CN111303291A

CN111303291A - Monoclonal antibody for detecting tobacco adenosine triphosphatase and application thereof

Info

Publication number: CN111303291A
Application number: CN202010102554.8A
Authority: CN
Inventors: 陈穗云; 廖菊够; 陶柯良; 魏雪梅
Original assignee: Yunnan University YNU
Current assignee: Yunnan University YNU
Priority date: 2020-02-19
Filing date: 2020-02-19
Publication date: 2020-06-19

Abstract

A monoclonal antibody of tobacco ATPase, wherein the heavy chain variable region sequence of the monoclonal antibody comprises the amino acid sequence ISYDGSN of CDR2 shown in the amino acid sequence GYSITSGYY, SEQ ID NO.4 of CDR1 shown in SEQ ID NO.2 and the amino acid sequence FGKGY of CDR3 shown in SEQ ID NO. 6; the light chain variable region sequence comprises the amino acid sequence KVS of CDR2 shown in amino acid sequence QSIVHSNGNTY, SEQ ID NO.11 of CDR1 shown in SEQ ID NO.9 and the amino acid sequence FQGSHVPWT of CDR3 shown in SEQ ID NO. 13. The invention provides a monoclonal antibody of tobacco adenosine triphosphatase (APY) and application thereof, aiming at solving the defects in the prior art.

Description

Monoclonal antibody for detecting tobacco adenosine triphosphatase and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody for detecting tobacco adenosine triphosphatase and application thereof.

Background

Apyrases (Apyrases, APY, EC 3.6.1.5) responsible for the catalytic hydrolysis of the phosphoanhydride linkages in Nucleoside Triphosphates (NTPs) and Nucleoside Diphosphates (NDPs) to form Nucleoside Monophosphates (NMPs); the enzyme is widely present in bacteria, fungi, animals and plants, and its function is most widely studied in mammals, among which potato (solanumtureosum), Arabidopsis thaliana (Arabidopsis), cotton (Gossypium hirsutum), pea (Pisum sativum), soybean (glycnesoja) and alfalfa (Medicago truncatula) are reported. The results of the studies in the above plants show that: the Apyrases plays multiple roles in regulating and controlling the growth and development of plants, such as regulating and controlling fiber growth, stomata opening and closing, cold resistance, pollen germination and growth and the like. Studies have reported that extracellular Apyrases are expressed in large quantities in rapidly growing cells, such as root apex growth zone cells, hypocotyl cells and pollen tube cells (Clark et al, 2010), which transport intracellular nucleotides to the outside, and are capable of timely breakdown and regulation of extracellular nucleotide concentrations (Wu et al, 2007).

Currently, no targeting antibody specific to tobacco adenosine triphosphatase (APY) 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 APY at the protein level is rapidly and sensitively detected, so that the APY can be used for functional research of the APY in cell growth and reproductive pollination of tobacco and other plants.

Disclosure of Invention

The invention provides a monoclonal antibody of tobacco adenosine triphosphatase (APY) and application thereof, aiming at solving the defects in the prior art.

In order to achieve the purpose, the invention adopts the technical scheme that:

a monoclonal antibody of tobacco adenosine triphosphatase (APY),

a) the heavy chain variable region sequence of the monoclonal antibody comprises an amino acid sequence GYSITSGYY of a CDR1 shown in SEQ ID NO.2, an amino acid sequence ISYDGSN of a CDR2 shown in SEQ ID NO.4 and an amino acid sequence FGKGY of a CDR3 shown in SEQ ID NO. 6;

b) the light chain variable region sequence of the monoclonal antibody comprises the amino acid sequence KVS of the CDR2 shown in the amino acid sequence QSIVHSNGNTY, SEQ ID NO.11 of the CDR1 shown in the SEQ ID NO.9 and the amino acid sequence FQGSHVPWT of the CDR3 shown in the SEQ ID NO. 13.

Further, the monoclonal antibody of the tobacco adenosine triphosphatase (Apyrases, APY) is applied as follows: the method is used for detecting the expression quantity and the positioning of the ATPase of different tissues of the tobacco.

Further, the monoclonal antibody of the tobacco adenosine triphosphatase (Apyrases, APY) is applied as follows: the method is used for detecting the expression quantity and distribution change of the pistil adenosine triphosphatase of different pollination combinations and researching the correlation between the expression of the adenosine triphosphatase and the pollination affinity.

Further, the monoclonal antibody of the tobacco adenosine triphosphatase (Apyrases, APY) is applied as follows: the method is used for detecting the expression of the ATPase in various plant tissues and is used for the expression and function research of the ATPase in plants.

Further, the monoclonal antibody of the tobacco adenosine triphosphatase (Apyrases, APY) is applied as follows: the method is used for developing co-immunoprecipitation in the pistils of the tobacco to identify the interaction protein of the ATPase and researching the interaction protein regulation network of the ATPase.

Further, the specific method for detecting the expression and the location of the ATPase of different tissues of the tobacco comprises the following steps:

1) taking tissue and cell materials such as tobacco roots, stems, leaves, pistils, stamens, pollen tubes and the like, fixing by 4% paraformaldehyde, and carrying out conventional paraffin slicing treatment;

2) processing the slices according to an immunofluorescence step, and respectively incubating an anti-ATPase primary antibody and a fluorescent secondary antibody in the invention;

3) the expression and localization of atpase in different tissues was observed by fluorescence microscopy.

Further, the specific method for detecting the expression of ATPase in various plants comprises the following steps: taking leaves of different plants as materials, extracting total protein by a BPP method, and carrying out an immunoblotting experiment.

Further, the specific method for detecting the change of the expression level of the pistil adenosine triphosphatase of different pollination combinations comprises the following steps: taking pistils with different pollination combinations, fixing by 4% paraformaldehyde, processing by a conventional paraffin section, processing the section according to an immunofluorescence step, and observing the expression quantity and positioning of protein by a fluorescence microscope.

Further, the specific method for identifying the adenosine triphosphatase interacting protein comprises the steps of carrying out co-immunoprecipitation by using total protein of the tobacco pistil as a material, and identifying the protein by mass spectrum, wherein the adenosine triphosphatase interacts with α subunits of ATP synthetase in the tobacco pistil.

Further, the detection tool comprises the monoclonal antibody of the tobacco ATPase, is used for detecting the ATPase, and is a kit, a chip or test paper.

Compared with the prior art, the invention has at least the following beneficial effects:

the invention uses prokaryotic expression tobacco APY protein as immunogen to immunize mouse to obtain lymphocyte, and uses hybridoma cell fusion technique to prepare fusion cell, and obtains cell strain capable of producing monoclonal antibody with high affinity and high specificity and monoclonal antibody secreted by cell strain by immunoblotting method, limiting dilution method and ELISA method.

The monoclonal antibody of the tobacco ATPase has high affinity and high specificity to the ATPase, and can be widely used for detecting the ATPase, particularly for expressing and positioning the ATPase of different tissue parts of tobacco and expressing the ATPase in different plant leaves; in addition, the monoclonal antibody provides a tool for researching the target protein, so that the expression of the ATPase can be researched by an immunoblotting method, an enzyme-linked immunosorbent assay, an immunohistochemistry method and an immunofluorescence method, and the research on an ATPase protein interaction regulation network can be deeply carried out by utilizing technologies such as co-immunoprecipitation and the like.

Drawings

FIG. 1 is a graphical representation of the results of Western blot WB validation of anthers and pollinating pistil antibodies from different tobacco species;

FIG. 2 is a graph showing the effect of Anti-APY antibody on the chip recognition of a target protein;

FIG. 3 is an immunofluorescence method for detecting APY expression of Anti-APY antibody in different tissues of tobacco;

FIG. 4 shows that Anti-APY antibody detects APY expression of different plant species leaves by an immunoblotting method.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

As shown in the figure:

example 1: preparation of monoclonal antibody hybridoma of ATPase and preparation and purification of anti-ATPase monoclonal antibody

1.1 preparation of antigens

Pronucleus expresses atpase holoprotein as immunogen, total protein couples VLP and immunogenicity enhancing factor of traditional KLH system.

1.2 immunization of mice

6 Balb/c mice (8-12 weeks old) were immunized with antigen and their serum titers were monitored to determine the optimal number of immunizations. The optimized adjuvant and immunization method can produce high affinity antibodies (IgG subtype) against most antigenic polypeptides. After the initial immunization, 3 to 4 times of boosting are carried out, and after the boosting, the serum of the mice is taken to detect the titer (recombinant protein of adenosine triphosphatase is coated as an anti-antigen). Mice that are eligible for titer will be bumped once and used for fusion, and ineligible mice will continue to be boosted one to two times until the titer is highest and fused.

1.3 serum detection and screening

The immunized mice were bled from the orbit and the serum titers were measured by ELISA (recombinant protein of ATPase as antigen coating). The serum titer needs to be more than 10K, otherwise the boosting is continued.

1.4 fusion and screening

Lymph nodes from whole spleens and 1/2 were taken and fused with myeloma SP2/0 cell line. The process is optimized PEG fusion. The fused cells were plated on 4 384-well plates (cells 102 to 104 per well) and cultured. 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 assayed for reaction with soluble fragment assay by ELISA. Positive wells further tested different dilutions of soluble fragment for binding to assay for affinity sequencing. The 20 parent clones with the highest affinity for each polypeptide immunogen entered subcloning. The 60 parental clones with the highest immunogenic affinity per soluble fragment entered subcloning.

1.5 subcloning and screening

Subcloning was performed by limiting dilution and ELISA screening to obtain monoclonal hybridoma cells. Cells were plated in 96-well plates and cultured to cover the bottom of about 1/6. ELISA detects the reaction of the supernatant of each hole to the soluble fragment detection antigen and the corresponding polypeptide detection antigen, and two holes with high OD values and good cell states are selected to enter the next round of subcloning. The above procedure was repeated until the positive rate of cell lines in the wells was 100%. At this point we obtained a monoclonal cell line. After the final round of subcloning, all positive cells were immediately expanded, one part was frozen for later use, and the other part was subjected to supernatant or ascites preparation.

1.6 preparation and purification of antibody supernatants

Monoclonal cell lines were finally obtained and injected ventrally into F1 mice for antibody production. The ascites fluid produced was purified with Protein A/G and used for subsequent detection.

Example 2: validation of anti-ATPase monoclonal antibodies

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.

2.1 ELISA (enzyme-Linked immunosorbent assay) verification of antibody and antigen polypeptide

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 1:

TABLE 1 antibody ELISA test data

2.2 endogenous protein immunoblot (WB) validation of antibodies

Using different tobacco species gynoecium holoprotein lysates, the antibody dilution concentration is 1: 1000 WB verification was performed. The experimental result shows that anti-APY can specifically identify a 50KD 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 figure 1. The antibody of the invention has specificity to the ATPase, can be used as a detection antibody for specially detecting the expression of the ATPase, and can be further developed into a detection kit.

2.3 antibody chip assay

Using a chip spotting instrument, anti-APY antibody and control antibody were spotted on a glass plate with NC membrane as a substrate 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 results are shown in fig. 2, the Anti-APY 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.

Example 3: sequencing of anti-ATPase monoclonal antibody

Culturing hybridoma cell strains of the anti-APY antibody, 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.

Example 4: immunofluorescence detection of expression and location of ATPase protein of different tissues of tobacco

The antibody of the invention has specificity to adenosine triphosphatase, and can be applied to in-situ detection of protein expression and positioning 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. Sections were processed according to an immunofluorescence procedure and primary and secondary anti-atpase antibodies and fluorescent antibodies of this patent were incubated separately. The expression and localization of atpase in different tissues was observed by fluorescence microscopy. As can be seen in FIG. 3, the antibody was effective in detecting ATPase expression in various tissues and cells of tobacco, and was able to make an initial determination of ATPase expression and localization in these tissues.

Example 5: application experiment of tobacco adenosine triphosphatase 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 ATPase antibody in other plant materials is explored. Extracting protein from leaves of different plants, and performing experiment. FIG. 4 shows that the antibody of the present invention can be used in assays for detecting ATPase expression in a plurality of plants, and provides guidance for cross-species use of the antibody of the present invention.

Example 6 identification of interacting proteins by Co-immunoprecipitation of tobacco anti-ATPase antibodies in tobacco pistils

The adenosine triphosphatase antibody can be used for performing co-immunoprecipitation in tobacco and identifying the interaction protein of the adenosine triphosphatase, wherein the co-immunoprecipitation is performed by taking the total protein of the pistil of the tobacco as a material, and the protein obtained by mass spectrum identification shows that the adenosine triphosphatase and the α subunit (A0A0M4JJI5) of ATP synthetase interact in the pistil of the tobacco.

Example 7 detection of the amount of ATPase expression before and after different pollination of pistils

Selfing pollination (compatibility) and hybrid pollination (incomplete wave tobacco, Stokes' tobacco pollen as a male parent and incompatibility) are carried out by using the cultivated tobacco pistil as a female parent. Taking pistils (stigma and style) 10h after pollination, fixing% paraformaldehyde, processing by a conventional paraffin section, processing the section according to an immunofluorescence step, observing the expression amount and positioning of protein by a fluorescence microscope, and displaying that the expression amount of adenosine triphosphatase in the self-compatible pistils is higher than that of the hybridization-incompatible pistils, which shows that the adenosine triphosphatase can participate in the regulation and control of pollen-pistil affinity.

The english abbreviations and key terms appearing in the present invention are set forth in the following table:

the preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A monoclonal antibody of tobacco ATPase, which is characterized in that,

1) the heavy chain variable region sequence of the monoclonal antibody comprises an amino acid sequence GYSITSGYY of a CDR1 shown in SEQ ID NO.2, an amino acid sequence ISYDGSN of a CDR2 shown in SEQ ID NO.4 and an amino acid sequence FGKGY of a CDR3 shown in SEQ ID NO. 6;

2) the light chain variable region sequence of the monoclonal antibody comprises the amino acid sequence KVS of the CDR2 shown in the amino acid sequence QSIVHSNGNTY, SEQ ID NO.11 of the CDR1 shown in the SEQ ID NO.9 and the amino acid sequence FQGSHVPWT of the CDR3 shown in the SEQ ID NO. 13.

2. The monoclonal antibody of tobacco ATPase according to claim 1, wherein the monoclonal antibody is used as follows: the method is used for detecting the expression quantity and the positioning of the ATPase of different tissues of the tobacco.

3. The monoclonal antibody of tobacco ATPase according to claim 1, wherein the monoclonal antibody is used as follows: the method is used for detecting the expression quantity and distribution change of the pistil adenosine triphosphatase of different pollination combinations and researching the correlation between the expression of the adenosine triphosphatase and the pollination affinity.

4. The monoclonal antibody of tobacco ATPase according to claim 1, wherein the monoclonal antibody is used as follows: the method is used for detecting the expression of the ATPase in various plant tissues and is used for the expression and function research of the ATPase in plants.

5. The monoclonal antibody of tobacco ATPase according to claim 1, wherein the monoclonal antibody is used as follows: the method is used for identifying the interaction protein of the adenosine triphosphatase in the pistil of the tobacco through co-immunoprecipitation and researching the interaction protein regulation network of the adenosine triphosphatase.

6. The monoclonal antibody of tobacco atpase according to claim 2, characterized in that the specific method for detecting the expression and localization of atpase in different tissues of tobacco is as follows:

7. The monoclonal antibody of tobacco atpase according to claim 3, characterized in that the specific method for detecting atpase expression in various plants is:

taking leaves of different plants as materials, extracting total protein by a BPP method, and carrying out an immunoblotting experiment.

8. The monoclonal antibody of tobacco atpase according to claim 4, characterized in that the specific method for detecting the change of the expression level of the pistil atpase of different pollination combinations comprises:

taking pistils with different pollination combinations, fixing by 4% paraformaldehyde, processing by a conventional paraffin section, processing the section according to an immunofluorescence step, and observing the expression quantity and positioning of protein by a fluorescence microscope.

9. The monoclonal antibody of tobacco atpase according to claim 5, characterized in that the specific method for identifying the atpase interacting protein is:

carrying out co-immunoprecipitation by using total protein of the tobacco pistil as a material, and identifying the protein by mass spectrum, wherein in the tobacco pistil, adenosine triphosphatase and α subunits of ATP synthetase interact.

10. An assay kit comprising a monoclonal antibody to tobacco atpase according to claim 1 for detecting atpase in the form of a kit, chip or strip.