CN111217914B - Monoclonal antibody for detecting tobacco polyphenol oxidase and application thereof - Google Patents
Monoclonal antibody for detecting tobacco polyphenol oxidase and application thereof Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody for detecting tobacco polyphenol oxidase and application thereof, wherein a heavy chain variable region sequence of the monoclonal antibody comprises an amino acid sequence GYTFTDYY of CDR1 shown in SEQ ID NO.1, an amino acid sequence INPYNGDT of CDR2 shown in SEQ ID NO.2 and an amino acid sequence AREDYSGSSYVANFDY of CDR3 shown in SEQ ID NO. 3; the light chain variable region sequence of the monoclonal antibody comprises an amino acid sequence QMS of a CDR2 shown in an amino acid sequence KSLLHSNGITY, SEQ ID NO.5 of a CDR1 shown in an SEQ ID NO.4 and an amino acid sequence AQNLELWT of a CDR3 shown in an SEQ ID NO. 6. The invention provides a monoclonal antibody for detecting tobacco polyphenol oxidase and application thereof, and aims to realize deeper research on PPO protein expression change and protein network maps of different tissues and different physiological conditions of tobacco and other plants by providing a tool for targeted protein research.
Description
Technical Field
The invention relates to the technical field of monoclonal antibody preparation, in particular to a monoclonal antibody for detecting tobacco polyphenol oxidase and application thereof.
Background
Polyphenol Oxidase (PPO) is capable of oxidizing phenols or polyphenols by molecular oxygen to form the corresponding quinones. In 1938, Keilin and Mann first isolated and purified PPO from mushrooms, and initiated a study of this enzyme. PPO is a class of Cu2+Binding proteins, formed by the coding of nuclear genes, are mainly localized in the thylakoid membrane of chloroplasts, in the cytoplasm and in the vacuole (Ono et al, 2006),There are also distributions on organelles such as golgi (Olmedo et al, 2018), mitochondria, etc. Depending on the specificity and the self-structure of the catalytic substrate, PPO can be classified into catechol oxidase (EC 1.10.3.2), Tyrosinase (EC 1.14.18.1) and laccase (lacase, EC 1.10.3.1), wherein Tyrosinase is mainly distributed in animals, laccase is mainly distributed in microorganisms, and PPO in plants is mainly catechol oxidase. PPO in plants utilizes oxygen to complete the catalytic action on phenols through two different reactions, wherein the first reaction is to catalyze monophenols to generate hydroxylation reaction to form bisphenol, and the second reaction is to catalyze bisphenol to generate oxidation reaction to form quinones. The formation of quinones is a consequence of plant senescence, mechanical damage and pathogen infestation (thipyopong, Stout,&Attajarusit,2007)
with the development of molecular biology, genes of polyphenol oxidase in many plants have been cloned, and from the viewpoint of the genes of polyphenol oxidase, the genes belong to gene families. 19 ppo genes (Li, Li, Li, Shao, & Lu,2017) were identified in Salvia miltiorrhiza. Potatoes contain 7 members of PPO (Goldman, Seurinck, Marins, Goldman, & Mariani,1998) and tobacco contains 14 members of PPO. There are no sequences similar to PPO in the arabidopsis genome (Tran, Taylor, & Constabel, 2012). The expression of these PPO members is spatio-temporally different and tissue specific, suggesting that the genes for polyphenol oxidase play different roles in plants. As in tobacco, two PPO members are highly expressed in pistils, suggesting that they may play a role in tobacco pollination and pollen tube growth regulation. The PPO enzyme activity detection method is commonly used by a pressure detection method and a spectrophotometry method. The former uses polyphenol oxidase (PPO) to catalyze the oxidation-reduction reaction of substrates such as catechin and the like under the aerobic condition, and according to the principle that the oxidation rate of the substrates is in direct proportion to the concentration of unit enzyme and the oxygen consumption in unit time, the oxygen consumption in the reaction process is measured by a Wacker to obtain the PPO activity. The color substance of the color-developing substance has maximum absorption at 460 nm, the change of the absorption value in unit time is in direct proportion to the unit enzyme activity, and the PPO activity intensity is calculated. The operation method is simple and convenient, and the reproducibility is good.
At present, no targeting antibody specially aiming at Plant Polyphenol Oxidase (PPO) exists in the market, so that an effective antibody tool is lacked in the research field on the protein level, and the expression change of the polyphenol oxidase at the protein level is rapidly and sensitively detected, so that the protein-based targeting antibody is used for functional research of the polyphenol oxidase in plant disease resistance and reproductive pollination.
Disclosure of Invention
The invention provides a monoclonal antibody for detecting tobacco polyphenol oxidase and application thereof, and aims to realize deeper research on PPO protein expression change and protein network maps of different tissues and different physiological conditions of tobacco and other plants by providing a tool for targeted protein research.
In order to solve the technical problems, the invention adopts the following technical scheme:
a monoclonal antibody for detecting tobacco polyphenol oxidase is disclosed, wherein the heavy chain variable region sequence of the monoclonal antibody comprises the amino acid sequence GYTFTDYY of CDR1 shown in SEQ ID NO.1, the amino acid sequence INPYNGDT of CDR2 shown in SEQ ID NO.2 and the amino acid sequence AREDYSGSSYVANFDY of CDR3 shown in SEQ ID NO. 3; the light chain variable region sequence of the monoclonal antibody comprises an amino acid sequence QMS of a CDR2 shown in an amino acid sequence KSLLHSNGITY, SEQ ID NO.5 of a CDR1 shown in an SEQ ID NO.4 and an amino acid sequence AQNLELWT of a CDR3 shown in an SEQ ID NO. 6.
Further, the method comprises the following steps of; the preparation and purification method of the monoclonal antibody comprises the following steps:
(1) preparation of antigens
Prokaryotic expression polyphenol oxidase holoprotein is used as immunogen, and immune prototobacco is extracted from the total protein of the pistil and pistil of grass;
(2) immunization of mice
Immunizing 6 Balb/c mice with the antigen, monitoring the serum titer of the mice to determine the optimal immunization times, and strengthening for 3-4 times after primary immunization;
(3) serum detection and screening
The eye sockets of the immunized mice are bled, and the serum titer is detected by ELISA;
(4) fusion and screening
Taking lymph nodes of whole spleen and 1/2, fusing with myeloma SP2/0 cell line, laying fused cells on 4 pieces of 384-well plates for culture, collecting supernatants of all holes, screening polypeptide detection sources by ELISA, transferring positive holes with cells by microscopic examination to 96-well plates for continuous culture, after several days of growth, collecting supernatants of all holes, detecting the reaction of the supernatants and the soluble fragment detection sources by ELISA, and further detecting the combination of the soluble fragment detection sources with different dilutions by the positive holes;
(5) subcloning and screening
Carrying out subcloning to obtain monoclonal hybridoma cells, laying the cells on a 96-well plate, culturing until the bottoms of the cells cover about 1/6, detecting the reaction of supernatant of each well aiming at a soluble fragment detection source and a corresponding polypeptide detection source by ELISA, taking two holes with high OD values and good cell states, entering the next round of subcloning, repeating the steps until the positive rate of cell strains in the holes is 100 percent, immediately carrying out expanded culture on all positive cells, freezing one part of the positive cells for later use, and preparing the supernatant or ascites for the other part of the positive cells;
(6) antibody supernatant preparation and purification
Monoclonal cell lines were obtained, injected ventrally into immunized mice for antibody production, and the resulting ascites fluid was purified with ProteinA/G and used for subsequent detection.
Further, the method comprises the following steps of; the monoclonal antibody is used for ELISA verification of an antibody and an antigen polypeptide;
the ELISA verifies that:
(1) taking an ascites antibody to be paired to coat a 96-hole ELISA plate, incubating, washing, sealing skim milk overnight, washing with PBS, and storing at 4 ℃ for later use;
(2) incubating antigen polypeptide, washing by PBS, and setting a control;
(3) adding an HRP (horse radish peroxidase) -labeled detection antibody into the ELISA plate incubated with the antibody in the step (1);
(4) TMB color reaction, reading by a microplate reader.
Further, the method comprises the following steps of; the monoclonal antibody is used for WB verification of an endogenous protein of the antibody;
the WB verification: using different tobacco species of full-female and male pistils; white lysate, antibody dilution concentration 1: 1000 WB verification was performed.
Further, the method comprises the following steps of; the monoclonal antibody is used for an antibody chip detection experiment;
the chip detection experiment:
(1) using a chip spotting instrument to spot anti-PPO antibody and a control antibody on a glass sheet taking an NC membrane as a matrix to form an antibody spot with the diameter of 100 um;
(2) carrying out biotin labeling on the tobacco stamen and stamen holoprotein, incubating on an antibody chip according to the concentration of 2ug/ml, and incubating for half an hour at room temperature;
(3) the cells were gently washed three times with PBS, incubated with CY3-SA fluorescent secondary antibody, washed three times with PBS, and the chip was scanned 523nm using a GenePix fluorescent chip scanner.
Further, the method comprises the following steps of; the monoclonal antibody is used for sequencing.
Further, the method comprises the following steps of; the sequencing comprises the following steps:
(1) culturing hybridoma cell strains of Anti-PPO antibodies, extracting total RNA, and reversely transcribing mRNA into first-chain cDNA;
(2) amplifying heavy chain and light chain genes through PCR, cloning the amplified genes to a sequencing vector, and performing sequencing on a plurality of positive clones to obtain a final sequence result.
Further, the method comprises the following steps of; the monoclonal antibody is used for detecting the expression and the positioning of polyphenol oxidase proteins of different tissues of tobacco.
Further, the method comprises the following steps of; the expression and positioning method of the monoclonal antibody for detecting phenol oxidase protein comprises the following steps:
(1) taking tobacco root, stem, leaf, pistil, stamen and pollen tube tissues and cell materials, fixing by 4% paraformaldehyde, and carrying out conventional paraffin section treatment;
(2) processing the slices according to an immunofluorescence step, and respectively incubating an anti-polyphenol oxidase primary antibody and a fluorescent secondary antibody;
(3) the expression and localization of polyphenol oxidase in different tissues were observed by fluorescence microscopy.
Further, the method comprises the following steps of; the monoclonal antibody is used for immunoblotting application in other plant materials.
Compared with the prior art, the invention has at least the following beneficial effects:
1. the monoclonal antibody of polyphenol oxidase has high affinity and high specificity to polyphenol oxidase, and can be widely used for detecting polyphenol oxidase, particularly expressing and positioning polyphenol oxidase at different tissue parts of tobacco and expressing polyphenol oxidase in different plant leaves; in addition, the monoclonal antibody of the invention provides a tool for researching a target protein, so that the expression of polyphenol oxidase can be researched by an immunoblotting method, an enzyme-linked immunosorbent assay, an immunohistochemistry method and an immunofluorescence method.
2. The invention extracts the total protein of the stamens and stamens of the tobacco, takes the total protein as immunogen, immunizes a mouse to obtain lymphocyte, and utilizes hybridoma cell fusion technology to prepare fusion cell, and ELISA method obtains cell strain which can produce monoclonal antibody with high affinity and high specificity, and monoclonal antibody secreted by the cell strain, and the most effective antibody is determined by enzyme-linked immunity, protein imprinting and other verification. And identifying the monoclonal antibody of polyphenol oxidase (PPO) by using immunoprecipitation mass spectrometry.
3. The invention also discloses the application of the monoclonal antibody of the polyphenol oxidase in detecting plant polyphenol oxidase, which comprises the steps of detecting the change of the expression quantity of the polyphenol oxidase before and after different pollination treatments of pistils and different tissues of tobacco; and is applied to detecting the expression of polyphenol oxidase in a plurality of plant leaves.
Drawings
FIG. 1 is a schematic diagram showing the result of Western blot WB validation of anthers and pollinating pistil antibodies of different tobacco species;
FIG. 2 is a graph showing the chip recognition effect of Anti-PPO antibody on target protein;
FIG. 3 is a diagram showing the detection of PPO expression in different tissues of tobacco by Anti-PPO antibody through an immunofluorescence method;
FIG. 4 is a PPO expression diagram of Anti-PPO antibody in different plant species detected by an immunoblotting method.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
a monoclonal antibody for detecting tobacco polyphenol oxidase is disclosed, wherein the heavy chain variable region sequence of the monoclonal antibody comprises the amino acid sequence GYTFTDYY of CDR1 shown in SEQ ID NO.1, the amino acid sequence INPYNGDT of CDR2 shown in SEQ ID NO.2 and the amino acid sequence AREDYSGSSYVANFDY of CDR3 shown in SEQ ID NO. 3;
the light chain variable region sequence of the monoclonal antibody comprises an amino acid sequence QMS of a CDR2 shown in an amino acid sequence KSLLHSNGITY, SEQ ID NO.5 of a CDR1 shown in an SEQ ID NO.4 and an amino acid sequence AQNLELWT of a CDR3 shown in an SEQ ID NO. 6; the method comprises the steps of extracting total protein of the tobacco stamens and stamens, immunizing a mouse by taking the total protein as immunogen to obtain lymphocytes, preparing fusion cells by utilizing a hybridoma cell fusion technology, obtaining a cell strain capable of producing a monoclonal antibody with high affinity and high specificity by an immunoblotting method, a limiting dilution method and an ELISA method, and determining the most effective antibody by enzyme-linked immunosorbent assay, western blot and other verification. And identifying the monoclonal antibody of polyphenol oxidase (PPO) by adopting a co-immunoprecipitation mass spectrum.
Example 2:
on the basis of example 1, the preparation and purification method of monoclonal antibody:
(1) preparation of antigens
Prokaryotic expression polyphenol oxidase holoprotein is used as immunogen, immune prototobacco is extracted from the total protein of the pistil and pistil of grass, and the total protein is coupled with VLP and immunogenicity enhancing factor of the traditional KLH system;
(2) immunization of mice
Immunizing 6 Balb/c mice with the antigen, selecting the mice with the age of 8-12 weeks, monitoring the serum titer of the mice to determine the optimal immunization times, and strengthening for 4 times after the initial immunization; the serum titer is monitored to determine the optimal immunization times, the optimized adjuvant and the optimized immunization method can generate an antibody (IgG subtype) with high affinity for most antigen polypeptides, the antibody can be enhanced for 4 times after primary immunization, the serum of a mouse is taken to detect the titer (prokaryotic expression protein of polyphenol oxidase is used as anti-antigen coating) after the enhancement, the mouse with qualified titer impacts once and is used for fusion, the mouse with unqualified titer is continuously enhanced for one to two times until the titer is the highest, and then the fusion is carried out;
(3) serum detection and screening
The eye sockets of the immunized mice are bled, and the serum titer is detected by ELISA; prokaryotic expression protein of polyphenol oxidase in serum is used as antigen coating, the serum titer is required to be more than 10K, otherwise, the immunity is continuously strengthened;
(4) fusion and screening
Taking the lymph nodes of the whole spleen and 1/2, fusing with a myeloma SP2/0 cell line, paving fused cells on 4 384-well plates for culture, collecting supernatants of all the wells, screening polypeptide detection sources by using ELISA, transferring positive wells with cells subjected to microscopic examination to 96-well plates for continuous culture, after several days of growth, collecting supernatants of all the wells, detecting the reaction of the supernatants and the soluble fragment detection sources by using ELISA, and further detecting the combination of the soluble fragment detection sources with different dilutions by using the positive wells; the lymph nodes from the whole spleen and 1/2 were taken and fused with myeloma SP2/0 cell line by optimized PEG fusion, and the fused cells were plated on 4 384-well plates for culture. Supernatants from all wells were collected, screened for the polypeptide assay by ELISA, and positive wells with microscopic cells were transferred to 96-well plates for further culture. After several days of growth, supernatants from all wells were collected and tested for reaction with soluble fragment assay antigen by ELISA, and positive wells were further tested for binding of different dilutions of soluble fragment assay antigen for affinity ranking, with 20 parent clones with the highest immunogenic affinity per polypeptide entered the subclones and 60 parent clones with the highest immunogenic affinity per soluble fragment entered the subclones;
(5) subcloning and screening
ELISA screening and subcloning to obtain monoclonal hybridoma cells, paving 96 pore plates with the cells, culturing until the bottoms of the cells cover about 1/6, detecting the reaction of supernatant of each pore aiming at a soluble fragment detection source and a corresponding polypeptide detection source by ELISA, taking two pores with high OD values and good cell states, entering a next round of subcloning, repeating the steps until the positive rate of cell strains in the pores is 100%, immediately performing expanded culture on all positive cells, freezing one part of the positive cells for later use, and preparing the supernatant or ascites on the other part of the positive cells; repeating the steps until the positive rate of the cell strain in the hole is 100%, obtaining a monoclonal cell strain, performing the final round of subcloning, immediately performing the expanded culture of all positive cells, freezing one part for later use, and performing the preparation of supernatant or ascites on the other part.
(6) Antibody supernatant preparation and purification
Monoclonal cell lines were obtained, injected ventrally into immunized mice for antibody production, and the resulting ascites fluid was purified with ProteinA/G and used for subsequent detection.
Example 3:
on the basis of examples 1-2, the preparation and purification method of monoclonal antibodies:
(1) preparation of antigens
Prokaryotic expression polyphenol oxidase holoprotein is used as immunogen, immune prototobacco is extracted from the total protein of the pistil and pistil of grass, and the total protein is coupled with VLP and immunogenicity enhancing factor of the traditional KLH system;
(2) immunization of mice
Immunizing 6 Balb/c mice with the antigen, selecting the mice with the age of 8-12 weeks, monitoring the serum titer of the mice to determine the optimal immunization times, and strengthening for 3 times after the initial immunization; the serum titer is monitored to determine the optimal immunization times, the optimized adjuvant and the optimized immunization method can generate an antibody (IgG subtype) with high affinity for most antigen polypeptides, the antibody can be strengthened for 3 times after primary immunization, the serum of a mouse is taken to detect the titer (prokaryotic expression protein of polyphenol oxidase is used as anti-antigen coating) after strengthening, the mouse with qualified titer impacts once and is used for fusion, the unqualified mouse is continuously strengthened for one to two times until the titer is the highest, and then the fusion is carried out;
(3) serum detection and screening
The eye sockets of the immunized mice are bled, and the serum titer is detected by ELISA; prokaryotic expression protein of polyphenol oxidase in serum is used as antigen coating, the serum titer is required to be more than 10K, otherwise, the immunity is continuously strengthened;
(4) fusion and screening
Taking the whole spleen and 1/2 lymph nodes, fusing with myeloma SP2/0 cell line, spreading fused cells on 4 384-well plates for culture, collecting supernatants of all the wells, screening polypeptide detection sources by ELISA, switching positive wells with microscopic cells to 96-well plates for continuous culture, after several days of growth, collecting supernatants of all the wells, detecting the reaction of the supernatants and the soluble fragment detection sources by ELISA, and further detecting the combination of the soluble fragment detection sources with different dilutions by the positive wells; the lymph nodes from the whole spleen and 1/2 were taken and fused with myeloma SP2/0 cell line by optimized PEG fusion, and the fused cells were plated on 4 384-well plates for culture. Supernatants from all wells were collected, screened for the polypeptide assay by ELISA, and positive wells with microscopic cells were transferred to 96-well plates for further culture. After several days of growth, supernatants from all wells were collected and tested for reaction with soluble fragment assay antigen by ELISA, and positive wells were further tested for binding of different dilutions of soluble fragment assay antigen for affinity ranking, with 20 parent clones with the highest immunogenic affinity per polypeptide entered the subclones and 60 parent clones with the highest immunogenic affinity per soluble fragment entered the subclones;
(5) subcloning and screening
ELISA screening and subcloning to obtain monoclonal hybridoma cells, paving 96 pore plates with the cells, culturing until the bottoms of the cells cover about 1/6, detecting the reaction of supernatant of each pore aiming at a soluble fragment detection source and a corresponding polypeptide detection source by ELISA, taking two pores with high OD values and good cell states, entering a next round of subcloning, repeating the steps until the positive rate of cell strains in the pores is 100%, immediately performing expanded culture on all positive cells, freezing one part of the positive cells for later use, and preparing the supernatant or ascites on the other part of the positive cells; repeating the steps until the positive rate of the cell strain in the hole is 100%, obtaining a monoclonal cell strain, performing the final round of subcloning, immediately performing the expanded culture of all positive cells, freezing one part for later use, and performing the preparation of supernatant or ascites on the other part.
(6) Antibody supernatant preparation and purification
Monoclonal cell lines were obtained, injected ventrally into immunized mice for antibody production, and the resulting ascites fluid was purified with ProteinA/G and used for subsequent detection
Example 4:
on the basis of the examples 1-3, the monoclonal antibody is used for ELISA verification of the antibody and the antigen polypeptide;
and (3) ELISA verification:
(1) taking an ascites antibody to be paired to coat a 96-hole ELISA plate, incubating, washing, sealing skim milk overnight, washing with PBS, and storing at 4 ℃ for later use;
(2) incubating antigen polypeptide, washing by PBS, and setting a control;
(3) adding an HRP (horse radish peroxidase) -labeled detection antibody into the ELISA plate incubated with the antibody in the step (1);
(4) TMB color reaction, and reading by an enzyme-linked immunosorbent assay (ELISA) instrument; antibody titers were monitored.
Example 5:
on the basis of examples 1 to 4, monoclonal antibodies were used for WB validation of the endogenous protein of the antibodies;
WB verification: using different tobacco species of full-female and male pistils; white lysate, antibody dilution concentration 1: 1000, performing WB verification; detecting the expression quantity difference of the protein in different anthers.
Example 6:
on the basis of examples 1-5, monoclonal antibodies were used in antibody chip detection experiments;
chip detection experiment:
(1) using a chip spotting instrument to spot anti-PPO antibody and a control antibody on a glass sheet taking an NC membrane as a matrix to form an antibody spot with the diameter of 100 um;
(2) carrying out biotin labeling on the tobacco stamen and stamen holoprotein, incubating on an antibody chip according to the concentration of 2ug/ml, and incubating for half an hour at room temperature;
(3) the PBS was gently washed three times, incubated with CY3-SA fluorescent secondary antibody, washed three times with PBS, and the chip was scanned 523nm using a GenePix fluorescent chip scanner; and detecting that the Anti-PPO antibody has an enrichment binding effect on the target protein.
Example 7:
on the basis of examples 1 to 6, monoclonal antibodies were used for sequencing; and sequencing a plurality of positive clones to obtain a final sequence result.
Example 8:
on the basis of examples 1 to 7, sequencing comprised:
(1) culturing hybridoma cell strains of Anti-PPO antibodies, extracting total RNA, and reversely transcribing mRNA into first-chain cDNA;
(2) amplifying heavy chain and light chain genes through PCR, cloning the amplified genes to a sequencing vector, and performing sequencing on a plurality of positive clones to obtain a final sequence result.
Example 9:
on the basis of the embodiments 1-8, the monoclonal antibody is used for detecting the expression and the location of polyphenol oxidase protein of different tissues of tobacco; the antibody can effectively detect the expression of polyphenol oxidase in different tissues and cells of tobacco, and can preliminarily judge the expression and the positioning of the polyphenol oxidase in the tissues.
Example 10:
on the basis of examples 1 to 9, the monoclonal antibody was used for the expression and localization method for detecting phenol oxidizing enzyme protein:
(1) taking tobacco root, stem, leaf, pistil, stamen and pollen tube tissues and cell materials, fixing by 4% paraformaldehyde, and carrying out conventional paraffin section treatment;
(2) processing the slices according to an immunofluorescence step, and respectively incubating an anti-polyphenol oxidase primary antibody and a fluorescent secondary antibody;
(3) observing the expression and the location of polyphenol oxidase in different tissues by a fluorescence microscope; the antibody can effectively detect the expression of polyphenol oxidase in different tissues and cells of tobacco, and can preliminarily judge the expression and the location of the polyphenol oxidase in the tissues.
Example 11:
based on examples 1-10, monoclonal antibodies were used for immunoblotting applications in other plant materials, and antibodies could be used for assays that detect polyphenol oxidase expression in multiple plants, providing guidance for cross-species applications of the antibodies of the present application.
Validation of anti-Polyphenol oxidase monoclonal antibody
And carrying out enzyme-linked immunosorbent assay, immunoblotting, co-immunoprecipitation and mass spectrometry, antibody chip verification and the like on the obtained monoclonal antibody cell strain to determine the effectiveness of the antibody.
(1) ELISA (immuno-enzyme-Linked) validation of antibodies and antigen polypeptides
And (3) coating a 96-hole ELISA plate with the ascites antibody to be paired, incubating, washing, sealing the degreased milk overnight, washing with PBS, and storing at 4 ℃ for later use. Antigen polypeptide incubation, PBS wash, with controls. HRP-labeled detection antibody was added to the ELISA plate incubated with the aforementioned. TMB color reaction, reading by a microplate reader. Antibody titers are shown in table one:
antibody titer of the Table-antibody ELISA
(2) Endogenous protein immunoblot (WB) validation of antibodies
Different tobacco species gynoecium holoprotein lysates, antibody dilution concentration 1: 1000 WB verification was performed. The experimental result shows that anti-PPO can specifically identify a 65KD band in WB verification, the size of the band is consistent with the expected size, and meanwhile, the expression amount of the protein in different anthers is obviously different, as shown in FIG. 1. The antibody of the invention has specificity to polyphenol oxidase, can be used as a detection antibody for specially detecting the expression of polyphenol oxidase, and can be further developed into a detection kit.
(3) Antibody chip detection experiment
Using a chip spotting instrument, anti-PPO antibody and control antibody were spotted on a glass plate with NC membrane as a matrix to form antibody spots with a diameter of 100 μm. The tobacco stamen and stamen holoprotein is labeled by biotin, incubated on an antibody chip according to the concentration of 2ug/ml, and incubated for half an hour at room temperature. The cells were gently washed three times with PBS, incubated with CY3-SA fluorescent secondary antibody, washed three times with PBS, and the chip was scanned 523nm using a GenePix fluorescent chip scanner.
The experimental result is shown in fig. 2, the Anti-PPO antibody has obvious enrichment binding effect on the target protein and stronger fluorescence intensity, while the control antibody does not have antigen-antibody binding reaction.
(4) Sequencing of anti-polyphenol oxidase monoclonal antibody
Culturing hybridoma cell strains of Anti-PPO antibodies, extracting total RNA, and reversely transcribing mRNA into first-chain cDNA; amplifying heavy chain and light chain genes through PCR, cloning the amplified genes to a sequencing vector, and performing sequencing on a plurality of positive clones to obtain a final sequence result.
(5) Immunofluorescence detection of expression and location of polyphenol oxidase protein of different tissues of tobacco
The antibody of the invention has specificity to polyphenol oxidase, and can be applied to in situ detection of protein expression and location at the tissue level. Taking tissue and cell materials of tobacco roots, stems, leaves, pistils, stamens, pollen tubes and the like, fixing with 4% paraformaldehyde, and carrying out conventional paraffin section treatment. The sections were processed according to an immunofluorescence procedure to incubate the primary anti-polyphenol oxidase antibody and the secondary fluorescent antibody of this patent, respectively. The expression and localization of polyphenol oxidase in different tissues were observed by fluorescence microscopy. As can be seen in FIG. 3, the antibody was effective in detecting polyphenol oxidase expression in various tissues and cells of tobacco, and was able to preliminarily determine the expression and localization of polyphenol oxidase in these tissues.
(6) Application experiment of tobacco polyphenol oxidase resisting immunoblotting in other plant materials
Based on the homology of the same protein among different plant species, the immunoblotting application of the anti-tobacco polyphenol oxidase antibody in the present invention in other plant materials was investigated. Extracting protein from leaves of different plants, and carrying out experiments. FIG. 4 shows, in which K represents tobacco lamina; MLS, potato; FQ, tomato; QZ, eggplant; KF, coffee; PT, grape; CD, kidney bean; BM, castor 2; BLH, macleaya cordata; HG, cucumber; cha, tea; WJ, lettuce; KG, balsam pear this application antibody can apply to the detection that detects polyphenol oxidase expression in a plurality of plants, provides the guidance for the cross inter-species application of this application antibody.
Although the invention has been described herein with reference to a number of illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More specifically, various variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure and claims of this application. In addition to variations and modifications in the component parts and/or arrangements, other uses will also be apparent to those skilled in the art.
<110> Yuxi Zhong tobacco seeds Limited liability company, Yunnan university
<120> a monoclonal antibody for detecting tobacco polyphenol oxidase
<160> 6
<210> 1
<211> 8
<212> amino acid
<213> Artificial sequence
<400> 1
Gly Tyr Thr Phe Thr Asp Tyr Tyr
<210> 2
<211>8
<212> amino acid
<213> Artificial sequence
<400> 2
Ile Asn Pro Tyr Asn Gly Asp Thr
<210> 3
<211> 16
<212> amino acid
<213> Artificial sequence
<400> 3
Ala Arg Glu Asp Tyr Ser Gly Ser Ser Tyr Val Ala Asn Phe Asp Tyr
<210> 4
<211> 11
<212> amino acid
<213> Artificial sequence
<400> 4
Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr
<210> 5
<211> 3
<212> amino acid
<213> Artificial sequence
<400> 5
Gln Met Ser
<210> 6
<211> 8
<212> amino acid
<213> Artificial sequence
<400> 6
Ala Gln Asn Leu Glu Leu Trp Thr
Claims (1)
1. A monoclonal antibody for detecting tobacco polyphenol oxidase is characterized in that: the heavy chain variable region sequence of the monoclonal antibody comprises an amino acid sequence GYTFTDYY of CDR1 shown in SEQ ID NO.1, an amino acid sequence INPYNGDT of CDR2 shown in SEQ ID NO.2 and an amino acid sequence AREDYSGSSYVANFDY of CDR3 shown in SEQ ID NO. 3;
the light chain variable region sequence of the monoclonal antibody comprises an amino acid sequence QMS of a CDR2 shown in an amino acid sequence KSLLHSNGITY, SEQ ID NO.5 of a CDR1 shown in an SEQ ID NO.4 and an amino acid sequence AQNLELWT of a CDR3 shown in an SEQ ID NO. 6.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1209839A (en) * | 1996-01-29 | 1999-03-03 | 诺沃挪第克公司 | Bacterial polyphenol oxidase from bacillus for use in oxidation of colored substance |
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| WO2005068625A1 (en) * | 2004-01-05 | 2005-07-28 | The Regents Of The University Of California | Plants transformed for elevated levels of gallic acid and methods of producing said plants |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1209839A (en) * | 1996-01-29 | 1999-03-03 | 诺沃挪第克公司 | Bacterial polyphenol oxidase from bacillus for use in oxidation of colored substance |
Non-Patent Citations (1)
| Title |
|---|
| 多酚氧化酶卵黄抗体的制备及其抑制虾类黑变效果研究;党昕;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20140315;B024-258 * |
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