CN110106152B

CN110106152B - Hybridoma cell strain YTT-2 and anti-cyclopiazonic acid monoclonal antibody generated by same

Info

Publication number: CN110106152B
Application number: CN201910361592.2A
Authority: CN
Inventors: 张奇; 李培武; 唐晓倩; 李慧; 张文
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2019-04-30
Filing date: 2019-04-30
Publication date: 2021-08-17
Anticipated expiration: 2039-04-30
Also published as: CN110106152A

Abstract

The invention relates to a hybridoma cell strain YTT-2 and an anti-cyclopiazonic acid monoclonal antibody generated by the same. The hybridoma cell strain YTT-2 provided by the invention has a preservation number of CCTCC NO: C201871, can be used for preparing a high-titer anti-cyclopiazonic acid monoclonal antibody, and the titer can reach 1.2 multiplied by 10 when the anti-cyclopiazonic acid ascites antibody is measured by an indirect non-competitive enzyme-linked immunosorbent assay (ELISA)⁵. The anti-cyclopiazonic acid monoclonal antibody provided by the invention has high sensitivity and good specificity, and 50% inhibition concentration IC of cyclopiazonic acid₅₀The concentration is 0.84ng/mL, the cross reaction rate to aflatoxin and variegated aflatoxin is less than 0.1%, and the method can be applied to residue detection of cyclopiazonic acid.

Description

Hybridoma cell strain YTT-2 and anti-cyclopiazonic acid monoclonal antibody generated by same

Technical Field

The invention relates to a hybridoma cell strain YTT-2 and an anti-cyclopiazonic acid monoclonal antibody generated by the same.

Background

Cyclopianidic acid (CPA) is a secondary metabolite produced by penicillium and aspergillus fungi, has good stability, general storage conditions and processing procedures cannot destroy the cyclopiazonic acid, and the toxicity of the cyclopiazonic acid is mainly shown as gastrointestinal ulcer, spleen atrophy, kidney disease, liver and myocardial necrosis. At the same time, cyclopiazonic acid is a specific inhibitor of calcium-dependent ATPases in the sarcoplasmic reticulum, resulting in Ca²⁺The concentration is increased, so that the muscle contraction is increased, the cell membrane permeability is increased, the neuron skeleton is damaged, the nerve filaments are decomposed, and the nerve is micro-depolymerized, so that the neuron necrosis is caused. Cyclopianianic acid can contaminate peanut, corn, cottonseed, feed and other agricultural products, and can be transferred into milk and eggs through feed. The cyclopiazonic acid can be produced simultaneously with the aflatoxin and is generally suitable for producing the aflatoxinThe conditions are also suitable for the generation of the cyclopiazonic acid, so that the agricultural product and food safety problems caused by the cyclopiazonic acid can be covered by the aflatoxin which is simultaneously generated, and the researches at present consider that the turkey X disease and Kodua poisoning are caused by the cyclopiazonic acid and the aflatoxin. Some agricultural products without aflatoxin detection may contain cyclopiazonic acid with high concentration, which causes health hazards to people and livestock. Therefore, there is a need to establish a fast and reliable immunological detection technique for CPA in agricultural products. Therefore, the method for analyzing the cyclopiazonic acid in agricultural products and food has important significance.

The existing analysis and detection method of cyclopiazonic acid mainly comprises thin-layer chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry and the like. In the thin-layer chromatography, organic solvents and toxins can generate toxic action on operators in the pretreatment operation process, and the sensitivity and accuracy of the measurement result are not high. Although high-performance liquid chromatography, liquid chromatography-mass spectrometry and other instrumental analysis methods are commonly used qualitative and quantitative analysis methods, the methods have high sensitivity and high stability, but require a large amount of time for complex pretreatment, and require strict laboratory environment, expensive instruments and equipment and professional operators, so that the field pollution detection cannot be satisfied. The immunoassay is based on the specific binding reaction of an antigen and an antibody, and is used for qualitatively and quantitatively detecting a substance to be detected by combining markers such as enzyme, radioactive elements, colloidal gold and the like with the antigen or the antibody through an immunolabeling technology, wherein the antibody is a core reagent, the quality of the antibody determines the sensitivity and specificity of the immunoassay to a great extent, and only the high-quality antibody can meet the immunoassay. At present, CPA detection methods at home and abroad are mainly instrument detection, and a needle ring pimaric acid immunological detection technology is reported. Therefore, the research of the anti-cyclopiazonic acid monoclonal antibody can open up a new idea for the rapid detection of cyclopiazonic acid, and has important significance for realizing the quantitative immunoassay of cyclopiazonic acid.

Disclosure of Invention

The invention aims to solve the problem of providing a hybridoma cell strain YTT-2 and an anti-cyclopiazonic acid monoclonal antibody generated by the same.

The invention provides a hybridoma cell strain YTT-2 which is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 3 and 23 months, wherein the preservation address is China, Wuhan and Wuhan university, and the preservation number is CCTCC NO. C201871. It has the encoding gene sequence of the heavy chain variable region of the anti-cyclopiazonic acid monoclonal antibody shown in SEQ ID NO. 1 in the sequence table and the encoding gene sequence of the light chain variable region of the anti-cyclopiazonic acid monoclonal antibody shown in SEQ ID NO. 2 in the sequence table.

The anti-cyclopiazonic acid monoclonal antibody is secreted and produced by hybridoma cell strain YTT-2 with the preservation number of CCTCC NO. C201871. The heavy chain variable region has an amino acid sequence shown as SEQ ID NO.3 in the sequence table; the light chain variable region has an amino acid sequence shown as SEQ ID NO.4 in the sequence table. The anti-cyclopiazonic acid monoclonal antibody can specifically recognize cyclopiazonic acid, and the 50% inhibition concentration IC50 of the cyclopiazonic acid is 0.84 ng/mL.

The application of the anti-cyclopiazonic acid monoclonal antibody in the measurement of cyclopiazonic acid content.

The hybridoma cell strain YTT-2 provided by the invention is obtained by adopting a two-step screening method, and comprises the following specific steps: a complete antigen CPA-KLH is synthesized by a Mannich reaction between cyclopiazonic acid (CPA) and hemocyanin (KLH), emulsified and immunized into female BALB/c mice for 4-5 times, then boosted with the same immune dose of non-emulsified CPA-KLH, and subjected to cell fusion after 3 days. Screening hybridoma cells by adopting a two-step gradient method: firstly, screening out positive holes which resist cyclopiazonic acid but not resist carrier protein KLH by using an indirect non-competitive ELISA method; and step two, detecting the culture solution of the positive wells screened in the step one by adopting an indirect competitive ELISA method, and selecting the positive wells with higher light absorption values and sensitivity by using CPA as a competitive source. Diluting cells by adopting a limiting dilution method, culturing subcloned cells by using a semi-solid culture medium, transferring each cell colony into a liquid culture medium after cloning until the cell colony grows to a size visible to naked eyes, culturing and screening by adopting the same two-step screening method. And repeating the subcloning for 2-3 times, and finally screening to obtain the hybridoma cell strain YTT-2.

The preparation method of the anti-cyclopiazonic acid monoclonal antibody provided by the invention comprises the following steps: and (3) injecting the obtained hybridoma cell strain YTT-2 into a BALB/c mouse body which is treated by Freund incomplete adjuvant in advance, collecting ascites of the mouse, and purifying to obtain the anti-cyclopiazonic acid monoclonal antibody.

According to the scheme, the purification treatment method is an octanoic acid-ammonium sulfate method, and the specific operations are as follows: filtering ascites of mice by using double-layer filter paper, centrifuging the filtered ascites at 4 ℃ and 12000r/min for more than 15min, sucking a supernatant, mixing the supernatant with 4 times of acetate buffer solution, slowly adding n-octanoic acid while stirring, wherein the volume of the n-octanoic acid required by each milliliter of ascites is 30-35 mu L, mixing at room temperature for 30-60 min, standing at 4 ℃ for more than 2h, centrifuging at 4 ℃ and 12000r/min for more than 30min, discarding precipitates, filtering the obtained supernatant by using the double-layer filter paper, adding 1/10 phosphate buffer solution with the molar concentration of 0.1mol/L and the pH of 7.4, adjusting the pH of the mixed solution to 7.4 by using 2mol/L sodium hydroxide solution, precooling at 4 ℃, slowly adding ammonium sulfate until the final concentration is 0.277g/mL, standing at 4 ℃ for more than 2h, centrifuging at 4 ℃ and 12000r/min for more than 30min, discarding supernatant, resuspending the obtained precipitate with 0.01mol/L phosphate buffer solution with original ascites volume of 1/10, placing into dialysis bag, dialyzing with pure water, freezing the fully dialyzed protein solution in refrigerator at-70 deg.C, freeze-drying with freeze vacuum drier, collecting freeze-dried powder to obtain purified anti-cyclopiazonic acid monoclonal antibody, and placing the antibody in refrigerator at-20 deg.C for use.

According to the scheme, the acetate buffer solution is as follows: 0.29g of sodium acetate and 0.141mL of acetic acid were added to 100mL of water with ultrapure water.

According to the scheme, the 0.1mol/L phosphate buffer solution is as follows: 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate were added to a volume of 100mL with ultrapure water.

The invention has the beneficial effects that:

(1) the hybridoma cell strain YTT-2 provided by the invention can be used for preparing a high-titer anti-cyclopiazonic acid monoclonal antibody, and the titer can reach 1.2 multiplied by 10 when the anti-cyclopiazonic acid ascites antibody is measured by an indirect non-competitive enzyme-linked immunosorbent assay (ELISA) method⁵。

(2) The anti-cyclopiazonic acid monoclonal antibody provided by the invention has high sensitivity and good specificity, and can inhibit 50% of concentration IC of cyclopiazonic acid₅₀0.84ng/mL, for aflatoxin B₁，B₂，G₁，G₂，M₁，P₁The cross reaction rate of the compound and the variegated aspergillus toxin is less than 0.1 percent.

(3) The anti-cyclopiazonic acid monoclonal antibody provided by the invention can be applied to the content determination of cyclopiazonic acid.

Detailed Description

Example 1: screening of hybridoma cell line YTT-2

1. Antigen synthesis and animal immunization

The method is characterized in that a commercially available cyclopiazonic acid standard is purchased for complete antigen synthesis, and the specific synthesis steps are as follows: dissolve 1mg CPA in 1mL 0.05M NaHCO₃50% aqueous methanol solution; adding 0.4mL 3M sodium acetate into 2mg hemocyanin (KLH), dropwise adding 0.2mL formaldehyde within 1min under stirring at room temperature, and continuously stirring for 10 min; CPA was slowly added dropwise to KLH with constant stirring at room temperature for more than 16 h. The final reaction product CPA-KLH was placed in a dialysis bag of appropriate size and dialyzed in PBS at 4 ℃ for three days. The original CPA-OVA is synthesized and detected by the same method.

6 female Balb/c mice at 6 weeks of age were purchased and immunized against the self-synthesized cyclopiazonic acid complete antigen CPA-KLH at a dose of 100. mu.g/mouse. The complete antigen CPA-KLH is mixed and emulsified with Freund's complete adjuvant for the first immunization, and the immunization is carried out on the back by subcutaneous multipoint injection. The priming was performed 3 weeks apart, followed by 2 weeks apart, and immunization was performed using Freund's incomplete adjuvant emulsion. And (3) after one week from the third immunization, tail vein blood collection is carried out, serum is separated, the titer of the antibody of the serum of the mouse is monitored by adopting an indirect ELISA method, the sensitivity of the serum of the mouse is measured by adopting an indirect competitive ELISA method, the mouse corresponding to the serum with higher titer and sensitivity is selected to carry out the last sprint immunization, and 100 mu g of immunogen is taken 3 days before fusion and dissolved in 200 mu L PBS for direct injection into the abdominal cavity. Freund's adjuvant was purchased from Sigma-Aldrich.

2. Cell fusion

After 3 days of the last sprint immunization, 50 percent (weight percentage) of polyethylene glycol (PEG) is adopted as a fusion agent, and cell fusion is carried out according to a conventional method, which comprises the following steps:

killing the immunized mouse by cervical dislocation, taking out spleen under aseptic condition, grinding and separating spleen cells, mixing with murine myeloma cell SP2/0 at a ratio of 5:1, washing the mixed cells with RPMI-1640 basic culture solution, fusing with 50% PEG for 1min, then adding RPMI-1640 basic culture solution, centrifuging, removing supernatant, re-suspending the fusion cells formed by mouse splenocytes and mouse myeloma cells SP2/0 with 72mLRPMI-1640 basic culture solution, dripping the re-suspended cells into 96-well cell culture plates, culturing at 2 drops/well in a carbon dioxide incubator at 37 ℃, the RPMI-1640 basic culture solution contains 20% (volume percentage) of fetal bovine serum, 2% (weight percentage) of growth factor and 1% (weight percentage) of hypoxanthine-aminopterin-thymidine, namely HAT. The SP2/0 was purchased from Shanghai Panko Biotech Co., Ltd; RPMI-1640 basal medium was purchased from Hyclone; 1% hypoxanthine-aminopterin-thymidine, HAT, was purchased from Sigma-Aldrich.

3. Screening and cloning of cell lines

And about 12 days after cell fusion, the cell colony grows to the size of 1/2 area of the bottom of the hole, and the culture solution turns yellow, so that the antibody detection can be carried out. Screening culture holes with hybridoma cells growing by adopting an ELISA method, wherein the screening is carried out in two steps, and in the first step, positive holes which resist cyclopiazonic acid but not resist carrier protein KLH are screened out by adopting an indirect non-competitive ELISA method; and a second step of detecting the positive holes screened in the first step by adopting an indirect competitive ELISA method, taking cyclopiazonic acid as a competitive antigen, and selecting holes with higher light absorption value and sensitivity (the higher light absorption value means that the final measured value of the holes with the competitive antigen of 0, namely the positive control holes, is higher, and the higher sensitivity means that the competitive antigen concentration when the inhibition rate is 50%, namely the IC₅₀Small value), cloning by limiting dilution method, detecting by the same two-step method about 10 days after cloning, repeating cloning for 2-3 times to obtain hybridoma cell strain YTT-2, and storing in Chinese dictionaryThe type culture collection center (CCTCC) has a collection address of China, Wuhan and Wuhan university with a collection number of CCTCC NO. C201871.

Example 2: determination of sequence of variable region of anti-cyclopiazonic acid monoclonal antibody hybridoma cell line YTT-2 antibody

(1) Extracting total RNA: total RNA extraction kit of Tiangen company is adopted and total RNA capable of generating hybridoma cell strain YTT-2 is extracted according to the instruction.

(2) Synthesis of cDNA: oligo (dT) using the total RNA obtained in step 1 as a template₁₅As primers, according to SuperScript^TM-2II reverse transcriptase instructions for reverse transcription to synthesize first strand cDNA; primer oligo (dT)₁₅Purchased from Invitrogen;

(3) cloning of variable region genes by PCR: designing a primer according to a conserved site of a mouse antibody gene sequence in GENBANK, and amplifying antibody heavy chain and light chain variable region genes by using CDNA as a template. The PCR procedure was: amplification is carried out for 30 cycles at 94 ℃ for 30s, 50s at 55 ℃ for 1min at 72 ℃ and finally for 10min at 72 ℃. After 1% (weight percentage) agarose gel electrophoresis separation of the PCR product, using the kit to purify and recover DNA fragment, connecting the DNA fragment to the vector pMD18-T, transforming Escherichia coli DH5 alpha competent cell, selecting positive clone, and sending to Suzhou Hongxi biotechnology Limited for sequencing. Wherein the sequences of the primers are respectively as follows: heavy chain variable region primers were 5 '-AGG TSM ARC TGC AGS AGT CWG G-3' (22mer) and 5'-TGA GGA GACGGT GAC CGT GGT CCC TTG GCC CC-3' (32mer) where S, M, R and W are degenerate bases, M ═ a/C, R ═ a/G, S ═ C/G, W ═ a/T, and light chain variable region primers were 5'-GAC ATT GAG CTC ACC CAG CTT GGT GCC-3' (24mer) and 5'-CCG TTT CAG CTC CAG CTT GGT CCC-3' (24 mer).

Results of the gene sequences obtained: the length of the gene sequence of the heavy chain variable region coding gene is 360bp, the sequence is shown as SEQ ID NO. 1, the heavy chain variable region coded by the gene sequence is deduced according to the obtained gene sequence and consists of 120 amino acids, and the sequence is shown as SEQ ID NO. 3. The light chain variable region coding gene sequence has the length of 322bp and is shown as SEQ ID NO. 2, the light chain variable region coded by the gene sequence is deduced according to the obtained gene sequence and consists of 107 amino acids, and the sequence is shown as SEQ ID NO. 4.

Example 3: preparation, purification, subtype and characteristic identification of anti-cyclopiazonic acid monoclonal antibody

The cyclopiazonic acid monoclonal antibody hybridoma cell strain YTT-2 obtained in the example 2 is injected into a BALB/c mouse body which is treated by Freund's incomplete adjuvant in an abdominal cavity, ascites of the mouse is collected, and the antibody is purified by adopting an octanoic acid-ammonium sulfate method, which comprises the following specific operations: filtering ascites of mice by using double-layer filter paper, centrifuging the filtered ascites at 4 ℃ and 12000r/min for more than 15min, sucking supernatant, mixing the supernatant with 4 times of acetate buffer solution, slowly adding n-octanoic acid while stirring, wherein the volume of the n-octanoic acid required by each milliliter of ascites is 30-35 mu L, mixing at room temperature for 30-60 min, standing at 4 ℃ for more than 2h, centrifuging at 4 ℃ and 12000r/min for more than 30min, discarding precipitates, filtering the obtained supernatant by using the double-layer filter paper, adding 1/10 phosphate buffer solution with the molar concentration of 0.1mol/L and the pH of 7.4, adjusting the pH of the mixed solution to 7.4 by using 2mol/L sodium hydroxide solution, precooling at 4 ℃, slowly adding ammonium sulfate until the final concentration is 0.277g/mL, standing at 4 ℃ for more than 2h, centrifuging at 4 ℃ and 12000r/min for more than 30min, discarding the supernatant, resuspending the obtained precipitate with 0.01mol/L phosphate buffer solution with original ascites volume of 1/10, placing into a dialysis bag, dialyzing with pure water, freezing the fully dialyzed protein solution in a refrigerator at-70 ℃, then freeze-drying with a freeze vacuum drier, collecting the freeze-dried powder to obtain a purified anti-cyclopiazonic acid monoclonal antibody, and placing the antibody in a refrigerator at-20 ℃ for later use;

the acetate buffer solution is 0.29g of sodium acetate, and 0.141mL of acetic acid is obtained by adding water to a constant volume of 100 mL; the 0.01mol/L phosphate buffer solution is prepared by adding water to a constant volume of 100mL, wherein the phosphate buffer solution is 0.9g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of monopotassium phosphate.

The subtype of the anti-cyclopiazonic acid monoclonal antibody secreted by the hybridoma cell strain YTT-2 is identified to be IgG2a type by using a commercial subtype identification kit.

The titer of the mouse ascites antibody of YTT-2 can reach 1.2 multiplied by 10 measured by the conventional indirect non-joint competitive enzyme-linked immunosorbent assay (ELISA)⁵I.e. byDilution of murine ascites antibody 1.2X 10⁵The results of the solution assay at time of doubling were positive. Sensitivity (IC) of cyclopiazonic acid identified by conventional indirect competitive ELISA method₅₀) The concentration is 0.84ng/mL, and the cross reaction rate to aflatoxin B1, B2, G1, G2, M1 and variegated aflatoxin is less than 0.1%.

Example 4: antibody applications

The application of the anti-cyclopiazonic acid monoclonal antibody secreted by the hybridoma cell strain YTT-2 in an addition recovery test of cyclopiazonic acid specifically comprises the following steps:

(1) coating an enzyme label plate with 2 mu g/mL artificial antigen CPA-OVA, wherein each well is 100 mu L, is kept overnight at 4 ℃, is washed by 0.01mol/L pH7.4 PBST and is dried;

(2) preparing 4% skimmed milk powder into sealing liquid, sealing, washing at 37 deg.C for 1 hr, and drying;

(3) preparing 0, 0.050805, 0.15242, 0.45725, 1.3717, 4.1152, 12.3457, 37.037, 111.11, 333.33 and 1000ng/mL cyclopiazonic acid standard solution (phosphate buffer solution containing 10% methanol), sequentially adding 50 muL of CPA standard solution to be detected into each well of the first row, taking other wells as detection wells, repeating the steps for each sample in three times, incubating 50 muL of anti-cyclopiazonic acid monoclonal antibody (diluted by 9000 times) in each well for 1h at 37 ℃, washing and drying;

(4) adding 1 to each hole: 100 mu L of 5000 goat anti-mouse IgG-HRP, incubating for 1h at 37 ℃, washing and drying;

(5) substrate color development reaction: adding 100 μ L of substrate color developing solution into each well (the preparation of the substrate color developing solution: 1mg/mL of tetramethylbenzidine 0.5mL, the preparation of substrate buffer solution 9.5mL, 1% H)₂O₂37 mu L for use in the preparation), reacting at 37 ℃ for 15min in the dark, wherein 50 mu L of the solution is 2mol/L H per well₂SO₄Stopping the reaction, and measuring the light absorption value at 450 nm;

(6) a standard curve was plotted, and the concentration at which cyclopiazonic acid inhibited the antigen-antibody binding reaction by 50% (IC) was calculated₅₀)。

(7) Addition recovery test: 5g of the powder sample are weighed into 50mL centrifuge tubes, and 20mL of methanol/2% NaHCO are added₃Shaking the aqueous solution for 1h, centrifuging and collecting the supernatant. Adding 50mL of n-hexane in portions, oscillating, mixing and extractingTaking, layering and collecting a lower aqueous phase. 5ml of 10% potassium chloride solution are added and adjusted to pH 2-3 with HCl. Adding 30mL of chloroform in portions, shaking and mixing for multiple times, and collecting the lower layer of chloroform. Chloroform was removed by rotary evaporation, then 2mL of methanol was added and dissolved thoroughly, and filtered through a 0.45 μm organic filter. Accurately adding 50ng/mL, 100ng/mL and 200ng/mL cyclopiazonic acid into the filtrate respectively, performing an addition recovery test by adopting an indirect competitive ELISA method, and determining that the addition recovery rate of the rice, corn and peanut samples is between 83.6 and 97.7 percent, and the repeatability and the accuracy are better.

Preparing a solution required by ELISA:

phosphate buffer (pH7.4PBS) Na₂HPO₄·12H₂O 8.7g，NaCl 24g，KCl 0.6g，KH₂PO₄0.6g, and the volume was adjusted to 3000mL with ultrapure water.

Coating buffer (PH9.6 carbonate buffer): 1.59g Na₂CO₃，2.93g NaHCO₃And the volume is adjusted to 1000mL by using ultrapure water.

0.5% tween 20 in PBS (PBST solution): na (Na)₂HPO₄.12H₂O 8.7g，NaCl 24g，KCl 0.6g，KH₂PO₄0.6g, Tween-201.5 mL, and the volume is adjusted to 3000mL by ultrapure water.

Sealing liquid: 4g of skim milk powder was dissolved in 100mL of PBST.

Substrate buffer: na (Na)₂HPO₄·12H₂O1.843 g and citric acid 0.933g, and the volume is adjusted to 100mL by ultrapure water.

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Claims

1. Hybridoma cell line YTT-2, characterized in that: it is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C201871.

2. An anti-cyclopiazonic acid monoclonal antibody, which is characterized in that: the hybridoma cell strain YTT-2 with the preservation number of CCTCC NO: C201871 as claimed in claim 1.

3. The method for producing an anti-cyclopiazonic acid monoclonal antibody according to claim 2, characterized in that: the hybridoma cell strain YTT-2 of claim 1, which is injected into BALB/C mice treated with Freund's incomplete adjuvant in advance, ascites of the mice are collected, and the anti-cyclopiazonic acid monoclonal antibody is obtained after purification treatment.

4. Use of the anti-cyclopiazonic monoclonal antibody of claim 2 in cyclopiazonic content determination.