CN109776667B - One breeder Nrf2 protein antibodies and its immunogene, immunogenic polypeptide, detection kit and application - Google Patents
One breeder Nrf2 protein antibodies and its immunogene, immunogenic polypeptide, detection kit and application Download PDFInfo
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Abstract
One breeder Nrf2 protein antibodies and its immunogene, immunogenic polypeptide, detection kit and application, belong to virological immunology technical field.For the antibody and coherent detection kit for lacking effectively detection chicken Nrf2 protein expression situation in the prior art, the present invention provides a kind of immunogenic polypeptides, and amino acid sequence is as shown in SEQ ID NO:1.This section of immunogenic polypeptide is former by coupling agent and carrier protein couplet adaptive immune, and utilize immunity inoculation rabbit after the immunogene and adjuvant mixing and emulsifying, the chicken Nrf2 protein antibodies prepared after taking the blood of immune rear rabbit purified, also provided is a kind of ELISA detection kits for detecting chicken Nrf2 albumen.The present invention can also be used to monitor the expression and distribution of chicken Nrf2 albumen in the cell.
Description
Technical field
The invention belongs to virological immunology technical fields, and in particular to a breeder Nrf2 protein antibodies and its immunogene are exempted from
Epidemic disease antigenic polypeptide, detection kit and application.
Background technique
Cellular redox balance has important and complicated influence for body health.On the one hand, energetic supersession is urged
Raw a large amount of active oxygen radicals, the serious damaging cells of the accumulation of these active oxygen radicals in vivo are to cause aging, allergy anti-
It answers, the pathophysiological basis of numerous diseases such as diabetes, liver diseases and cardiovascular disease.On the other hand, work as pathogenic microorganism
When infecting body, cell will trigger the response to oxidative stress of proper level, and the active oxygen of generation not only can be with direct killing cause of disease
Body can regulate and control body innate immune responses system with indirect induced.In order to ensure external environment changes opportunity in the cell
Body can accurately regulating cell oxidation-reduction process in time, form into the cell a set of with nuclear factor-erythroid 2-related factor 2
(Nrf2, also referred to as NFE2L2) is the complicated oxidative stress answering system of core regulatory factor.Intracellular oxygen is maintained as body
Change the central regulation person of reduction stable state, Nrf2 is opened in II phase detoxication enzyme in a manner of transcription regulatory factor in nucleus
Sub-area ARE is combined, and regulates and controls a series of expression of the gene of oxidative stress relevant enzymes and anti-oxidant albumen, including reduced form cigarette
Amide adenine-dinucleotide phosphoric acid (NAD (P) H), oxidoreducing enzyme (NQO1), glutathione S-transferase (GST), gluathione
Peptide peroxidase (GSH), peroxidase (POD), Glutamate-cysteine ligase (GCL), glutathione epoxides
Hydrolase (EH) and heme oxidase (HO-1) etc. make cell be in stable state, maintain body redox dynamic equilibrium.
It is currently known chicken Nrf2 protein amino acid sequence, the Serial No. BAA08364.1 in GenBank, by 582
Amino acid composition.However, Nrf2 correlative study concentrates on people and mammal, birds include that chicken Nrf2 studies basic blank.
There has been no expression and distribution situation that the antibody of identification chicken Nrf2 albumen and suitable reagent can detect chicken Nrf2 albumen.
Summary of the invention
For the antibody and coherent detection kit for lacking effectively detection chicken Nrf2 protein expression situation in the prior art, originally
Invention provides a kind of immunogenic polypeptide, and amino acid sequence is as shown in SEQ ID NO:1.
The present invention also provides a kind of for obtaining the immunogene of chicken Nrf2 protein antibodies, is by amino acid sequence such as SEQ
Immunogenic polypeptide shown in ID NO:1 is obtained by coupling agent and carrier protein couplet.
It is to be immunized after above-mentioned immunogene and adjuvant mixing and emulsifying the present invention also provides a breeder Nrf2 protein antibodies
It is inoculated with rabbit, is prepared after taking the blood of immune rear rabbit purified.
The present invention also provides the preparation methods of above-mentioned chicken Nrf2 protein antibodies, include the following steps:
1) it prepares immunogene: by amino acid sequence polypeptide as shown in SEQ ID NO:1, passing through coupling agent and carrier protein
Coupling, obtains chicken Nrf2 immunogene；
2) immunity inoculation: after chicken Nrf2 immunogene and adjuvant mixing and emulsifying that step 1) is obtained, inoculation rabbit is immunized；
3) after inoculation one month, the dosage used referring to first immunisation halves carry out secondary immunity；
4) it prepares thick antibody: after secondary immunity one month, chicken Nrf2 immunogene, injection is injected intravenously under the conditions of no adjuvant
Dosage is 0.1-1mg/, hereafter, carries out booster immunization every two weeks, adds up booster immunization 2-5 times, each booster immunization 1 week
After take rabbit blood to be centrifuged, collect supernatant, obtain the thick antibody of chicken Nrf2 albumen；
5) amino acid sequence polypeptide as shown in SEQ ID NO:1 antibody purification: is coupled to mercapto preactivated in column
Antigen affinity column is prepared on base albumen agarose binding resin, then carries out column equilibration with PBS buffer solution；By filtered chicken
The antigen affinity column after balance is added in the thick antibody of Nrf2 albumen, then carries out column equilibration again with PBS buffer solution, makes after eluting
It is standby to obtain chicken Nrf2 protein antibodies.
It further limits, the step 1) coupling agent is 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic acid
Base succinimide ester sodium salt, N- succinimide -3- (2- pyridine two is thio) propionic ester or glutaraldehyde；The carrier protein is
Yue hole relative hemocyanin, ovalbumin, bovine serum albumin(BSA) or artificial synthesized poly-D-lysine.
It further limits, the step 2) immunity inoculation is by 0.25-1.5mg immunogene and 0.1-1mg muramyl two
After the mixing of peptide adjuvant, adds PBS to be settled to 1mL, be then inoculated with rabbit with after isometric Freund's complete adjuvant mixing and emulsifying；The rabbit is
Two month female New Zealand White Rabbit；The inoculation position is that 4 points of rabbit two sides groin and oxter and 4 points of the nape of the neck are subcutaneous；It connects
Kind of dosage is 0.1-1mg immunogene/only.
It further limits, the step 5) elution, which refers to, uses the 100mM glycine solution of pH2.5 as antibody elution liquid
Antigen affinity column is eluted, merges and collects the eluent that absorbance at 280nm wavelength is greater than 1.0, be with PBS buffer solution
Analysis liquid is dialysed, and the chicken Nrf2 protein antibodies are obtained.
The present invention also provides a kind of ELISA detection kit for detecting chicken Nrf2 albumen, the ELISA detection kits
Component includes ELISA Plate, coating buffer, confining liquid, ELIAS secondary antibody, TMB developing solution, terminate liquid, detection antigen and above-mentioned chicken
Nrf2 protein antibodies, the detection antigen be using glutaraldehyde as coupling agent, amino acid sequence is more as shown in SEQ ID NO:1
What peptide and bovine serum albumin(BSA) coupling prepared.
It further limits, the 50mM carbonate that the coating buffer is pH9.6 is coated with buffer；The confining liquid is
The BSA solution that mass fraction is 1%；The ELIAS secondary antibody is Fc sections of secondary antibodies of goat anti-rabbit antibody of horseradish peroxidase-labeled；
The terminate liquid is the aqueous sulfuric acid of 2M.
The present invention also provides the chicken Nrf2 protein antibodies the expression in the cell of monitoring chicken Nrf2 albumen and point
Application in cloth.
Beneficial effect
Chicken Nrf2 can regulate and control the redox equilibrium of chicken cell, the health and anti-microbial infection for chicken to Guan Chong
It wants, is the important regulating and controlling factor of the antimicrobial innate immune response of chicken, be expected to become the new of chicken anti-pathogenic and breeding for disease resistance
Target.
Applicant is compared by BLAST and is found, chicken Nrf2 albumen differs greatly with people and mouse Nrf2 protein sequence, homologous
Property is respectively 65.6% and 62.3%.Applicant further compares several main antibody suppliers currently used for preparing commercially available knowledge
Immune peptide and chicken Nrf2 albumen the corresponding sequence discovery of the antibody of others and mouse Nrf2 albumen, therebetween homology generally compared with
It is low.Applicant has detected a variety of commercially available anti-human or mouse Nrf2 antibody, can not identify chicken Nrf2 (see Fig. 2).
The present invention has synthesized chicken Nrf2 Argine Monohydrochloride 569 to 582, amino acid according to chicken Nrf2 protein sequence information
Polypeptide is as immunogene.Selected polypeptide is in chicken Nrf2 PROTEIN C end, amino acid sequence: IFLVPKSRKAETKL.Utilize this
Immunogene obtains chicken Nrf2 antibody and ELISA detection kit, achieves following beneficial technical effect:
The present invention is inoculated with the antibody of rabbit acquisition using selected polypeptide as immunogene, being capable of specific recognition chicken Nrf2 egg
White, selected polypeptid acid sequence is also only consistent with chicken Nrf2 protein amino acid sequence height by BLAST comparison.
It is provided by the invention a kind of for detecting the ELISA detection kit of chicken Nrf2 albumen, have to chicken Nrf2 albumen
Specific recognition capability and higher sensitivity are able to detect the expression of chicken Nrf2 albumen in sample.
The present invention also passes through immunofluorescence experiment and verifies to antibody, and Nrf2 protein antibodies of the present invention can as the result is shown
For Immunofluorescence test, realize that monitor chicken Nrf2 albumen simultaneously expresses and distribution dynamic in the cell, with compared with high specific.
The present invention can be used for the fields such as biological therapy, diagnosis identification, biological study, and prepared antibody can use system
For the drug of diagnosis, prevention and/or treatment individual, such as: one or more in chicken body and Nrf2 associated disease；And it can be used for
Adjust the purposes having in the anti-oxidant of chicken Nrf2 mediation, anti-inflammatory, proteasome function and anti-infective innate immune response.
Detailed description of the invention
The measurement of Fig. 1 ELISA kit detection range, abscissa are peptide concentration (pg/mL), and ordinate is at 450nm
Light absorption value；
The ELISA testing result of Nrf2 expression quantity, abscissa are followed successively by control and difference in Fig. 2 chicken macrophage HD11
Antibody, ordinate are the light absorption value at 450nm, and wherein Abcam-ab62352 is rabbit-anti people Nrf2 monoclonal antibody (Abcam
Ab62352), wherein Abcam-ab156883 is rabbit-anti mouse Nrf2 polyclonal antibody (Abcam ab156883), Sigma-
SAB4501984 is rabbit-anti people/mouse Nrf2 polyclonal antibody (Sigma SAB4501984)；
The Immunofluorescence test result that Nrf2 is expressed in Fig. 3 chicken macrophage HD11；
After Fig. 4 TMUV infected chicken macrophage HD11, the expression and distribution situation of Nrf2 albumen in the cell, wherein DAPI
Refer to that nucleus indicator, Nrf2 refer to that chicken Nrf2 albumen, Merge are that DAPI nucleus cue mark and chicken Nrf2 albumen are copolymerized
Coke is in the same visual field.
Specific embodiment
Invention is further described in detail combined with specific embodiments below.Following case study on implementation is with the technology of the present invention
Premised under implemented, now provide detailed embodiment and specific operating process illustrate the present invention it is creative,
But protection scope of the present invention embodiment not limited to the following.Unless otherwise noted it is conventional practices in example, is made
Reagent or instrument and equipment enter specified otherwise, can be bought and be obtained by commercial sources.
1%BSA solution: it is also referred to as 1%BSA confining liquid in the present invention, is the bovine serum albumin(BSA) that mass fraction is 1%
Solution is bought from Sigma-Aldrich, article No. A1933.
Chicken macrophage HD11: it can be bought and be obtained by commercial sources, chicken macrophage HD11 used in the present invention comes
From in Harbin Veterinary Medicine Inst., China Academy of Agriculture.
DMEM culture medium (10%FBS, 1%sp): wherein DMEM culture medium: purchase is from Sigma-Aldrich, article No.
D0819。
10%FBS: fetal calf serum is bought from Sigma-Aldrich, article No. 12006C, volume fraction in the medium
It is 10%.
1%sp: the dual anti-solution of Pen .- Strep is bought from Invitrogen, article No. 15140122, in the medium
Volume fraction be 1%.
Fowl tembusu virus (TMUV): the strain is recorded in Sun L, Li Y, Zhang Y, Han Z, Xu Y, Kong X,
Liu S.Adaptation and attenuation of duck Tembusu virus strain Du/CH/LSD/
110128following serial passage in chicken embryos.Clin Vaccine
Immunol.2014Aug；21 (8): the strain that the 1046-53. present invention uses is from Chinese Academy of Agricultural Sciences Harbin animal doctor
Research institute.
Embodiment 1. is used to obtain the immunogene of chicken Nrf2 protein antibodies.
Immunogene described in the present embodiment is prepared via a method which to obtain:
1) according to chicken Nrf2 protein sequence information, (Serial No. BAA08364.1 in GenBank), integrated application biology letter
Breath learns analysis method and predicts and screen the polypeptide that antigenicity is good, solubility is high and is readily synthesized.569, final choice amino acid is extremely
582 polypeptides of amino acid are as immunogene, and amino acid sequence is as shown in SEQ ID NO:1.The segment polypeptide is in chicken Nrf2 PROTEIN C
End, amino acid sequence: IFLVPKSRKAETKL.
2) chemical synthesis aforementioned polypeptides, using 4- (N- maleimidomehyl) hexamethylene -1- carboxylic acid sulfonic group succinyl
Imines ester sodium salt is coupled as coupling agent, by above-mentioned 10mg polypeptide and 25mg Yue hole relative hemocyanin in room temperature, then uses 10kD
Bag filter in the PBS (pH7.2) dialysis remove the free polypeptide not being coupled, obtain described for obtaining chicken Nrf2 protein antibodies
Immunogene, be denoted as: KLH569.
The preparation method of 2. chicken Nrf2 protein antibodies of embodiment.
Chicken Nrf2 protein antibodies described in the present embodiment are to mix the immunogene KLH569 that embodiment 1 prepares with adjuvant
Immunity inoculation rabbit after emulsifying is closed, is prepared after taking the blood of immune rear rabbit purified.The preparation method is as follows:
1) it prepares immunogene: by amino acid sequence polypeptide as shown in SEQ ID NO:1, passing through coupling agent and carrier protein
Coupling, obtains chicken Nrf2 immunogene KLH569, referring to preparation method described in embodiment 1.
2) immunity inoculation: after chicken Nrf2 immunogene and adjuvant mixing and emulsifying that step 1) is obtained, inoculation rabbit is immunized；
It is specific as follows: 0.4mg KLH569 being mixed with 0.1mg muramyl dipeptide adjuvant, PBS is added to be settled to 1mL.Then again with equal bodies
After product Freund's complete adjuvant (1mL) mixing and emulsifying, in 4 points of two month female New Zealand White Rabbit two sides groins and oxter and neck
4, back subcutaneous immunizations, dosage of inoculation be 0.1mg immunogene/only.
3) after inoculation one month, the dosage used referring to first immunisation halves carry out secondary immunity.
4) it prepares thick antibody: after secondary immunity one month, being injected intravenously PBS under the conditions of no adjuvant and be settled to 0.1mL's
Hereafter 0.1mg KLH569 carries out booster immunization every two weeks, add up booster immunization 5 times.Each booster immunization takes after 1 week
20mL rabbit blood is centrifuged, and supernatant is collected, and obtains the thick antibody of chicken Nrf2 albumen；The booster immunization, referred to according to the last time
Dosage is inoculated with again, and the dosage of booster immunization is 0.1mL KLH569 (KLH569 containing 0.1mg) in the present embodiment.
5) amino acid sequence polypeptide as shown in SEQ ID NO:1 antibody purification: is coupled to mercapto preactivated in column
Antigen affinity column is prepared on base albumen agarose binding resin, then carries out column equilibration with PBS buffer solution；By filtered chicken
The antigen affinity column after balance is added in the thick antibody of Nrf2 albumen, then carries out column equilibration again with PBS buffer solution, makes after eluting
It is standby to obtain chicken Nrf2 protein antibodies.Specifically:
Firstly, amino acid sequence polypeptide as shown in SEQ ID NO:1 is coupled to sulfydryl albumen preactivated in column
Antigen affinity column is prepared on agarose binding resin；Then the thick antibody of filtered chicken Nrf2 albumen is added and is buffered with PBS
The antigen affinity column that liquid has balanced, then carries out column equilibration with PBS buffer solution again；It is finally molten with the 100mM glycine of pH2.5
Liquid elutes antigen affinity column as antibody elution liquid, merges and collects the elution that absorbance at 280nm wavelength is greater than 1.0
Liquid is dialysed by dialyzate of PBS buffer solution, obtains the chicken Nrf2 protein antibodies, in the present invention also referred to as chicken Nrf2-
569 protein antibodies.
The detection of 3. antibody mass of embodiment.
The purpose of the present embodiment is that detection the chicken Nrf2 protein antibodies obtained of embodiment 2 quality, specific method and
Steps are as follows.
1) antigen coat:
Using glutaraldehyde as coupling agent, by 10mg amino acid sequence polypeptide as shown in SEQ ID NO:1 and 20mg cow's serum
The coupling of albumin shaken at room temperature is as detection antigen.Antigen is detected with carbonate coating buffer (pH 9.6) dissolution of 50mM,
Adjustment antigen concentration is 4 μ g/mL, and every hole adds 100 holes μ L/ to 96 hole elisa Plates, and 4 DEG C stand overnight.
2) it closes:
It discards coating buffer within second day, is washed 3 times with PBS, the 1%BSA solution of 150 μ L is added in every hole, and 37 DEG C of closings 1 are small
When.
3) antigen-antibody combines:
Confining liquid is discarded, after PBS is washed 3 times, the anti-chicken Nrf2 after purification of 100 μ L difference doubling dilution degree is added in every hole
Protein antibodies or commercially available people and mouse Nrf2 antibody, as negative control, 37 DEG C are incubated for 2 hours not immune rabbit anteserum.
4) it is incubated for ELIAS secondary antibody:
PBS pats ELISA Plate several times after washing 5 times, and the 100 μ L diluted horseradish peroxidase of confining liquid 1:200 is added
Fc sections of secondary antibodies of goat anti-rabbit antibody of enzyme label, 37 DEG C are incubated for 1 hour.
5) it develops the color:
PBS pats ELISA Plate several times after washing 5 times, and the TMB developing solution of 100 μ L is added, and 37 DEG C are incubated for 20 minutes.
6) it reads:
The 2M sulfuric acid terminate liquid of 50 μ L is added in every hole, and the ELISA Plate terminated after reacting is read at 450nm in microplate reader
Light absorption value, testing result is as shown in the table.
1. chicken Nrf2-569 antibody ELISA testing result of table
It is specific and sensitive well to can be seen that the isolated antibody of the method for the present invention has from 1 test data of table
Property, the immune antibody titer obtained of the segment polypeptide is at 1:512000 times or more.
The ELISA detection kit of the detection chicken Nrf2 albumen of embodiment 4..
The ELISA detection kit component includes ELISA Plate, coating buffer, confining liquid, ELIAS secondary antibody, TMB colour developing
Liquid, terminate liquid, detection antigen and chicken Nrf2 protein antibodies as described in example 2, the detection antigen are with glutaraldehyde for coupling
Agent, amino acid sequence polypeptide as shown in SEQ ID NO:1 and bovine serum albumin(BSA) coupling are prepared.
The ELISA detection kit constituent and quality measurements of the detection chicken Nrf2 albumen of citing description below.
1.ELISA detection kit composition
ELISA detection kit component described in the present embodiment specifically can be by as follows at being grouped as: 96 hole elisa Plates, pH9.6
50mM carbonate coating buffer, 1%BSAr solution, horseradish peroxidase-labeled goat anti-rabbit antibody Fc sections of secondary antibodies, TMB
Developing solution and 2M sulfuric acid terminate liquid, using glutaraldehyde as coupling agent, by amino acid sequence polypeptide and ox as shown in SEQ ID NO:1
Seralbumin coupling is (as by 10mg amino acid sequence polypeptide as shown in SEQ ID NO:1 and 20mg bovine serum albumin(BSA) room
Temperature oscillation coupling) prepared by detection antigen and chicken Nrf2-569 protein antibodies as described in example 2.
2, ELISA kit quality testing
Specific method and steps are as follows.
2.1 antigen coats:
It is coupling agent respectively by amino acid sequence polypeptide and bovine serum albumin(BSA) as shown in SEQ ID NO:1 using glutaraldehyde
Coupling is as detection antigen.With the carbonate of 50mM coating buffer (pH 9.6) dissolution detection antigen, antigen concentration is from 2 μ g/
ML doubling dilution is to 15.6pg/mL, and every hole adds 100 μ L, and 4 DEG C stand overnight.
2.2 closings:
It discards coating buffer within second day, is washed 3 times with PBS, the 1%BSA of 150 μ L is added in every hole, and 37 DEG C are closed 1 hour.
2.3 antigen-antibodies combine:
Confining liquid is discarded, PBS is washed 3 times, and anti-chicken Nrf2 protein antibodies concentration after purification is adjusted to 1mg/mL, every hole
100 μ L are added by the diluted antibody of 1:2000,37 DEG C are incubated for 2 hours.
2.4 are incubated for ELIAS secondary antibody:
PBS pats ELISA Plate several times after washing 5 times, and the 100 μ L diluted horseradish peroxidase of confining liquid 1:200 is added
Fc sections of secondary antibodies of goat anti-rabbit antibody of enzyme label, 37 DEG C are incubated for 1 hour.
2.5 colour developings:
PBS pats ELISA Plate several times after washing 5 times, and the TMB developing solution of 100 μ L is added, and 37 DEG C are incubated for 20 minutes.
2.6 readings:
The 2M sulfuric acid terminate liquid of 50 μ L is added in every hole, and the ELISA Plate terminated after reacting is read at 450nm in microplate reader
Light absorption value, testing result is as shown in Figure 1.
As can be seen that the ELISA kit of the method for the present invention development has wider detection range from Fig. 1 test data
With preferable sensitivity and stability.Commercially available chicken Nrf2 protein ELISA kit and relevant report there is no to can be used for and this reagent
Box is compared.
5. chicken Nrf2 protein antibodies of embodiment are monitoring the application in the expression and distribution of chicken Nrf2 albumen in the cell.
1. utilizing Nrf2 expression in chicken Nrf2 protein antibodies detection chicken macrophage HD11
More commercially available people Nrf2 antibody, the knowledge of mouse Nrf2 antibody and chicken Nrf2-569 antibody of the present invention to chicken Nrf2
Not Shi Foucun difference, specific method and steps are as follows.
1) cell culture:
Every hole inoculation 5 × 10 in 96 porocyte culture plates4A HD11 cell, DMEM culture medium (10%FBS, 1%sp)
Culture, 37 DEG C, 5%CO2。
2) fixed:
After attached cell is cultivated 24 hours, supernatant is abandoned, and washed 3 times with PBS, the 4% poly first of 150 μ L is added in every hole
Aldehyde, 37 DEG C are fixed 1 hour.
3) permeable membrane:
PBS is washed 3 times, and the 0.25%triton-100 permeable membrane liquid of 200 μ L is added in every hole, is incubated at room temperature 0.5 hour.
4) it closes:
PBS is washed 3 times, and the 1%BSA confining liquid of 200 μ L is added in every hole, and 37 DEG C are incubated for 1 hour.
5) antigen-antibody combines:
Confining liquid is discarded, PBS is washed 3 times, will use the diluted commercially available people Nrf2 antibody of confining liquid, mouse Nrf2 antibody and sheet
The chicken Nrf2 antibody is invented by the concentration incubated cell of 0.01 μ g/mL, not immune rabbit anteserum is as negative control, 37 DEG C of incubations
2 hours.
6) it is incubated for ELIAS secondary antibody:
PBS is washed 3 times, and the goat anti-rabbit antibody of the 100 μ L diluted horseradish peroxidase-labeleds of confining liquid 1:500 is added
Fc sections of secondary antibodies, 37 DEG C are incubated for 1 hour.
7) it develops the color:
PBS is washed 5 times, and the TMB developing solution of 100 μ L is added, and 37 DEG C are incubated for 20 minutes.
8) it reads:
The 2M sulfuric acid terminate liquid of 50 μ L is added in every hole, and the ELISA Plate terminated after reacting is read at 450nm in microplate reader
Light absorption value, testing result is as shown in Figure 2.
It can be seen that the antibody that the method for the present invention obtains can preferably detect the table of chicken Nrf2 from Fig. 2 test data
It reaches, however the three plants of commercially available people and mouse Nrf2 antibody that detect cannot identify chicken Nrf2.
2. Immunofluorescence test chicken Nrf2 is expressed
Can determine antibody of the present invention be used for chicken Nrf2 Immunofluorescence test, specific method and steps are as follows.
1) cell culture:
Every hole 5 × 10 is pressed in 6 porocyte culture plates5A cell inoculation HD11, DMEM culture medium (10%FBS, 1%sp)
Culture, 37 DEG C, 5%CO2。
2) fixed:
After attached cell is cultivated 24 hours, supernatant is abandoned, and washed 3 times with PBS, the 4% poly first of 750 μ L is added in every hole
Aldehyde, 37 DEG C are fixed 1 hour.
3) permeable membrane:
PBS is washed 3 times, and the 0.25%triton-100 permeable membrane liquid of 1mL is added in every hole, is incubated at room temperature 0.5 hour.
4) it closes:
PBS is washed 3 times, and the 1%BSA confining liquid of 1mL is added in every hole, and 37 DEG C are incubated for 1 hour.
5) antigen-antibody combines:
Confining liquid is discarded, PBS is washed 3 times, 6 orifice plates are added by 0.02 μ g/mL concentration in chicken Nrf2 antibody of the present invention,
As negative control, 37 DEG C are incubated for 2 hours not immune rabbit anteserum.
6) it is incubated for ELIAS secondary antibody:
PBS is washed 3 times, Fc sections of secondary antibodies of goat anti-rabbit antibody of the addition 1mL diluted FITC label of confining liquid 1:500, and 37
DEG C be incubated for 1 hour.
7) fluorescence detection:
PBS is washed 3 times, and DAPI dyeing liquor contaminates core 10 minutes；Anti- fluorescence quenching is sufficiently added after cleaning, fluorescence is inverted aobvious
It observes and takes pictures under micro mirror (Olympus IX81), testing result is as shown in Figure 3.
Antibody Nrf2-569 of the present invention can be used for the Immunofluorescence test of chicken Nrf2 albumen as can be seen from Figure 3,
Specificity with higher.
3. Immunofluorescence test chicken Nrf2 is distributed into the cell
Can antibody of the present invention be detected be used for the expression and distribution of immunofluorescence monitoring chicken Nrf2 albumen in the cell
Dynamically, specific method and steps are as follows.
1) cell culture and virus infection:
5 × 10 are inoculated in 35mm culture vessel with glass bottom5A HD11 cell, DMEM culture medium (10%FBS, 1%sp) training
It supports, 37 DEG C, 5%CO2.After attached cell is cultivated 24 hours, fowl tembusu virus (TMUV) is inoculated with by infection multiplicity MOI=1.
2) fixed:
After TMUV infects 24 hours, supernatant is abandoned, and washed 3 times with PBS, 4% paraformaldehyde of 750 μ L of every hole addition, 37
DEG C fix 1 hour.
3) permeable membrane:
PBS is washed 3 times, and the 0.25%triton-100 permeable membrane liquid of 1mL is added in every hole, is incubated at room temperature 0.5 hour.
4) it closes:
PBS is washed 3 times, and the 1%BSA confining liquid of 1mL is added in every hole, and 37 DEG C are incubated for 1 hour.
5) antigen-antibody combines:
Confining liquid is discarded, PBS is washed 3 times, and culture is added by 0.02 μ g/mL concentration in chicken Nrf2 antibody of the present invention
Ware, 37 DEG C are incubated for 2 hours.
6) it is incubated for ELIAS secondary antibody:
PBS is washed 3 times, Fc sections of secondary antibodies of goat anti-rabbit antibody of the addition 1mL diluted FITC label of confining liquid 1:500, and 37
DEG C be incubated for 1 hour.
7) fluorescence detection:
PBS is washed 3 times, and DAPI dyeing liquor contaminates core 10 minutes；Anti- fluorescence quenching, confocal fluorescent sufficiently is added after cleaning
It observes and takes pictures under microscope (Zeiss LSM880), testing result is as shown in Figure 4.
From fig. 4 it can be seen that Nrf2 albumen overall expression level does not obviously change after TMUV infected chicken macrophage HD11
Become, but Nrf2 albumen is significantly assembled into nucleus, the oxidation for prompting virus infection to trigger host cell Nrf2 mediation is answered
Swash regulated and control network.
The above results show, antibody of the present invention can be used for monitoring simultaneously the expression of chicken Nrf2 albumen in the cell and
Distribution dynamic.
The preparation method of 6. chicken Nrf2 protein antibodies of embodiment.
Embodiment 2 is repeated, is with the difference of embodiment 2, step 2) dosage of inoculation is immune for 1mg in the preparation method
Former/only；Step 4) is described when preparing thick antibody, after secondary immunity one month, is injected intravenously PBS under the conditions of no adjuvant and is settled to
The 1mg KLH569 of 0.4mL.Antibody manufactured in the present embodiment can be equally used for Immunofluorescence test, realizes while monitoring chicken
Nrf2 albumen is expressed in the cell and distribution dynamic, has compared with high specific.
Sequence table
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture (China Animal Health and Epidemiology Center Harbin
Branch center)
<120>one breeder Nrf2 protein antibodies and its immunogene, immunogenic polypeptide, detection kit and application
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 14
<212> PRT
<213>immunogenic polypeptide
<400> 1
Ile Phe Leu Val Pro Lys Ser Arg Lys Ala Glu Thr Lys Leu
1 5 10
Claims (8)
1. a kind of for obtaining the immunogene of chicken Nrf2 protein antibodies, which is characterized in that be by amino acid sequence such as SEQ ID
Polypeptide shown in NO:1 is obtained by coupling agent and carrier protein couplet.
2. a breeder Nrf2 protein antibodies, which is characterized in that be after immunogene described in claim 1 and adjuvant mixing and emulsifying
Immunity inoculation rabbit prepares after taking the blood of immune rear rabbit purified, and the specific method is as follows:
1) immunogene is prepared: even by coupling agent and carrier protein by amino acid sequence polypeptide as shown in SEQ ID NO:1
Connection, obtains chicken Nrf2 immunogene；
2) immunity inoculation: after chicken Nrf2 immunogene and adjuvant mixing and emulsifying that step 1) is obtained, inoculation rabbit is immunized；
3) after inoculation one month, the dosage used referring to first immunisation halves carry out secondary immunity；
4) it prepares thick antibody: after secondary immunity one month, chicken Nrf2 immunogene, injection dosage is injected intravenously under the conditions of no adjuvant
Only for 0.1-1mg/, hereafter, booster immunization is carried out every two weeks, add up booster immunization 2-5 times, each booster immunization takes after 1 week
Rabbit blood is centrifuged, and supernatant is collected, and obtains the thick antibody of chicken Nrf2 albumen；
5) amino acid sequence polypeptide as shown in SEQ ID NO:1 antibody purification: is coupled to sulfydryl egg preactivated in column
Antigen affinity column is prepared on white agarose binding resin, then carries out column equilibration with PBS buffer solution；By filtered chicken Nrf2 egg
The antigen affinity column after balance is added in white thick antibody, then carries out column equilibration again with PBS buffer solution, prepares after eluting
Chicken Nrf2 protein antibodies.
3. the preparation method of chicken Nrf2 protein antibodies as claimed in claim 2, which comprises the steps of:
1) immunogene is prepared: even by coupling agent and carrier protein by amino acid sequence polypeptide as shown in SEQ ID NO:1
Connection, obtains chicken Nrf2 immunogene；
2) immunity inoculation: after chicken Nrf2 immunogene and adjuvant mixing and emulsifying that step 1) is obtained, inoculation rabbit is immunized；
3) after inoculation one month, the dosage used referring to first immunisation halves carry out secondary immunity；
4) it prepares thick antibody: after secondary immunity one month, chicken Nrf2 immunogene, injection dosage is injected intravenously under the conditions of no adjuvant
Only for 0.1-1mg/, hereafter, booster immunization is carried out every two weeks, add up booster immunization 2-5 times, each booster immunization takes after 1 week
Rabbit blood is centrifuged, and supernatant is collected, and obtains the thick antibody of chicken Nrf2 albumen；
5) amino acid sequence polypeptide as shown in SEQ ID NO:1 antibody purification: is coupled to sulfydryl egg preactivated in column
Antigen affinity column is prepared on white agarose binding resin, then carries out column equilibration with PBS buffer solution；By filtered chicken Nrf2 egg
The antigen affinity column after balance is added in white thick antibody, then carries out column equilibration again with PBS buffer solution, prepares after eluting
Chicken Nrf2 protein antibodies.
4. preparation method according to claim 3, which is characterized in that the step 1) coupling agent is 4- (N- maleimide
Amine methyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium salt, N- succinimide -3- (2- pyridine two is thio) propionic acid
Ester or glutaraldehyde；Carrier protein Wei Yue hole relative hemocyanin, ovalbumin, bovine serum albumin(BSA) or artificial synthesized poly
Lysine.
5. preparation method according to claim 3, which is characterized in that the step 2) immunity inoculation is by 0.25-1.5mg
After immunogene is mixed with 0.05-0.5mg muramyl dipeptide adjuvant, PBS is added to be settled to 1mL, is then helped completely with isometric Freund
Rabbit is inoculated with after agent mixing and emulsifying；The rabbit is two month female New Zealand White Rabbit；The inoculation position be rabbit two sides groin and
4 points of oxter and 4 points of the nape of the neck it is subcutaneous；Dosage of inoculation be 0.1-1mg immunogene/only.
6. preparation method according to claim 3, which is characterized in that the step 5) elution refers to the 100mM with pH2.5
Glycine solution elutes antigen affinity column as antibody elution liquid, merges absorbance at collection 280nm wavelength and is greater than 1.0
Eluent, dialyse by dialyzate of PBS buffer solution, obtain the chicken Nrf2 protein antibodies.
7. a kind of ELISA detection kit for detecting chicken Nrf2 albumen, which is characterized in that the ELISA detection kit component
Including ELISA Plate, coating buffer, confining liquid, ELIAS secondary antibody, TMB developing solution, terminate liquid, detection antigen and claim 2 institute
The chicken Nrf2 protein antibodies stated, the detection antigen are using glutaraldehyde as coupling agent, by amino acid sequence such as SEQ ID NO:1 institute
What polypeptide and the bovine serum albumin(BSA) coupling shown prepared.
8. ELISA detection kit according to claim 7, which is characterized in that the coating buffer is pH9.6's
50mM carbonate is coated with buffer；The confining liquid is the BSA solution that mass fraction is 1%；The ELIAS secondary antibody is horseradish mistake
Fc sections of secondary antibodies of goat anti-rabbit antibody of oxide enzyme label；The terminate liquid is the aqueous sulfuric acid of 2M.
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| CN103074348A (en) * | 2013-02-06 | 2013-05-01 | 四川农业大学 | Recombinant carp Nrf2 (NF-E2-related factor 2) gene, protein, preparation and detection methods and application of recombinant carp Nrf2 gene |
| CN106699899A (en) * | 2016-12-27 | 2017-05-24 | 重庆大学 | Immunogen for obtaining Nrf1D protein antibody, Nrf1D protein antibody and Elisa detection kit |
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| CN103074348A (en) * | 2013-02-06 | 2013-05-01 | 四川农业大学 | Recombinant carp Nrf2 (NF-E2-related factor 2) gene, protein, preparation and detection methods and application of recombinant carp Nrf2 gene |
| CN106699899A (en) * | 2016-12-27 | 2017-05-24 | 重庆大学 | Immunogen for obtaining Nrf1D protein antibody, Nrf1D protein antibody and Elisa detection kit |
Non-Patent Citations (1)
| Title |
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| Cloning and characterization of a novel erythroid cell-derived CNC family transcription factor heterodimerizing with the small Maf family proteins;Itoh,K.等;《Mol. Cell. Biol.》;19950831;第15卷(第8期);全文 * |
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