CN108409858B - Tomato fruit transcription factor CNR polyclonal antibody and preparation method thereof - Google Patents

Abstract

The invention provides a tomato fruit transcription factor CNR polyclonal antibody and a preparation method thereof, wherein the method comprises the following steps: (1) construction of a peptide containing SEQ ID No: 1, transforming the recombinant expression vector into escherichia coli competent cells to obtain a CNR protein antigen; (2) the polyclonal antibody of the tomato CNR protein is obtained by immunizing animals with the CNR protein antigen and separating and purifying the serum, and the polyclonal antibody prepared by the method provided by the invention can specifically identify the CNR protein of tomato fruits, but cannot identify other proteins of the tomato fruits, and has wide application in detection and functional identification of the CNR protein and research thereof. The preparation of the CNR protein antibody has important significance for detecting the expression condition of the tomato transcription factor CNR in the fruit ripening process and researching the function of the CNR protein.

Description

Tomato fruit transcription factor CNR polyclonal antibody and preparation method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a tomato fruit transcription factor CNR polyclonal antibody and a preparation method thereof.

Background

Tomato (tomato lycopersicum Mill.) has a long history of cultivation, and is a model organism for researching the development and maturation of fleshy fruits due to its small genome (950Mb), short growth cycle (fruit setting to maturation of about 60d), and long genetic relationship with model plants such as arabidopsis, rice and corn. A large number of tomato germplasm resources, mutant libraries, high-density genetic maps and EST resources are obtained, and the transient expression and stable transformation technology is mature. In addition, the fine sequence analysis of the whole genome of cultivated tomatoes, which is completed in 5 months of 2012, greatly promotes the research of tomato functional genomics and molecular genetics. The fruit ripening process involves regulation of a large number of metabolic pathways and significant change of physiological and biochemical properties, and requires precise transcriptional regulation of time-sequence expression of a series of genes, so that the functional research of tomato ripening related transcription factors becomes one of the research hotspots in the field of botany.

The fruit ripening process is accomplished by the individual or coordinated regulation of specific downstream genes by a number of transcription factors (Giovannoni 2007), one class of which is the SBP box family. SBP box transcription factors are involved in many aspects of plant development (penhua 2016), including phase transformation, flower organ establishment, fruit ripening, phytohormone signaling and copper homeostasis, among others (hiying 2009). CNR is a special member of SBP box transcription factor family, which can play an important role in the development and maturation of fruit. At present, the tomato transcription factor CNR is mainly studied at the transcription level, however, there is a post-translational modification after protein synthesis, and the protein level is the level at which the transcription factor CNR plays a role, and no report is found about antibodies of the CNR protein at present.

There is therefore a need to provide polyclonal antibodies against the tomato CNR protein, thus providing a powerful tool for studying and utilizing the biological functions of this protein.

Disclosure of Invention

The invention aims to fill the blank of the existing antibody research on the CNR protein and develop a polyclonal antibody aiming at the tomato CNR protein and a preparation method thereof.

A method for preparing a tomato fruit transcription factor CNR polyclonal antibody comprises the following steps:

(1) construction of a peptide containing SEQ ID No: 1, transforming the recombinant expression vector into escherichia coli competent cells to obtain a recombinant protein antigen;

(2) immunizing animals with the CNR protein antigen, and separating and purifying the serum to obtain the tomato CNR protein polyclonal antibody.

Wherein, the SEQ ID No: the nucleotide sequence shown in 1 is obtained by designing a primer according to a published tomato CNR gene mRNA sequence (NM-001319308.1) in GenBank by the inventor, respectively inserting EcoRI and Xho I double enzyme cutting sites at the 5' ends of an upstream primer and a downstream primer, extracting mRNA from a tomato fruit of the variety Lycosporan esculentum cv.

Optionally, the recombinant expression vector is obtained by cloning a fragment containing the nucleotide sequence shown in SEQ ID No.1 to a prokaryotic expression vector.

Optionally, the CNR protein antigen contains an amino acid sequence shown in SEQ ID No. 2.

The type of the escherichia coli competent cell used in the present invention is not particularly limited as long as it is suitable for efficient expression of the recombinant expression vector in the cell, and preferably, the escherichia coli competent cell is escherichia coli Rosetta competent cell in order to obtain a better expression effect.

In the present invention, prokaryotic expression vectors suitable for recombinant protein antigens, which are conventionally used in the art, may be used, and preferably, when the prokaryotic expression vector is pET-30a, the expression effect of recombinant protein antigens is better.

In the method provided by the invention, the step of immunizing the animal comprises 3 times of immunization, wherein the 1 st time of immunization is carried out after the purified recombinant protein antigen is mixed with complete Freund's adjuvant, and then the 2 nd-3 rd time of immunization is carried out after the purified recombinant protein antigen is mixed with non-complete Freund's adjuvant, and the time interval of each immunization is 14 d.

In the present invention, the dose of the immunization and the mixing ratio with the adjuvant used for immunizing an animal can be performed according to a method conventional in the art. The immunized animal includes, but is not limited to, a mouse.

And 10d after 3 rd immunization, taking animal blood, collecting antiserum, and obtaining the tomato CNR protein polyclonal antibody with the purity of not less than 80% by using a purification reagent.

The invention also provides a tomato CNR protein polyclonal antibody prepared by the method. The polyclonal antibody can specifically recognize a specific amino acid fragment SEQ ID No.2 in the CNR protein, and is a specific antibody of the CNR protein.

The invention also provides application of the tomato CNR protein polyclonal antibody. The application comprises the preparation of a reagent or a kit containing the tomato CNR protein polyclonal antibody, and the reagent or the kit is used for detecting the expression condition of a tomato transcription factor CNR in the fruit ripening process or carrying out functional identification.

The method provided by the invention obtains the DNA sequence of the coding CNR protein by cloning through a PCR method, thereby obtaining the specific polyclonal antibody. The polyclonal antibody prepared by the method provided by the invention can specifically identify the tomato CNR protein, but cannot identify other tomato proteins, and has wide application in the detection, functional identification and research of the CNR protein. The preparation of the CNR protein antibody has important significance for detecting the expression condition of the transcription factor CNR in tomato and researching the function of the CNR protein.

Drawings

FIG. 1 is agarose gel electrophoresis of total RNA from tomato fruit.

FIG. 2 is a PCR gel electrophoresis diagram of engineering bacteria liquid carrying expression vector pET-30 a-CNR. M: marker; 1: a fragment of interest.

FIG. 3 is the gel electrophoresis diagram of recombinant plasmid pET-30a-CNR, M: marker; 1: plasmid double digestion.

Fig. 4 is a gel electrophoresis diagram of a small amount of inducible expressed protein of CNR protein, M: marker; 1-3: performing single cloning; 4: no induction, 15% SDS-PAGE was used.

Fig. 5 is a gel electrophoresis diagram of CNR protein expression at an induction temperature of 37 ℃, M: marker; 1-4: the supernatant fraction of the cell lysate; 5-8: pellet fraction of cell lysate: 1 and 50.1mmol/L IPTG induction; 2 and 6: inducing by 0.4mmol/L IPTG; 3 and 7: inducing by 0.8mmol/L IPTG; 4 and 8: 1mmol/L IPTG induction.

Fig. 6 is an affinity purification gel electrophoresis of His-tagged CNR protein 1, M: marker; 1: 1.0mmol/L IPTG induced thallus lysate supernatant is a purified sample; 2-7: respectively is protein eluted after the elution buffer solution passes through the column, namely the binding buffer solution, the rinsing buffer solution, the elution buffer solution I, II, III and IV.

Fig. 7 is an affinity purification gel electrophoresis of His-tagged CNR protein fig. 2, M: marker; 1-8: the concentration of imidazole is 500mmol/L, and the protein is eluted after the elution buffer solution passes through the column.

Fig. 8 is an affinity purification gel electrophoresis of His-tagged CNR protein fig. 3, M: marker; 1-7: the concentration of imidazole is 500mmol/L, and the protein is eluted after the elution buffer solution passes through the column.

Fig. 9 is an affinity purification gel electrophoresis of His-tagged CNR protein fig. 4, M: marker; 1-8: the concentration of imidazole is 500mmol/L, and the protein is eluted after the elution buffer solution passes through the column.

FIG. 10 is a gel electrophoresis diagram of protein expression of different competent cells transformed by recombinant vector, M: marker; 1 and 2: monoclonal (BL21 plysS); 3: non-induced (BL21 plysS); 4 and 5: monoclonal (Codon plus); 6: uninduced (Codon plus); 7 and 8: monoclonal (Rosetta); 9: not induced (Rosetta).

Fig. 11 is a graph of CNR protein polyclonal antibody titers.

For a person skilled in the art, other relevant figures can be obtained from the above figures without inventive effort.

FIG. 12 is a specific electrophoretogram of Western blot for detecting serum, 1: purified CNR protein.

FIG. 13 is a gel electrophoresis image of total protein from tomato fruit at different time periods, M: marker; 1: the immature period; 2: green ripe period; 3: a color breaking period; 4: a pink period; 5: red ripe period.

Fig. 14 is a western blot analysis of CNR protein expression during fruit ripening in tomato, 1: the immature period; 2: green ripe period; 3: a color breaking period; 4: a pink period; 5: red ripe period.

Detailed Description

In order to make the technical solution of the present invention better understood, the technical solution of the present invention is further described below with reference to specific examples.

Example 1

1. Extraction of total RNA from tomato fruit

The tomato variety Lycopepsicon esculentum cv. Ailsa Craig was stored in the laboratory. The method comprises the steps of extracting total RNA of tomato fruits by a TRIzol method, carrying out 2% agarose gel electrophoresis, and detecting the quality of the extracted total RNA of the tomato fruits, wherein as shown in figure 1, the extracted total RNA of the tomato is particularly good in integrity, free from degradation and DNA pollution, and can be used as an ideal reverse transcription template in the next step.

2. Reverse transcription

The tomato total RNA obtained in the previous step is used as a template to carry out reverse transcription to obtain cDNA.

3. Design and synthesis of CNR gene primer

A pair of primers is designed in Premier5.0 according to the published tomato CNR gene mRNA sequence (NM-001319308.1) in GenBank, and primers EcoRI and XhoI double-restriction sites are inserted at the 5' ends of an upstream primer and a downstream primer respectively. The primers used in this experiment were synthesized by Shanghai Biotech.

The nucleotide sequence of the primer is as follows:

an upstream primer: GGAATTCATGGAAACTAACAAATGGGAAGGG (SEQ ID NO.3)

A downstream primer: CCGCTCGAGGCCCAAATTTTCTCCATGAGAGTC (SEQ ID NO.4)

4. Obtaining target gene by RT-PCR

The cDNA obtained by reverse transcription is taken as a template, the primer is utilized to obtain the target CNR, the nucleotide sequence of the CNR gene is shown as SEQ ID NO.1, and the specific steps are as follows:

GACTACGATAGGGCGATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTGCCCTTGGAATTCATGGAAACTAACAAATGGGAAGGGAAGAGAAGCATTACTGAAGCTGAAAAGGAAGAGGATGAACATGGAAGTGTTGAAGAGGATAGCAAAAGAAAAAGGGTATTGACTCTCTCTGGTAGGAAGCTAGTTGGTGAAGGGTCGGCACATCCTTCTTGCCAGGTCGATCAGTGCACTGCAGATATGGCAGATGCCAAGCCATACCATCGCCGCCACAAGGTGTGTGAGTTCCATTCAAAGTCTCCAATAGTACTTATTAGTGGACTCCAGAAGCGATTCTGTCAGCAATGTAGCAGATTTCATCTGTTAGCAGAGTTTGATGATGCTAAGAGGAGTTGCCGAAGGCGTTTGGCAGGTCACAATGAGCGCCGCCGTAAAATTACATATGACTCTCATGGAGAAAATTTGGGCCTCGAGCGGAAGGGCAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGG

5. EcoRI and Xho I double enzyme digestion

The product obtained by RT-PCR and the expression vector pET-30a are all cut by EcoR I and Xho I.

6. Gel electrophoresis and gel recovery

(1) And (3) performing gel electrophoresis on the enzyme digestion product in the last step, cutting off a gel block of a target strip contained in the agarose gel by using a blade according to the molecular weight of the target fragment and referring to a Marker, taking out the gel block cut off by using the blade, and carefully moving the gel block into a new clean centrifugal tube.

(2) Weighing the gel block, weighing about 200g, transferring into a proper centrifuge tube, adding 400 mu L of the binging Buffer solution into the centrifuge tube, putting the centrifuge tube into a water bath for water bath (55-60 ℃) until the agarose gel is completely melted.

(3) The completely melted solution was transferred to a collection tube UNIQ-10 column using a pipette and centrifuged at room temperature for 2 min.

(4) The UNIQ-10 column was removed, the waste liquid at the bottom of the collection tube was discarded, the UNIQ-10 column was returned to the collection tube from which the waste liquid was discarded, and 500. mu.L of Washing Solution was added to the tube, followed by centrifugation (10000rpm, 30 s).

(5) Repeating the step (3) once.

(6) The UNIQ-10 column was removed, the liquid discarded at the bottom of the tube was completely decanted, and the column was returned to the collection tube and centrifuged (10000rpm, 15 s).

(7) The UNIQ-10 column was removed and placed in a new centrifuge tube, and then an Elution Buffer, 30. mu.L or so, was added to the center of the membrane of the column, and the column was left at room temperature for 2 min.

(8) And centrifuging at room temperature, wherein the liquid left in the centrifuge tube is the DNA fragment recovered at this time.

7. Connection of

Directionally recombining the purified and recovered RT-PCR product into an expression vector pET-30a, wherein a connection system is as follows:

reaction conditions are as follows: ligation was carried out at 16 ℃ for 4 h.

8. Transformation of

(1) Competent cells Rosetta stored at-80 ℃ in an ultra-low temperature freezer were removed, placed on ice and thawed slowly.

(2) 200 μ L of the competent cells were placed in a 1.5ml centrifuge tube, then 10 μ L of the ligation product was added to the tube, mixed well with a pipette, and then placed on ice for 30 min.

(3) The tube was placed in a water bath for water bath (42 ℃ C., 90s), taken out, and immediately placed on ice for 2 min.

(4) Adding 800 μ L LB liquid culture medium without Carna into the centrifuge tube, turning upside down, mixing, and culturing in 37 deg.C constant temperature shaking incubator for 45 min.

(5) Centrifugation (5000rpm, 3min) was carried out to discard most of the supernatant solution, leaving about 10. mu.l, to resuspend the cells, and then plating (Carna-resistant LB solid medium plate) was carried out.

(6) Air-drying, placing in 37 deg.C incubator, standing for 30min, and then culturing overnight by inverting.

9. PCR of bacterial liquid

And (3) selecting a monoclonal on the transformed engineering bacteria plate, transferring the monoclonal to a 15ml centrifuge tube, carrying out overnight culture (12-14h) in a constant-temperature shaking incubator at 37 ℃, then carrying out 2% agarose gel electrophoresis, and detecting the size of the target fragment. As shown in FIG. 2, it can be seen that the size of the target fragment coincides with the size of the expected result of the experiment.

10. Enzyme digestion identification of recombinant plasmid pET-30a-CNR

Extracting plasmids from the transformed engineering bacteria, carrying out double enzyme digestion on the recombinant plasmid pET-30a-CNR by adopting Xho I and EcoR I, and then carrying out 2% agarose gel electrophoresis, as shown in figure 3, obtaining a band with the size of about 420bp, wherein the size of the band is consistent with the size of the CNR gene expected by the experiment, and the success of the construction of the pET-30a-CNR recombinant vector can be preliminarily determined. And simultaneously performing sequencing analysis, and comparing a sequencing result with a published CNR sequence (NM-001319308.1) by using the biological software DNAMAN, wherein the comparison result completely accords with the sequence.

11. Small expression of CNR proteins

Picking monoclonals on a flat plate cultured with engineering bacteria, transferring the picked monoclonals to a centrifugal tube (the tube is added with LB liquid culture medium containing 100mg/L canana) with the volume of 1.5ml, culturing overnight, then carrying out amplification culture until the OD value is 0.5-0.7, adding IPTG inducer for induction, centrifuging, removing supernatant, carrying out ice bath ultrasonic wave crushing, then quantifying protein, and analyzing the expression result of the protein by 15% SDS-PAGE electrophoresis, as shown in figure 4, the bacterial liquid induced by adding IPTG inducer has protein expression at 27KD, the bacterial liquid without adding IPTG inducer has no protein expression at 27KD, the size of the expressed protein is consistent with the expected result of the test, and the expression condition is better.

The amino acid sequence of the CNR protein is specifically as follows (shown as SEQ ID NO. 2):

METNKWEGKRSITEAEKEEDEHGSVEEDSKRKRVLTLSGRKLVGEGSAHPSCQVDQCTADMADAKPYHRRHKVCEFHSKSPIVLISGLQKRFCQQCSRFHLLAEFDDAKRSCRRRLAGHNERRRKITYDSHGENLG

12. IPTG inducible expression

And picking a Rosetta single positive colony containing the recombinant plasmid pET-30a-CNR into a LB liquid culture medium containing Kan (100mg/mL) for overnight culture, and performing protein expression after the next day of transfer and expansion. IPTG induction concentrations are 0.1, 0.4, 0.8 and 1.0mmol/L respectively, thalli are collected after expression for 3h at 37 ℃, and after ultrasonic cracking, supernatant and sediment are reserved. After the protein was quantified by BCA kit, the expression of the protein was analyzed by 12% SDS-PAGE electrophoresis, and the total protein loading was 15. mu.g per sample. As shown in FIG. 5, the protein expression level was significantly increased at a molecular weight of 27kD in the supernatant of the cell lysate, but was not significantly expressed in the precipitate. The molecular weight of the expressed protein is consistent with that of the CNR fusion protein expressed by experimental design. When the IPTG induction concentration is 1.0mmol/L, the content of CNR protein in the supernatant of the thallus lysate is obviously improved (figure 5, lane 4), which shows that the optimal induction concentration of the IPTG inducer is 1.0 mmol/L.

13. Purification of CNR proteins

The collected bacteria after induction expression are resuspended with PBS (containing lysozyme), the bacteria are cracked by ultrasonic wave, and Ni Sepharose is added into the supernatant solution filtered by 0.22 mu m filter membrane^TMPurification was performed in a 6Fast Flow affinity column. As shown in FIG. 6, after the supernatant was applied to the column, all proteins were bound to the affinity column, and no 27kD protein was present in the effluent. The protein is not eluted after being washed by a rinsing buffer solution with the imidazole concentration of 20mmol/L, so that the CNR protein is still firmly combined with the Ni column; when the rinsing buffer with imidazole concentration of 50mmol/L and the elution buffers I-IV of 100-400mmol/L were added, the hetero-protein was not eluted yet (FIG. 7, lanes 3-7). The sample was eluted with elution buffer V having the highest imidazole concentration (imidazole concentration 500mmol/L) and collected at 200. mu.l/tube. As can be seen from fig. 8, the hetero protein begins to be eluted, and the content of the hetero protein begins to decrease as the elution and collection of the elution buffer solution are continued; as shown in fig. 9, a large amount of hetero-proteins had been washed away, and CNR protein bands began to become single with less and less hetero-protein content; and (4) continuing to elute, completely eluting the foreign protein, and enabling the target protein band to become single, which indicates that the CNR protein with high purity is collected.

Comparative example 1

The procedure used was the same as in example 1, except that BL21 plysS was used as the competent cells, and the expression level of the protein was as shown in FIG. 10, in which the expression level of the target protein (at 27 KD) in both the induced and non-induced transformants was lower than that of the transformant in which Rosetta was used as the competent cells, and therefore, the expression level of the CNR protein was increased by transformation using Rosetta as the competent cells.

Comparative example 2

The procedure used was the same as in example 1, except that Codon plus was used as the competent cells, and the expression level of the protein was as shown in FIG. 10, in which the expression level of the target protein (at 27 kD) was lower in both the induced and non-induced transformants than in the transformant in which Rosetta was used as the competent cells, and thus the expression level of the CNR protein was increased by transformation using Rosetta as the competent cells.

14. Preparation of antiserum

Untreated mice, venous blood 10. mu.l from the rat tail was taken, diluted with PBS, centrifuged (1500rpm, 10min), and the supernatant was taken and stored at-20 ℃ for further use.

(1) Preparation of antigen: taking the purified protein, carrying out 12% SDS-PAGE electrophoresis, taking a single collection tube with a single strip, and measuring the protein concentration by using a BCA method, wherein the purified protein with the protein concentration of more than or equal to 1.0 mu g/mu l is used as an antigen.

(2) Basic immunity: the purified protein and complete Freund's adjuvant are mixed and emulsified in equal amount, and then the mixture is respectively immunized and inoculated into four mice (same batch). The inoculation was performed by abdominal subcutaneous injection, and each mouse was injected with 100. mu.g of antigen mixed with an equal volume of complete Freund's adjuvant.

(3) And (3) boosting immunity: after 2 weeks of primary immunization, 50 μ g of purified protein was mixed with an equal volume of Freund's incomplete adjuvant, emulsified, and inoculated into mice by abdominal subcutaneous injection.

(4) Secondary boosting immunization: method (3)

(5) Three booster immunizations: method (3)

(6) Collecting serum: after 10 days of the third booster injection, whole blood of the mice is taken, the whole blood is collected, placed at 37 ℃ for 2 hours, and then kept overnight at 4 ℃, centrifuged, and the supernatant is taken. Then subpackaging at-80 ℃ for later use.

15. Indirect ELISA method for determining antibody titer

(1) Coating antigen: the CNR protein antigen was diluted to 1. mu.g/mL with phosphate coating buffer at 0.05mol/L, pH 9.6. 0.1mL of antigen was added to each well of the 96-well microplate using a pipette gun at 4 ℃ overnight.

(2) The next day, the 4 ℃ microplate was removed, the well solutions were spun off, patted clean, and washed with 200. mu.l of wash buffer per well for 4 times, 3 min/time.

(3) And (3) sealing: wash buffer was spun down, blocked with 3% bovine serum albumin, and 0.1mL was added to each well for incubation at 37 ℃ for 2 h.

(4) Washing: the blocking solution in the wells was drained and washed with pipette and wash buffer 200. mu.l per well for 3 min/4 times.

(5) Specific antibodies against CNR protein diluted in fold: diluting with diluent at a multiple ratio (1:4 × 10, 1:8 × 10)³，1:1.6×10⁴，1:3.2×10⁴，1:6.4×10⁴，1:1.28×10⁵，1:2.56×10⁵，1:5.12×10⁵，1:1.024×10⁶) Specific antibodies against CNR proteins.

(6) The anti-CNR protein specific antibody diluted in multiple times was added to a 96-well plate at 100. mu.l/well, and each gradient was repeated 4 times. And (3) incubation: 37 ℃ for 1 h.

(7) Washing: the antibody in the well was spun off and washed with pipette plus wash buffer at 200. mu.l/well for 3 min/time for 4 times.

(8) Adding an enzyme-labeled secondary antibody: diluting the enzyme-labeled secondary antibody by 1000 times of the diluent, adding the diluted enzyme-labeled secondary antibody into a 96-well enzyme-labeled plate, incubating at 37 ℃ for 1h, wherein each well comprises 100 mu l.

(9) Washing: throwing off enzyme-labeled secondary antibodies in the holes, washing by adding washing buffer solution into a pipette gun, and washing for 3 times (200 mu l/hole and 3 min/time); throwing off, adding distilled water for cleaning, 200 μ l/hole, 3 min/time, 1 time totally.

(10) Adding a substrate color development liquid: adding substrate developing solution into 96-well enzyme label plate, 100 μ l/well, incubating in dark for 15 min.

(11) And (3) terminating the reaction: the reaction was stopped by adding 2mol/L sulfuric acid, 50. mu.l/well, to a 96-well plate.

(12) And (4) determining the result: and (3) placing the 96-well enzyme label plate in an enzyme label instrument, adjusting zero at 450nm by taking a blank well as a reference, and determining the OD value of each well.

Antiserum was diluted from 1:4000 to 1:2048000 fold according to the above procedure, and antiserum titers were measured by indirect ELISA with 4 replicates per dilution gradient. The results show that the titers of four mice were all positive before the gradient 1:128000 (ratio of measured OD to negative control OD after dilution > 2). As can be seen from FIG. 11, the titer of mice No.1 and 2 was 1:128000, and the titer of mice No.3 and 4 was 1: 256000. The titer of the prepared polyclonal antibody of the CNR protein is higher.

16. Western blot detection of polyclonal antibodies

(1) Electrophoresis

(2) And (3) rotating the die: A. and (5) after electrophoresis is finished, closing the switch and taking down the rubber plate. Placing the rubber plate on the other side of the white porcelain plate, lightly pushing in the rubber plate by using the tip of a green scraper, opening the small glass plate, removing concentrated glue, cutting a target gel strip by using the green scraper according to a 27KDa strip of a Marker, and cutting the upper left corner of the PVDF membrane; B. the sandwich was placed in an electric rotating cell with the black plate against the black side. Film transferring conditions: 300mA is transferred into the film for 2 h.

(3) And (3) sealing: preparing sealing solution (weighing 5g of milk powder, adding TBS to the scale of a small bottle, and electromagnetically stirring) and TBST solution (800. mu.L of Tween 20+35mL of TBS +700mL of water) half an hour before the membrane conversion is finished, taking down the sandwich after the membrane conversion is finished, putting the membrane into a culture dish containing the sealing solution, and sealing for 1 hour.

(4) Incubating the primary antibody: A. diluting polyclonal antibody solution of CNR protein with TBST at ratio of 1:1000, 1:5000, 1:10000, 1:50000, 1: 100000; B. removing the sealing liquid, putting the PVDF membrane into a polyclonal antibody solution diluted by TBST, sealing the membrane into a hybridization bag, marking the hybridization bag, sealing, and standing overnight at 4 ℃ to ensure that the antigen-antibody generates immune reaction.

(5) Incubation of secondary antibody: A. the secondary antibody-goat anti-mouse IgG was diluted with TBST according to instructions; B. taking out the PVDF membrane in the miscellaneous gel bag, putting into a culture dish poured with TBST, washing the membrane for 3 times, 10min each time; after washing, the PVDF membrane is put into a diluted second antibody reaction solution, namely goat anti-mouse IgG, and placed on a shaking table to be incubated and reacted for 1h at room temperature.

(6) Exposure: after incubation, washing the PVDF membrane with TBST solution for three times, 10min each time; and (3) preparing ECL Plus hypersensitive luminescent liquid (liquid A: liquid B: 1) while cleaning, transferring the PVDF membrane to a gel imager after cleaning, photographing and storing.

As shown in fig. 12, the results indicate that the polyclonal antibody and the CNR protein can generate specific antigen-antibody reaction, and the detected band corresponds to the molecular weight expected by the experiment and is single, so that the specificity of the polyclonal antibody of the CNR protein is better.

17. Extraction of total protein from tomato fruit

Taking out the tomato jelly samples in the immature stage, the green stage, the color breaking stage, the pink stage and the red stage, using a liquid nitrogen precooling mortar and a pestle, weighing 1g of the sample, putting the sample into the mortar, adding liquid nitrogen to grind the sample into powder, and transferring the powder to a precooled 1.5ml centrifuge tube (note: the sample is always kept in a frozen state).

(1) Adding 1ml of prepared extracting solution, and uniformly mixing by oscillation to fully dissolve the sample.

(2) The mixture was centrifuged (12000rpm, 15min) to remove the supernatant.

(3) The mixture was centrifuged again (12000rpm, 15min) to remove the supernatant.

(4) 100. mu.L of the supernatant after centrifugation was taken out for quantification of the protein, and the remaining supernatant solution was aliquoted and stored at-20 ℃.

Extracting total protein of tomato fruit at immature stage, green mature stage, color breaking stage, pink stage and red mature stage respectively according to the above steps, and performing SDS-PAGE electrophoresis on the parts to detect the quality, wherein the sample loading amount is 15 μ g/hole as shown in FIG. 13; immunoblotting experiments were then performed on the CNR protein, all at 20 μ g/well. As shown in FIG. 14, the CNR protein was expressed in the highest amount in the breaker stage of tomato fruits, but was hardly expressed in the red-ripe stage. Therefore, the accumulation of the CNR protein is closely related to the development and maturation of tomato fruits.

And (4) conclusion: the experimental results show that the tomato CNR protein polyclonal antibody prepared in the invention can identify the CNR protein in tomato and detect the CNR protein, but does not react with other proteins in tomato. The polyclonal antibody can be used for detecting the expression condition of the tomato transcription factor CNR in the fruit ripening process, and has important significance for functional identification and research of CNR protein.

The invention has been described in an illustrative manner, and it is to be understood that any simple variations, modifications or other equivalent changes which can be made by one skilled in the art without departing from the spirit of the invention fall within the scope of the invention.

Sequence listing

<110> Tianjin college of agriculture

<120> tomato fruit transcription factor CNR polyclonal antibody and preparation method thereof

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 949

<212> DNA

<213> tomato (Lycopersicon esculentum cv. Ailsa Craig)

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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

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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

<212> DNA

<213> Artificial sequence

<400> 3

ggaattcatg gaaactaaca aatgggaagg g 31

<210> 4

<211> 33

<212> DNA

<213> Artificial sequence

<400> 4

ccgctcgagg cccaaatttt ctccatgaga gtc 33

Claims (6)

1. A preparation method of a tomato fruit transcription factor CNR polyclonal antibody is characterized by comprising the following steps:

(1) construction of a peptide containing SEQ ID No: 1, transforming the recombinant expression vector into escherichia coli Rosetta competent cells, and transferring the escherichia coli Rosetta competent cells into a pET-30a prokaryotic expression vector;

(2) after a large amount of expression and purification, CNR protein antigen is obtained;

(3) immunizing animals with the CNR protein antigen, and separating and purifying the serum to obtain the polyclonal antibody of the tomato CNR protein.

2. The method according to claim 1, wherein the recombinant expression vector is obtained by cloning a fragment containing the nucleotide sequence shown in SEQ ID No.1 into a prokaryotic expression vector.

3. The method according to claim 1, wherein the CNR protein antigen has an amino acid sequence as shown in SEQ ID No. 2.

4. The method as set forth in any one of claims 1, 2 and 3, wherein the step of immunizing the animal comprises 3 immunizations, wherein the 1 st immunization is carried out after mixing the purified CNR protein antigen with complete freund's adjuvant, and the 2 nd to 3 rd immunization is carried out after mixing the purified CNR protein antigen with non-complete freund's adjuvant, and the time interval between each immunization is 14 days.

5. A tomato CNR protein polyclonal antibody prepared by the method of any one of claims 1-4.

6. Use of the tomato CNR protein polyclonal antibody of claim 5 in the preparation of a kit for detecting tomato CNR protein.

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