High-affinity anti-carbendazim monoclonal antibody and full-length IgG recombinant antibody expression plasmid thereof
By using genetic engineering amplification and mammalian cell expression systems to prepare high-affinity full-length IgG recombinant antibodies against carbendazim, the problems of batch variation and storage difficulties caused by hybridoma cells were solved, enabling stable large-scale production and high-sensitivity detection of antibodies.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-01-27
- Publication Date
- 2026-05-19
AI Technical Summary
Existing anti-carbendazim antibodies rely on hybridoma cells, which carries the risk of gene loss and mutation, leading to batch-to-batch differences in recognition performance and affecting detection stability. Furthermore, the stringent storage requirements make it difficult to mass-produce antibodies with good uniformity and high homogeneity.
Using genetic engineering technology, the heavy and light chain variable region sequences of a high-affinity anti-carbendazim monoclonal antibody were amplified and constructed. Full-length IgG recombinant antibodies were prepared using a mammalian cell expression system. Heavy and light chain expression vectors were constructed through homologous recombination technology to achieve stable secretion and mass production.
The obtained full-length IgG recombinant antibody has the same sensitivity as the ascites monoclonal antibody, realizing the immortalization of antibody production and providing a stable core reagent for the rapid detection of carbendazim residues.
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Figure CN122060071A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to a high-affinity monoclonal antibody against carbendazim and its full-length IgG recombinant antibody expression plasmid. Background Technology
[0002] Carbendazim is a systemic benzimidazole carbamate fungicide with broad protective and curative effects against fungal diseases caused by ascomycetes, basidiomycetes, and deuteromycetes. Its mechanism of action involves interfering with DNA synthesis, particularly the inhibition of nucleoside production. During pathogen cell division, carbendazim binds to microtubule proteins in spindle fibers, thus interfering with mitosis. Due to its high efficiency and low cost, carbendazim is one of the most widely used pesticides globally. However, in recent years, excessive and high-dose use of carbendazim has led to significant residues in the environment and agricultural products, posing potential hazards to ecosystems and human health, drawing widespread attention. In particular, its degradation product, 2-aminobenzimidazole, is highly toxic and can inhibit cell proliferation by blocking nuclear division. Currently, carbendazim is widely recognized as a potential human carcinogen. Furthermore, this pesticide can negatively impact soil microorganisms, phytoplankton, and zooplankton. Many countries and regions, such as Australia, the United States, and Europe, have banned the use of carbendazim in fruits and vegetables; however, its use still exists in developing countries such as China and India. Given the risks of carbendazim to human health and the environment, strengthening the detection of its residues is particularly important.
[0003] Compared to traditional large-scale laboratory analytical instruments, immunoassay methods based on antigen-antibody specific reactions have gradually become the mainstream technology for rapid on-site screening of small molecule pollutants such as pesticides due to their advantages of simple operation, high sensitivity, and high specificity. Antibodies, as the core reagents of immunoassay methods, directly affect the sensitivity, specificity, and stability of the detection and analysis methods. Currently, most publicly available anti-carbendazim antibodies are ascites monoclonal antibodies developed based on specific hybridoma cell lines. The preparation of these antibodies is highly dependent on hybridoma cell lines. However, hybridoma cells are at risk of gene loss and mutation during passage culture, which can lead to differences in the recognition performance of different batches of monoclonal antibodies, thus affecting the stability of the detection performance of downstream immunoassay methods. Furthermore, hybridoma cells have strict requirements for storage temperature and are prone to failure to be revived, which not only results in the loss of valuable antibody resources but also hinders the development and promotion of corresponding immunoassay methods.
[0004] Recombinant antibodies prepared using genetic engineering and in vitro recombinant expression technologies can effectively overcome the limitations of traditional hybridoma cell-derived monoclonal antibodies. By storing antibody genes in computers and plasmids, we can rapidly and massively produce antibodies with good uniformity and high homogeneity when needed, using in vitro recombinant expression systems. This not only achieves antibody immortalization but also provides a solid foundation for developing stable and reliable rapid detection products for pesticide residues. Based on their structural characteristics, recombinant antibodies can be divided into fragment recombinant antibodies (e.g., single-chain antibodies) and full-length immunoglobulin G (IgG) recombinant antibodies. Compared to fragment recombinant antibodies, full-length IgG recombinant antibodies are prepared through mammalian cell expression systems, undergoing a modification and folding process similar to that of natural antibodies produced by B cells and hybridoma cells. Therefore, full-length IgG recombinant antibodies possess a natural dimer structure, which makes them excellent in terms of structural stability, soluble expression, and affinity, effectively addressing many challenges that fragment recombinant antibodies may face. Currently, there are no publicly reported full-length IgG recombinant antibodies against carbendazim. Summary of the Invention
[0005] To overcome the shortcomings of the prior art, the present invention provides a high-affinity monoclonal antibody against carbendazim and its full-length IgG recombinant antibody expression plasmid.
[0006] The technical solution adopted by the present invention to solve its technical problem is: a high-affinity monoclonal antibody against carbendazim, comprising a heavy chain constant region, a heavy chain variable region, a light chain constant region, and a light chain variable region, wherein the amino acid sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO:2; and the amino acid sequence of the gene encoding the light chain variable region is shown in SEQ ID NO:4.
[0007] Preferably, the nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO:1.
[0008] Preferably, the nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO:3.
[0009] The present invention also discloses a heavy chain expression plasmid for a full-length anti-carbendazim IgG recombinant antibody, containing the above-mentioned heavy chain variable region and mouse heavy chain IgG1 constant region nucleotide sequence, which can express the heavy chain protein of the full-length anti-carbendazim IgG recombinant antibody; or, containing the above-mentioned light chain variable region and mouse light chain Kappa constant region nucleotide sequence, which can express the light chain protein of the full-length anti-carbendazim IgG recombinant antibody.
[0010] This invention, based on the hybridoma cell line DJL-E5-mAb which stably secretes carbendazim antibodies, successfully amplified, sequenced, and synthesized its heavy and light chain variable region genes. Expression vectors for the heavy and light chains were constructed using homologous recombination technology and co-transfected into mammalian HEK 293(F) cells. After culture and purification, a full-length anti-carbendazim IgG recombinant antibody was obtained. Using carbendazim as the detection target, the antibody's recognition activity was tested using an indirect competitive ELISA method. The results showed that the full-length IgG recombinant antibody had the same sensitivity as the mouse parental ascites monoclonal antibody, thus confirming the correctness and effectiveness of the obtained antibody variable region sequence. The antibody variable region sequence of this invention can be applied to the stable multi-batch production of high-affinity anti-carbendazim full-length IgG recombinant antibodies, providing strong support for the development of carbendazim residue immunoassay methods and technologies.
[0011] Compared with existing technologies, this invention has the following advantages: This invention discloses a variable region sequence for a high-affinity anti-carbendazim monoclonal antibody, comprising heavy chain variable region and light chain variable region sequences; this invention uses a mammalian HEK293(F) cell recombinant expression system to verify the reliability and accuracy of the variable region sequence. By constructing a heavy chain gene expression vector (containing heavy chain variable region genes and heavy chain constant region genes) and a light chain gene expression vector (containing light chain variable region genes and light chain constant region genes) dual plasmids, this invention obtains a full-length anti-carbendazim IgG recombinant antibody (IC). 50 (1.88 ng / mL) was confirmed by indirect competitive ELISA to have a similar effect to ascites monoclonal antibodies (IC50). 50 The sensitivity is comparable to 4.49 ng / mL. The variable region sequence of the anti-carbendazim antibody of this invention can be applied to the large-scale and stable production of recombinant antibodies, and the variable region sequence of the antibody heavy and light chains and its recombinant expression plasmid can be applied to the immortalization production of full-length anti-carbendazim IgG recombinant antibodies. This provides reliable and stable core reagents for the construction of immunoassay methods for carbendazim residues in the environment and agricultural products and the development of accurate and rapid detection products. Attached Figure Description
[0012] Figure 1 The ELISA competition curves of the ascites monoclonal antibody (A) and the full-length IgG recombinant antibody (B) prepared in this invention for detecting carbendazim are shown.
[0013] Figure 2 This is an electrophoresis diagram of total RNA from hybridoma cells prepared in this invention.
[0014] Figure 3 This is the result of agarose gel electrophoresis of the antibody variable region amplified by PCR using cDNA obtained by reverse transcription as a template (electrophoresis diagram of antibody light and heavy chain variable region fragment size). Detailed Implementation
[0015] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the embodiments are all conventional methods.
[0016] This invention relates to a novel monoclonal antibody against carbendazim, the amino acid sequences of which are shown in SEQ ID NO:2 for the heavy chain variable region and in SEQ ID NO:4 for the light chain variable region. The variable region sequence of this monoclonal antibody can be applied to the large-scale and standardized production of full-length IgG recombinant antibodies, providing a reliable and stable core material for rapid detection of carbendazim residues.
[0017] 1. Preparation and performance characterization of anti-carbendazim monoclonal antibody
[0018] Based on traditional hybridoma technology, this invention utilizes a previously prepared artificial antigen, carbendazim hapten-BSA conjugate, and screens a hybridoma cell line, DJL-E5, capable of stably secreting anti-carbendazim monoclonal antibodies through mouse immunization, titer testing, cell fusion, and limiting dilution. The cell line contains IgG1 and Kappa subtypes in its heavy and light chains, respectively. This cell line was inoculated into the peritoneal cavity of F1 mice injected with northane, and the ascites fluid was purified using the octanoic acid-ammonium sulfate precipitation method to obtain the ascites fluid monoclonal antibody, DJL-E5-mAb. The sensitivity of the antibody was characterized using an indirect competitive ELISA method. Figure 1 A) It was found to have a high affinity for carbendazim, inhibiting the medium concentration IC50. 50 The concentration was 4.49 ng / mL, and the limit of detection (IC50) was 4.49 ng / mL. 20 It was 0.81 ng / mL.
[0019] 2. Amplification and Identification of Antibody Variable Region Genes
[0020] Total RNA was extracted from the hybridoma cell line DJL-E5 using a one-step Trizol reagent method. The extracted total RNA samples were identified by 1% agarose gel electrophoresis, and the purity and integrity of the extracted RNA samples were good. Figure 2This met the requirements for subsequent experiments. Using the prepared total RNA as a template, cDNA was synthesized via reverse transcription using the PrimeScript 1st strand cDNA synthesis Kit. Antibody subtype-specific primers were used for PCR amplification, successfully obtaining the heavy chain variable region (VH) and light chain variable region (VL) of the anti-carbendazim monoclonal antibody.
[0021] The procedure for PCR amplification is as follows:
[0022]
[0023] The agarose gel electrophoresis results of the amplification products are shown in the figure. Figure 3 The target DNA fragment was purified using a gel extraction kit. The purified product was cloned into a Blunt zero cloning vector containing an ampicillin resistance tag, transformed with *E. coli*, and then sequenced using Sanger sequencing. The VH and VL genes with complete sequences, matching subtypes, and correct expression frames were identified using NCBI igBLAST alignment analysis.
[0024] The functional heavy and light chain variable region DNA and its amino acid sequence were screened as follows:
[0025] 1) The DJL-E5-mAb heavy chain variable region DNA sequence is as follows:
[0026] CAGGTCCAGTTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGATATCCTGCAAGGCTTCAGGCTACACCTTCACTAACTATTGGCTCGGTTGGGTAAAGCAGAGGCCTGGACATGGACTTGAGTGGATTGGAGATACTTATCCTGGAGGTGGTTATACT AAGTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACCGCCTACATGCAGTTGAGTAGCCTGACATCTGAGGACTCTGCTGTCTATTTCTGTGCAAGAGGAAACCAAGGGGCTTACTGGGGCCAAGGGACTCTGGTCTCTGTCTCTGCA (SEQ ID NO:1)
[0027] The functional heavy chain variable region is 345 bases long, and the domains start from the first base and encode 115 amino acids.
[0028] The structural domain is defined using the IMGT method, and the specific structural domain is divided as follows:
[0029]
[0030] 2) The amino acid sequence of the variable region of the DJL-E5-mAb heavy chain is as follows:
[0031] QVQLQQSGAELVRPGTSVKISCKASGYTFTNYWLGWVKQRPGHGLEWIGDTYPGGGYTKYNEKFKGKATLTADTSSSTAYMQLSSLTSEDSAVYFCARGNQGAYWGQGTLVSVSA (SEQ ID NO: 2)
[0032] 3) The DNA sequence of the variable region of the light chain of the DJL-E5-mAb antibody is as follows:
[0033] GAAAATGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAAAAGGTCTCCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCGCTGGTACCAGCAGAAGTCAAGCACCTCCCCCAAACTCTGGATTTATGACACATCCAAACTGGC TTCTGGAGTCCCAGGTCGCTTCAGTGGCAGTGGGTCTGGAAACTCTTACTCTCTCACGATCAGCAGCATGGAGGCTGAAGATGTTGCCACTTATTACTGTTTTCAGGCGAGTGGGTACCCACTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO:3)
[0034] The functional light chain variable region is 318 bases long, and the domains start from the first base and encode 106 amino acids.
[0035] The structural domain is defined using the IMGT method, and the specific structural domain is divided as follows:
[0036]
[0037] 4) The amino acid sequence of the variable region of the light chain of the DJL-E5-mAb antibody is as follows:
[0038] ENVLTQSPAIMSASLGEKVSMTCSASSSVSYMRWYQQKSSTSPKLWIYDTSKLASGVPGRFSGSGSGNSYSLTISSMEAEDVATYYCFQASGYPLTFGSGTKLEIK (SEQ ID NO: 4)
[0039] 3. Construction of full-length IgG recombinant antibody expression plasmid
[0040] Homologous recombination technology was used to construct heavy and light chain expression plasmids containing variable and constant regions. The VH and VL genes were cloned into the linearized expression plasmids pCDNA3.4-Mouse-IgG1-CH and pCDNA3.4-Mouse-C, respectively. κ Above. Among them, pCDNA3.4-Mouse-IgG1-CH (HindIII / EcoRI) contains the mouse IgG1 heavy chain constant region gene; pCDNA3.4-Mouse-C κ The (HindIII / EcoRI) plasmid contains the mouse Kappa light chain constant region gene. The constructed expression plasmid was transformed into competent *E. coli* cells, activated by shaking, and subjected to antibiotic resistance screening. Recombinants were selected for sequencing. The sequencing results were analyzed, and the bacterial culture corresponding to the plasmid with the correct sequence was expanded. Endotoxin-free expression plasmids were then extracted using a kit.
[0041] 4. Evaluation of expression and activity of full-length IgG recombinant antibody
[0042] This invention utilizes a mammalian cell expression system to prepare full-length IgG recombinant antibodies. An indirect competitive ELISA method was used to test the recognition performance of the recombinant antibody against ascites monoclonal antibodies, evaluating the accuracy and effectiveness of the antibody variable region sequence in this invention. The steps are as follows: Frozen cells were thawed and expressed at 2 × 10⁻⁶ cells / mL. 5 Inoculate cells at a density of 3 × 10⁶ cells / mL into culture flasks and incubate at 37°C, 130 rpm, and 5% CO₂; wait until cells reach the logarithmic growth phase (density reaches 3 × 10⁶ cells / mL). 6 (number / mL), at 3×10 5 Subcultured twice more at a density of cells / mL; cells in good growth condition (95% > cell viability) were then passaged at a density of 1.5 × 10⁻⁶ cells / mL. 6 Inoculate at a density of cells / mL and culture in suspension for 2 h; add heavy chain expression plasmid and light chain expression plasmid at a mass ratio of 2:3 to pre-prepared cell culture medium containing transfection reagent and incubate at 37°C for 15 min; then, add the plasmid mixture dropwise to the cell culture medium, suspend and culture at 130 rpm for 5 days, and collect the supernatant; elute the protein using a Protein A affinity chromatography column, and dialyze with 0.01M PBS to obtain the full-length IgG recombinant antibody product.
[0043] Indirect competitive ELISA results showed that the full-length IgG recombinant antibody DJL-E5-rAb detected the IC50 of carbendazim. 50 The concentration was 1.88 ng / mL, which is comparable to the sensitivity of ascites monoclonal antibodies (IC50). 50:The result (4.49 ng / mL) indicates that the antibody variable region sequence is accurate and effective, and can be applied to the large-scale stable production of full-length IgG recombinant antibodies. Table 1 lists the IC50 values of previously reported anti-carbendazim ascites monoclonal antibodies. 50 The anti-carbendazim antibodies DJL-E5-mAb and DJL-E5-rAb developed in this invention also have advantages in sensitivity. More importantly, the antibody sequences disclosed in this invention can be applied to the immortalization production of full-length anti-carbendazim IgG recombinant antibodies, which provides a reliable and stable core reagent for the construction of immunoassay methods and rapid detection product development for carbendazim residues in the environment and food.
[0044] Table 1. Sensitivity (IC50) of developed anti-carbendazim antibodies to carbendazim in indirect competitive ELISA. 50, (ng / mL)
[0045]
[0046] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.
Claims
1. A high-affinity monoclonal antibody against carbendazim, comprising a heavy chain constant region, a heavy chain variable region, a light chain constant region, and a light chain variable region, characterized in that: The amino acid sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO:2; the amino acid sequence of the gene encoding the light chain variable region is shown in SEQ ID NO:
4.
2. The anti-carbendazim monoclonal antibody according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO:
1.
3. The anti-carbendazim monoclonal antibody according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO:
3.
4. A heavy chain expression plasmid for a full-length recombinant IgG antibody against carbendazim, characterized in that: A heavy chain protein containing the heavy chain variable region and the mouse heavy chain IgG1 constant region as described in any one of claims 1-2 can express a recombinant antibody against full-length carbendazim IgG; or, a light chain protein containing the light chain variable region and the mouse light chain Kappa constant region as described in any one of claims 1 and 3 can express a recombinant antibody against full-length carbendazim IgG.