An anti-acetamiprid monoclonal antibody and a recombinant full-length IgG expression plasmid thereof

Abstract

The application discloses an anti-acetamiprid high-affinity and high-specificity monoclonal antibody, an amino acid sequence of a heavy chain variable region of mAb-QA1 is shown as SEQ ID NO: 2, and an amino acid sequence of a light chain variable region of mAb-QA1 is shown as SEQ ID NO: 4. The application also discloses an expression plasmid of an anti-acetamiprid full-length IgG recombinant antibody. The variable region sequence genes obtained by the application are connected to expression vectors containing heavy chain constant region genes and light chain constant region genes respectively, double-plasmid transfection is adopted, and the anti-acetamiprid full-length IgG recombinant antibody is obtained based on mammalian cell expression and purification, and it is proved by indirect competitive ELISA analysis that the expressed recombinant antibody has similar detection activity to the parent monoclonal antibody. The antibody heavy and light chain variable region sequences and the recombinant expression plasmids thereof can be applied to the stable production of batches of the anti-acetamiprid full-length IgG recombinant antibody, and provide reliable and stable core reagents for the construction of an immune analysis method for acetamiprid residues in the environment and food and the development of precise and rapid measurement products.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to an anti-acetamiprid monoclonal antibody and its recombinant full-length IgG expression plasmid. Background Technology

[0002] Acetamiprid is a neonicotinoid insecticide developed and commercialized by Nippon Soda Co., Ltd. in 1996, and was first registered for use on crops and livestock in 2002. Due to the unique mechanism of action of neonicotinoid insecticides, represented by acetamiprid, they exhibit almost no cross-resistance to organophosphates, pyrethroids, and carbamates, making them a good alternative to these insecticides. Acetamiprid and other neonicotinoid insecticides act on the neonicotinic acetylcholine receptor (nAChR) on the postsynaptic membrane of the insect nervous system, blocking neurotransmitter transmission, altering membrane potential, and thus continuously stimulating the postsynaptic membrane with neurotransmitters. This continuous excitation ultimately leads to neuronal overexcitation, paralysis, and death. Currently, acetamiprid has a high market share in my country and is one of the fifteen most important insecticides in the country, playing a vital role in plant protection and increasing grain yields. However, the harmful effects of acetamiprid on mammals and other non-target organisms have also attracted widespread global attention. Multiple studies have shown that acetamiprid increases reactive oxygen species levels in mammals and significantly damages and breaks DNA and RNA. Simultaneously, research indicates that long-term exposure to acetamiprid alters social and anxiety behaviors in rats, leading to memory loss and impaired learning abilities. Furthermore, acetamiprid poses potential harm to beneficial insects such as bees, lacewings, Trichogramma wasps, and ladybugs, affecting lifespan and egg production, thus posing a potential risk to ecosystems. In recent years, numerous countries and organizations have issued bans restricting the use or registration of acetamiprid and other neonicotinoid products. For example, in 2018, France added five neonicotinoid pesticides—acetamiprid, imidacloprid, thiamethoxam, thiamethoxam, and thiamethoxam—to its prohibited list. Over the past decade or so, acetamiprid has been widely used in agriculture, households, and public health activities, and researchers both domestically and internationally have reported finding high levels of acetamiprid residues in rivers, surface soils, and agricultural products. Given the health and ecological risks of acetamiprid, it is crucial to develop detection methods for acetamiprid residues.

[0003] Currently, the analysis of acetamiprid residues mainly uses traditional instrumental analysis methods, such as GC-MS and LC-MS. However, due to limitations such as expensive equipment, specialized operation, and cumbersome sample pretreatment processes, traditional instrumental analysis methods are difficult to apply to rapid on-site detection and large-scale sample screening. In contrast, immunoassay methods based on the specific binding of antigens and antibodies are characterized by simple operation, high sensitivity, and high specificity, thus showing great application potential and promotional value for rapid on-site screening of large batches of samples. Immunoassay methods such as colloidal gold immunochromatography and enzyme-linked immunosorbent assay (ELISA) have been widely used in the detection of pesticide and other small molecule pollutants. Regardless of the immunoassay method, the core reaction element is the antibody. The performance of the antibody directly affects the sensitivity, specificity, and detection stability of the analytical method. Typically, researchers obtain monoclonal antibodies through hybridoma technology; however, hybridoma cells may experience gene loss or mutation during passage culture. These factors can lead to batch-to-batch variations in the detection performance of immunoassay methods based on different batches of monoclonal antibodies.

[0004] Currently, in vitro expressed recombinant antibodies, known as third-generation antibodies, effectively circumvent the drawbacks of traditional monoclonal antibodies, such as large batch-to-batch variability, poor stability, and long production cycles. Furthermore, they allow for large-scale production of antibodies with good uniformity and high homogeneity without the use of laboratory animals. This is of significant guiding importance for the development of stable and reliable rapid detection products for pesticide residues. Recombinant antibodies can be classified into fragment recombinant antibodies and full-length immunoglobulin G (IgG) recombinant antibodies based on their structure. Compared to the former, full-length IgG recombinant antibodies have significant advantages in affinity, half-life, and stability due to their conformation similar to natural immunoglobulins. Currently, there are no publicly reported full-length IgG recombinant antibodies against acetamiprid. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides an anti-acetamiprid monoclonal antibody and its recombinant full-length IgG expression plasmid.

[0006] The technical solution adopted by the present invention to solve its technical problem is: an anti-acetamiprid monoclonal antibody, the amino acid sequence of the heavy chain variable region of mAb-QA1 is shown in SEQ ID NO:2; the amino acid sequence of the light chain variable region of mAb-QA1 is shown in SEQ ID NO:4.

[0007] Furthermore, the nucleotide sequence of the mAb-QA1 heavy chain variable region encoding gene is shown in SEQ ID NO:1.

[0008] Furthermore, the nucleotide sequence of the mAb-QA1 light chain variable region encoding gene is shown in SEQ ID NO:3.

[0009] The present invention also discloses an expression plasmid for a recombinant full-length IgG antibody against acetamiprid, containing the nucleotide sequences of the heavy chain variable region and the mouse heavy chain IgG1 constant region as described above, expressing the heavy chain protein of the recombinant full-length IgG antibody against acetamiprid; and simultaneously containing the nucleotide sequences of the light chain variable region and the mouse light chain Lambda constant region as described above, expressing the light chain protein of the recombinant full-length IgG antibody against acetamiprid.

[0010] The variable region sequence gene obtained in this invention was ligated into an expression vector containing both the heavy chain constant region gene and the light chain constant region gene. Double plasmid transfection was used, and a full-length IgG recombinant antibody against acetamiprid was obtained through expression and purification in mammalian cells. Indirect competitive ELISA analysis confirmed that the expressed recombinant antibody possessed detection activity similar to the parent monoclonal antibody. The aforementioned antibody heavy and light chain variable region sequences and their recombinant expression plasmids can be used for stable multi-batch production of the full-length IgG recombinant antibody against acetamiprid, providing a reliable and stable core reagent for the construction of immunoassay methods and the development of accurate and rapid detection products for acetamiprid residues in the environment and food.

[0011] This invention focuses on a hybridoma cell line (clone number QA1) that stably secretes antibodies recognizing acetamiprid. The variable region genes of the heavy and light chains were amplified, sequenced, and synthesized within this cell line. Heavy and light chain expression vectors were constructed using homologous recombination technology. These vectors were transfected into mammalian HEK 293(F) cells, cultured, and purified to obtain a full-length IgG recombinant antibody against acetamiprid. Using acetamiprid as the detection target, the accuracy of the variable region sequence was verified using ELISA. The results demonstrated that the full-length IgG recombinant antibody against acetamiprid exhibited the same high sensitivity as the parental mouse ascites monoclonal antibody, indicating that the variable region sequence in the anti-acetamiprid monoclonal antibody is effective and reliable. The antibody variable region sequence of this invention can be applied to the stable multi-batch production of high-specificity, high-affinity full-length IgG recombinant antibodies against acetamiprid, providing long-term assurance for the development of immunoassay methods and technologies for acetamiprid residues.

[0012] Compared with existing technologies, this invention has the following advantages: This invention discloses the variable region sequence of an anti-acetamiprid monoclonal antibody, including 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-acetamiprid IgG recombinant antibody (IC). 50 (4.97 ng / mL) was confirmed by ELISA to contain a monoclonal antibody against ascites fluid (IC50). 50With a sensitivity similar to 3.24 ng / mL and a low cross-reactivity with structural analogs (<9%), this antibody exhibits high affinity and high specificity. The variable region sequence of the anti-acetamiprid antibody of this invention can be applied to the large-scale and stable production of recombinant antibodies, providing a reliable and stable core reagent for the construction of immunoassay methods and the development of accurate and rapid detection products for acetamiprid residues in the environment and agricultural products. Attached Figure Description

[0013] Figure 1 Standard curves for the detection of acetamiprid and thiamethoxam using ascites monoclonal antibody and full-length IgG recombinant antibody in heterologous indirect competitive ELISA of this invention were constructed. (A) Ascites monoclonal antibody mAb-QA1; (B) Full-length IgG recombinant antibody rAb-QA1.

[0014] Figure 2 The recognition of eight neonicotinoid pesticides by the ascites monoclonal antibody mAb-QA1 prepared for this invention in a heterologous indirect competitive ELISA method.

[0015] 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

[0016] 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.

[0017] This invention relates to a novel monoclonal antibody with high specificity and high affinity for acetamiprid. The amino acid sequences of the heavy chain variable region encoding gene are shown in SEQ ID NO:2, and the amino acid sequences of the light chain variable region encoding gene are shown in SEQ ID NO:4. The variable region sequence of this monoclonal antibody can be used for the large-scale and standardized production of recombinant antibodies, providing a reliable and stable core reagent for the rapid detection of acetamiprid residues.

[0018] 1. Preparation and performance characterization of monoclonal antibodies against acetamiprid

[0019] Based on traditional hybridoma technology, this invention utilizes the previously prepared artificial antigen DCM-BSA. Through steps including mouse artificial antigen assay, titer testing, cell fusion, and limiting dilution, a hybridoma cell line QA1 capable of stably secreting monoclonal antibodies against acetamiprid was obtained. The light and heavy chain subtypes of this cell line are IgG1 and Lambda, respectively. This cell line was inoculated into the peritoneal cavity of F1 mice injected with norfloxacin, and the ascites fluid was purified using the octanoic acid-ammonium sulfate precipitation method to obtain the monoclonal antibody. The sensitivity and specificity of the antibody were characterized using a heterologous indirect competitive ELISA method (coated with a conjugate of thiamethoxam hapten and OVA).

[0020] (1) Sensitivity: A 96-well ELISA plate was coated with a heterologous coating agent (0.37 mg / L), incubated, and blocked. 50 μL of serially diluted acetamiprid standard and 50 μL of antibody at the optimal working concentration were added to each well. After the competitive reaction, the signal was amplified using enzyme-labeled rabbit anti-mouse secondary antibody, with 3,3',5,5'-tetramethylbenzidine (TMB) as the reaction substrate. After terminating the reaction, the absorbance of each well was measured at 450 nm, and an ELISA standard curve was established. Figure 1 As shown, the ic-ELISA standard curve established by mAb-QA1 using the calibrated method reflects the sensitivity (IC50) of mAb-QA1 to acetamiprid. 50 The effective concentration was 3.24 ng / mL, and the linear range for recognition was 1.10–9.57 ng / mL.

[0021] (2) Specificity: This invention employs a heterologous indirect competitive ELISA single-point inhibition method, evaluating antibody specificity by testing the inhibition rates of eight common neonicotinoid pesticides (at a concentration of 1 mg / L). For example... Figure 2 As shown, the results indicate that the antibody prepared in this invention possesses high specificity recognition performance. Besides recognizing acetamiprid, it only shows slight cross-recognition with thiamethoxam and no significant cross-recognition with other neonicotinoid compounds. Subsequently, a heterologous indirect competitive ELISA standard curve for thiamethoxam was established, and the cross-reactivity rates of other compounds were calculated with reference to the sensitivity of acetamiprid. The calculation formula is CR(%) = IC50. 50 (Acetamiprid) / IC 50 (Other acetamiprid pesticides) × 100%. Results are as follows: Figure 1 An indirect competitive ELISA method was established using the highly specific monoclonal antibody developed in this invention. The sensitivity IC50 of mAb-QA1 for detecting thiamethoxam was [not specified]. 50 The concentrations were 42.65 ng / mL, with a cross-reactivity rate of 7.60%. Therefore, this monoclonal antibody can recognize acetamiprid with high sensitivity and high specificity.

[0022] 2. Amplification and identification of antibody variable region genes

[0023] Total RNA was extracted from hybridoma cell line QA1 using a one-step Trizol reagent method; the extraction procedure can be found in the Takara kit instructions. The extracted total RNA sample was confirmed by 1% agarose gel electrophoresis to have good purity and integrity, meeting 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. Using antibody subtype-specific primers, PCR amplification successfully yielded the heavy chain variable region (VH) and light chain variable region (VL) of the monoclonal antibody against acetamiprid pesticide.

[0024] The procedure for PCR amplification is as follows:

[0025]

[0026] 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 the pUC19-T vector containing an ampicillin resistance tag, transformed with E. coli, and then subjected to Sanger sequencing. The VH and VL genes with complete sequences, matching subtypes, and correct expression frames were identified using NCBI igBLAST alignment analysis.

[0027] The functional light and heavy chain variable region DNA and its amino acid sequence were screened as follows:

[0028] 1) The DNA sequence of the heavy chain variable region of the acetamiprid-QA1 highly specific monoclonal antibody is: CAGGTTACTCTAAAAGAGTCTGGCCCTGGGATTTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGTACAGTTATGATACAGGAGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGC AAACATTTGGTGGAATGGTTATAAATTCTATAACACAGACCTGAGGAGCCGACTCACAATCTCCAAGGATCCCTCCAACAACCAGGTTTCCTCACGATCGCCAATGTGAACACTGCAGATACTTCCACATATTGTGTTCGAACCTATAGGTCCGACGGGGGACGGGTCTATACTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 1)

[0029] The functional heavy chain variable region is 372 bases long, and the domains start from the first base and encode 124 amino acids.

[0030] The structural domain is defined using the IMGT method, and the specific structural domain is divided as follows:

[0031]

[0032] 2) The amino acid sequence of the heavy chain variable region of the acetamiprid-QA1 high-specificity monoclonal antibody is: QVTLKESGPGILQPSQTLSLTCSFSGFSLYSYDTGVGWIRQPSGKGLEWLANI WWNGYKFYNTDLRSRLTISKDPSNNQVFLTIANVNTADTSTYYCVRTYRSDG GRVYTMDFWGQGTSVTVSS (SEQ ID NO:2)

[0033] 3) The DNA sequence of the variable region of the light chain of the acetamiprid-QA1 high-specificity monoclonal antibody is: CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTTCTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAGCCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAGGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTTCAGCAACCTTTGGATATTCGGTGGAGGAACCAAACTGACTGTCCTA (SEQ ID NO:3)

[0034] The variable region of the functional light chain is 327 bases in length, and the domains start from the first base and encode 109 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 acetamiprid-QA1 high-specificity monoclonal antibody is: QAVVTQESALTTSPGETVTLTCRSSSGAVTTSNYANWVQEKPDHLFTGLIGG TNSRAPGVPARFSGSLIGDRAALTITGAQTEDEAIYFCALWFSNLWIFGGGTK LTVL (SEQ ID NO:4)

[0038] 3. Construction of recombinant full-length antibody expression plasmid

[0039] Homologous recombination technology was used to construct heavy and light chain recombinant plasmids containing variable and constant regions. VH and VL were cloned into the double-digested linearized expression vectors pCDNA3.4-Mouse-IgG1-CH and pCDNA3.4-Mouse-C, respectively. λ Above. pCDNA3.4-Mouse-IgG1-CH (HindIII / EcoRI) contains the mouse IgG1 heavy chain constant region gene. λ The (Hind III / BamHI) plasmid contains the mouse Lambda light chain constant region gene. The constructed expression plasmid was transformed into competent E. coli cells, cultured in a shaker, and subjected to resistance screening. Recombinants were selected for sequencing.

[0040] 4. Expression and activity evaluation of recombinant full-length antibody

[0041] This invention uses HEK293(F) cells as the expression system for the preparation of full-length IgG recombinant antibodies. The detection performance of the recombinant antibody and ascites monoclonal antibody was analyzed using a heterologous indirect competitive ELISA method to evaluate the effectiveness and authenticity of the antibody variable region sequence in this invention. The steps are as follows: HEK293(F) cells in good growth condition and passaged twice were seeded into culture flasks at a seeding density of 1.5 × 10⁻⁶ cells / year. 6 Cells / mL. Before transfection, the cells were incubated in suspension with shaking for 2 hours (37℃, 120 rpm, 5% CO2). Based on the sequencing results, the bacterial culture corresponding to the plasmid with the correct sequence was expanded and cultured further. After extracting the endotoxin-free plasmid using a kit, the heavy chain expression plasmid and light chain expression plasmid were added to the pre-prepared cell culture medium containing transfection reagent at a mass ratio of 2:3 and incubated at 37℃ for 15 minutes. Subsequently, the plasmid mixture was added dropwise to the cell culture medium, and the cells were incubated in suspension with shaking at 120 rpm for 5 days. The supernatant was then collected. Protein was eluted using a Protein A affinity chromatography column, and the recombinant full-length antibody product was obtained by dialysis with 0.01M PBS.

[0042] Heterogeneous indirect competitive ELISA results showed that the full-length IgG recombinant antibody rAb-QA1 detected the IC50 of acetamiprid.50 The concentration was 4.97 ng / mL, which is similar to the sensitivity of ascites monoclonal antibodies (IC50). 50: The concentration of 3.24 ng / mL indicates that the variable region sequence of the antibody obtained by sequencing is accurate and has recognition activity, and can be applied to the large-scale stable production of full-length IgG recombinant antibodies. Table 1 lists previously reported anti-acetamiprid monoclonal antibodies obtained using the same acetamiprid immunization hapten, showing cross-reactivity rates (10.6%–43.8%) with the acetamiprid structural analogue thiamethoxam. In contrast, the anti-acetamiprid antibody mAb-QA1 and its rAb-QA1 developed in this invention not only have high affinity but also strong specificity (cross-reactivity rate with thiamethoxam is less than 9%), making them highly suitable for the highly sensitive, accurate, and rapid detection of acetamiprid residues.

[0043] Table 1. Detection sensitivity of developed ELISAs to acetamiprid and cross-reactivity to thiamethoxam.

[0044]

[0045] 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 monoclonal antibody against acetamiprid, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:2; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

2. An expression plasmid for a full-length recombinant IgG antibody against acetamiprid, characterized in that: The nucleotide sequence contains a nucleotide sequence encoding the mouse heavy chain IgG1 constant region and a nucleotide sequence encoding the heavy chain variable region as described in claim 1; and also contains a nucleotide sequence encoding the mouse light chain Lambda constant region and a nucleotide sequence encoding the light chain variable region as described in claim 1.

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