CN110845608B - Tomato ringspot virus monoclonal antibody and preparation method thereof - Google Patents

Tomato ringspot virus monoclonal antibody and preparation method thereof Download PDF

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CN110845608B
CN110845608B CN201911141637.1A CN201911141637A CN110845608B CN 110845608 B CN110845608 B CN 110845608B CN 201911141637 A CN201911141637 A CN 201911141637A CN 110845608 B CN110845608 B CN 110845608B
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monoclonal antibody
ringspot virus
tomato ringspot
chain variable
variable region
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CN110845608A (en
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张蓬军
俞晓平
孙凯
杨倩倩
刘光富
郝培应
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China Jiliang University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1009—Picornaviridae, e.g. hepatitis A virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Abstract

The invention provides a tomato ringspot virus monoclonal antibody and a preparation method thereof, wherein a BALB/c mouse is immunized by an antigenic peptide CQAAQQAGKNPFGRG to obtain a hybridoma cell strain, a cell strain capable of generating a specific recognition antigenic peptide monoclonal antibody is screened out from the hybridoma cell strain, and a monoclonal antibody capable of specifically recognizing a tomato ringspot virus coat protein QAAQQAGKNPFGRG peptide segment is obtained by an ascites preparation method, wherein the lengths of a heavy chain variable region and a light chain variable region of the monoclonal antibody are 181 amino acids and 118 amino acids respectively; the yield of the monoclonal antibody is 500-5000mg/L ascites, and the purity is 85-99.5%. The monoclonal antibody has the characteristic of specifically recognizing the coat protein of the tomato ringspot virus, and has wide application prospect in the aspects of import and export inspection and quarantine of the tomato ringspot virus.

Description

Tomato ringspot virus monoclonal antibody and preparation method thereof
Technical Field
The invention relates to a monoclonal antibody, in particular to a tomato ringspot virus monoclonal antibody and a preparation method thereof.
Background
Tomato ringspot virus (ToRSV) is a member of the genus Nepovirus (Comoviridae) of the Comoviridae family, and the virus particles are isodiametric icosahedron with a diameter of about 30 nm. The genome contains two positive-sense single-stranded RNAs which respectively code for replicase, Motor Protein (MP) and Coat Protein (CP). ToRSV is widely distributed in Europe, America and the state of the America, can infect more than 150 crops such as soybean, grape, peach, plum, apple, cherry, tomato, tobacco and the like, causes various diseases and leads to outcropping in serious cases. There are several modes of ToRSV transmission, and the vectors of the nematode (Xiphinema) and graft transmission in the field are important modes of ToRSV transmission and prevalence, while long-distance transmission is mainly based on seed (seedling) regulation. The seed-borne poison plants are many, and some have high seed-borne rate, such as 76% of soybean, 76% of globe amaranth, 11% of elderberry, 24% of dandelion, 3-7% of red clover and 3% of tomato. The virus is a high-risk virus and is a quarantine pest prohibited from entry in China. Since ToRSV is a high-risk quarantine harmful organism which can be remotely transmitted along with the transportation of seed seedlings, establishing an effective rapid detection method is very important for preventing the virus from entering China. At present, the means used for inspection and quarantine against ToRSV at home and abroad include electron microscope observation, differential host reaction (biological method), serological test and molecular biological means. Since ToRSV is a spherical virus with the diameter of 28nm, the virus content in a sample is low, and the virus is difficult to be observed and identified under an electron microscope; the inoculation of the differential hosts requires a special isolation greenhouse, takes a long time and influences the identification result due to the occult phenomenon of viruses. The serology method is a necessary means for rapidly detecting the plant virus, the virus detection of the port depends on antiserum purchased abroad at present, the price is high, and the ToRSV serum detection kit for the imported plant seedlings is not reported at home.
Aiming at the current situation that a large number of seedlings imported in China possibly carry tomato ringspot virus, the invention screens and obtains a monoclonal antibody cell strain by taking tomato ringspot virus coat protein as a detection target through the selection and synthesis of a tomato ringspot virus specific universal antigen peptide segment, cross-linking immune mice, cell fusion, antibody activity detection and other series of steps. And obtaining antigen recognition region sequences in the antibody gene, namely heavy chain variable region sequences and light chain variable region sequences by an RT-PCR method. Finally obtaining the monoclonal antibody with known sequence for specifically recognizing the target antigen, the cell strain for producing the antibody and the corresponding antibody production method. Compared with the method for obtaining the monoclonal antibody by immunizing a mouse with the purified virus particles, the monoclonal antibody obtained by the invention has definite recognition epitope, and the monoclonal antibody obtained by immunizing the virus particles has indefinite recognition epitope. In addition, the invention firstly separates out a conserved region from the tomato ringspot virus coat protein sequence, then selects and designs a candidate antigen peptide segment from the conserved region, and then adopts a chemical synthesis method to prepare the antigen peptide segment, so that the acquisition of the antigen is not limited by a source of a virus-carrying sample, and the operation permission range of an imported inspection and quarantine substance is not violated. The specific recognition epitope also enables the monoclonal antibody of the invention to have specific and easily recognized characteristics with the existing antibodies on the market and the related research report antibodies.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a tomato ringspot virus monoclonal antibody which has the reaction specificity of identifying and combining the specific peptide segment of the tomato ringspot virus coat protein and lacks the combination specificity to other viruses and plant tissues, thereby laying a foundation for further developing and developing a tomato ringspot virus serum detection kit.
The invention firstly discloses a tomato ringspot virus monoclonal antibody, wherein the sequence of a heavy chain variable region of the antibody is SEQ ID NO: 1, the light chain variable region sequence is SEQ ID NO: 2, respectively.
The length of the heavy chain variable region and the length of the light chain variable region of the monoclonal antibody are 181 amino acids and 118 amino acids respectively.
As a preferred embodiment of the present invention, the nucleotide sequences encoding the heavy and light chain variable regions are SEQ ID NOs: 3 and SEQ ID NO: 4, respectively. The 181 amino acid residue sequence in the heavy chain variable region was analyzed by BLASTP and the first matched sequence similarity was 90.34%; the 118 amino acid residue sequence of the light chain was analyzed by BLASTP for first matched sequence similarity of 77.78%.
The monoclonal antibody is prepared by an ascites induction method, the yield is 500-5000mg/L, and the purity is 85-99.5%.
The invention also discloses a hybridoma cell strain (mouse hybridoma cell 6A5C4) for secreting the tomato ringspot virus monoclonal antibody, which is preserved in the China general microbiological culture Collection center (CGMCC), and the preservation numbers are as follows: CGMCC No.18321, with a preservation date of 2019, 8 months and 15 days, and a preservation unit address of No. 3 Xilu No.1 of Beijing, Chaoyang.
The invention also discloses application of the tomato ringspot virus monoclonal antibody in preparation of a tomato ringspot virus detection reagent or kit.
The tomato ringspot virus monoclonal antibody is prepared by the following steps:
(1) searching and obtaining a plurality of coat protein sequences (including protein sequences translated by gene sequences) of tomato ringspot viruses from Genbank, and selecting conserved segments from the coat protein sequences by sequence comparison;
(2) selecting a proper antigen sequence aiming at the conserved segment to obtain an optimized antigen peptide segment QAAQQAGKNPFGRG which is shown as SEQ ID NO. 5;
(3) synthesizing the antigen peptide segment CQAAQQAGKNPFGRG with 1 cysteine residue added at the N terminal by a chemical synthesis method;
(4) crosslinking the antigen peptide segment with KLH;
(5) immunizing a mouse with the cross-linked antigenic peptide fragment;
(6) preparing hybridoma cells and screening cell strains which produce antibodies which specifically recognize the peptide fragments of SEQ ID NO. 5 from the hybridoma cells;
(7) adopting an RT-PCR method to clone and measure the variable region sequences of the heavy chain and the light chain of the monoclonal antibody in the monoclonal antibody cell strain;
(8) preparing a monoclonal antibody which can identify the peptide segment of SEQ ID NO. 5 and make positive reaction on a sample carrying the tomato ringspot virus by adopting an ascites induction method;
(9) the monoclonal antibody of the invention is purified by ammonium sulfate precipitation and protein G affinity layer washing.
The preparation process of the ascites induction method of the monoclonal antibody specifically comprises the following steps:
(1) injecting 0.1-0.5mL of sterilized paraffin into the abdominal cavity of a BALB/c mouse for 8-15 weeks;
(2)5-10 days later, the hybridoma cells were inoculated intraperitoneally with 5X 10 of PBS suspension5-5×106A/only;
(3) collecting ascites 2-4 times after 5-10 days;
(4) centrifuging 1000 Xg ascites for 10min, sucking upmost adipose tissue, removing cell components and precipitate, and collecting supernatant;
(5) 3mL of ascites supernatant is taken, added with 3mL of 0.06M sodium acetate-acetic acid buffer solution with the pH value of 4.0 to 4.6; adding 99uL n-octanoic acid dropwise, stirring at 0-10 deg.C for 10-60min, and clarifying for 10-60 min; centrifuging at 14000 Xg for 20min at 4 deg.C, removing precipitate, adding 1/10 volume of 10 XPBS (0.01M, pH7.4) into the supernatant, and adjusting pH to 7.2 with 1M NaOH; dropwise adding equal amount of saturated ammonium sulfate (pH6.0-8.0) solution into the supernatant, stirring for 30min, and standing for 30 min; centrifuging at 14000 Xg for 30min at 4 deg.C, discarding the supernatant, dissolving the precipitate in 1.2mL PBS (0.01M, pH7.4), dialyzing with 0.01M, pH7.4 PBS at 0-10 deg.C overnight, and concentrating with 5% PEG20000 to 5 mL;
(6) and (3) passing the concentrated solution through protein G purification resin, washing 10-15 column volumes by using pre-elution solution, eluting the monoclonal antibody by using 5-10 column volumes of elution buffer solution, and concentrating to obtain the monoclonal antibody.
Preferably, the pre-elution solution in step (6) comprises 0.1-0.15M NaCl and 10-40mM Na2HPO4pH 7.0; the elution buffer solution is 0.1-0.3M glycine, and the pH value is 3.0.
The term "monoclonal antibody (mab)" as used herein refers to an antibody obtained from a substantially homogeneous population of cells, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. The monoclonal antibody specifically recognizes a peptide segment shown in SEQ ID NO. 5 derived from tomato ringspot virus coat protein, has a unique and definite recognition sequence, is different from a conventional monoclonal antibody with an unknown recognition sequence obtained by immunizing a mouse with tomato ringspot virus, and is also definitely different from a tomato ringspot virus polyclonal antibody.
The invention has the following beneficial effects: the results of potency determination and enzyme-linked immunoassay of the tomato ringspot virus monoclonal antibody show that the monoclonal antibody for identifying the peptide segment shown in SEQ ID NO. 5 has the specificity of reaction with tomato ringspot virus, can be used as an antibody for detecting the tomato ringspot virus, can lay a good foundation for the subsequent development of a kit for detecting the serum of the tomato ringspot virus, and serves the requirements of entry-exit inspection and quarantine work.
Drawings
FIG. 1 shows the antigen sequence and the position of candidate antigenic peptide 7 on the coat protein of tomato ringspot virus.
Detailed Description
The invention is further illustrated by the following examples:
example 1
(1) Tomato ringspot virus protein sequences (sequence accession number: NP-733973) are called from Genbank, the length of the sequences is 562 amino acid residues (namely 562aa), and the number of theoretical candidate antigen peptide fragments is 549 according to the requirement of the length of 14 amino acid residues (14aa) antigen peptide fragments. 7 candidate antigenic peptides are obtained through the analysis and prediction of solubility, synthesis difficulty and immunogenicity, and the sequences of the candidate antigenic peptides 1-6 are shown in SEQ ID NO: 6-11, the sequence of candidate antigenic peptide 7 is shown in SEQ ID NO:5, wherein the antigen sequence and the position of the candidate antigen peptide 7 on the coat protein of the tomato ringspot virus are shown in figure 1. For coupling with the carrier protein, hemocyanin KLH, 1 cysteine (C) is added at the N-terminal or C-terminal, and after 1 cysteine is added, the candidate antigen peptides are as follows:
candidate antigenic peptide 1: LLRYKEWQRQGFLHC
Candidate antigenic peptide 2: GPTEIDLTSTPAPNC
Candidate antigenic peptide 3: KLVDRLSVNVILQEC
Candidate antigenic peptide 4: GQGAFSLPISTPHAC
Candidate antigenic peptide 5: CLLRYKEWQRQGFLH
Candidate antigenic peptide 6: CDDKSEVLLRQHPLS
Candidate antigenic peptide 7: CQAAQQAGKNPFGRG are provided.
(2) The 7 antigenic polypeptides described in step (1) were prepared by solid-state synthesis from commercial polypeptide synthesis companies, and the purity of the polypeptides was 98% or more.
(3) The candidate antigenic peptide was coupled to KLH by a two-step reaction (this example is described as "candidate antigenic peptide 7: CQAAQQAGKNPFGRG"). The coupling reaction steps are as follows:
a) 20mg of SMCC [ sodium 4- (N-maleimidomethyl) cyclohexane-1-carboxylate sulfosuccinimidyl ester ] was dissolved in 2mL of DMF (N, N-dimethylformamide).
b) 0.8ml of KLH (hemocyanin) was added to a 25ml round bottom flask, supplemented with 1 XPBS (pH7.2) to give a final concentration of 15mg/ml protein.
c) The dissolved SMCC solution is slowly added into a 120mg KLH protein system in a dropwise manner, and the reaction is stirred at room temperature for 1 h.
d) Dialyzed against 1L of 1 XPBS (pH 7.4) solution at 4 ℃ for 6 hours.
e) The dialyzed KLH protein was poured into a 50mL centrifuge tube and its volume (at a density of 1 g/cm) was determined by weighing3Calculation), the concentration of the protein after dialysis was calculated from the amount of KLH protein added before the reaction, and then 2.5mg of the KLH-SMCC solution was transferred to a 5ml centrifuge tube according to its concentration.
f) 3.0mg of "candidate antigenic peptide 7: CQAAQQAGKNPFGRG' was dissolved in 0.6ml of 1 XPBS (pH 7.2).
g) Thiol groups in polypeptides were detected with Ellman's reagent: adding 100 μ l Ellman reagent stock solution into 96-well plate, adding 10 μ l polypeptide solution, measuring ultraviolet absorption value at λ 412nm, and performing the next step if OD value is greater than 0.15; the OD value is less than 0.15 and more than 0.05, and the polypeptide is added until the requirement is met;
h) the candidate antigen peptide 7 solution is dripped into a KLH-SMCC tube, and is mixed and reacted for 4 hours by a vertical mixer at room temperature.
i) Thiol groups in polypeptides were detected with Ellman's reagent: mu.l of Ellman reagent stock solution was added to a 96-well plate, 10. mu.l of the conjugated polypeptide solution was added, and the UV absorbance was measured at λ 412 nm. The OD value is less than 0.03, which indicates that the crosslinking rate of the polypeptide and the KLH protein reaches more than 80 percent; OD >0.03 and then adding activated KLH protein of SMCC to continue crosslinking. Obtaining the coupled antigen 'coupled candidate antigen peptide 7-KLH'.
(4) Immunization of mice
a) For primary immunization, 1.5mL of coupled candidate antigen peptide 7-KLH (the total amount is 150ug) is uniformly mixed with equal volume of Freund's complete adjuvant, subcutaneous multipoint injection is carried out according to the amount of 1.5mL per mouse, and 3 mice are immunized simultaneously.
b) The second immunization is carried out 3 weeks after the first immunization, the dosage route is the same as above, and Freund's incomplete adjuvant is added at intervals of 3 weeks.
c) The third immunization, dose as above, without adjuvant, intraperitoneal injection, 7 days later blood sampling to test the titer, and the test of the immunization effect, with 3 weeks interval.
d) The immunization is strengthened, the dosage is 50 ug/mouse, and the injection is carried out in the abdominal cavity.
e) After 3 days, the mouse with the highest titer is selected to take the spleen for fusion
(5) Hybridoma cell preparation
a) Will be 1 × 108Spleen cells and 1X 107Myeloma cells SP2/0 were mixed in a 50mL fusion tube, supplemented with incomplete medium to 30mL, and mixed well.
b) Centrifuging at 1000r/min for 10min, and sucking the supernatant as clean as possible.
c) Flicking the fusion tube bottom on the palm to loosen and uniform the precipitated cells;
d) add preheated 50% PEG 1mL over 30s with a 1mL pipette with gentle stirring.
e) The suction tube was left standing for 1 min.
f) The PEG reaction was terminated by adding the preheated incomplete culture medium, and 1mL, 2mL, 3mL, 4mL, 5mL, and 10mL were added every 2min in succession.
g)800rpm, 5 minutes; the supernatant was discarded.
h) Adding 5mL of complete culture medium, gently sucking the precipitated cells, suspending and mixing the cells uniformly, and then supplementing the complete culture medium to 40-50 mL. Subpackaging 96-well cell culture plates at 100uL per well, and placing the culture plates at 37 ℃ and 5% CO2Culturing in an incubator.
i) After 6h, the selection medium was supplemented. 50uL per well, half-change with selection medium after 3 days.
j) The growth of the hybridoma cells is often observed, and when the hybridoma cells grow to a bottom area of the wells above 1/10, the supernatant is aspirated for antibody detection.
(6) Cloning of hybridoma cells (limiting dilution method)
a) Mouse splenocytes were prepared as feeder cells.
b) Hybridoma cell suspensions to be cloned were prepared and diluted with HT medium containing 20% serum to 3 different dilutions containing 5, 10 and 20 cells per ml.
c) Adding 1X 10 per ml5The ratio of cells, abdominal cavity macrophage is added into the hybridoma cell suspension。
d) Each hybridoma cell was divided into 96-well plates at 100ul per well.
e)37℃、5％CO2Culturing for 6 days, and detecting the antibody by macroscopic cloning; wells in which only a single clone grew were marked by observation under an inverted microscope and the supernatant was taken for antibody detection.
f) And (4) taking the cells of the antibody detection positive hole, carrying out expanded culture and freezing and storing.
(7) Preparation of monoclonal antibody by ascites induction method
a) Injecting 0.1mL of sterilized paraffin into the abdominal cavity of a BALB/c mouse for 8 weeks;
b)5 days later, peritoneal inoculation with PBS suspension hybridoma cells 5 x 105A/only;
c) collecting ascites 2-4 times after 5 days;
d) centrifuging 1000 Xg ascites for 10min, sucking upmost adipose tissue, removing cell components and other precipitates, and collecting supernatant;
e) 3mL of ascites supernatant is taken, added with 3mL of 0.06M sodium acetate-acetic acid buffer solution with the pH value of 4.0, and the pH value is adjusted to 4.6; adding 99uL n-octanoic acid dropwise, stirring at 0 deg.C for 10min, and clarifying for 10-60 min; centrifuging at 14000 Xg for 20min at 4 deg.C, removing precipitate, adding 1/10 volume of 10 XPBS (0.01M, pH7.4) into the supernatant, and adjusting pH to 7.2 with 1M NaOH; dropwise adding equal amount of saturated ammonium sulfate (pH6.0-8.0) solution into the supernatant, stirring for 30min, and standing for 30 min; centrifuging at 14000 Xg for 30min at 4 deg.C, discarding the supernatant, dissolving the precipitate in 1.2mL PBS (0.01M, pH7.4), dialyzing with 0.01M, pH7.4 PBS at 0 deg.C overnight, and concentrating with 5% PEG20000 to 5 mL;
f) the concentrate was passed through protein G purification resin to pre-elute (0.1M NaCl,10mM Na)2HPO4pH7.0), eluting the column by 10 column volumes, eluting the monoclonal antibody by 5 column volumes of elution buffer solution (0.1M glycine, pH 3.0), concentrating to obtain the monoclonal antibody of the invention, wherein the yield is 1200mg/L ascites, the purity is 85%, detecting tomato leaves carrying inactivated tomato ringspot virus, and the detection signal is positive. The antibody is negative to tomato sample not infected by tomato ringspot virus, and can be used for resisting tobacco mosaic virus, T7 bacteriophage, arabis mosaic virus and kidney bean podThe reaction of mottle virus, etc. is negative.
Example 2
Steps (1) - (6) of example 2 are the same as example 1.
(7) Preparation of monoclonal antibody by ascites induction method
a) Injecting 0.5 mL/mouse of sterilized paraffin into the abdominal cavity of a BALB/c mouse for 15 weeks;
b)10 days later, peritoneal inoculation with PBS suspension hybridoma cells 5 x 106A/only;
c) ascites was collected 4 times after 10 days;
d) centrifuging 1000 Xg ascites for 10min, sucking upmost adipose tissue, removing cell components and other precipitates, and collecting supernatant;
e) 3mL of ascites supernatant is taken, added with 3mL of 0.06M sodium acetate-acetic acid buffer solution with the pH value of 4.0, and the pH value is adjusted to 5.0; adding 99uL n-octanoic acid dropwise, stirring at 0-10 deg.C for 60min, and clarifying for 60 min; centrifuging at 14000 Xg for 20min at 4 deg.C, removing precipitate, adding 1/10 volume of 10 XPBS (0.01M, pH7.4) into the supernatant, and adjusting pH to 7.2 with 1M NaOH; dropwise adding equal amount of saturated ammonium sulfate (pH8.0) solution into the supernatant, stirring for 30min, and standing for 30 min; centrifuging at 14000 Xg for 30min at 4 deg.C, discarding the supernatant, dissolving the precipitate in 1.2mL PBS (0.01M, pH7.4), and adding 0.01M, pH7.4
PBS was dialyzed overnight at 10 ℃ and concentrated to 5mL with 5% PEG 20000;
f) the concentrate was passed through protein G purification resin to pre-elute (00.15M NaCl,40mM Na)2HPO4pH7.0), eluting the column by 15 column volumes, eluting the monoclonal antibody by 10 column volumes of elution buffer solution (0.3M glycine, pH 3.0), concentrating to obtain the monoclonal antibody of the invention, wherein the yield is 500mg/L ascites, the purity is 87.2 percent, detecting tomato leaves carrying inactivated tomato ringspot virus, and the detection signal is positive. The antibody is negative to tomato samples which are not infected by tomato ringspot virus, and negative to tobacco mosaic virus, T7 phage, arabis mosaic virus, bean pod mottle virus and the like.
Example 3
Steps (1) - (6) of example 3 are the same as example 1.
(7) Preparation of monoclonal antibody by ascites induction method
a) Injecting 0.3mL of sterilized paraffin into the abdominal cavity of a BALB/c mouse for 11.5 weeks;
b) intraperitoneal inoculation 7 days later hybridoma cells suspended in PBS 1.25X 106A/only;
c) ascites was collected 3 times 7 days later;
d) centrifuging 1000 Xg ascites for 10min, sucking upmost adipose tissue, removing cell components and other precipitates, and collecting supernatant;
e) 3mL of ascites supernatant is taken, added with 3mL of 0.06M sodium acetate-acetic acid buffer solution with the pH value of 4.0, and the pH value is adjusted to 4.8; adding 99uL n-octanoic acid dropwise, stirring at 5 deg.C for 10-60min, and clarifying for 35 min; centrifuging at 14000 Xg for 20min at 4 deg.C, removing precipitate, adding 1/10 volume of 10 XPBS (0.01M, pH7.4) into the supernatant, and adjusting pH to 7.2 with 1M NaOH; dropwise adding equal amount of saturated ammonium sulfate (pH7.0) solution into the supernatant, stirring for 30min, and standing for 30 min; centrifuging at 14000 Xg for 30min at 4 deg.C, discarding the supernatant, dissolving the precipitate in 1.2mL PBS (0.01M, pH7.4), dialyzing with 0.01M, pH7.4 PBS at 5 deg.C overnight, and concentrating with 5% PEG20000 to 5 mL;
f) the concentrate was passed through protein G purification resin to pre-elute (0.125M NaCl,25mM Na)2HPO4pH7.0), eluting the column by 13 column volumes, eluting the monoclonal antibody by 7 column volumes of elution buffer solution (0.2M glycine, pH 3.0), concentrating to obtain the monoclonal antibody of the invention, wherein the yield is 500-5000mg/L ascites, the purity is 93.5 percent, detecting tomato leaves carrying inactivated tomato ringspot virus, and the detection signal is positive. The antibody is negative to tomato samples which are not infected by tomato ringspot virus, and negative to tobacco mosaic virus, T7 phage, arabis mosaic virus, bean pod mottle virus and the like.
Example 4
Steps (1) - (6) of example 4 are the same as example 1.
(7) Preparation of monoclonal antibody by ascites induction method
a) Injecting 0.3mL of sterilized paraffin into the abdominal cavity of a BALB/c mouse for 12 weeks;
b) intraperitoneal inoculation of hybridoma cells suspended in PBS 1X 10 after 7 days6A/only;
c) ascites was collected 3 times 7 days later;
d) centrifuging 1000 Xg ascites for 10min, sucking upmost adipose tissue, removing cell components and other precipitates, and collecting supernatant;
e) 3mL of ascites supernatant is taken, added with 3mL of 0.06M sodium acetate-acetic acid buffer solution with the pH value of 4.0, and the pH value is adjusted to 4.8; adding 99uL n-octanoic acid dropwise, stirring at 4 deg.C for 20min, and clarifying for 40 min; centrifuging at 14000 Xg for 20min at 4 deg.C, removing precipitate, adding 1/10 volume of 10 XPBS (0.01M, pH7.4) into the supernatant, and adjusting pH to 7.2 with 1M NaOH; dropwise adding equal amount of saturated ammonium sulfate (pH7.3) solution into the supernatant, stirring for 30min, and standing for 30 min; centrifuging at 14000 Xg for 30min at 4 deg.C, discarding the supernatant, dissolving the precipitate in 1.2mL PBS (0.01M, pH7.4), dialyzing with 0.01M, pH7.4 PBS at 4 deg.C overnight, and concentrating with 5% PEG20000 to 5 mL;
f) the concentrate was passed through protein G purification resin to pre-elute (0.12M NaCl,30mM Na)2HPO4pH7.0), eluting the monoclonal antibody by using elution buffer solution (0.15M glycine, pH 3.0) with 7 column volumes, concentrating to obtain the monoclonal antibody of the invention, wherein the yield is 5000mg/L ascites, the purity is 99.5%, the prepared antibody is used for detecting tomato leaves carrying inactivated tomato ringspot virus by an indirect ELISA method, and a detection signal is positive. The antibody is negative to tomato samples which are not infected by tomato ringspot virus, and negative to tobacco mosaic virus, T7 phage, arabis mosaic virus, bean pod mottle virus and the like.
Example 5
The procedure was as in example 4, and the antigenic peptide was "candidate antigenic peptide 1: LLRYKEWQRQGFLHC' and the monoclonal antibody generated by the hybridoma is detected by indirect ELISA method, and is positive to unconjugated candidate antigen peptide 1 and negative to tomato leaves carrying inactivated tomato ringspot virus. Monoclonal antibodies that can be used for detection of native viral particles are not available.
Example 6
The procedure was as in example 4, and the antigenic peptide was "candidate antigenic peptide 2: GPTEIDLTSTPAPNC' and the monoclonal antibody generated by the hybridoma is detected by indirect ELISA method, and is positive to unconjugated candidate antigen peptide 2 and negative to tomato leaves carrying inactivated tomato ringspot virus. Monoclonal antibodies that can be used for detection of native viral particles are not available.
Example 7
The procedure was as in example 4, and the antigenic peptide was "candidate antigenic peptide 3: KLVDRLSVNVILQEC ', the monoclonal antibody generated by hybridoma is detected by indirect ELISA method, and is positive to unconjugated candidate antigen peptide 3' and negative to tomato leaf with inactivated tomato ringspot virus. Monoclonal antibodies that can be used for detection of native viral particles are not available.
Example 8
The procedure was as in example 4, and the antigenic peptide was "candidate antigenic peptide 4: GQGAFSLPISTPHAC' and the monoclonal antibody generated by the hybridoma is detected by indirect ELISA method, and is positive to the unconjugated candidate antigen peptide 4 and negative to tomato leaves carrying inactivated tomato ringspot virus. Monoclonal antibodies that can be used for detection of native viral particles are not available.
Example 9
The procedure was as in example 4, and the antigenic peptide was "candidate antigenic peptide 5: CLLRYKEWQRQGFLH' and the monoclonal antibody generated by the hybridoma is detected by indirect ELISA method, and is positive to the unconjugated candidate antigen peptide 5 and negative to tomato leaves carrying inactivated tomato ringspot virus. Monoclonal antibodies that can be used for detection of native viral particles are not available.
Example 10
The procedure was as in example 4, and the antigenic peptide was "candidate antigenic peptide 6: CDDKSEVLLRQHPLS' and the monoclonal antibody generated by the hybridoma is detected by indirect ELISA method, and is positive to the unconjugated candidate antigen peptide 6 and negative to tomato leaves carrying inactivated tomato ringspot virus. Monoclonal antibodies that can be used for detection of native viral particles are not available.
Sequence listing
<110> China metering university
<120> tomato ringspot virus monoclonal antibody and preparation method thereof
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Met Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Ala
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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr
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Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
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Gly Ala Ile Tyr Pro Gly Asn Ser Asp Thr Asn Tyr Asn Gln Lys Phe
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Lys Gly Lys Ala Lys Leu Thr Ala Val Thr Ser Ala Ser Thr Ala Tyr
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Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys
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Thr Arg Leu Ser Met Leu Gly Arg Ser Tyr Tyr Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Ala Thr Pro Pro Ser
115 120 125
Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser Ser Val
130 135 140
Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Ser Val Thr Val
145 150 155 160
Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser Val His Thr Phe Pro Asp
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Ser Cys Ser Phe Thr
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Met Ala Cys Arg Pro Ser Ile Val Leu Thr Gln Ser Pro Ser Ser Leu
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Ala Met Ser Val Gly Gln Arg Val Thr Met Ser Cys Lys Ser Ser Gln
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Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Cys Gln
35 40 45
Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Glu Ser Gly Val Pro Asp
50 55 60
Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
65 70 75 80
Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Leu His Tyr
85 90 95
Ser Thr Pro His Val Arg Cys Trp Glu Gln Ala Gly Asp Pro Thr Lys
100 105 110
Leu Val Arg Ala Thr Arg
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atggtacaac tgcaacaatc tggtaccgtt ctggcacgtc ctggtgcatc cgtgaaaatg 60
agctgcaaag caagcggcta ctccttcact agctactgga tgcactgggt taagcagcgt 120
ccgggccagg gtctggaatg gatcggtgca atctatccgg gcaatagcga cacgaactac 180
aatcagaaat tcaaaggcaa ggctaaactg accgcggtaa cctccgcctc tactgcatac 240
atggagctgt ccagcctgac caatgaagat tccgcggttt actattgcac ccgcctgtct 300
atgctgggtc gtagctacta ttttgactat tggggccaag gtaccaccct gaccgtttct 360
agcgctaaag ctacgccacc gtccgtgtat ccgctggctc caggttgtgg tgataccacg 420
ggttctagcg ttaccctggg ttgcctggtt aaaggctact tcccagagag cgttacggtc 480
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ggacagaggg tcactatgag ctgcaagtcc agtcagagcc ttttaaatag tagcaatcaa 120
aagaactatt tggcctggtg ccagcagaga ccaggacagt ctcctaaact tctggaatct 180
ggggtccctg atcgcttcat aggcagtgga tctgggacag atttcactct taccatcagc 240
agtgtgcagg ctgaagacct ggcagattac ttctgtcagc tacattatag cactcctcac 300
gttcggtgct gggaacaagc tggagatcca acgaagcttg taagggcgac acgc 354
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<211> 14
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<213> Tomato ringspot virus (Tomato ringspot virus)
<400> 9
Gly Gln Gly Ala Phe Ser Leu Pro Ile Ser Thr Pro His Ala
1 5 10
<210> 10
<211> 14
<212> PRT
<213> Tomato ringspot virus (Tomato ringspot virus)
<400> 10
Leu Leu Arg Tyr Lys Glu Trp Gln Arg Gln Gly Phe Leu His
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<210> 11
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<213> Tomato ringspot virus (Tomato ringspot virus)
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Claims (7)

1. A tomato ringspot virus monoclonal antibody is characterized in that: the sequence of the heavy chain variable region of the antibody is SEQ ID NO: 1, the light chain variable region sequence is SEQ ID NO: 2, respectively.
2. The monoclonal antibody of claim 1, characterized in that: the length of the heavy chain variable region and the length of the light chain variable region of the monoclonal antibody are 181 amino acids and 118 amino acids respectively.
3. A nucleic acid molecule encoding the monoclonal antibody of claim 1, comprising a nucleotide sequence encoding a light chain variable region and a nucleotide sequence encoding a heavy chain variable region as set forth in SEQ ID NO: 3, the nucleotide sequence of the coding light chain variable region is shown as SEQ ID NO: 4, respectively.
4. The hybridoma cell strain capable of secreting the tomato ringspot virus monoclonal antibody is characterized by being preserved in China general microbiological culture Collection center (CGMCC), wherein the preservation numbers are as follows: CGMCC No. 18321.
5. A method for producing the monoclonal antibody of claim 1, comprising the steps of:
(1) injecting 0.1-0.5mL of sterilized paraffin into the abdominal cavity of a BALB/c mouse for 8-15 weeks;
(2) intraperitoneal inoculation of 5-10 days later, hybridoma cells of claim 4 suspended in PBS, 5 x 105-5×106A/only;
(3) collecting ascites 2-4 times after 5-10 days;
(4) centrifuging 1000 Xg ascites for 10min, sucking upmost adipose tissue, removing cell components and precipitate, and collecting supernatant;
(5) 3mL of ascites supernatant is taken, added with 3mL of 0.06M sodium acetate-acetic acid buffer solution with the pH value of 4.0 to 4.6; adding 99uL n-octanoic acid dropwise, stirring at 0-10 deg.C for 10-60min, and clarifying for 10-60 min; centrifuging at 14000 Xg for 20min at 4 deg.C, removing precipitate, adding 1/10 volume of 10 XPBS into supernatant, and adjusting pH to 7.2 with 1M NaOH; dropwise adding equivalent saturated ammonium sulfate solution into the supernatant, continuously stirring for 30min, and standing for 30 min; centrifuging at 14000 Xg for 30min at 4 deg.C, discarding the supernatant, dissolving the precipitate in 1.2mL PBS, dialyzing with 0.01M, pH7.4 PBS at 0-10 deg.C overnight, and concentrating with 5% PEG20000 to 5 mL;
(6) and (3) passing the concentrated solution through protein G purification resin, washing 10-15 column volumes by using pre-elution solution, eluting the monoclonal antibody by using 5-10 column volumes of elution buffer solution, and concentrating to obtain the monoclonal antibody.
6. The method of claim 5, wherein the step of preparing the composition is carried out in a batch processThe pre-elution solution in the step (6) of (1) comprises 0.1 to 0.15M NaCl and 10 to 40mM Na2HPO4pH 7.0; the elution buffer solution is 0.1-0.3M glycine, and the pH value is 3.0.
7. The use of the tomato ringspot virus monoclonal antibody of claim 1 in the preparation of a tomato ringspot virus detection reagent or kit.
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