CN108409858A - A kind of tamato fruit transcription factor CNR polyclonal antibodies and preparation method thereof - Google Patents
A kind of tamato fruit transcription factor CNR polyclonal antibodies and preparation method thereof Download PDFInfo
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Abstract
The present invention provides a kind of tamato fruit transcription factor CNR polyclonal antibodies and preparation method thereof, and this method includes：(1) structure contains SEQ ID No：Recombinant expression carrier conversion competent escherichia coli cell is obtained CNR proteantigens by the recombinant expression carrier of nucleotide sequence shown in 1；(2) animal is immunized in the CNR proteantigens, menses, which isolate and purify clearly, obtains tomato CNR protein polyclone antibodies, the polyclonal antibody prepared using method provided by the present invention, it is capable of the CNR albumen of specific recognition tamato fruit, and cannot identify other albumen of tamato fruit, there is extensive use in the detection of CNR albumen and Function Identification and its research.The function of being prepared as expressions and research CNR albumen of the detection tomato transcription factor CNR in ripening of fruits of CNR protein antibodies is of great significance.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of tamato fruit transcription factor CNR polyclonal antibodies and its
Preparation method.
Background technology
Tomato (Solanum lycopersicum Mill.) cultivation history is long, because its genome is small (950Mb), growth
Period is short (bearing fruit to maturation about 60d), and with arabidopsis, rice, corn isotype plant affiliation farther out and as research
Fleshy fruits are developed and ripe model organism.A large amount of Tomato Germplasm Resources, mutant library, dense genetic map are obtained
Spectrum, EST resources, and transient expression and stable conversion technology maturation.In addition, the cultivated tomato full-length genome that in May, 2012 completes
Fine sequence analysis has greatly pushed tomato functional genomics and molecule genetics research.Ripening of fruits is related to greatly
The adjusting of metabolic pathway and significantly changing for Physiology and biochemistry attribute are measured, needs to carry out essence to the timing expression of series of genes
Close transcriptional control, therefore the functional study of Tomato Ripening associated transcription factor becomes one of the research hotspot in botany field.
The maturation of fruit regulates and controls specific downstream gene to complete alone or synergistically by many transcription factors
(Giovannoni 2007), one type transcription factor are SBP box families.SBP box transcription factors take part in development of plants
Many aspects (Peng Hua 2016), including phase transition, the building up of floral organ, fruit maturation, Plant hormone signal transduction and copper
Homeostasis etc. (high English 2009).CNR is a more special member in SBP box transcription factor families, it can be in fruit
It plays a significant role in real development, maturation.Currently, research about tomato transcription factor CNR is mainly in transcriptional level,
However deposit modification horizontal upon translation after albumen synthesis, protein level is only the level that transcription factor CNR plays a role, and mesh
The preceding antibody about CNR albumen has not been reported.
It is therefore desirable to provide a kind of polyclonal antibody for tomato CNR albumen, to be to study and utilize the albumen
Biological function provide powerful.
Invention content
It is an object of the invention to fill up the blank of the existing antibody research for CNR albumen, exploitation is a kind of to be directed to tomato
The polyclonal antibody and preparation method thereof of CNR albumen.
A kind of preparation method of tamato fruit transcription factor CNR polyclonal antibodies, this approach includes the following steps：
(1) structure contains SEQ ID No：The recombinant expression carrier of nucleotide sequence shown in 1, by the recombinant expression carrier
It converts competent escherichia coli cell and obtains recombinant protein antigen；
(2) animal is immunized in the CNR proteantigens, menses, which isolate and purify clearly, obtains tomato CNR protein polyclone antibodies.
Wherein, the SEQ ID No：Nucleotides sequence shown in 1 is classified as inventor according to the tomato delivered in GenBank
CNR gene mRNA sequences (NM_001319308.1) design primer, and be inserted into respectively at 5 ' ends of sense primer and downstream primer
I double enzyme site of EcoR I and Xho, with the tomato that kind is Lycopersicon esculentum cv.Ailsa Craig
The cDNA that reverse transcription obtains after fruit extraction mRNA is template, is obtained through PCR amplification.
Optionally, the recombinant expression carrier is will to be cloned containing the segment of nucleotide sequence shown in SEQ ID No.1
It is obtained to prokaryotic expression carrier.
Optionally, the CNR proteantigens contain the amino acid sequence as shown in SEQ ID No.2.
The present invention is not particularly limited the type of used competent escherichia coli cell, as long as can be suitble to
Recombinant expression carrier effective expression in cell, in the case of preferred, in order to obtain better expression effect, Escherichia coli
Competent cell is Escherichia coli Rosetta competent cells.
In the present invention, this field can be utilized routinely to be suitable for the prokaryotic expression carrier of recombinant protein antigen, preferably
In the case of, when the prokaryotic expression carrier is pET-30a, the expression effect of recombinant protein antigen is more preferable.
In method provided by the present invention, the step of animal is immunized includes immune 3 times, first by the recombinant protein of purifying
1st immune animal after antigen is mixed with complete Freund's adjuvant then helps the recombinant protein antigen of purifying and non-fully Freund
The 2-3 times is carried out after agent mixing to be immunized, immune time interval is 14d every time.
In the present invention, used immunizing dose and can be according to ability with the mixed proportion of adjuvant when animal is immunized
The method of domain routine carries out.Immune animal includes but not limited to mouse.
10d after being immunized at the 3rd time takes animal blood, collects antiserum, and obtaining purity using purified reagent is not less than
80% tomato CNR protein polyclone antibodies.
The present invention also provides the tomato CNR protein polyclone antibodies prepared using method of the present invention.It is described more
Clonal antibody is capable of the specific amino acid segment SEQ ID No.2 in the identification CNR albumen of specificity, is the spy of CNR albumen
Heterogenetic antibody.
The present invention also provides the applications of the tomato CNR protein polyclone antibodies.The application includes preparing to contain
The reagent or kit of the tomato CNR protein polyclone antibodies, for detecting tomato transcription factor CNR in ripening of fruits
In expression or carry out Function Identification.
Method provided by the present invention clones to obtain the DNA sequence dna of coding CNR albumen by PCR method, to obtain spy
Anisotropic polyclonal antibody.The polyclonal antibody prepared using method provided by the present invention, being capable of specific recognition tomato CNR
Albumen, and cannot identify other albumen of tomato, there is extensive use in the detection of CNR albumen and Function Identification and research.
The function of being prepared as detecting the expression of transcription factor CNR in tomato and study CNR albumen of CNR protein antibodies has weight
Want meaning.
Description of the drawings
Fig. 1 is tamato fruit total serum IgE agarose gel electrophoresis figure.
Fig. 2 is the engineering bacteria bacterium solution PCR gel electrophoresis figures for carrying expression vector pET-30a-CNR.M：Marker；1：Mesh
Segment.
Gel electrophoresis figure, M are identified in the digestion that Fig. 3 is recombinant plasmid pET-30a-CNR：Marker；1：Plasmid double digestion.
Fig. 4 is a small amount of induction expression protein gel electrophoresis figures of CNR albumen, M：Marker；1-3：Monoclonal；4：It does not lure
It leads, using 15% SDS-PAGE.
Fig. 5 is the CNR protein expression gel electrophoresis figures that inducing temperature is 37 DEG C, M：marker；1-4：Cell pyrolysis liquid
Supernatant fraction；5-8：The sediment fraction of cell pyrolysis liquid：1 and 50.1mmol/L IPTG inductions；2 and 6：0.4mmol/L IPTG
Induction；3 and 7：0.8mmol/L IPTG inductions；4 and 8：1mmol/L IPTG inductions.
Fig. 6 is affinity purification gel electrophoresis Fig. 1, M of the CNR albumen with His labels：marker；1：1.0mmol/L
The cellular lysate liquid supernatant of IPTG inductions is purification of samples；2-7：Respectively combination buffer, wash buffer, elution buffer
I, II, III, IV elution buffer crosses the albumen eluted after column.
Fig. 7 is affinity purification gel electrophoresis Fig. 2, M of the CNR albumen with His labels：marker；1-8：Imidazole concentration
500mmol/L elution buffers cross the albumen eluted after column.
Fig. 8 is affinity purification gel electrophoresis Fig. 3, M of the CNR albumen with His labels：marker；1-7：Imidazole concentration
500mmol/L elution buffers cross the albumen eluted after column.
Fig. 9 is affinity purification gel electrophoresis Fig. 4, M of the CNR albumen with His labels：marker；1-8：Imidazole concentration
500mmol/L elution buffers cross the albumen eluted after column.
Figure 10 is the protein expression gel electrophoresis figure that recombinant vector converts different feeling state cell, M：marker；1 and 2：It is single
It clones (BL21 plysS)；3：It does not induce (BL21 plysS)；4 and 5：Monoclonal (Codon plus)；6：(Codon is not induced
plus)；7 and 8：Monoclonal (Rosetta)；9：It does not induce (Rosetta).
Figure 11 is the potency figure of CNR protein polyclone antibodies.
It for those of ordinary skill in the art, without creative efforts, can be according to above attached
Figure obtains other relevant drawings.
Figure 12 is that Western blot detect serum specificity electrophoretogram, 1：CNR albumen after purification.
Figure 13 is the gel electrophoresis figure of tamato fruit different times total protein, M：Marker；1：The underdone phase；2：Green ripe stage；
3：The broken color phase；4：The pink phase；5：The red ripe phase.
Figure 14 be Fruit Ripening of Tomato during CNR expressing quantities western blot analysis figure, 1：The underdone phase；2：It is green ripe
Phase；3：The broken color phase；4：The pink phase；5：The red ripe phase.
Specific implementation mode
In order to enable those skilled in the art to better understand the solution of the present invention, furtherly with reference to specific embodiment
Bright technical scheme of the present invention.
Embodiment 1
1, the extraction of tamato fruit total serum IgE
Tomato variety is Lycopersicon esculentum cv.Ailsa Craig, is stored in this laboratory.Using
TRIzol methods extract tamato fruit total serum IgE, then carry out 2% agarose gel electrophoresis, the tamato fruit total serum IgE of Detection and Extraction
Quality, as shown in Figure 1, it can be seen that the integrality of the tomato total serum IgE of extraction is especially good, does not degrade, is also not affected by DNA
Pollution, ideal reverse transcription template in next step can be used as.
2, reverse transcription
Reverse transcription is carried out using the tomato total serum IgE that previous step obtains as template obtains cDNA.
3, the design and synthesis of CNR gene primers
According to the tomato CNR gene mRNA sequences (NM_001319308.1) delivered in GenBank in Premier5.0
Middle design pair of primers is inserted into primer EcoR I and I double enzyme sites of Xho respectively at 5 ' ends of sense primer and downstream primer.
This experiment primer used is synthesized by Shanghai Sheng Gong biotech firms.
Primer nucleotide sequences are specific as follows：
Sense primer：GGAATTCATGGAAACTAACAAATGGGAAGGG(SEQ ID NO.3)
Downstream primer：CCGCTCGAGGCCCAAATTTTCTCCATGAGAGTC(SEQ ID NO.4)
4, RT-PCR obtains target gene
The cDNA obtained using reverse transcription obtains the nucleotide sequence of purpose CNR, CNR gene using above-mentioned primer as template
It is specific as follows as shown in SEQ ID NO.1：
GACTACGATAGGGCGATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCA
GAATTGCCCTTGGAATTCATGGAAACTAACAAATGGGAAGGGAAGAGAAGCATTACTGAAGCTGAAAAGGAAGAGGA
TGAACATGGAAGTGTTGAAGAGGATAGCAAAAGAAAAAGGGTATTGACTCTCTCTGGTAGGAAGCTAGTTGGTGAAG
GGTCGGCACATCCTTCTTGCCAGGTCGATCAGTGCACTGCAGATATGGCAGATGCCAAGCCATACCATCGCCGCCAC
AAGGTGTGTGAGTTCCATTCAAAGTCTCCAATAGTACTTATTAGTGGACTCCAGAAGCGATTCTGTCAGCAATGTAG
CAGATTTCATCTGTTAGCAGAGTTTGATGATGCTAAGAGGAGTTGCCGAAGGCGTTTGGCAGGTCACAATGAGCGCC
GCCGTAAAATTACATATGACTCTCATGGAGAAAATTTGGGCCTCGAGCGGAAGGGCAATTCCAGCACACTGGCGGCC
GTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATC
CGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTC
ACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCA
ACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTC
GGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAG
AACATGTGAGCAAAAGGCCAGCAAAAGGCCAGG
5, I double digestion of EcoR I and Xho
The product that RT-PCR is obtained carries out digestion with expression vector pET-30a with EcoR I and Xho I.
6, gel electrophoresis and glue recycle
(1) digestion products in previous step are subjected to gel electrophoresis, according to the molecular size range of target fragment and reference
Marker is cut the gel piece for the purpose band for including in Ago-Gel with blade, and the blob of viscose cut with blade is taken out,
Careful moves on in new clean centrifuge tube.
(2) weighing of blob of viscose weight, weighs 200g or so, is transferred in suitable centrifuge tube, 400 are added into centrifuge tube
Centrifuge tube is put into water-bath and carries out water-bath (55-60 DEG C), until Ago-Gel by the Bingding Buffer solution of μ L
Melt completely.
(3) complete solution will be melted to be transferred in collecting pipe UNIQ-10 columns with liquid-transfering gun, centrifuge 2min at normal temperatures.
(4) UNIQ-10 columns are removed, outwells the liquid waste for collecting bottom of the tube, UNIQ-10 columns is put back into and outwell waste liquid
Collecting pipe, and the Washing Solution of 500 μ L are added into pipe, are centrifuged (10000rpm, 30s).
(5) it is primary to repeat step (3).
(6) UNIQ-10 columns are removed, the liquid that bottom of the tube is discarded all are outwelled, the column is then put back into collecting pipe,
It is centrifuged (10000rpm, 15s).
(7) UNIQ-10 columns are removed, new centrifuge tube is positioned over, Elution then is added to the film center of pillar
Buffer, 30 μ L or so, places 2min at ambient temperature.
(8) it is centrifuged at ambient temperature, the liquid left in centrifuge tube at this time is the DNA fragmentation of this recycling.
7, it connects
The RT-PCR products orientation of purifying, recycling is recombinated into expression vector pET-30a, linked system is as follows：
Reaction condition：16 DEG C of connection 4h.
8, it converts
(1) the competent cell Rosetta preserved in -80 DEG C of ultra low temperature freezers is taken out, is placed on ice, is slowly solved
Freeze.
(2) it takes the competent cell of 200 μ L to be put into 1.5ml centrifuge tubes, 10 μ L of connection product is then added into pipe, then use
Liquid-transfering gun mixing is then placed within 30min on ice.
(3) centrifuge tube is put into water-bath and carries out water-bath (42 DEG C, 90s), taken out, be immediately put into centrifuge tube on ice
2min。
(4) it takes 800 μ L there is no the LB liquid medium of Jia Kana, the centrifuge tube is added, turn upside down mixing, is put into 37 DEG C
45min in constant-temperature shaking incubator culture.
(5) (5000rpm, 3min) is centrifuged, discards most supernatant solution, leaves about 10 μ l or so, carry out thalline
It is resuspended, then carries out coated plate (containing the LB solid medium tablets for blocking that resistance).
(6) be placed on after drying in 37 DEG C of incubator, first just placement 30min, be then inverted overnight incubation again.
9, bacterium solution PCR
The picking monoclonal on the engineering bacteria tablet that conversion is completed, is transferred to 15ml centrifuge tubes, is trained in 37 DEG C of constant temperature oscillations
It supports in case and is incubated overnight (12-14h), then carry out 2% agarose gel electrophoresis, the size of testing goal segment.Such as
Shown in Fig. 2, it can be seen that the size of target fragment is consistent with experiment expected results size.
10, the digestion identification of recombinant plasmid pET-30a-CNR
Plasmid is extracted from the engineering bacteria that conversion is completed, by recombinant plasmid pET-30a-CNR, using Xho I and EcoR I
Then double digestion carries out 2% agarose gel electrophoresis, as shown in figure 3, obtain the band of size about 420bp, the band it is big
The small size with the expected CNR genes of experiment is consistent, and can primarily determine the success of pET-30a-CNR construction of recombinant vector.While into
Row sequencing analysis is used in combination biosoftware DNAMAN to carry out sequencing result and the CNR sequences (NM_001319308.1) delivered
It compares, comparison result is consistent completely.
11, a small amount of expression of CNR albumen
Monoclonal is chosen on the tablet that culture has engineering bacteria, and the monoclonal of picking is gone into the centrifuge tube of 1.5ml (in pipe
Added with that LB liquid medium of card containing 100mg/L), it is incubated overnight, then spreads cultivation, until when OD values are 0.5-0.7, be added
IPTG derivants induce, centrifugation, remove supernatant, ice-bath ultrasonic wave is broken, is then quantified to albumen, with 15% SDS-PAGE
Electrophoresis analyzes the expression of results of albumen, such as Fig. 4, and the bacterium solution being added after the induction of IPTG derivants has albumen table at 27KD
It reaches, without adding the bacterium solutions of IPTG derivants no protein expression at 27KD, and the albumen size expressed and the expected knot of experiment
Fruit is consistent, and expression is preferable.
The amino acid sequence of CNR albumen is (as shown in SEQ ID NO.2) specific as follows：
METNKWEGKRSITEAEKEEDEHGSVEEDSKRKRVLTLSGRKLVGEGSAHPSCQVDQCTADMADAKPYHR
RHKVCEFHSKSPIVLISGLQKRFCQQCSRFHLLAEFDDAKRSCRRRLAGHNERRRKITYDSHGENLG
12, IPTG induced expressions
The single positive bacterias of Rosetta that picking contains recombinant plasmid pET-30a-CNR are dropped down onto containing Kan (100mg/mL) LB
It is incubated overnight in fluid nutrient medium, switching in second day carries out protein expression after spreading cultivation.The induced concentration of IPTG is 0.1 respectively,
0.4, it collects thalline respectively after 0.8 and 1.0mmol/L, 37 DEG C of expression 3h, after ultrasonic treatment, retains supernatant precipitation.Using
After BCA RNA isolation kits are to protein quantification, with the expression of 12%SDS-PAGE electrophoretic analysis albumen, on each sample total protein
Sample amount is 15 μ g.As shown in figure 5, in cellular lysate liquid supernatant at molecular weight 27kD, expressing quantity obviously increases, heavy
Unobvious are expressed in shallow lake.The molecular weight of the albumen given expression to is consistent with the CNR fusion protein molecule amounts that experimental design is expressed.When
When IPTG induced concentrations are 1.0mmol/L, CNR albumen content in the supernatant of cellular lysate liquid significantly improves (Fig. 5, swimming lane 4),
Illustrate that the best induced concentration of IPTG derivants is 1.0mmol/L.
13, the purifying of CNR albumen
The thalline collected after induced expression is resuspended with PBS (lysozyme), ultrasonic treatment thalline, is filtered through 0.22 μm
Ni Sepharose are added in the supernatant solution of membrane filtrationTMIt is purified in 6Fast Flow affinity columns.On as shown in fig. 6,
After clear upper prop, albumen is all combined with affinity column, does not have the albumen of 27kD in efflux.It is 20mmol/L by imidazole concentration
Wash buffer washing, albumen is not eluted, it can be seen that it is more firm that CNR albumen is still combined with Ni columns；It is added
When elution buffer I-IV of wash buffer and 100-400mmol/L that imidazole concentration is 50mmol/L, foreign protein does not still have
It is eluted (Fig. 7, swimming lane 3-7).Again with the highest elution buffer of imidazole concentration (imidazole concentration 500mmol/L) V
Sample is eluted and is collected, 200 μ l/ pipes.As seen from Figure 8, foreign protein starts to be eluted, with elution buffer
The continuous elution of liquid is collected, and foreign protein content starts to reduce；As shown in figure 9, a large amount of foreign protein has been washed off, CNR albumen
Band starts to become single, and foreign protein content is fewer and fewer；Continue to elute, foreign protein is washed away completely, destination protein band
Become single, illustrates to have collected the very high CNR albumen of purity.
Comparative example 1
Method therefor with embodiment 1, the difference is that, select BL21 plysS as competent cell, the table of albumen
See Figure 10 up to amount, is below by the expression quantity of destination protein in induction and the transformant without induction (being located at 27KD) in figure
Expression quantity of the Rosetta as the transformant of competent cell, selecting Rosetta to carry out conversion as competent cell thus can
Improve the expression quantity of CNR albumen.
Comparative example 2
Method therefor with embodiment 1, the difference is that, select Codon plus as competent cell, the table of albumen
See Figure 10 up to amount, is below by the expression quantity of destination protein in induction and the transformant without induction (being located at 27KD) in figure
Expression quantity of the Rosetta as the transformant of competent cell, selecting Rosetta to carry out conversion as competent cell thus can
Improve the expression quantity of CNR albumen.
14, sero-fast preparation
Untreated mouse is diluted from 10 μ l of rat-tail extracting vein blood with PBS, is centrifuged (1500rpm, 10min), is taken
Clearly, it saves backup for -20 DEG C.
(1) preparation of antigen：Albumen after purification is taken, 12%SDS-PAGE electrophoresis is carried out, takes the collecting pipe that band is single
Albumen concentration is measured with BCA methods, and using the purifying protein of the μ g/ μ l of albumen concentration >=1.0 as antigen.
(2) fundamental immunity：It is emulsified with complete Freund's adjuvant mixed in equal amounts with albumen after purification, is immunized, is inoculated into respectively
Four mouse (same batch) are internal.Vaccination ways are abdominal part hypodermic, every mouse inject 100 μ g antigens with it is isometric
The mixed liquor of complete Freund's adjuvant.
(3) booster immunization：After initial immunity 2 weeks, the albumen of 50 μ g after purification and isometric incomplete Freund are taken
Agent mixed in equal amounts, emulsification, is inoculated into Mice Body, vaccination ways are abdominal part hypodermic.
(4) secondary booster immunization：Method is the same as (3)
(5) booster immunization three times：Method is the same as (3)
(6) serum collection：After third time booster immunization injects 10d, Mouse whole blood, acquisition is taken to finish, 37 DEG C of placement 2h,
Then it stays overnight for 4 DEG C, centrifugation takes supernatant.Then it is dispensed, -80 DEG C save backup.
15, indirect elisa method measures antibody titer
(1) envelope antigen：Phosphate coating buffer solution with a concentration of 0.05mol/L, pH 9.6 is dilute by CNR proteantigens
It is interpreted as 1 μ g/mL.0.1mL antigens are added with liquid-transfering gun in the reacting hole of each 96 hole elisa Plates, 4 DEG C overnight.
(2) 4 DEG C of ELISA Plate is taken out, gets rid of solution in hole by next day, is patted dry only, clear with liquid-transfering gun plus washing buffer
It washes, per 200 μ l of hole, 3min/ times, totally 4 times.
(3) it closes：Washing buffer is got rid of to the greatest extent, is closed with 3% bovine serum albumin(BSA), 0.1mL is added per hole and is incubated,
37 DEG C, 2h.
(4) it washs：Confining liquid in hole is got rid of to the greatest extent, is cleaned with liquid-transfering gun plus washing buffer, per hole 200 μ l, 3min/
It is secondary, totally 4 times.
(5) specific antibody of the anti-CNR albumen of doubling dilution：With dilution doubling dilution (1:4 × 10,1:8×103, 1:
1.6×104, 1:3.2×104, 1:6.4×104, 1:1.28×105, 1:2.56×105, 1:5.12×105, 1:1.024×
106) anti-CNR albumen specific antibody.
(6) 96 hole elisa Plates, 100 holes μ l/, each gradient is added in the specific antibody of the anti-CNR albumen of doubling dilution
It is repeated 4 times.It is incubated：37 DEG C, 1h.
(7) it washs：Antibody in hole is got rid of to the greatest extent, is cleaned with liquid-transfering gun plus washing buffer, 200 holes μ l/, 3min/ times, altogether
4 times.
(8) add ELIAS secondary antibody：With 1000 times of dilution ELIAS secondary antibodies of dilution, 96 hole enzymes are added in the ELIAS secondary antibody diluted
Target, 100 holes μ l/ are incubated 37 DEG C, 1h.
(9) it washs：ELIAS secondary antibody in hole is got rid of to the greatest extent, is cleaned with liquid-transfering gun plus washing buffer, 200 holes μ l/, 3min/
It is secondary, totally 3 times；It gets rid of to the greatest extent, distilled water is added to clean, 200 holes μ l/, 3min/ times, totally 1 time.
(10) add substrate developing solution：Substrate developing solution is added in 96 hole elisa Plates, 100 holes μ l/ are protected from light incubation, 15min.
(11) reaction is terminated：2mol/L sulfuric acid is added in 96 hole elisa Plates, 50 holes μ l/ terminate reaction.
(12) result measures：96 hole elisa Plates are placed in microplate reader, at 450nm, are carried out afterwards as a contrast with blank well
Zeroing, measures each hole OD values.
According to above-mentioned steps, by antiserum from 1:4000-1:2048000 doubling dilutions pass through the method pair of indirect ELISA
Antiserum titre is detected, and each dilution gradient does 4 repetitions.The results show that the potency of four mouse is in gradient 1:
Positive (the ratio between surveyed OD values and negative control OD value after dilution are presented before 128000>2).As seen from Figure 11, No. 1,2
The potency of number mouse is 1:The potency of 128000, No. 3, No. 4 mouse can reach 1:256000.Illustrate CNR albumen obtained
Polyclonal antibody potency it is higher.
16, the Western blot detections of polyclonal antibody
(1) electrophoresis
(2) revolving die：A, electrophoresis terminates, and closes switch, removes offset plate.Offset plate is put in ceramic whiteware disk another side, with green scraper plate
Tip slide into, rise and open small glass plate, remove concentration glue, according to the 27KDa bands of Marker, it is solidifying to cut purpose with green scraper plate
Adhesive tape band cuts off the pvdf membrane upper left corner；B, sandwich is put in electric turn trough, black plate puts black flour.Transferring film condition：300mA
Transferring film 2h.
(3) it closes：The preceding half an hour that transferring film terminates soon makes confining liquid and (weighs 5g milk powder, add TBS to bottle scale, electricity
Magnetic stirs) and TBST solution (800 μ L soil temperature 20+35mLTBS+700mL water), to the end of transferring film, sandwich is removed, film is put into
In the culture dish for filling confining liquid, 1h is closed.
(4) it is incubated primary antibody：A, 1 is pressed with TBST:1000、1:5000、1:10000、1:50000、1:100000 dilution CNR eggs
White Anti-TNF-α liquid solution；B, confining liquid is removed, pvdf membrane is put into in the diluted Anti-TNF-α liquid solutions of TBST, by film
It seals in hybridization bag, hybridization bag marks, sealing, and 4 DEG C overnight, makes antigen-antibody that immune response occur.
(5) it is incubated secondary antibody：A, with TBST two anti-sheep anti-mouse iggs are diluted according to specification；B, by the pvdf membrane in miscellaneous poly-bag
It takes out, has been put into the culture dish of TBST, has washed film 3 times, each 10min；Pvdf membrane is put into the secondary antibody diluted after washing
Reaction solution --- sheep anti-mouse igg is placed on shaking table, at room temperature incubation reaction 1h.
(6) it exposes：Incubation finishes, and pvdf membrane is cleaned three times with TBST solution, each 10min；While cleaning, prepare
Super quick luminescent solution (the A liquid of ECL Plus:Liquid=1 B:1), cleaning finishes, and pvdf membrane is gone to gel imager, is taken pictures, and preserves.
As shown in figure 12, the results showed that with CNR albumen specific antigen-antibody reaction can occur for polyclonal antibody, detect
To band be consistent with the expected molecular size range of experiment, and band is single, it can be seen that the Anti-TNF-α of CNR albumen
The specificity of body is preferably.
17, the extraction of tamato fruit total protein
Underdone phase, green ripe stage, broken color phase, pink phase, red ripe phase tomato jelly sample are taken out, with Liquid nitrogen precooler mortar and pestle, title
It takes 1g samples to be put into mortar, liquid nitrogen grinding is added to powdered, powder is moved into pre-cooled 1.5ml centrifuge tubes (note：Sample
Remain freezing state).
(1) the configured good extracting solutions of 1ml are added, vibrate mixing, keep sample dissolving abundant.
(2) (12000rpm, 15min) is centrifuged, takes out supernatant solution.
(3) primary (12000rpm, 15min) is centrifuged again, takes out supernatant solution.
(4) 100 μ L quantifying for albumen is taken out from the supernatant after centrifugation, remaining supernatant solution is dispensed, and -20
DEG C preserve.
Extracted respectively according to above-mentioned steps the underdone phase, green ripe stage, the broken color phase, the pink phase, red ripe phase tamato fruit total egg
In vain, its quality of part progress SDS-PAGE electrophoresis detections is taken, as shown in figure 13, applied sample amount is 15 holes μ g/；Then CNR is carried out
The immunoblotting analysis of albumen is tested, and applied sample amount is 20 holes μ g/.As a result as shown in figure 14, broken color phase of the CNR albumen in tamato fruit
Expression quantity highest, and in the red ripe phase almost without expression.It can thus be appreciated that the accumulation of CNR albumen and the development of tamato fruit and maturation
It is in close relations.
Conclusion：It is above-mentioned the experimental results showed that, the tomato CNR protein polyclone antibodies that prepare can identify tomato in the present invention
In CNR albumen, CNR albumen can be detected, without having an effect with other albumen in tomato body.The polyclonal antibody, can
For the detection of tomato transcription factor CNR expressions in ripening of fruits, the Function Identification to CNR albumen and research
Have great importance.
Illustrative description has been done to the present invention above, it should explanation, the case where not departing from core of the invention
Under, any simple deformation, modification or other skilled in the art can not spend the equivalent replacement of creative work equal
Fall into protection scope of the present invention.
Sequence table
<110>TanJin Agricultural College
<120>A kind of tamato fruit transcription factor CNR polyclonal antibodies and preparation method thereof
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 949
<212> DNA
<213>Tomato（Lycopersicon esculentum cv.Ailsa Craig）
<400> 1
gactacgata gggcgattgg gccctctaga tgcatgctcg agcggccgcc agtgtgatgg 60
atatctgcag aattgccctt ggaattcatg gaaactaaca aatgggaagg gaagagaagc 120
attactgaag ctgaaaagga agaggatgaa catggaagtg ttgaagagga tagcaaaaga 180
aaaagggtat tgactctctc tggtaggaag ctagttggtg aagggtcggc acatccttct 240
tgccaggtcg atcagtgcac tgcagatatg gcagatgcca agccatacca tcgccgccac 300
aaggtgtgtg agttccattc aaagtctcca atagtactta ttagtggact ccagaagcga 360
ttctgtcagc aatgtagcag atttcatctg ttagcagagt ttgatgatgc taagaggagt 420
tgccgaaggc gtttggcagg tcacaatgag cgccgccgta aaattacata tgactctcat 480
ggagaaaatt tgggcctcga gcggaagggc aattccagca cactggcggc cgttactagt 540
ggatccgagc tcggtaccaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 600
ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 660
gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 720
gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 780
tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 840
gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 900
ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagg 949
<210> 2
<211> 136
<212> PRT
<213>Tomato（Lycopersicon esculentum cv.Ailsa Craig）
<400> 2
METNKWEGKR SITEAEKEED EHGSVEEDSK RKRVLTLSGR KLVGEGSAHP SCQVDQCTAD 60
MADAKPYHRR HKVCEFHSKS PIVLISGLQK RFCQQCSRFH LLAEFDDAKR SCRRRLAGHN 120
ERRRKITYDS HGENLG 136
<210> 3
<211> 31
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<213>Artificial sequence
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ggaattcatg gaaactaaca aatgggaagg g 31
<210> 4
<211> 33
<212> DNA
<213>Artificial sequence
<400> 4
ccgctcgagg cccaaatttt ctccatgaga gtc 33
Claims (8)
1. a kind of preparation method of tamato fruit transcription factor CNR polyclonal antibodies, which is characterized in that this method includes following step
Suddenly：
(1) structure contains SEQ ID No：The recombinant expression carrier of nucleotide sequence shown in 1 converts the recombinant expression carrier
Competent escherichia coli cell obtains CNR proteantigens；
(2) animal is immunized in the CNR proteantigens, menses isolate and purify clearly the polyclonal antibody for obtaining tomato CNR albumen.
2. preparation method according to claim 1, which is characterized in that the recombinant expression carrier is will to contain SEQ ID
The segment of nucleotide sequence shown in No.1 is cloned into prokaryotic expression carrier acquisition.
3. preparation method according to claim 1, which is characterized in that the CNR proteantigens contain such as SEQ ID No.2
Shown in amino acid sequence.
4. preparation method according to claim 1, which is characterized in that the competent escherichia coli cell is Escherichia coli
Rosetta competent cells.
5. preparation method according to claim 1, which is characterized in that the prokaryotic expression carrier is pET-30a.
6. according to the preparation method described in any one of claim 1,2,3,4 and 5, which is characterized in that the step of immune animal
Rapid includes immune 3 times, and the 1st immune animal after first mixing the CNR proteantigens of purifying with complete Freund's adjuvant then will
The CNR proteantigens of purifying carry out the 2-3 times and are immunized after being mixed with non-fully Freund's adjuvant, immune time interval is 14 every time
It.
7. the tomato CNR protein polyclone antibodies prepared using the method described in any one of claim 1-6.
8. the application of the tomato CNR protein polyclone antibodies described in claim 7.
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Cited By (3)
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| CN114478776A (en) * | 2022-02-21 | 2022-05-13 | 天津农学院 | Polyclonal antibody for resisting chicken TLR15 protein and preparation method thereof |
| CN114807217A (en) * | 2022-05-06 | 2022-07-29 | 中国农业大学 | Application of CNR gene and protein coded by CNR gene in regulating and controlling synthesis of flavonoid compounds in plant fruits |
| CN116622766A (en) * | 2023-06-16 | 2023-08-22 | 西南大学 | Application of poplar SPL16 and SPL23 genes in regulation and control of transformation in dormancy period of poplar |
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Cited By (5)
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| CN114478776A (en) * | 2022-02-21 | 2022-05-13 | 天津农学院 | Polyclonal antibody for resisting chicken TLR15 protein and preparation method thereof |
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| CN116622766A (en) * | 2023-06-16 | 2023-08-22 | 西南大学 | Application of poplar SPL16 and SPL23 genes in regulation and control of transformation in dormancy period of poplar |
| CN116622766B (en) * | 2023-06-16 | 2024-04-02 | 西南大学 | Application of poplar SPL16 and SPL23 genes in regulation and control of transformation in dormancy period of poplar |
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