Ractopamine monoclonal antibody or antigen-binding portion thereof, preparation method and application

Abstract

The present application provides a ractopamine monoclonal antibody or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three complementarity determining regions with amino acid sequences of 26-32, 50-56 and 95-104 of SEQ ID No. 1 respectively; the light chain variable region comprising three complementarity determining regions with amino acid sequences of 27-35, 53-55 and 92-97 of SEQ ID No. 2 respectively; and a preparation method and application thereof are provided. The ractopamine monoclonal antibody or antigen-binding portion thereof, preparation method and application provided by the present application are obtained by expressing the monoclonal antibody through mammalian cells, and the ractopamine monoclonal antibody has single product composition, good antibody specificity, and an IC50 of 0.21 ppb, and is suitable for immunological detection of ractopamine residue and meets the actual application needs.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to ractopamine monoclonal antibody or its antigen-binding portion, preparation method and application. Background Technology

[0002] Ractopamine (RAC) is a synthetic β-adrenergic receptor agonist that redistributes nutrients in the body, reducing fat accumulation, increasing protein content, improving lean meat percentage, enhancing meat quality, and promoting growth in animals. Clinically, RAC is used to treat conditions such as muscular dystrophy and congestive heart failure. However, driven by profit, the misuse of ractopamine in animal husbandry and livestock farming is becoming increasingly serious. If humans consume food containing high levels of ractopamine residues, it can cause rapid heartbeat and breathing, arrhythmia, chest tightness, abdominal pain, numbness in the limbs, and in severe cases, can be life-threatening. Therefore, many countries have banned the use of ractopamine as feed and feed additives.

[0003] Currently, classic methods for RAC detection include high-performance liquid chromatography-mass spectrometry (HPLC-MS), gas chromatography-mass spectrometry (GC-MS), and high-performance liquid chromatography (HPLC). These methods can be used for qualitative and quantitative analysis. However, these methods have drawbacks such as high detection cost, long detection time, high requirements for test samples, and complex operation, which do not meet the requirements for rapid detection and hinder widespread adoption. Typically, laboratory testing of large numbers of samples uses immunoassay techniques, which utilize the specific reaction between antigen and antibody. To establish an immunoassay method for RAC, it is necessary to prepare mass-producible, highly specific ractopamine antibodies.

[0004] The commonly used method for producing ractopamine antibodies on the market is to prepare hybridoma cell lines. Mice are immunized with ractopamine antigen, and mouse spleen cells are fused with SP2 / 0 myeloma cells to obtain hybridoma cells. Hybridoma cells are obtained by screening with ELISA to obtain hybridoma cell lines that stably express monoclonal antibodies. Then, mice are injected to collect ascites fluid. The advantages of this method are that the technology is mature, the antibody concentration in the ascites fluid is high, and the titer is good. In 2019, Peng et al. (Peng D, Zhao L, Zhao L, et al. A Novel IndirectCompetitive Enzyme-Linked Immunosorbent Assay Format for the Simultaneous Determination of Ractopamine and Phenylethanolamine A Residues in Swine Urine[J]. Food Analytical Methods, 2019, 12(5): 1077-1085.) prepared a monoclonal antibody against RAC and constructed an ic-ELISA to detect RAC residues in animal samples. The IC50 reached 1.15±0.037μg / L. However, the method of generating antibodies by injecting mice with hybridoma cells has a major drawback: the antibodies are mixed with mouse proteins (including Ig), and even with purification, it is difficult to obtain high-purity single antibodies. These unknown components can interfere with the test results and increase the probability of false positives. Summary of the Invention

[0005] To address the aforementioned technical limitations, this invention proposes a ractopamine monoclonal antibody or its antigen-binding moiety, its preparation method, and its application; which overcomes the deficiencies and defects mentioned in the background art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The inventive point of this invention is to provide a ractopamine monoclonal antibody or its antigen-binding portion thereof, wherein the monoclonal antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity-determining regions of amino acid sequences at positions 26-32, 50-56, and 95-104 of SEQ ID No. 1; and the light chain variable region comprises three complementarity-determining regions of amino acid sequences at positions 27-35, 53-55, and 92-97 of SEQ ID No. 2.

[0007] Optionally, in the above-mentioned ractopamine monoclonal antibody or its antigen-binding portion, the amino acid sequence of the heavy chain variable region is SEQ ID No. 1; and the amino acid sequence of the light chain variable region is SEQ ID No. 2.

[0008] The amino acid sequence of the heavy chain variable region, SEQ ID No. 1, is: QVRLQQPGAELVKPGASVKLSCKASGYFTTYWIQWVRQRPGQGLEWI GEINNNGRTEFNEKFKRKATLTVDTSSTTAFMELSSLTSEDSAVYFCARYIPYA MEYWGQGTSVTVSS.

[0009] The amino acid sequence of the light chain variable region, SEQ ID No. 2, is: DVLMTQSPLSLPVSLGDQVSISCRSSQSHSSGNTYLEWYLQKPGQSPRL LIYKQSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSHVPPTCGG GTKLEIK.

[0010] Optionally, in the above-mentioned ractopamine monoclonal antibody or its antigen-binding portion, both the heavy chain variable region and the light chain variable region contain a frame region, which is derived from mice.

[0011] The second inventive point of this invention is to provide a nucleic acid molecule that encodes the aforementioned ractopamine monoclonal antibody or its antigen-binding portion.

[0012] Optionally, the above-mentioned nucleic acid molecules may encode the gene nucleotide sequence of the heavy chain variable region of the ractopamine monoclonal antibody or its antigen-binding portion as shown in SEQ ID No. 3; and the gene nucleotide sequence of the light chain variable region of the ractopamine monoclonal antibody or its antigen-binding portion as shown in SEQ ID No. 4.

[0013] The nucleotide sequence of the heavy chain variable region, SEQ ID No. 3, is: caggtccgactgcagcagcctggggctgaactggtgaagcctggggcttcagtgaagctgtcctgcaaggcttctggctacttcaccacctactggatacagtgggtgaggcagaggcctggacaaggccttgagtggattggagagattaataataacggtcgtactgagtt taatgagaagttcaagaggaaggccacactgttgacacatcctccactacagccttcatggaactcagcagtctgacatctgaggactctgcggtctatttctgtgcaagatatatcccgtatgctatggagtactggggtcaaggaacctcagtcaccgtctcctca.

[0014] The nucleotide sequence of the light chain variable region, SEQ ID No. 4, is as follows: gatgttttgatgacccaaagtccactctccctgcctgtcagtcttggggatcaagtctccatctcttgcagatctagtcagagccacagtagtggaaacacctatttagaatggtacctgcagaaaccaggccagtctccaaggctcctgatctacaaacagtc caaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactgctttcaatcacatgttcctccgacgtgcggtggaggcaccaagttggaaatcaaa.

[0015] The third inventive point of this invention is to provide a recombinant expression vector comprising the above-described nucleic acid molecules.

[0016] The fourth inventive point of this invention is to provide a host cell containing the above-described recombinant expression vector, or whose genome integrates the above-described nucleic acid molecules.

[0017] The fifth inventive point of this invention is to provide a method for preparing the above-mentioned ractopamine monoclonal antibody or its antigen-binding portion, the method comprising the following steps: culturing the above-mentioned host cells under appropriate conditions.

[0018] The sixth inventive point of this invention is to provide the application of the above-mentioned ractopamine monoclonal antibody or its antigen-binding moiety in the preparation of ractopamine residue detection reagents or kits.

[0019] The specific preparation method of ractopamine monoclonal antibody includes the following steps: 1) Preparation of ractopamine-BSA antigen: First, ractopamine derivatives are synthesized, and then BSA is coupled to obtain ractopamine-BSA antigen; 2) Hybridoma cell line screening: Hybridoma cells were prepared by immunizing mice, and ractopamine monoclonal antibodies were obtained through screening and sequencing. 3) Expression in mammalian cells: A mammalian expression plasmid for RAC monoclonal antibody was constructed, and the plasmid was transformed into HEK293FT_S. After five days of culture, the culture supernatant was collected. 4) Indirect ELISA assay for antibody titer; 5) Indirect competitive ELISA assay to determine antibody titer.

[0020] Compared with the prior art, the present invention has the following advantages: This invention provides a ractopamine monoclonal antibody or its antigen-binding moiety, a preparation method, and applications. The method for obtaining the monoclonal antibody is reproducible, produces high-purity products, and is suitable for mass production. By expressing the monoclonal antibody in mammalian cells, a single-component ractopamine monoclonal antibody is obtained, exhibiting high antibody specificity (IC50 of 0.21 ppb). This monoclonal antibody is suitable for the immunological detection of ractopamine residues, meeting practical application needs. The preparation method of the ractopamine monoclonal antibody and the produced ractopamine monoclonal antibody possess high specificity and sensitivity, making them suitable for establishing immunological detection methods for ractopamine content. Attached Figure Description

[0021] Figure 1 This is the regression curve for detecting the inhibition rate of ractopamine monoclonal antibody in this embodiment of the invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. However, it should be understood that the description herein is merely illustrative and not intended to limit the scope of the invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention. All reagents and instruments used herein are commercially available, and the characterization methods involved are described in relevant prior art and will not be repeated herein.

[0024] To further understand the present invention, the present invention will be described in detail below with reference to the preferred embodiments.

[0025] Example 1

[0026] The ractopamine monoclonal antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises three complementarity-determining regions (CDRs) of amino acid sequences at positions 26-32, 50-56, and 95-104 of SEQ ID No. 1, respectively. The light chain variable region comprises three CDRs of amino acid sequences at positions 27-35, 53-55, and 92-97 of SEQ ID No. 2, respectively.

[0027] The amino acid sequence of the heavy chain variable region is SEQ ID No. 1; the amino acid sequence of the light chain variable region is SEQ ID No. 2.

[0028] The amino acid sequence of the heavy chain variable region, SEQ ID No. 1, is: QVRLQQPGAELVKPGASVKLSCKASGYFTTYWIQWVRQRPGQGLEWI GEINNNGRTEFNEKFKRKATLTVDTSSTTAFMELSSLTSEDSAVYFCARYIPYA MEYWGQGTSVTVSS.

[0029] The amino acid sequence of the light chain variable region, SEQ ID No. 2, is: DVLMTQSPLSLPVSLGDQVSISCRSSQSHSSGNTYLEWYLQKPGQSPRL LIYKQSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSHVPPTCGG GTKLEIK.

[0030] Both the heavy chain variable region and the light chain variable region contain a framework region, which is derived from mice.

[0031] The present invention also provides a nucleic acid molecule encoding the above-mentioned ractopamine monoclonal antibody or its antigen-binding moiety. The nucleotide sequence of the gene encoding the heavy chain variable region of the ractopamine monoclonal antibody or its antigen-binding moiety is shown in SEQ ID No. 3; the nucleotide sequence of the gene encoding the light chain variable region of the ractopamine monoclonal antibody or its antigen-binding moiety is shown in SEQ ID No. 4.

[0032] The nucleotide sequence of the heavy chain variable region, SEQ ID No. 3, is: caggtccgactgcagcagcctggggctgaactggtgaagcctggggcttcagtgaagctgtcctgcaaggcttctggctacttcaccacctactggatacagtgggtgaggcagaggcctggacaaggccttgagtggattggagagattaata ataacggtcgtactgagtttaatgagaagttcaagaggaaggccacactgttgacacatcctccactacagccttcatggaactcagcagtctgacatctgaggactctgcggtctatttctgtgcaagatatatcccgtatgctatggagtactggggtcaaggaacctcagtcaccgtctcctca.

[0033] The nucleotide sequence of the light chain variable region, SEQ ID No. 4, is as follows: gatgttttgatgacccaaagtccactctccctgcctgtcagtcttggggatcaagtctccatctcttgcagatctagtcagagccacagtagtggaaacacctatttagaatggtacctgcagaaaccaggccagtctccaaggctcctgatctacaaacagtc caaccgattttctggggtcccagacaggttcagtggcagtggatcagggacagatttcacactcaagatcagcagagtggaggctgaggatctgggagtttattactgctttcaatcacatgttcctccgacgtgcggtggaggcaccaagttggaaatcaaa.

[0034] The present invention also provides a recombinant expression vector comprising the above-described nucleic acid molecules.

[0035] The present invention also provides a host cell comprising the above-mentioned recombinant expression vector, or a host cell in which the above-mentioned nucleic acid molecules are integrated into the genome.

[0036] The present invention also provides a method for preparing the above-mentioned ractopamine monoclonal antibody or its antigen-binding portion, comprising the following steps: culturing the above-mentioned host cells under appropriate conditions.

[0037] The present invention also provides the application of the above-mentioned ractopamine monoclonal antibody or its antigen-binding moiety in the preparation of ractopamine residue detection reagents or kits.

[0038] The specific preparation method of ractopamine monoclonal antibody includes the following steps: 1) Preparation of ractopamine-BSA antigen: First, synthesize ractopamine derivatives, and then couple them with BSA to obtain ractopamine-BSA antigen; 2) Hybridoma cell line screening: Hybridoma cells were prepared by immunizing mice, and ractopamine monoclonal antibodies were obtained through screening and sequencing. 3) Expression in mammalian cells: A mammalian expression plasmid for RAC monoclonal antibody was constructed, and the plasmid was transformed into HEK293FT-S. After five days of culture, the culture supernatant was collected. 4) Indirect ELISA assay for antibody titer; 5) Indirect competitive ELISA assay to determine antibody titer.

[0039] Example 2

[0040] I. Preparation of ractopamine-BSA antigen: 1. Anhydrous potassium carbonate was added to anhydrous acetone, followed by the addition of p-hydroxybutyric acid (p-hydroxyphenylbutanone) and magnetic stirring at room temperature for 1 hour. Ethyl 4-bromobutyrate was then added and the mixture was stirred overnight. Acetone was removed by rotary evaporation under reduced pressure. Ultrapure water was added to the residue, and the mixture was extracted with ethyl acetate. Excess sodium sulfate was used to remove water, and the supernatant was evaporated to dryness. The residue was purified by column chromatography to obtain intermediate product A.

[0041] 2. Take intermediate product A, octylamine hydrochloride, dissolve it in methanol, mix well, add triethylamine and sodium cyanoborohydride dropwise, and stir the reaction overnight. Vacuum evaporate the solvent, add hydrochloric acid and ultrapure water, and wash once with ethyl acetate. Take the aqueous phase and adjust the pH with sodium hydroxide. Extract three times with ethyl acetate until 12-13. Combine the organic phases and remove water from the organic phase with anhydrous sodium sulfate. Allow to stand, pour off the supernatant, and evaporate to dryness. Purify the residue by column chromatography to obtain intermediate B.

[0042] 3. Dissolve intermediate product B in methanol, add sodium hydroxide, and stir the reaction mixture at room temperature for 16 hours. Vacuum evaporate the reaction solution to dryness. Dissolve the residue in ultrapure water, adjust the pH to 3-4 with hydrochloric acid, precipitate the suspension, centrifuge, collect the precipitate, and dry it with phosphorus pentoxide. The target product is the ractopamine derivative.

[0043] 4. Activation: Dissolve the ractopamine derivative in DMF with stirring, add NHS and EDC, and react overnight at room temperature. Coupling: Weigh BSA and dissolve it in PBS buffer, then slowly add the activation solution dropwise to the above solution, and react overnight. Purification: Centrifuge to remove the precipitate, and dialyze with PBS to obtain ractopamine-BSA antigen.

[0044] II. Hybridoma cell line screening: 1. Immunization of mice: Balb / C mice were used as immunization animals. The immunogen was RAC_BSA. The immunization dose was 50-100 μg / mouse per immunization, and a total of three immunizations were performed.

[0045] 2. Preparation of hybridoma cells: Spleen cells from the immunized mice described above were used as a fusion agent, and immunized spleen cells and SP2 / 0 myeloma cells were fused at a ratio of 7:1 at 37°C. Mouse feeder cells were collected beforehand to promote the growth of the fused cells.

[0046] 3. The culture supernatant of surviving fusion cells was detected using an indirect ELISA method. Highly positive clones were subjected to limiting dilution, and the culture supernatant from wells showing monoclonal growth was examined. Positive wells were selected for further expansion culture, followed by a second limiting dilution. The culture supernatant from wells showing monoclonal growth was then examined again to obtain a highly specific hybridoma cell line. Sequencing was performed to obtain the heavy and light chain sequences of the ractopamine monoclonal antibody.

[0047] III. Expression in mammalian cells: 1. Plasmid preparation: The sequence obtained from sequencing was synthesized into a plasmid vector, and heavy and light chain plasmid powders were obtained through sequence synthesis (Genewiz). The powders were diluted to 10 ng / μl with sterile water. Competent cells were thawed at -80℃ and placed on ice for 15 min. 50 μl of competent cells were mixed with 1 μl of plasmid by gentle tapping and incubated on ice for 30 min. Centrifuge tubes were placed in a 42℃ water bath for 90 s, then immediately placed on ice for 3 min. 950 μl of LB broth without antibiotics was added to the centrifuge tubes, and the cells were incubated at 37℃ for 1 h using a shaker. One-sixth of the transformed cells were plated and incubated overnight at 37℃. Single colonies were picked and cultured overnight in LB broth (with ampicillin). The next morning, plasmids were extracted using a kit (FastPure EndoFree Plasmid Mini Kit) to obtain the heavy and light chain plasmids.

[0048] 2. Cell transfection and culture: Before HEK293FT_S transfection, the cells were passaged once, seeded at 5×10⁵ cells / mL on day 3, and at 2.5×10⁵ cells / mL on day 4 in Expi293 expression medium. On the day of transfection, the cell density reached 5–6×10⁶ cells / mL, with a viability >95%. The cells were diluted to 3×10⁶ cells / mL with the preheated Expi293 expression medium at 37 °C, and 25 mL was reserved. ExpiFectamine 293Reagent was gently inverted 4–5 times beforehand to mix thoroughly. Dilute 80 μL of ExpiFectamine 293Reagent with 1.4 mL of Opti_EME Reduce Serum Medium and incubate at room temperature for 5 minutes to activate the transfection reagent. Dilute 25 μg of plasmid (heavy to light chain mass ratio 1:2) with 1.5 mL of Opti_EME Reduce Serum Medium. Add the diluted plasmid to the ExpiFectamine 293Reagent, mix well, and incubate at room temperature for 15 minutes to form a plasmid-transfection reagent complex. Then, add this complex to a prepared 25 mL cell culture container. After transfection, culture the cells in a shaker at 37°C, 5% CO2, 120 rpm, and 80% humidity. 18–22 hours after transfection, add 150 μL of Enhancer 1 and 1.5 mL of Enhancer 2. On the fifth day after transfection, centrifuge at 12000 rpm for 10 minutes to collect the culture supernatant.

[0049] IV. Identification of ractopamine monoclonal antibodies: 1. Indirect ELISA assay for antibody titer: Dilute the antigen to 0.1 ppm with PBS, and use 100 μL / well for coating. Incubate at 37°C for 2 h. Wash three times with PBST and blot dry. Add 250 μL of BSA blocking buffer and incubate overnight at 4°C. Blot dry the next day and set aside. Dilute the culture supernatant with PBS at dilution factors of 100, 500, 1000, 2000, 4000, 8000, and 16000 times, and set up a blank control (PBS). Add 100 μL / well of the supernatant to the wells of the coated plate, incubate at 37°C for 30 min, then wash the plate 5 times with PBST and blot dry. Add 100 μL / well of enzyme-labeled secondary antibody (goat anti-mouse, 10000-fold dilution), incubate at 37°C for 30 min, wash the plate 5 times with PBST and blot dry. Add 100 μL of substrate chromogenic solution to each well and incubate at 37°C for 15 min. Add 50 μL of stop solution to each well, gently shake to mix, and measure the absorbance at 450 nm within 5 minutes. The results of the monoclonal antibody titer determination are shown in Table 1. An OD value greater than twice that of the negative control is considered positive.

[0050] Table 1 4.000 1:100 2.230 1:500 1.422 1:1000 0.727 1:2000 0.386 1:4000 0.271 1:8000 0.197 1:16000 0.209 PBS (Polysphingomyelin) Results analysis: As shown in Table 1 (monoclonal antibody potency determination), the potency of this monoclonal antibody is 1:2000.

[0051] 2. Indirect competitive ELISA determination of IC50: On the coated plate, add 50 μL of standard solution first, followed by 100-fold diluted transfection supernatant. Dilute the standards to 0.1, 0.3, 0.9, 2.7, and 8.1 ppb with PBS, replacing the first row with 50 μL of PBS. Incubate at 37°C for 30 min, then wash the plate 5 times with PBST and pat dry. Add 100 μL of substrate chromogenic solution to each well and incubate at 37°C for 15 min. Add 50 μL of stop solution to each well, gently vortex to mix, and measure the absorbance at 450 nm within 5 minutes. The IC50 results are shown in Table 2.

[0052] Table 2

[0053]

[0054] Inhibition rate = (3.200_OD value) / 3.200 × 100%.

[0055] The concentration of the RAC standard at an inhibition rate of 50% (i.e., an OD value of 1.600) is the IC50. A regression curve is plotted with the standard concentration on the x-axis and absorbance on the y-axis, as shown below. Figure 1 As shown in the figure. The regression curve shows that the IC50 of this monoclonal antibody is 0.21 ppb.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A ractopamine monoclonal antibody or its antigen-binding moiety, characterized in that, The monoclonal antibody or its antigen-binding portion comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises three complementarity-determining regions (CDRs) of amino acid sequences 26-32, 50-56, and 95-104 of SEQ ID No.

1. The light chain variable region comprises three CDRs of amino acid sequences 27-35, 53-55, and 92-97 of SEQ ID No.

2.

2. The ractopamine monoclonal antibody or its antigen-binding moiety according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is SEQ ID No. 1; the amino acid sequence of the light chain variable region is SEQ ID No.

2.

3. The ractopamine monoclonal antibody or its antigen-binding moiety according to any one of claims 1-2, characterized in that, Both the heavy chain variable region and the light chain variable region contain a framework region, which is derived from mice.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the ractopamine monoclonal antibody or its antigen-binding portion as described in any one of claims 1-2.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleotide sequence of the gene encoding the heavy chain variable region of the ractopamine monoclonal antibody or its antigen-binding portion is shown in SEQ ID No. 3; the nucleotide sequence of the gene encoding the light chain variable region of the ractopamine monoclonal antibody or its antigen-binding portion is shown in SEQ ID No.

4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 4 or 5.

7. A host cell, characterized in that, The host cell contains the recombinant expression vector as described in claim 6, or has a nucleic acid molecule as described in claim 4 or 5 integrated into its genome.

8. A method for preparing the ractopamine monoclonal antibody or its antigen-binding moiety as described in any one of claims 1-2, characterized in that, The method includes the following steps: culturing the host cell as described in claim 7 under appropriate conditions.

9. The use of the ractopamine monoclonal antibody or its antigen-binding moiety as described in claim 1 or 2 in the preparation of ractopamine residue detection reagents or kits.

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