CN111690623B - Tea tree flavanonol 4-reductase protein antigen polypeptide, antibody, detection kit and application thereof - Google Patents
Tea tree flavanonol 4-reductase protein antigen polypeptide, antibody, detection kit and application thereof Download PDFInfo
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Abstract
The invention provides a tea tree flavanonol 4-reductase protein antigen polypeptide, an antibody, a detection kit and application thereof, belonging to the technical field of protein detection. The invention provides tea tree flavanonol 4-reductase protein antigen polypeptide, the amino acid sequence of which is shown as SEQ ID No. 1; the invention relates to an immune antigen of tea tree flavanonol 4-reductase protein, which is obtained by coupling polypeptide and carrier protein through amido bond. Immunizing an animal with the immunizing antigen to produce an antibody against the tea tree flavanonol 4-reductase protein antigen. The invention also provides a tea tree flavanonol 4-reductase detection kit based on an immunoreaction technology, which comprises the detection antigen and/or the antibody. The DFR antibody prepared by the invention not only lays a foundation for the functional research of the protein, but also has positive reference significance for the subsequent research on metabolism of flavonoids and other substances in tea trees.
Description
Technical Field
The invention belongs to the technical field of protein detection, and particularly relates to a flavanonol 4-reductase protein antigen polypeptide of tea trees, an antibody, a detection kit and application thereof.
Background
Dihydroflavonol 4-reductase (DFR) belongs to a single gene coding or small gene family, and is a short-chain reductase with NADPH dependence. The protein encoded by DFR has a binding region for binding NADPH, and a domain consisting of 25 amino acids around amino acids 132-157 of the protein encoded by DFR. DFR, one of the structural enzymes of the flavonoid biosynthetic metabolic pathway, can catalyze Dihydrokaempferol (DHK), Dihydroquercitrin (DHQ), Dihydromyricetin (DHM), leucospidin (leucospiadinidin), leucocyanidin (leucocyanidin), and leucodelphinidin (leucospidin), respectively, which then synthesize various anthocyanidins under the action of anthocyanin synthase (anthocyanidin synthase) and flavonoid 3-O-glycosyltransferase (flavonoid 3-O-amylosyltransferase, 3 GT). The colorless anthocyanin can synthesize 4 simple catechin monomers under the action of colorless anthocyanin reductase (LAR), anthocyanin synthase (ANS) and anthocyanin reductase (ANR), so that DFR not only is a key enzyme for synthesizing anthocyanin in plants, but also plays a key role in regulating the synthesis and accumulation of catechin in plants. The content of flavonoid substances such as anthocyanin and catechin in tea trees can be different due to different growth conditions, varieties and tissue parts, which can be caused by the difference of DFR enzyme gene expression or DFR protein expression.
The protein expression level has important value for subsequent research and explanation of different tea plant varieties or content difference of anthocyanin, catechin and the like of different tissues and parts of the tea plant. However, currently, research on the protein level of the DFR is still limited, no commercial antibody aiming at the DFR is sold, the expression condition of the DFR on the protein level cannot be detected, and the regulation effect of the DFR on the synthesis of substances such as anthocyanin and catechin of different varieties, different tissues and parts of tea trees cannot be revealed on the protein expression level.
Disclosure of Invention
In view of the above, the present invention aims to provide an antigenic polypeptide of tea tree flavanonol 4-reductase protein, an antibody and an application thereof, which provide an effective detection substance for detecting tea tree flavanonol 4-reductase on a protein expression level.
The invention also provides a detection kit for tea tree flavanonol 4-reductase, which realizes the purpose of commercial rapid detection.
The invention provides tea tree flavanonol 4-reductase protein antigen polypeptide, and an amino acid sequence of the polypeptide is shown as SEQ ID No. 1.
The invention provides an immune antigen of tea tree flavanonol 4-reductase protein, which is obtained by coupling polypeptide and carrier protein through amido bond;
the carrier protein comprises ovalbumin or hemocyanin.
The invention provides a detection antigen of tea tree flavanonol 4-reductase protein, wherein the detection antigen is obtained by coupling polypeptide and carrier protein through amido bond;
the carrier protein comprises bovine serum albumin.
The invention provides an antibody for resisting a tea tree flavanonol 4-reductase protein antigen, wherein the titer of the antibody generated by immunizing an animal by the immune antigen is not lower than that of the antibody generated by immunizing an animal by the immune antigen: 80000.
preferably, the antibody includes monoclonal and polyclonal antibodies.
The present invention provides a hybridoma cell secreting the antibody.
The invention provides a tea tree flavanonol 4-reductase detection kit based on an immunoreaction technology, which comprises a detection antigen and/or an antibody.
Preferably, the immunoreaction technology comprises immunochromatography, ELISA, western-blot, immunofluorescence in situ hybridization or immunohistochemistry.
The invention provides application of the polypeptide, the detection antigen or the antibody in a flavanonol 4-reductase detection kit.
The invention provides an application of a tea tree flavanonol 4-reductase detection kit based on an immunoreaction technology in detection of expression protein in tea tree cells, tea tree tissues, tea tree organs and tea tree near-source plants.
The invention provides tea tree flavanonol 4-reductase protein antigen polypeptide, and an amino acid sequence of the polypeptide is shown as SEQ ID No. 1. The invention finds out a consistent amino acid sequence based on the analysis of conserved domains of DFR genes of different tea trees, and analyzes the immunogenicity, hydrophilicity and hydrophobicity, surface accessibility and the like of the consistent amino acid sequence to determine the amino acid sequence of a proper section of intracellular polypeptide as an antigen. The verification proves that the immune antigen polypeptide can immunize animals to generate specific antibodies, and the antibody titer is ideal, so that the antigen polypeptide provided by the invention has stronger immunogenicity.
The invention provides an immune antigen of tea tree flavanonol 4-reductase protein, which is obtained by coupling polypeptide and carrier protein through amido bond; the carrier protein comprises ovalbumin or hemocyanin. The polypeptide is artificially synthesized, and then coupled with a maleimide activated carrier KLH, and the coupled product is subjected to desalting column purification, so that an antibody which has ideal potency and can specifically recognize DFR protein can be generated by immunizing a New Zealand white rabbit.
The invention provides an antibody for resisting a tea tree flavanonol 4-reductase protein antigen, and an antibody produced by immunizing an animal by the immune antigen. After a new zealand white rabbit is immunized by the immunizing antigen for multiple times, the titer of the antibody is detected by an ELISA method by the obtained rabbit-derived serum, the immune rabbit-derived serum is collected after the titer reaches an ideal value, and the antibody is purified by a Protein G Protein purification column; and (3) carrying out specificity reaction identification on the purified antibody, wherein the identification result shows that the generated antibody can specifically recognize the DFR protein. The method for preparing the DFR antibody has great significance, not only can lay a foundation for the functional research of the protein, but also has positive reference significance for the subsequent research on metabolism of flavonoids and other substances in tea plants.
Drawings
FIG. 1 is a flow chart of a method for producing a polypeptide of a DFR protein antigen provided in an embodiment of the present invention;
FIG. 2 shows the Western-blot analysis results of DFR protein, in which A, M: standard pre-staining protein, 1-2: tea tree DFR protein; tea tree Actin protein (for internal reference, detected by Actin antibody).
Detailed Description
The invention provides tea tree flavanonol 4-reductase protein antigen polypeptide, and an amino acid sequence of the polypeptide is shown as SEQ ID No.1 (C-KLMREKWPEYNVPTEF-NH 2). The source of the polypeptide is not particularly limited in the present invention, and any source of the polypeptide known in the art may be used. In the present example, the polypeptide is synthesized by a polypeptide synthesis company.
In the present invention, the screening method of the DFR protein antigen polypeptide is shown in FIG. 1, and comprises the following steps:
s101: according to two DFR gene sequences (SEQ ID No.2 and SEQ ID No.3) cloned in the tea trees of Yinghong Jiu and Nankunshan Mao tea, DFR genes in different tea tree resources are obtained by Blast search in GenBank.
S102: the conserved structural domains of the DFR protein sequences of different tea trees are obtained by using MEGA7 and Genedoc software for alignment analysis.
S103: using DNAstar software to find out all the consistent amino acid sequences of different tea trees from the conserved structural domain of DFR protein.
S104: DFR protein antigen polypeptide screening determination: the obtained consistent amino acid sequences are screened and determined to be antigen polypeptides by combining requirement analysis with stronger antigenicity, hydrophilicity and surface accessibility, and a 16-amino acid length peptide segment at the 273-288aa position of the DFR protein conserved domain is determined, wherein the polypeptide sequence is C-KLMREKWPEYNVPTEF-NH 2.
The invention provides an immune antigen of tea tree flavanonol 4-reductase protein, which is obtained by coupling polypeptide and carrier protein through amido bond by applying an SMCC method (refer to: Yejian, Yuhua, conrunmin, Yanyubao, Xiejing, Jujuan, Liaopo Danao, Zhongying, Xiaojijiang, Xiluo, Chibo, Yeyonggong, Jifeng, Lixing, Yilin, Panmeng, Weiheng, Dazhuojian. Enramycin artificial antigen synthesis and identification [ J ]. Hubei agrscience, 2019,58(07):92-95+ 156.); the carrier protein comprises ovalbumin or hemocyanin. The invention also provides a detection antigen of tea tree flavanonol 4-reductase protein, wherein the detection antigen is obtained by coupling the polypeptide and carrier protein through amido bond by applying glutaraldehyde method (Tan Xingmei preparation of cyclic guanosine monophosphate (cGMP) rabbit monoclonal antibody and establishment of competitive ELISA determination method [ D ]. Zhejiang university, 2016 ]); the carrier protein comprises bovine serum albumin.
In the present invention, since the chemically synthesized polypeptide antigen is a small molecule belonging to a hapten, it hardly has good antigenicity itself, and can induce only weak immune reaction in animals, it is important to crosslink with a carrier protein. The carrier protein contains many epitopes and is capable of stimulating T helper cells and thereby inducing B cell responses. There are many carrier proteins for cross-linking with polypeptides, the most commonly used carriers being Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), Ovalbumin (OVA) and bovine Thyroglobulin (THY). Experiments prove that KLH has higher antigenicity and has the strongest immunogenicity after being crosslinked with the DFR antigen polypeptide, so that the KLH is used as a coupling carrier protein to prepare the immune antigen. BSA is used as a polypeptide carrier protein, and because BSA is often used as a blocking agent for detection tests, the antibody produced by the method has certain application limitation, and therefore, BSA is used as the carrier protein for preparing detection antigens.
The invention provides an antibody for resisting a tea tree flavanonol 4-reductase protein antigen, and an antibody produced by immunizing an animal by the immune antigen. The antibodies preferably include monoclonal antibodies and polyclonal antibodies. The method for preparing the polyclonal antibody preferably comprises the following steps: diluting the immune antigen to 1mg/mL, emulsifying the immune antigen by using a Freund's complete adjuvant according to a volume ratio of 1:1, performing multipoint injection (1 mL for each injection) immunization under the skin of the back of the neck of two New Zealand white rabbits for 4 times at an interval of 2 weeks, collecting blood, and detecting the antibody titer by using an ELISA method; and (3) when the antibody titer reaches 1:80000, collecting serum, and purifying, ELISA and western blot identification to obtain the polyclonal antibody against DFR. When the antibody is a monoclonal antibody, the invention provides a hybridoma cell that secretes the antibody. Experiments such as ELISA, Western bot and immunofluorescence staining of DFR prove that the antibody can recognize DFR protein. The polyclonal antibody of the DFR protein lays a foundation for further researching the function of the DFR protein.
The invention provides a tea tree flavanonol 4-reductase detection kit based on an immunoreaction technology, which comprises a detection antigen and/or an antibody. The immunoreaction technology is not particularly limited, and all detection methods utilizing the combination of antigen and antibody to form a compound are applicable, and comprise an immunochromatography technology, an ELISA technology, a western-blot, immunofluorescence in situ hybridization or immunohistochemistry.
The invention provides application of the polypeptide or the detection antigen in a flavanonol 4-reductase detection kit. The invention provides application of the antibody in a flavanonol 4-reductase detection kit. The method for preparing the kit is not particularly limited, and the preparation method known in the art can be adopted. The method of using the kit is not particularly limited in the present invention, and a method of using a kit known in the art may be used.
The invention provides an application of a tea tree flavanonol 4-reductase detection kit based on an immunoreaction technology in detection of expression protein in tea tree cells, tea tree tissues, tea tree organs and tea tree near-source plants.
The tea tree flavanonol 4-reductase protein antigen polypeptide provided by the present invention and the antibody, detection kit and application thereof will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Design and synthesis of DFR protein polypeptide
1. Blast search of tea tree DFR gene: according to two DFR gene sequences (SEQ ID No.2 and SEQ ID No.3) cloned in the tea trees of Yinghong Jiu and Nankunshan Mao tea, DFR genes in different tea tree resources are obtained by Blast search in GenBank.
2. The conserved structural domains of the DFR protein sequences of different tea trees are obtained by analyzing and aligning MEGA7 and Genedoc software.
3. Using DNAstar software to find out all the consistent amino acid sequences of different tea trees from the conserved structural domain of DFR protein.
DFR protein antigen polypeptide screening determination: the obtained consistent amino acid sequences are screened and determined to be antigen polypeptides by combining requirement analysis with stronger antigenicity, hydrophilicity and surface accessibility, and a 16-amino acid length peptide segment at the 273-288aa position of the DFR protein conserved domain is determined, wherein the polypeptide sequence is C-KLMREKWPEYNVPTEF-NH 2.
Example 2
Preparation and immunization method of DFR protein antigen polypeptide
1 DFR protein antigen polypeptide: KLMREKWPEYNVPTE was synthesized by Qiaozhou Biometrics.
2 DFR antigen polypeptide, KLH and BSA coupling preparation of immune antigen and detection antigen
(1) Immune antigens were prepared by coupling DFR antigen polypeptides to KLH (Sulfo-SMCC as coupling agent) using SMCC kit (purchased from Thermo Fisher Scientific). 4mg of the DFR antigenic polypeptide was weighed, dissolved in 0.4mL of PBS (pH 7.2, the same applies hereinafter), 4mg of Sulfo-SMCC was added, and after incubation at 4 ℃ for 2 hours, 25mg of KLH (20mg/mL) was added to DFR-SMCC, reacted at 4 ℃ for 2 hours, dialyzed in PBS buffer for 24 hours, and stored at-20 ℃.
The concentration of DFR-KLH was determined using a BCA kit (purchased from Thermo Fisher Scientific) and Solution B and Solution A were formulated at a volume ratio of 1: 50. BSA standard protein concentrations were 0, 25, 125, 250, 500, 750 and 1000. mu.g/mL diluted with 1 XPBD. The absorbance was measured on a microplate reader at 562nm and a standard curve was drawn. mu.L of DFR-KLH antigen diluted to an appropriate amount was taken, 200. mu.L of BCA working solution was added, the absorbance at 562nm was measured, and the antigen concentration after the corresponding dilution was calculated according to the standard curve.
(2) The DFR antigen polypeptide is coupled with BSA (coupling agent is glutaraldehyde) to be used as a detection antigen.
Weighing mg of DFR antigen polypeptide and BSA protein, dissolving in 10mL of 0.2mol/L PBS buffer solution, adding 10mL of 1, 4-dioxane, and mixing. And (3) placing the beaker in a water bath stirrer at 25 ℃, stirring for 5min, dropwise adding 5mL of 0.25% glutaraldehyde solution, and continuously stirring for reacting for 4 h. Dialyzing at 4 deg.C for 72h, freeze-drying at-20 deg.C for 2d, and storing in-20 deg.C refrigerator.
3, diluting the immune antigen to 1mg/mL by using PBS buffer solution respectively, and subpackaging and freezing in a refrigerator with the temperature of-20 ℃.
4, on day 1, 1mL of DFR-KLH immune antigen is taken and added with 1mL of Freund's complete adjuvant, emulsification is carried out (the emulsification degree is checked, a drop of emulsified antigen liquid is dropped into physiological saline, if the emulsified antigen liquid is not dispersed, the requirement is met), 1mL of each of 2 New Zealand white rabbits (marked as rabbit A and rabbit B) is injected, and multiple-point (at least 8-point) injection is carried out subcutaneously on the back of the neck.
On day 15, 1mL of DFR-KLH immune antigen is taken and added with 1mL of Freund's incomplete adjuvant, emulsification is carried out (the emulsification degree is checked, a drop of emulsified antigen liquid is dripped into physiological saline, if the emulsified antigen liquid is not dispersed, the requirement is met), 1mL of 2 New Zealand white rabbits (marked as rabbit A and rabbit B) are respectively injected, and multiple (at least 8) injections are carried out subcutaneously on the back of the neck.
On the 29 th day 6, 1mL of DFR-KLH immune antigen is taken and added with 1mL of Freund's incomplete adjuvant, emulsification is carried out (the emulsification degree is checked, a drop of emulsified antigen liquid is dripped into physiological saline, if the emulsified antigen liquid is not dispersed, the requirement is met), 1mL of each of 2 New Zealand white rabbits (marked as rabbit A and rabbit B) is injected, and multiple (at least 8) injections are carried out subcutaneously on the back of the neck.
On day 7, on day 43, 1mL of DFR-KLH immune antigen was taken and added with 1mL of Freund's incomplete adjuvant, emulsification was carried out (the degree of emulsification was examined: one drop of emulsified antigen solution was dropped into physiological saline, and if not dispersed, it was found that the requirement was met), 1mL of each of 2 New Zealand white rabbits (labeled as Rabbit A and Rabbit B) were injected, and multiple injections (at least 8) were given subcutaneously on the back of the neck.
On day 8, day 53, carotid blood was taken and the rabbits were sacrificed.
The rabbit blood was left overnight at 4 ℃ and centrifuged (4 ℃ C., 10000rpm) for 30 minutes to collect the supernatant.
II, antibody purification process:
1 polypeptide was attached to activated Sulfolink Resin and an antigen affinity column was prepared, 1mL Sulfolink Resin was conjugated to 1mg polypeptide.
2, balancing the affinity column by using PBS (phosphate buffer solution) with 10 times of the column volume, and draining the solution; the rabbit serum was filtered through a 0.45 μm filter.
3, passing the serum through an antigen affinity column, draining the solution, and collecting the flow-through.
The 410 column volumes of PBS were equilibrated and the solution was drained.
5 add 5mL antibody eluent, collect eluent in tubes, 1mL each.
6 detecting the absorbance at 280nm of the collected eluent, combining the components with the absorbance more than 1.0, and dialyzing the mixture against PBS.
7 antibody identification after dialysis (protein concentration by ultraviolet absorption method, antibody titer by Elisa).
ElISA detection process
1 coating: the detection antigen was diluted to 1. mu.g/mL with CBS and added to the microplate at 100. mu.L per well overnight at 4 ℃.
2, sealing: the coating solution was discarded, and 200. mu.L of blocking solution (5% skim milk powder) was added to each well, followed by standing at 37 ℃ for 1.5 hours.
3 adding a sample: the blocking solution was discarded, and the sample (serum or antibody) was added thereto, and 100. mu.L of the microplate was added to each well, followed by standing at 37 ℃ for 1 hour.
4, washing: rinsed 10 times with tap water and patted dry.
5 adding a secondary antibody: the enzyme-labeled goat anti-rabbit is diluted to working concentration by using a confining liquid, an enzyme-labeled plate is added into each hole by 100 mu L, and the mixture is placed for 30 minutes at 37 ℃.
6, washing: rinsed 10 times with tap water and patted dry.
7 addition of TMB chromogenic substrate: add microplate to 100uL per well and incubate at 37 ℃ for 15 minutes.
8, adding a stop solution: add 50. mu.L of 2M H per well2SO4And (3) solution.
9 reading: enzyme-linked immunosorbent assay (OD)450nmAnd (6) reading.
Example 3
Identification of polyclonal antibodies against DFR
1. ELISA identification of polyclonal antibodies against DFR
The synthesized DFR polypeptide is used as a detection antigen, an enzyme label plate is coated, non-immune rabbit-derived rabbit serum diluted by 1:1250 is used as a negative control, the rabbit-derived serum and the purified antibody are diluted in a multiple ratio, an ELISA method is used for detection, and the antibody is judged to be positive when the ratio of the antibody to the negative serum is more than 2.1.
As a result, the titer of the purified anti-DFR antibody reached 1:80000 (see Table 1).
TABLE 1 anti-DFR antibody titers
2. Western Blot identification of polyclonal antibodies against DFR
1g of fresh tea leaves was taken and thoroughly ground in liquid nitrogen containing water-insoluble PVPP. 10mL of precooled 10% TCA/acetone (containing 0.07% beta-mercaptoethanol, 1mmol/L PMSF) was added and precipitated at-20 ℃ for 3 h. Followed by centrifugation at 12000 Xg for 15min at 4 ℃ and discarding the supernatant. The above acetone washing operation was repeated 2 to 3 times. Protein electrophoresis was performed using SDS-PAGE (12% separation gel and 5% concentrated gel) and the separation gel was stained colloidally with Coomassie Brilliant blue R-250 for 3-5 h. Transferring the protein in the gel to a PVDF membrane by a wet transfer printing technology, sealing the membrane by 5% of skimmed milk powder, and gently shaking for 10-14h at 4 ℃. Adding the prepared rabbit source antibody diluted by 1:5000, incubating for 10-14h at 4 ℃, and washing for 3 times by TBST. Adding a secondary antibody (goat anti-rabbit) diluted by 1:5000, shaking gently at 26 deg.C for 1h, washing with TBST for 3 times, adding appropriate amount of developer, and developing with a developer.
As a result, as shown in FIG. 2, the polyclonal antibody against DFR of the present invention detected a protein band around 40KD, which coincided with the molecular weight of the DFR protein.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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cgtgcaaccg ttcgcgatcc agcgaattta aagaaggtga agcacttgtt agacttgccg 180
aaagctgaca cgaacttgac actgtggaag gcggatttga atgaagaagg gagctttgat 240
gaggccattg agggttgctc tggagtgttt catgttgcca cacctatgga ttttgagtct 300
aaggaccctg agaatgaggt aatcaagccg acaatcaatg gtgtgttgag catcataagg 360
tcatgcacca aagctaagac agtgaagagg ctggtgttca catcctctgc tggaactgtt 420
aatgtccagg aacaccaaca acccgttttc gacgagaaca attggagtga cttggatttc 480
accaataaga agaagatgac tggctggatg tattttgttt caaaaacatt ggcagagaaa 540
gcagcatggg aagcagcaaa agagaacaac attgatttca ttagtatcat tcctacatta 600
gttgtaggac ctttcataat gccaacattc ccaccaagcc taatcactgc tctctccccc 660
atcactagga atgaaggaca ctactcgatc ataaagcaag ggcagtttgt gcaccttgat 720
gatctctgtg aatctcatat attcttgtat gagcgtcctc aggctgaggg cagatacatt 780
tgctcctccc atgatgctac catccatgat ttggccaaac tgatgagaga gaaatggccc 840
gagtacaatg tccccactga gtttaagggg atagataagg acttgccagt tgtgtcgttc 900
tcatcgaaga agttgatagg aatggggttt gaattcaagt atagcttgga ggacatgttc 960
agaggagcca ttgatacttg cagagagaag ggtttgcttc ctcactcttt tgcagaaaac 1020
cctgtcaatg gcaacaaggt ttaa 1044
Claims (9)
1. An immune antigen of tea tree flavanonol 4-reductase protein is characterized in that the immune antigen is obtained by coupling tea tree flavanonol 4-reductase protein antigen polypeptide and carrier protein through amido bond; the amino acid sequence of the polypeptide is shown as SEQ ID No. 1;
the carrier protein comprises ovalbumin or hemocyanin.
2. A detection antigen of tea tree flavanonol 4-reductase protein is characterized in that the detection antigen is obtained by coupling tea tree flavanonol 4-reductase protein antigen polypeptide with carrier protein through amido bond; the amino acid sequence of the polypeptide is shown as SEQ ID No. 1;
the carrier protein comprises bovine serum albumin.
3. An antibody against a tea tree flavanonol 4-reductase protein antigen, characterized in that the antibody produced by the immunizing animal with the immunizing antigen of claim 1 has a titer of not less than 1: 80000.
4. The antibody of claim 3, wherein said antibody comprises a monoclonal antibody and a polyclonal antibody.
5. A hybridoma cell secreting the antibody of claim 4.
6. An immunoassay-based kit for detecting flavanonol 4-reductase of Camellia sinensis, comprising the detection antigen of claim 2 and/or the antibody of claim 3 or 4.
7. The detection kit of claim 6, wherein the immunoreaction technology comprises immunochromatography, ELISA, western-blot, immunofluorescence in situ hybridization, or immunohistochemistry.
8. Use of the detection antigen of claim 2 or the antibody of claim 3 or 4 in a flavanonol 4-reductase detection kit.
9. Use of the kit for the detection of tea tree flavanonol 4-reductase based on immunoreaction technology of claim 6 or 7 for the detection of expression of flavanonol 4-reductase in tea tree cells, tea tree tissue, tea tree organs and tea tree proximal plants.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1294915A2 (en) * | 2000-06-29 | 2003-03-26 | Vlaams Interuniversitair Instituut voor Biotechnologie vzw. | Heterologous gene expression in plants |
| CN110904059A (en) * | 2019-11-18 | 2020-03-24 | 华中农业大学 | Anthocyanin synthetase epitope peptide, antibody and application thereof |
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2020
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1294915A2 (en) * | 2000-06-29 | 2003-03-26 | Vlaams Interuniversitair Instituut voor Biotechnologie vzw. | Heterologous gene expression in plants |
| CN110904059A (en) * | 2019-11-18 | 2020-03-24 | 华中农业大学 | Anthocyanin synthetase epitope peptide, antibody and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Molecular cloning of dihydroflavonol 4-reductase gene from grape berry and preparation of an anti-DFR polyclonal antibody;PING ZHANG等;《Vitis》;20080131;第141–145页 * |
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