammonium sulfate (4.1mol/L, 25 DEG C).
B. it precipitates
1. sample (serum) 20000 × g is centrifuged 30min, cell fragment is removed；
2. retaining supernatant and measuring volume；
3. being slowly added into isometric SAS while stirring into supernatant, final concentration of 1:1；
It stirs 6 hours or is stirred overnight (4 DEG C) 4. solution is placed on magnetic stirring apparatus, precipitate protein sufficiently.
C. it dialyses
1. 10000 × g of protein solution is centrifuged 30min (4 DEG C).It abandons supernatant and retains precipitating；
2. precipitating is dissolved in a small amount of (10~20mL) Sodium azide containing 0.2g/L PBS solution.Dialysis is put into after precipitating dissolution
It dialyses 24~48 hours (4 DEG C) in the bag PBS solution of Sodium azide containing 0.2g/L, it is primary to change elution buffer every 3~6 hours, with
Thoroughly remove sulfate of ammoniac；
3. dialyzate is centrifuged, protein content in supernatant is measured.
(2) Protein G affinity purification
Required reagent and special installation: 1.0mol/L Tris (pH8.0)；100mmol/L Tris(pH8.0)；10mmol/
L Tris(pH8.0)；50mmol/L glycine (pH3.0)；Column chromatography instrument.
Operating procedure:
A. the 1.0mol/L Tris (pH8.0) that 1/10 volume is added adjusts (serum, tissue culture supernatant containing antibody samples
Or ascites) pH value to 8.0.
B. antibody-solutions are passed through into albumin A or Protein G microballoon column.In these columns, every milliliter of wet microballoon can be in conjunction with about
10~20mL antibody (1 albumin A or Protein G microballoon molecule combine 2 molecular antibodies).The about volume of record dress column microballoon.Cause
The amount using cleaning and elution buffer is determined for the volume of microballoon column.
C. microballoon is washed with the 100mmol/L Tris (pH8.0) of 10 times of column volumes.
D. microballoon is washed with the 10mmo1/L Tris (pH8.0) of 10 times of column volumes.
E. microballoon column is eluted with 50mmol/L glycine (pH3.0), the buffer of about 1/2 column volume is added every time, by several times
It is added.Eluent is collected with the test tube of the 1mol/L Tris (pH8.0) containing 1/10 column volume, each pipe is slowly shaken up, its pH is made
Value is restored to neutrality.
F. the collecting pipe containing antibody is mixed, surveys total protein with SDS-PAGE and coomassie brilliant blue staining.
As a result: identifying (Fig. 2) through SDS-PAGE protein electrophoresis after antibody purification, ultraviolet specrophotometer measures antibody concentration
For 0.4mg/mL.Purified antibodies are stored in -20 DEG C, and in 0.01M PBS buffer solution, 40% glycerol and 0.5% are contained in solution
BSA。
Embodiment 4: the identification of the polyclonal antibody of anti-DRC3f
1. the ELISA of the polyclonal antibody of anti-DRC3f is identified
It is detection antigen with the DRC3f polypeptide of synthesis, coated elisa plate is made with the diluted non-immune serum of 1:800
For negative control, antibody doubling dilution by mice serum and after purification is detected using ELISA method, with negative serum
Ratio is greater than 2.1 and sentences section for the positive, calculates monoclonal antibody potency.
As a result, the potency of the anti-DRC3f antibody of purifying reaches 1:12000.
2. the Western-Blot of the polyclonal antibody of anti-DRC3f is identified
Rat liver tissue, after being cracked, lysate carries out 10%SDS-PAGE electrophoresis, referring to institute in " molecular cloning "
The western blotting method stated, electrotransfer condition are 100V electrophoresis 1h, and confining liquid is the TBST containing 5% skimmed milk power, and primary antibody is this
The polyclonal antibody (0.1 μ g/mL of final concentration) of anti-DRC3f in invention, secondary antibody are the sheep anti mouse of horseradish peroxidase-labeled
IgG antibody carries out immunoblotting assay by ECL Color Appearance System.
3. antibody specificity ELISA is detected
A. phosphate buffer (PBS) (10 × concentration): sodium chloride (NaCl) 50g, potassium chloride (KCl) 1.25g, di(2-ethylhexyl)phosphate
Hydrogen potassium (KH2PO4) 1.25g, disodium hydrogen phosphate (Na2HPO4·12H2O) 18.1g, distilled water add to 1000mL, with 1M HCl tune pH
To 7.2.
B. confining liquid: normal calf serum 10mL, 1 × PBS (being free of tween) 90mL.
C. washing buffer: Tween-20 (Tween 20) 0.2mL, 1 × PBS 1000mL.
D. substrate develop the color A liquid: sodium acetate (CH3COONa) 13.6g, citric acid (C6H8O7·H2O) 1.6g, hydrogen peroxide
(H2O230%) 0.3mL, distilled water add to 500mL.
E. substrate develop the color B liquid: disodium ethylene diamine tetraacetate (EDTANa2) 0.2g, citric acid (C6H8O7·H2O) 0.95g is sweet
Oil (C3H8O3) 50mL, tetramethyl benzidine (TMB) 0.2g, distilled water adds to 500mL.
F. terminate liquid (2M H2SO4): take dense H2SO427.62mL is added slowly in the distilled water of 473mL, and mixing is
It can.
G. antigen coat liquid (0.1M carbonate buffer solution): pH 9.6, sodium carbonate (Na2CO3) 1.59g, sodium bicarbonate
(NaHCO3) 2.93g, Sodium azide (NaN3) 0.2g, distilled water adds to 1000mL.
H. the goat anti-mouse igg (commercialization) of horseradish peroxidase-labeled.
(2) experimental procedure
A. it antigen coat: is coated with DRC3f, C3f and unrelated polypeptide (amino acid sequence for upsetting DRC3f) pure polypeptide respectively
Antigen final concentration is generally 12.5ug/mL, and 100 μ L is taken to be added in each hole of the enzyme-linked detection plate of polystyrene after being diluted with coating buffer, and 4
DEG C overnight after, washing lotion wash 3 times.
B. close: every hole adds 200 μ L confining liquids, after 4 DEG C are stayed overnight, washs 3 times, pats dry.4 DEG C of refrigerators are set to save backup.
C.ELISA detection
1. plus sample to be tested: PBS doubling dilution is used, 100 holes μ L/ are loaded onto the ELISA Plate being coated with, meanwhile, it chooses not
Immune serum is negative control, and with PBS doubling dilution, 4 DEG C of overnight incubations are washed 3 times, patted dry.
2. plus secondary antibody: according to the potency of ELIAS secondary antibody select extension rate 1:8000,100 holes μ L/, 37 DEG C of incubation 30min,
Washing 3 times, pats dry.
3. colour developing: substrate colour developing each hole 80 μ L/ of A, B liquid, 37 DEG C of colour developing 15min are added.
4. terminating: 80 hole μ L/ of terminate liquid is added.
4. 5. reading: measuring each hole OD value with 450nm Single wavelength, be greater than with the ratio (P/N) with negative control hole OD value
2.5 are limited, and as critical point that is positive or determining potency is judged as, the results are shown in Table 2.
As a result: the polyclonal antibody of the anti-DRC3f in the present invention cannot detect that 180KD size is left in cell pyrolysis liquid
The protein band of right C3, but it is able to detect that DRC3f is coupled KLH protein band, and C3 antibody can detect at 180KD
The protein band that C3 molecular size range is consistent, but cannot detect that DRC3f is coupled KLH protein band (Fig. 3).With conjunction in experiment
At polypeptide DRC3f as ELISA be coated with, it was demonstrated that its to DRC3f have specificity (using FITC coupling DRC3F stimulate cell
While, do immunohistochemistry).
Table 1
Table 2
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Hubei Institute For Nationalities
<120>DRC3f antigen polypeptide, the polyclonal antibody of anti-DRC3f and application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 48
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
agctccaaga tcacccaccg tatccactgg gaatctgcca gcctcctg 48
Claims (5)
1. a kind of DRC3f antigen polypeptide, which is characterized in that the amino acid sequence of the DRC3f antigen polypeptide is SEQ ID NO:
1。
2. a kind of DRC3f antigen polypeptide as described in claim 1 is that immunogene is that antigen immune balb/c mouse obtains
Polyclonal antibody.
3. a kind of preparation method of polyclonal antibody as claimed in claim 2, which is characterized in that the preparation of the polyclonal antibody
Animal will be immunized after the mouse DRC3f polypeptide and carrier protein couplet in method, collects immune animal blood and prepares antiserum, will
Antiserum isolates and purifies to obtain polyclonal antibody.
4. the preparation method of polyclonal antibody as claimed in claim 3, which is characterized in that the carrier protein is KLH.
5. the preparation method of polyclonal antibody as claimed in claim 3, which is characterized in that the potency of the polyclonal antibody is
1∶12000。
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| CN113151289A (en) * | 2021-03-09 | 2021-07-23 | 河南省农业科学院畜牧兽医研究所 | Preparation method of chicken PTF1a gene recombinant protein and polyclonal antibody |
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