CN112898422B - Tobacco ringspot virus monoclonal antibody and preparation method and application thereof - Google Patents

Tobacco ringspot virus monoclonal antibody and preparation method and application thereof Download PDF

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CN112898422B
CN112898422B CN202110390538.8A CN202110390538A CN112898422B CN 112898422 B CN112898422 B CN 112898422B CN 202110390538 A CN202110390538 A CN 202110390538A CN 112898422 B CN112898422 B CN 112898422B
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monoclonal antibody
ringspot virus
tobacco ringspot
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supernatant
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张蓬军
傅慧兰
刘光富
郝培应
俞晓平
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China Jiliang University
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Abstract

The invention discloses a tobacco ringspot virus monoclonal antibody, a preparation method and application thereof, and belongs to the technical field of immune engineering. The invention discloses a tobacco ringspot virus monoclonal antibody and a preparation method and application thereof.A BALB/c mouse is immunized by a recombined and expressed full-length tobacco ringspot virus coat protein TRSV (523aa) to obtain a hybridoma cell strain, a cell strain capable of generating a monoclonal antibody for specifically recognizing the antigen TRSV is screened out from the hybridoma cell strain, and the monoclonal antibody for specifically recognizing the tobacco ringspot virus coat protein TRSV is obtained by an ascites preparation method; the yield of the monoclonal antibody is 550-4500mg/L ascites, and the purity is 87.4-99.6%. The monoclonal antibody has the characteristic of specifically identifying the tobacco ringspot virus coat protein, and has wide application prospect in the aspects of import and export inspection and quarantine of the tobacco ringspot virus.

Description

Tobacco ringspot virus monoclonal antibody and preparation method and application thereof
Technical Field
The invention relates to the technical field of immune engineering, in particular to a tobacco ringspot virus monoclonal antibody and a preparation method and application thereof.
Background
Tobacco Ring Spot Virus (TRSV) is a member of the genus Nepovirus (Comoviridae) of the comovirus family, and the virus particles are isodiametric icosahedron with a diameter of about 28 nm. The genome contains two positive-sense single-stranded RNAs which respectively code for replicase, Motor Protein (MP) and Coat Protein (CP). TRSV is widely distributed in europe, the united states and the state of the oceanic province, and can infect more than 150 crops such as tobacco, grapes, soybeans, apples, cherries, tomatoes and the like to cause various diseases, and finally results in failure in severe cases. There are many kinds of transmission modes of TRSV, the vector of the field is the transmission and popular important mode of TRSV by the vector of the xiphonema (Xiphinema) and the propagation of grafting, and the long distance transmission mainly depends on the regulation and transportation of seeds (seedlings). The seed-borne poison plants are many, and some have high seed-borne poison rates, such as 76% of soybeans, 76% of globe amaranth, 11% of elderberry, 24% of dandelion, 3-7% of red clover and 3% of tomatoes. The virus is a high-risk virus and is a second class of quarantine pests prohibited from entry in China. Because TRSV is a high-risk epidemic-detection pest which can be remotely transmitted along with the distribution and transportation of seed nursery stocks, establishing an effective rapid detection method is very important for preventing the virus from entering China.
At present, the means used for the TRSV quarantine at home and abroad comprise electron microscope observation, differential host reaction (biological method), serological test and molecular biological means. Because the TRSV is a spherical virus with the diameter of 28nm, the virus content in a sample is low, and the TRSV is difficult to observe and identify 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 purchasing foreign antiserum at present, the price is high, and a TRSV serum detection kit for the imported plant seedlings is not reported at home.
Therefore, it is an urgent need to solve the problems of the art to provide a tobacco ringspot virus monoclonal antibody and a preparation method and application thereof.
Disclosure of Invention
In view of the above, the invention provides a tobacco ringspot virus monoclonal antibody and a preparation method and application thereof, the tobacco ringspot virus monoclonal antibody has reaction specificity for recognizing and combining with tobacco ringspot virus coat protein, and lacks the combination specificity for other viruses and plant tissues, thereby laying a foundation for further developing and developing a tobacco ringspot virus serum detection kit.
In order to achieve the purpose, the invention adopts the following technical scheme:
a monoclonal antibody of tobacco ringspot virus, wherein the heavy chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO.1, and the light chain variable region sequence is shown as SEQ ID NO. 2.
The variable regions of the heavy chain and the light chain of the monoclonal antibody have 185 and 121 amino acids in length respectively. The 185 amino acid residue sequence of the heavy chain variable region has a first matched sequence similarity of 88.33% as determined by BLASTP analysis; the light chain variable region has a first match sequence similarity of 85.86% by BLASTP analysis of the 121 amino acid residue sequence.
Further, a gene encoding a tobacco ringspot virus monoclonal antibody, comprising a nucleotide sequence encoding the heavy chain variable region and a nucleotide sequence encoding the light chain variable region; the nucleotide sequence for coding the heavy chain variable region is shown as SEQ ID NO.3, and the nucleotide sequence for coding the light chain variable region is shown as SEQ ID NO. 4.
Further, a hybridoma cell strain 12-1C4-D10 secreting monoclonal antibodies of tobacco ringspot virus, the preservation number is CGMCC No.20795, the hybridoma cell strain is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms (CGMCC for short), the institute of microbiology, China academy of sciences, No.3, West Lu 1 institute, North Cheng, the south China, the area of Tokyo, the Beijing, the preservation date is 2020, 09 months and 27 days, and the hybridoma cell strain is classified and named as mouse hybridoma cells.
Further, the tobacco ringspot virus monoclonal antibody is prepared by the following steps:
(1) the tobacco ringspot virus coat protein RNA sequence is called from Genbank (sequence accession number: KP144328.1, the length of the sequence is 1545bp, the recombinant gene sequence obtained by sequence optimization and separation label fusion is shown in SEQ ID NO.5, the corresponding protein sequence is shown in SEQ ID NO. 6. the SEQ ID NO.5 is inserted into pET28b vector and expressed in Escherichia coli BL21(DE3) strain, the induction expression condition is LB liquid culture medium, IPTG 1mM, 16 ℃ for 16h, 200 rpm. the fusion protein TRSV separation and purification method is nickel ion column affinity chromatography.
(2) Immunizing a mouse with the fusion protein TRSV;
(3) preparing hybridoma cells and screening cell strains which produce antibodies which specifically recognize the protein of SEQ ID NO.6 from the hybridoma cells;
(4) 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;
(5) preparing a monoclonal antibody capable of identifying the protein SEQ ID NO.6 and making a positive reaction on a tobacco ringspot virus-carrying sample by adopting an ascites induction method;
(6) the monoclonal antibody is purified by ammonium sulfate precipitation and protein G affinity layer washing.
The method for preparing the tobacco ringspot virus monoclonal antibody by the ascites induction method 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) intraperitoneal inoculation of 5-10 days later, hybridoma cells of claim 3 suspended in PBS, 5 x 10 5 -5×10 6 A/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 99 μ L of 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.
Further, the pre-elution solution in the step (6) comprises 0.1-0.15M NaCl and 10-40mM Na 2 HPO 4 pH 7.0; the elution buffer is 0.1-0.3M glycine, pH 3.0.
The monoclonal antibody is prepared by an ascites induction method, the yield is 550-4500mg/L ascites, and the purity is 87.4-99.6%.
Further, the application of the tobacco ringspot virus monoclonal antibody in preparing a tobacco ringspot virus detection reagent or a kit.
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 fusion protein shown by SEQ ID NO.6 derived from the tobacco ringspot virus coat protein.
According to the technical scheme, compared with the prior art, the tobacco ringspot virus monoclonal antibody and the preparation method and the application thereof are disclosed and provided, the result of the tobacco ringspot virus monoclonal antibody subjected to titer determination and enzyme-linked immunoassay shows that the monoclonal antibody for identifying the fusion protein shown in SEQ ID No.6 has the specificity of reaction with the tobacco ringspot virus, can be used as an antibody for detecting the tobacco ringspot virus, can lay a good foundation for the subsequent development of a tobacco ringspot virus serum detection kit, and serves the working requirements of entry-exit inspection and quarantine.
Aiming at the current situation that a large number of seedlings are imported in China and possibly carry tobacco ringspot virus, the invention screens and obtains the monoclonal antibody cell strain by taking the tobacco ringspot virus coat protein as a detection target through the series steps of whole gene fusion expression, separation and purification of the tobacco ringspot virus coat protein, cross-linked immune mice, cell fusion, antibody activity detection and the like. 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, 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 are obtained. Compared with the method for obtaining the monoclonal antibody by immunizing a mouse with the purified virus particles, the method does not need an active virus sample, directly obtains the virus coat protein by a whole gene synthesis and fusion expression method, and obtains the antigen without being limited by a source of a virus-carrying sample and violating the operation permission range of imported inspection and quarantine substances. The monoclonal antibody has definite variable region sequence, does not repeat with the existing antibody sequence, and has definite and easily-recognized characteristics with the existing antibodies on the market and related research reports.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1
(1) The tobacco ringspot virus coat protein RNA sequence is adjusted from Genbank (the sequence accession number is: the number of the KPs 144328.1 is KP144328.1, the length of the sequence is 1545bp, the recombination gene sequence obtained by sequence optimization and separation label fusion is shown in SEQ ID NO.5, the corresponding protein sequence is shown in SEQ ID NO.6, the SEQ ID NO.5 is inserted into NcoI-Not I site in pET28b vector, the insertion method is a recombinant Cloning method, the method is realized by adopting Clonexpress Ultra One Step Cloning Kit of Novonauzin, the ligation product adopts a heat shock method to transform Escherichia coli TOP10 strain, regenerated transformants obtain correct clones through sequencing verification, correct Cloning plasmids are extracted and sequenced, Escherichia coli BL21(DE3) strain is transformed, the correct transformants are sequenced, identified and induced expression of fusion protein TRSV is carried out, the induced expression condition is IPTG 1mM, 16 ℃ for 16h, 200rpm, and the separation and purification method of the fusion protein TRSV is nickel ion column affinity chromatography.
(2) Immunization of mice
a) For the initial immunization, 1.5mL of fusion protein TRSV (total amount is 150 μ g) is uniformly mixed with equal volume of Freund's complete adjuvant, and 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 boosted, the dosage is 50 mu g/mouse, and the injection is carried out in an abdominal cavity.
After 3 days, the mice with the highest titer were selected for splenic fusion.
(3) Hybridoma cell preparation
a) Will be 1 × 10 8 Spleen cells and 1X 10 7 Myeloma cells SP2/0 were mixed in a 50mL fusion tube, supplemented with incomplete medium (GIBCO MEM medium) to 30mL, and mixed well.
b) Centrifuging at 1000r/min for 10min, and sucking the supernatant as clean as possible.
c) The bottom of the fusion tube is flicked on the palm to loosen and homogenize the precipitated cells.
d) 1mL of preheated 50% PEG was added to the mixture over 30 seconds using a 1mL pipette with gentle stirring.
e) The suction tube was left standing for 1 min.
f) The PEG reaction was terminated by adding preheated incomplete 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 medium (GIBCO DEME high sugar medium), gently sucking the precipitated cells, suspending and mixing, and then supplementing the complete medium to 40-50 mL. Subpackaging 96-well cell culture plates at 100. mu.L per well, and placing the plates at 37 ℃ with 5% CO 2 Culturing in an incubator.
i) After 6h, selection medium (GIBCO HAT Supplement) was added. mu.L per well, half-changed with selection medium after 3 days.
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.
(4) 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 ml 5 The ratio of cells, abdominal cavity macrophage is added into the hybridoma cell suspension respectively.
d) Each hybridoma cell was dispensed into a 96-well plate in an amount of 100. mu.l per well.
e)37℃、5％CO 2 Culturing 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.
And (4) taking the cells of the antibody detection positive hole, carrying out expanded culture and freezing and storing.
(5) Preparation of monoclonal antibody by ascites induction method
a) Injecting 0.1mL of sterile paraffin into the abdominal cavity of a BALB/c mouse for 8 weeks;
b)5 days later, hybridoma cells suspended in PBS were inoculated intraperitoneally at 5X 10 5 A/only;
c) ascites was collected 2 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; dropwise adding 99 μ L of n-octanoic acid, stirring at 0 deg.C for 10min, and clarifying for 10 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) 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;
the concentrate was passed through a protein G purification resin to pre-elute (0.1M NaCl,10mM Na) 2 HPO 4 pH7.0), eluting the monoclonal antibody with 5 column volumes of elution buffer (0.1M glycine, pH3.0), concentrating to obtain the monoclonal antibody of the invention, wherein the yield is 1250mg/L ascites, and the purity is 87.4%. The prepared antibody is used for detecting the tobacco leaves carrying the inactivated tobacco 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 tobacco ringspot virus, and negative to tobacco mosaic virus, T7 phage, arabis mosaic virus, bean pod mottle virus and the like.
Example 2
Steps (1) to (4) of example 2 are the same as example 1.
(5) 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 10 6 A/only;
c) ascites was collected 4 times after 10 days;
d) centrifuging ascites at 1000 Xg for 10min, sucking the uppermost layer of adipose tissue, removing cell components and other precipitates, and collecting the 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 99 μ L of n-octanoic acid dropwise, stirring at 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), dialyzing with 0.01M, pH7.4 PBS at 10 deg.C overnight, and concentrating with 5% PEG20000 to 5 mL;
the concentrate was passed through a protein G purification resin to pre-elute (0.15M NaCl,40mM Na) 2 HPO 4 pH7.0), eluting the monoclonal antibody with 10 column volumes of elution buffer (0.3M glycine, pH3.0), and concentrating to obtain the monoclonal antibody of the invention, wherein the yield is 550mg/L ascites, and the purity is 89.8%. The prepared antibody is used for detecting the tobacco leaves carrying the inactivated tobacco ringspot virus by an indirect ELISA method, and a detection signal is positive; the antibody is negative to tobacco samples which are not infected by tobacco ringspot virus, and negative to tobacco mosaic virus, T7 bacteriophage, arabis mosaic virus, bean pod mottle virus and the like.
Example 3
Steps (1) to (4) of example 3 are the same as example 1.
(5) 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 10 6 A/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 99 μ L of n-octanoic acid dropwise, stirring at 5 deg.C for 35min, 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;
the concentrate was passed through a protein G purification resin to pre-elute (0.125M NaCl,25mM Na) 2 HPO 4 pH7.0), eluting the monoclonal antibody with 7 column volumes of elution buffer (0.2M glycine, pH3.0), concentrating to obtain the final productThe yield of the monoclonal antibody is 3137mg/L ascites, and the purity is 92.1%. The prepared antibody is used for detecting the tobacco leaves carrying the inactivated tobacco 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 tobacco ringspot virus, and negative to tobacco mosaic virus, T7 phage, arabis mosaic virus, bean pod mottle virus and the like.
Example 4
Steps (1) to (4) of example 4 are the same as example 1.
(5) 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 days 6 A/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 99 μ L of 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;
the concentrate was passed through a protein G purification resin to pre-elute (0.12M NaCl,30mM Na) 2 HPO 4 pH7.0), eluting the monoclonal antibody by using elution buffer solution (0.15M glycine, pH3.0) with 7 column volumes, and concentrating to obtain the monoclonal antibody of the invention, wherein the yield is 4500mg/L ascites, and the purity is 99.6%. The prepared antibody is used for detecting the tobacco leaves carrying the inactivated tobacco ringspot virus by an indirect ELISA method, and a detection signal is positive; the antibody reacts to tobacco samples not infected by tobacco ringspot virusNegative, negative to tobacco mosaic virus, T7 phage, arabis mosaic virus, bean pod mottle virus, etc.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
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Leu Ala Met Ser Val Gly Gln Arg Asn Val Thr Met Ser Cys Lys Ser
20 25 30
Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Ser Tyr Leu Ala
35 40 45
Trp Cys Gln Gln Arg Pro Gly Gln Ser Pro Lys Leu Leu Glu Ser Gly
50 55 60
Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
65 70 75 80
Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln
85 90 95
Leu His Tyr Ser Thr Pro His Val Arg Cys Trp Glu Gln Ala Gly Asp
100 105 110
Pro Thr Lys Leu Val Arg Ala Thr Arg
115 120
<210> 3
<211> 555
<212> DNA
<213> Artificial Sequence
<400> 3
atggtacaac tgcaacaatc tggtaccgtt ctgctggcac gtcctggtgc atccgtgaaa 60
atgagctgca aagcaagcgg ctactccttc ggtactagct actggatgca ctgggttaag 120
cagcgtccgg gtggccaggg tctggaatgg atcggtgcaa tctatccggg caaaaatagc 180
gacacgaact acaatcagaa attcaaaggc aaggctaaac tgaccgcggt aacctccgcc 240
tctactgcat acatggagct gtccagcctg accaatgaag attccgcggt ttactattgc 300
acccgcctgt ctatgctggg tcgtagctac tattttgact attggggcca aggtaccacc 360
ctgaccgttt ctagcgctaa agctacgcca ccgtccgtgt atccgctggc tccaggttgt 420
ggtgatacca cgggttctag cgttaccctg ggttgcctgg ttaaaggcta cttcccagag 480
agcgttacgg tcacctggaa ttccggtagc ctgtccagct ccgtgcatac cttcccagat 540
agctgctcct tcacg 555
<210> 4
<211> 363
<212> DNA
<213> Artificial Sequence
<400> 4
atggcgtgtc gcccttcgat tgtgctgtat actcaatctc catcctccct ggctatgtca 60
gtaggacaga ggtatgtcac tatgagctgc aagtccagtc agagcctttt aaatagtagc 120
aatcaaaaga actattgctt ggcctggtgc cagcagagac caggacagtc tcctaaactt 180
ctggaatctg gggtccctga tcgcttcata ggcagtggat ctgggacaga tttcactctt 240
accatcagca gtgtgcaggc tgaagacctg gcagattact tctgtcagct acattatagc 300
actcctcacg ttcggtgctg ggaacaagct ggagatccaa cgaagcttgt aagggcgaca 360
cgc 363
<210> 5
<211> 1572
<212> DNA
<213> Tobacco ringspot virus
<400> 5
atggccgctg tgacggttgt tcccgatccc acttgttgtg ggacattgtc ctttaaggtt 60
cccaaagatg cgaagaaagg aaagcatctt ggaacttttg acattcggca agccattatg 120
gaatatggtg gtttgcattc ccaagaatgg tgtgcaaagg gcattgttaa tcccactttt 180
acagtgagga tgcatgcccc acgcaacgcc tttgcaggtt tgtctatagc gtgcaccttt 240
gatgattaca aacgcataga cttaccagcg cttgggaatg aatgtcctcc ctccgagatg 300
tttgaactgc ctaccaaggt tttcatgctt aaagatgcag atgtgcatga atggcagttc 360
aactatgggg aacttacagg acatgggttg tgcaattggg caaatgtagt tacccagccc 420
acattgtact tttttgttgc gtccacaaat caagtgacga tggctgctga ttggcagtgt 480
attgttacta tgcatgtgga catggggccc gtcattgatc gttttgagtt agatccaact 540
atgacgtggc ctattcaatt gggtgacact ttcgccattg atagatatta tgaggcgaaa 600
gaaattaaac ttgacgggtc aacctccatg ttgtctatat cttataattt tggaggtccc 660
gtcaagcatt ctaagaaaca tgccatttca tattcccggg cagttatgtc taggaatctt 720
gggtggtctg gcactataag cggaagtgtc aagagtgttt cttctttatt ttgtaccgct 780
tcttttgtta tttttccatg ggaacacgaa gcacctccaa ccttacgtca ggtgttatgg 840
ggcccacatc agataatgca cggagatggc caatttgaaa ttgctatcaa aactcgtctt 900
cattcagctg ctacaactga agaagggttt ggtagacttg gtatactccc gctttctggg 960
cctatagctc ctgatgcaca tgttggatcg tacgagttta ttgtacatat agacacttgg 1020
cgacccgact ctcaggtgca tcctcccatg ttttctagtg cggagcttta taattggttc 1080
actttaacca atttgaaacc agatgcgaac actggcgtag tcaactttga tattcccgga 1140
tacatccatg acttcgcctc taaggacgca actgtgacgc tcgcatcaaa tcccctctct 1200
tggcttgtcg cagctactgg ctggcattat ggtgaggtgg atctctgcat ctcctggtca 1260
aggtccaaac aggcccaggc tcaggagggt agtgtttcca ttaccactaa ttatagagat 1320
tggggtgctt actggcaagg ccaggcccgg atttatgatt tgcggcgtac tgaagcggaa 1380
attcccatct tcttgggttc ttacgctggt gcgacgccat ctggtgcctt gggtaagcaa 1440
aactatgtcc ggatttcaat tgtcaatgct aaggacatag ttgcactgcg agtgtgtttg 1500
cgacccaaat ctataaagtt ctggggtcgc tccgccactt tgtttctcga gcaccaccac 1560
caccaccact ga 1572
<210> 6
<211> 523
<212> PRT
<213> Tobacco ringspot virus
<400> 6
Met Ala Ala Val Thr Val Val Pro Asp Pro Thr Cys Cys Gly Thr Leu
1 5 10 15
Ser Phe Lys Val Pro Lys Asp Ala Lys Lys Gly Lys His Leu Gly Thr
20 25 30
Phe Asp Ile Arg Gln Ala Ile Met Glu Tyr Gly Gly Leu His Ser Gln
35 40 45
Glu Trp Cys Ala Lys Gly Ile Val Asn Pro Thr Phe Thr Val Arg Met
50 55 60
His Ala Pro Arg Asn Ala Phe Ala Gly Leu Ser Ile Ala Cys Thr Phe
65 70 75 80
Asp Asp Tyr Lys Arg Ile Asp Leu Pro Ala Leu Gly Asn Glu Cys Pro
85 90 95
Pro Ser Glu Met Phe Glu Leu Pro Thr Lys Val Phe Met Leu Lys Asp
100 105 110
Ala Asp Val His Glu Trp Gln Phe Asn Tyr Gly Glu Leu Thr Gly His
115 120 125
Gly Leu Cys Asn Trp Ala Asn Val Val Thr Gln Pro Thr Leu Tyr Phe
130 135 140
Phe Val Ala Ser Thr Asn Gln Val Thr Met Ala Ala Asp Trp Gln Cys
145 150 155 160
Ile Val Thr Met His Val Asp Met Gly Pro Val Ile Asp Arg Phe Glu
165 170 175
Leu Asp Pro Thr Met Thr Trp Pro Ile Gln Leu Gly Asp Thr Phe Ala
180 185 190
Ile Asp Arg Tyr Tyr Glu Ala Lys Glu Ile Lys Leu Asp Gly Ser Thr
195 200 205
Ser Met Leu Ser Ile Ser Tyr Asn Phe Gly Gly Pro Val Lys His Ser
210 215 220
Lys Lys His Ala Ile Ser Tyr Ser Arg Ala Val Met Ser Arg Asn Leu
225 230 235 240
Gly Trp Ser Gly Thr Ile Ser Gly Ser Val Lys Ser Val Ser Ser Leu
245 250 255
Phe Cys Thr Ala Ser Phe Val Ile Phe Pro Trp Glu His Glu Ala Pro
260 265 270
Pro Thr Leu Arg Gln Val Leu Trp Gly Pro His Gln Ile Met His Gly
275 280 285
Asp Gly Gln Phe Glu Ile Ala Ile Lys Thr Arg Leu His Ser Ala Ala
290 295 300
Thr Thr Glu Glu Gly Phe Gly Arg Leu Gly Ile Leu Pro Leu Ser Gly
305 310 315 320
Pro Ile Ala Pro Asp Ala His Val Gly Ser Tyr Glu Phe Ile Val His
325 330 335
Ile Asp Thr Trp Arg Pro Asp Ser Gln Val His Pro Pro Met Phe Ser
340 345 350
Ser Ala Glu Leu Tyr Asn Trp Phe Thr Leu Thr Asn Leu Lys Pro Asp
355 360 365
Ala Asn Thr Gly Val Val Asn Phe Asp Ile Pro Gly Tyr Ile His Asp
370 375 380
Phe Ala Ser Lys Asp Ala Thr Val Thr Leu Ala Ser Asn Pro Leu Ser
385 390 395 400
Trp Leu Val Ala Ala Thr Gly Trp His Tyr Gly Glu Val Asp Leu Cys
405 410 415
Ile Ser Trp Ser Arg Ser Lys Gln Ala Gln Ala Gln Glu Gly Ser Val
420 425 430
Ser Ile Thr Thr Asn Tyr Arg Asp Trp Gly Ala Tyr Trp Gln Gly Gln
435 440 445
Ala Arg Ile Tyr Asp Leu Arg Arg Thr Glu Ala Glu Ile Pro Ile Phe
450 455 460
Leu Gly Ser Tyr Ala Gly Ala Thr Pro Ser Gly Ala Leu Gly Lys Gln
465 470 475 480
Asn Tyr Val Arg Ile Ser Ile Val Asn Ala Lys Asp Ile Val Ala Leu
485 490 495
Arg Val Cys Leu Arg Pro Lys Ser Ile Lys Phe Trp Gly Arg Ser Ala
500 505 510
Thr Leu Phe Leu Glu His His His His His His
515 520

Claims (6)

1. The monoclonal antibody for the tobacco ringspot virus is characterized in that the heavy chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO.1, and the light chain variable region sequence is shown as SEQ ID NO. 2.
2. A gene encoding the monoclonal antibody against tobacco ringspot virus according to claim 1, comprising a nucleotide sequence encoding the heavy chain variable region and a nucleotide sequence encoding the light chain variable region; the nucleotide sequence for coding the heavy chain variable region is shown as SEQ ID NO.3, and the nucleotide sequence for coding the light chain variable region is shown as SEQ ID NO. 4.
3. A hybridoma cell line 12-1C4-D10 secreting the tobacco ringspot virus monoclonal antibody of claim 1, characterized by the accession number of CGMCC No. 20795.
4. The method for preparing the tobacco ringspot virus monoclonal antibody of claim 1, which is characterized by comprising 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) intraperitoneal inoculation of 5-10 days later, hybridoma cells of claim 3 suspended in PBS, 5 x 10 5 -5×10 6 A/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 99 μ L of 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.
5. The method for preparing monoclonal antibody against tobacco ringspot virus according to claim 4, wherein the pre-elution solution of step (6) comprises 0.1-0.15M NaCl and 10-40mM Na 2 HPO 4 pH 7.0; the elution buffer is 0.1-0.3M glycine with a pH of 3.0.
6. The use of the monoclonal antibody against tobacco ringspot virus according to claim 1 in the preparation of a test agent or a kit for detecting tobacco ringspot virus.
CN202110390538.8A 2021-04-12 2021-04-12 Tobacco ringspot virus monoclonal antibody and preparation method and application thereof Expired - Fee Related CN112898422B (en)

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