CN105017417A - Arabidopsis thaliana ZFN3 protein polyclonal antibody and preparation method thereof - Google Patents
Arabidopsis thaliana ZFN3 protein polyclonal antibody and preparation method thereof Download PDFInfo
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Abstract
The invention provides a preparation method of a preparation method of an arabidopsis thaliana ZFN3 protein polyclonal antibody. The method comprises the following steps: (1) a recombinant expression vector comprising nucleotide sequences represented by SEQ ID No: 1 and SEQ ID No: 2 is constructed; the recombinant expression vector is used for transforming Escherichia coli competent cells, such that a recombinant protein antigen is obtained; (2) the recombinant protein antigen is used for immunizing animal; and serum separation purification is carried out, such that the arabidopsis thaliana ZFN3 protein polyclonal antibody is obtained. The polyclonal antibody prepared with the method can specifically recognize arabidopsis thaliana ZFN3 protein and cannot recognize other arabidopsis thaliana proteins, such that the polyclonal antibody can be widely applied in ZFN3 protein detection and function identification. The preparation of the ZFN3 protein antibody is significant in detecting ZFN3 protein in arabidopsis thaliana and in researching ZFN3 protein functions.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of Arabidopis thaliana ZFN3 protein polyclone antibody and preparation method thereof.
Background technology
Arabidopis thaliana is at plant science, comprises one of model animals in genetics and development of plants research.The advantage of Arabidopis thaliana be plant little, often short for the time, knot is many, vitality is strong.The genome of Arabidopis thaliana is minimum in current known plants genome.Its whole genome has been combined in 2000 Nian You international mouseearcress genome cooperative alliances, is also first Plant Genome be sequentially analyzed.Arabidopis thaliana is self-pollination plant, gene high homogenous, very high by chemical factors process mutation rate, easily obtains the defective type of various metabolic function.Owing to having these advantages above-mentioned, so Arabidopis thaliana is the ideal material carrying out genetics research, be described as " fruit bat in plant " by scientist.
Transcription factor is the albumen of one group of identification specific DNA sequence, can promote or suppress transcribing and expressing of some specific gene, thus cause the change of tissue cellularity and function.Transcription factor ZFN3 is a kind of special zinc finger protein (zinc finger protein), and it contains five CCCH zinc fingerses, and in Arabidopsis leaf aging course, expression amount raises gradually.The mutant of Arabidopis thaliana yuc6-1D, can by constantly accumulating growth hormone, postpone the aging of blade, shown by gene microarray analysis, ZFN3 gene is in this mutant, expression amount significantly declines, and ZFN3 protein coding gene is included the candidate gene into adjusting vane aging by Peking University Leaf SenescenceDatabase.But the antibody at present about ZFN3 albumen has no report.
Therefore be necessary to provide a kind of polyclonal antibody for Arabidopis thaliana ZFN3 albumen, thus for studying and utilizing the biological function of this albumen to provide powerful.
Summary of the invention
The object of the invention is to the blank filling up existing Arabidopis thaliana ZFN3 Protein Detection field, develop a kind of polyclonal antibody for Arabidopis thaliana ZFN3 albumen and preparation method thereof.
A preparation method for Arabidopis thaliana ZFN3 protein polyclone antibody, the method comprises the following steps:
(1) build the recombinant expression vector containing nucleotide sequence shown in SEQ ID No:1 and SEQ ID No:2, described recombinant expression vector transformation of E. coli competent cell is obtained recombinant protein antigen;
(2) by described recombinant protein antigen immune animal, the clear separation and purification of menses obtains Arabidopis thaliana ZFN3 protein polyclone antibody.
Wherein, nucleotides sequence shown in described SEQ ID No:1 and SEQ ID No:2 is classified as contriver by carrying out the coding DNA of a large amount of two specific polypeptide fragments found of analysing and comparing to Arabidopis thaliana ZFN3 protein sequence.
Optionally, described recombinant expression vector is containing, for example the nucleotide sequence shown in SEQ ID No.3.Wherein, nucleotide sequence shown in SEQ ID No.3 contains nucleotide sequence shown in SEQ ID No:1 and SEQ IDNo:2, in order to make the polypeptide fragment coded by the nucleotide sequence shown in SEQ ID No:1 and SEQ ID No:2 the expression of precise and high efficiency can obtain recombinant protein antigen in competent escherichia coli cell, contriver adds one section of GGGGGGGGGGGGTCC sequence between these two sections of sequences.
Optionally, described recombinant expression vector the fragment containing the nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2 is cloned into prokaryotic expression carrier to obtain.
Optionally, described recombinant protein antigen is containing, for example the aminoacid sequence shown in SEQ ID No.4 and SEQ ID No.5.
In preferred situation, described recombinant protein antigen is containing, for example the aminoacid sequence shown in SEQ ID No.6.
The present invention has no particular limits for the kind of used competent escherichia coli cell, as long as recombinant expression vector effective expression in cell can be applicable to, in preferred situation, in order to obtain better expression effect, competent escherichia coli cell is e. coli bl21 (DE3) competent cell.
In the present invention, this area routine can be utilized to be suitable for the prokaryotic expression carrier of recombinant protein antigen, and in preferred situation, when described prokaryotic expression carrier is pE-SUMOpro, the expression effect of recombinant protein antigen is better.
In method provided by the present invention, the step of immune animal comprises 4 immunity, first the 1st immune animal after the recombinant protein antigen of purifying being mixed with complete Freund's adjuvant, carry out the 2-4 time immunity after then being mixed with non-fully freund's adjuvant by the recombinant protein antigen of purifying, the timed interval of each immunity is 14 days.
In the present invention, the immunizing dose used during immune animal and can carrying out according to the method for this area routine with the blending ratio of adjuvant.The animal of immunity includes but not limited to new zealand white rabbit, small white mouse and big white mouse.
After the 4th immunity the 10th day, get animal blood, collect antiserum(antisera), utilize purified reagent to obtain Arabidopis thaliana ZFN3 polyclonal antibody that purity is not less than 80%.
One's duty invention additionally provides the Arabidopis thaliana ZFN3 protein polyclone antibody utilizing method of the present invention to prepare.Described polyclonal antibody can specific amino acid fragment SEQ ID No.4 in specific identification ZFN3 albumen and SEQ ID No.5, is the specific antibody of ZFN3 albumen.
Present invention also offers the application of described Arabidopis thaliana ZFN3 protein polyclone antibody.Described application comprises reagent or the test kit that preparation contains described Arabidopis thaliana ZFN3 protein polyclone antibody, for detecting Arabidopis thaliana ZFN3 albumen or carrying out Function Identification.
Method provided by the present invention obtains the coding DNA of two sections of specific fragments in Arabidopis thaliana ZFN3 encoding histone DNA by PCR method clone, thus obtains specific polyclonal antibody.Utilize polyclonal antibody prepared by method provided by the present invention, can specific recognition Arabidopis thaliana ZFN3 albumen, and other albumen of Arabidopis thaliana can not be identified, in the detection of ZFN3 albumen and Function Identification, research, there is extensive use.Being prepared as of ZFN3 protein antibodies in Arabidopis thaliana, to detect ZFN3 albumen and research ZFN3 protein function has certain meaning.
Accompanying drawing explanation
The pcr amplification result of DNA for the purpose of Fig. 1;
Fig. 2 is that prokaryotic expression carrier thalline PCR detects electrophorogram;
Fig. 3 is recombinant protein SDS-PAGE electrophorogram;
Fig. 4 is SDS-PAGE electrophorogram after recombinant protein purification, and wherein, M is albumen Marker; 1 and 2 is the albumen of purifying; The band of arrow instruction is target protein band;
Fig. 5 is that Arabidopis thaliana Western detects picture.
Embodiment
Below will be described in detail the specific embodiment of the present invention.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
The instrument used in following examples and reagent all can be obtained by the mode buying commercially available prod.
Embodiment 1
1, Arabidopis thaliana ZFN3 protein sequence obtains
In the method that Beijing Tian Yihuiyuan biotech company is synthesized by full genome, synthesis is used for the DNA sequence dna of Prokaryotic expression vector construction, namely the nucleotide sequence shown in SEQ ID No.3 is synthesized, nucleotide sequence shown in SEQ ID No.3 is cloned in carrier pGM-T (purchased from Beijing Tian Gen biotech firm), correct through DNA sequencing sequence, by its called after pGM-ZP plasmid.
2, Prokaryotic expression vector construction: design pair of primers is for building prokaryotic expression carrier primer:
ZP-f：5’-GAACAGATTGGAGGTAACTTGATGGTTTCTGTTCG-3’
ZP-r：5’-GTGAGACCGGTACCATCAGAATTCGAGTGATGTGGG-3’
With pGM-ZP plasmid for template, carry out pcr amplification.PCR program is: 95 DEG C of sex change 30s, and 60 DEG C of annealing 30s, 72 DEG C extend 30s, 25 circulations.PCR primer, through gel electrophoresis analysis, obtains the DNA band (Fig. 1) being about 250bp, and it is stand-by that product reclaims purifying.
Extract prokaryotic expression carrier pE-SUMOPro plasmid, SpeI enzyme cuts pE-SUMOPro, recovery, purifying digestion products.According to pE-SUMOPro carrier: PCR primer fragment is the ratio of 1:1,15min is connected in 50 DEG C with seamless link enzyme, product conversion E. coli competent BL21 (DE3) will be connected, after amoxicillin screening, picking mono-clonal bacterium colony carries out thalline PCR detection (Fig. 2), choose PCR and detect positive bacterium colony, called after SUMO-ZP.
3, expression of recombinant proteins and purifying:
By the Escherichia coli bacteria liquid containing SUMO-ZP, be seeded in LB nutrient solution, use constant-temperature table, 37 DEG C, 180rpm cultivates, and treats that thalline OD value reaches 0.6, adds the IPTG of final concentration 1mM, and temperature is adjusted to 30 DEG C, and 180rpm continues to cultivate 6h.Get 50ml Escherichia coli bacteria liquid, be placed on whizzer, 10000rpm, 1min, abandon supernatant and collect thalline, add 10ml PBS, Eddy diffusion thalline.The thalline of Eddy diffusion is placed in ice bath, uses Ultrasonic Cell Disruptor, carry out bacterial cell disruption.By the bacterium liquid after break process, place in centrifuges, 12000rpm, 10min, collect upper cleer and peaceful precipitation respectively, precipitation uses 10ml PBS Eddy diffusion, gets two kinds of solution 10 μ l respectively, adds 10 μ l 2 × SDS damping fluids, boiling water boils 10min, after centrifugal, get supernatant and carry out SDS-PAGE detection, detected result shows: the band (Fig. 3) containing a target protein size in supernatant.By supernatant solution, after Ni column purification, carry out SDS-PAGE detection, result shows: can be purified into target protein band, and with prediction target protein (Fig. 4) in the same size, its aminoacid sequence is for shown in SEQ ID No:6.
4, immune animal:
The recombinant protein of purifying is mixed with complete Freund's adjuvant 1:1, carry out first time immunity, latter 14th day of first time immunity, mixes the antigen of purifying with non-fully freund's adjuvant 1:1, carries out second time immunity, latter 14th day of second time immunity, the antigen of purifying is mixed with non-fully freund's adjuvant 1:1, carries out third time immunity, latter 14th day of third time immunity, the antigen of purifying is mixed with non-fully freund's adjuvant 1:1, carries out the 4th immunity and carry out;
5, antibody is separated and purifying:
After the 4th immunity the 10th day, by immunity with young new zealand white rabbit (body weight: 6-7 jin, purchased from Fang Yuan plant), adopt carotid artery bloodletting, blood is placed 30 minutes in 37 DEG C of thermostat containers, then spends the night 4 DEG C of placements.Blood clot is dialled from tube wall by medication shovel, and by blood transfer in plastic centrifuge tube, 4 DEG C, the centrifugal 10min of 10000g, collects supernatant liquor and be antiserum(antisera).Use Protein A+G Agarose reagent (article No.: the CW0349) antibody purification that health is ShiJi Co., Ltd, concrete steps:
(1) in 10ml antibody serum, add the fully resuspended Protein A+GAgarose of 100 μ l, 4 DEG C are slowly shaken 3 hours;
(2) centrifugal 5 minutes of 2500rpm, carefully absorbs supernatant;
(3) with PBS washing precipitation 5 times, the consumption of PBS is 10ml at every turn.Centrifugal 5 minutes of centrifugal condition during washing: 2500rpm, carefully absorbs supernatant.
(4) after completing last washing, remove supernatant, detect product purity, the purity of product antibodies is 86%.
6, antibody test:
Choose wildtype Arabidopsis thaliana and ZFN3 transgenation Arabidopis thaliana is experiment material, use the plant protein that health is ShiJi Co., Ltd to extract test kit (article No.: CW0885B), extract total protein respectively.Concrete steps are: choose Arabidopsis leaf and organize about 100mg, be placed in mortar, add extraction agent 0.5ml, carry out homogenized; After hatching 20 minutes on ice after homogenate, utilize low-temperature and high-speed refrigerated centrifuge 4 DEG C 12,000rpm, centrifugal 20 minutes; Collect the soluble proteins in supernatant, be placed on 4 DEG C, treat that next step is tested.
Extract protein, after SDS-PAGE electrophoresis, through half-dried transferring film instrument, be transferred on nitrocellulose filter, the skim-milk (TBST buffer solution) with 5%, under 4 DEG C of conditions, closes 2h; Add the polyclonal antibody (1:5000) of purifying, under 4 DEG C of conditions, reaction 12h; Add the goat anti-rabbit igg antibody (1:2000) of horseradish peroxidase mark, under 37 DEG C of conditions, reaction 1h; Employing chemoluminescence develops the color, and as shown in Figure 5, wildtype Arabidopsis thaliana has a single hybridising band clearly to detected result, and does not have detection signal (Fig. 5) in mutant.
Result proves: the polyclonal antibody prepared by the present invention, can identify the ZFN3 albumen in plant materials, and can specifically with Arabidopis thaliana ZFN3 protein binding.
Conclusion: above-mentioned experimental result shows, the Arabidopis thaliana ZFN3 protein polyclone antibody of preparation can identify can detect the ZFN3 albumen in Arabidopis thaliana ZFN3 albumen, and not have an effect with other albumen in plant materials.This polyclonal antibody, as ZFN3 protein assay reagent, can have great importance to the Function Identification of ZFN3 albumen.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a preparation method for Arabidopis thaliana ZFN3 protein polyclone antibody, the method comprises the following steps:
(1) build the recombinant expression vector containing nucleotide sequence shown in SEQ ID No:1 and SEQ ID No:2, described recombinant expression vector transformation of E. coli competent cell is obtained recombinant protein antigen;
(2) by described recombinant protein antigen immune animal, the clear separation and purification of menses obtains Arabidopis thaliana ZFN3 protein polyclone antibody.
2. preparation method according to claim 1, is characterized in that, described recombinant expression vector is containing, for example the nucleotide sequence shown in SEQ ID No.3.
3. preparation method according to claim 1 and 2, is characterized in that, described recombinant expression vector the fragment containing the nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2 is cloned into prokaryotic expression carrier to obtain.
4. the preparation method according to right 2, is characterized in that, described recombinant protein antigen is containing, for example the aminoacid sequence shown in SEQ ID No.4 and SEQ ID No.5.
5. the preparation method according to claim 1,2 or 4, is characterized in that, described recombinant protein antigen is containing, for example the aminoacid sequence shown in SEQ ID No.6.
6. preparation method according to claim 5, is characterized in that, competent escherichia coli cell is e. coli bl21 (DE3) competent cell.
7. according to the preparation method in claim 1,2,4 and 6 described in any one, it is characterized in that, the step of immune animal comprises 4 immunity, first the 1st immune animal after the recombinant protein antigen of purifying being mixed with complete Freund's adjuvant, carry out the 2-4 time immunity after then being mixed with non-fully freund's adjuvant by the recombinant protein antigen of purifying, the timed interval of each immunity is 14 days.
8. preparation method according to claim 7, is characterized in that, described prokaryotic expression carrier is pE-SUMOpro.
9. the Arabidopis thaliana ZFN3 protein polyclone antibody utilizing the method in claim 1-8 described in any one to prepare.
10. the application of Arabidopis thaliana ZFN3 protein polyclone antibody according to claim 9.
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| CN108409858A (en) * | 2018-05-29 | 2018-08-17 | 天津农学院 | A kind of tamato fruit transcription factor CNR polyclonal antibodies and preparation method thereof |
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| WO2003087153A1 (en) * | 2002-04-12 | 2003-10-23 | Molecular Engines Laboratories | Mouse kruppel-type zinc finger protein knox-25 and the use of thereof |
| CN1422864A (en) * | 2002-06-07 | 2003-06-11 | 武汉大学 | Zinc finger protein and its antibody preparation and use |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108409858A (en) * | 2018-05-29 | 2018-08-17 | 天津农学院 | A kind of tamato fruit transcription factor CNR polyclonal antibodies and preparation method thereof |
| CN108409858B (en) * | 2018-05-29 | 2021-06-18 | 天津农学院 | Tomato fruit transcription factor CNR polyclonal antibody and preparation method thereof |
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