A specific monoclonal antibody against chlorpyrifos and its recombinant full-length antibody expression plasmid

Abstract

The present invention discloses a specific monoclonal antibody against chlorpyrifos: 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 of the monoclonal antibody is shown in SEQ ID NO:4. The present invention also discloses an antibody expression plasmid. The variable region sequence genes obtained by the present invention are respectively connected to expression vectors containing heavy chain constant region genes and light chain constant region genes, and two recombinant full-length antibodies (rAb‑13C7 and rAb‑14G4) are obtained based on mammalian cell expression and purification by double plasmid transfection. Indirect competitive ELISA analysis shows that both have detection activities similar to those of the parent ascites antibody. The above variable region sequence can be applied to the stable production of multiple batches of anti-chlorpyrifos recombinant antibodies, providing a reliable and stable core reagent for the construction of immunoassay methods for chlorpyrifos residues in the environment and food and the development of precise and rapid test products.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a specific monoclonal antibody against chlorpyrifos and a recombinant full-length antibody expression plasmid thereof. Background Art

[0002] As an efficient organophosphorus pesticide, chlorpyrifos has contact, stomach and fumigation effects on mites, chewing and sucking pests, etc., and is widely used in a variety of important economic crops such as corn, soybean, wheat, rice, cotton, etc. This type of insecticide inhibits acetylcholinesterase (AChE) in the body, affects the hydrolysis of the neurotransmitter acetylcholine, and then causes its accumulation in the nerve synapse, resulting in continuous excitation of the postsynaptic membrane, and ultimately causing a series of poisoning symptoms such as abnormal excitation, spasm, and death in the target organism. In recent years, the harm of chlorpyrifos to the health of humans and non-target organisms (such as pollinating insects like bees) has attracted wide global attention. However, due to unreasonable use and illegal addition of hidden components, etc., it has been found through research that the chlorpyrifos residues in environmental and food samples such as surface water and fruits and vegetables in many countries exceed the standard, which poses a threat to the ecological environment diversity and human health. Since 2016, the UK has completely banned products containing chlorpyrifos. Subsequently, the states of Hawaii and California in the United States and the European Union have successively banned chlorpyrifos, and other states in the United States, Canada, Australia, Brazil and other countries have also imposed certain restrictions on its use. In February 2022, the US Environmental Protection Agency revoked the residue limit for chlorpyrifos and will comprehensively ban the food uses related to chlorpyrifos. The Announcement No. 2032 of the Ministry of Agriculture of China shows that since December 31, 2013, China has stopped accepting the registration of chlorpyrifos on vegetables. Therefore, strengthening the detection of chlorpyrifos residues is of great significance for protecting the ecological environment, ensuring food safety, and guiding the scientific use of pesticides.

[0003] At present, the detection methods for chlorpyrifos mainly rely on traditional instrumental analysis methods such as liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Instrumental analysis methods have high precision, accuracy, and sensitivity. However, due to the limitations of expensive instrument equipment, professional technical operations, and cumbersome sample pretreatment processes, they are difficult to apply to on-site rapid detection. Pesticide immunoassay methods based on the principle of specific recognition of antigen and antibody are accurate, rapid, and highly sensitive, and are very suitable for rapid screening of pesticide residues in large-scale samples. Antibodies are the core reaction elements in pesticide immunoassay methods, directly affecting the sensitivity and specificity of detection methods. At present, most immunoassay methods mainly use monoclonal antibodies (mAbs), which have disadvantages such as long production cycles and high R & D costs. Moreover, long-term culture faces bottleneck problems such as chromosome loss or mutation, which affect the stability between batches of mAbs and the reproducibility and reliability of detection methods. In recent years, the international community has widely called for replacing traditional ascites mAbs with "non-animal-derived" recombinant antibodies. With the continuous development of genetic engineering technology, a new generation of recombinant antibodies has gradually become a research hotspot.

[0004] Expressing exogenous antibody genes using model organisms such as bacteria, yeast, animal cells, and plant cells is the mainstream idea for the preparation of current recombinant antibodies. Mammalian cell systems represented by human embryonic kidney cells (HEK293) can correctly direct the folding of antibody structures and provide processes such as post-translational processing and modification, and can stably express active proteins close to natural antibody molecules. This not only provides an effective and reliable way to verify the accuracy of the variable region sequences of parental antibodies, but also avoids the disadvantages of large differences between batches, poor stability, and long production cycles of traditional antibodies. It can mass-produce antibodies with good uniformity and high homogeneity under the condition of avoiding the use of a large number of experimental animals, which has important guiding significance for the R & D of stable and reliable rapid pesticide residue detection products. According to different structures, recombinant antibodies are further divided into recombinant full-length antibodies and fragment antibodies (such as single-chain antibodies and Fab fragment antibodies). Compared with the latter, the former has obvious advantages in affinity, half-life, and stability due to its configuration similar to that of natural immunoglobulins. At present, there is no public report on recombinant full-length antibodies against chlorpyrifos. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the present invention provides a specific monoclonal antibody against chlorpyrifos and an antibody expression plasmid.

[0006] The technical solution adopted by the present invention to solve its technical problems is as follows: The amino acid sequence of the heavy chain variable region of the specific monoclonal antibody against chlorpyrifos: mAb-13C7 is shown in SEQ ID NO:2; the amino acid sequence of the heavy chain variable region of mAb-14G4 is shown in SEQ ID NO:6.

[0007] Preferably, the nucleotide sequence of the encoding gene of the heavy chain variable region of mAb-13C7 is shown in SEQ ID NO:1; the nucleotide sequence of the encoding gene of the heavy chain variable region of mAb-14G4 is shown in SEQ ID NO:5.

[0008] Preferably, the amino acid sequence of the encoding gene of the light chain variable region of mAb-13C7 is shown in SEQ ID NO:4; the amino acid sequence of the encoding gene of the light chain variable region of mAb-14G4 is shown in SEQ ID NO:8.

[0009] Preferably, the nucleotide sequence of the encoding gene of the light chain variable region of mAb-13C7 is shown in SEQ ID NO:3; the nucleotide sequence of the encoding gene of the light chain variable region of mAb-14G is shown in SEQ ID NO:7.

[0010] Preferably, mAb-13C7 and mAb-14G4 are derived from the same artificial antigen of chlorpyrifos, and the heavy and light chain subtypes are both IgG1 and Kappa.

[0011] The present invention also discloses an antibody expression plasmid, which contains the nucleotide sequences of the heavy chain variable region and the mouse heavy chain IgG1 constant region as described above, and can express the heavy chain protein of the specific recombinant full-length antibody against chlorpyrifos.

[0012] Preferably, it contains the nucleotide sequences of the light chain variable region and the mouse light chain Kappa constant region as described above, and can express the light chain protein of the specific recombinant full-length antibody against chlorpyrifos.

[0013] The present invention is applicable to the construction of immunoassay methods for the determination of chlorpyrifos residues in environmental and food samples and the development of precise and rapid detection products.

[0014] The present invention takes two hybridoma cell lines (clone numbers 13C7 and 14G4) that can stably secrete antibodies recognizing chlorpyrifos as the object, and completes the development and purification of monoclonal antibodies in mouse ascites for this cell line, as well as the amplification, sequencing and synthesis of the variable region genes of the heavy and light chains of the antibody. Based on homologous recombination technology, heavy chain and light chain expression vectors are constructed respectively, and the two plasmids are transfected into mammalian cells HEK 293(F). After cultivation and purification, two recombinant full-length antibodies against chlorpyrifos are obtained. Using chlorpyrifos as the detection object, the accuracy of the variable region sequence is verified by the indirect competitive ELISA (Indirect competitive ELISA, ic-ELISA) method. The results show that both recombinant full-length antibodies against chlorpyrifos (rAb-13C7 and rAb-14G4) have the same high affinity as the mouse parental ascites monoclonal antibodies (mAb-13C7 and mAb-14G4), indicating that the variable region sequence of the specific monoclonal antibody against chlorpyrifos is effective and reliable. At the same time, the cross-reactivity rate of one of the recombinant full-length antibodies, rAb-13C7, against methyl chlorpyrifos is less than 5%, which belongs to a highly specific antibody.

[0015] The variable region sequence genes obtained in the present invention are respectively ligated to expression vectors containing the heavy chain constant region gene and the light chain constant region gene. By using double plasmid transfection, two recombinant full-length antibodies (rAb-13C7 and rAb-14G4) are obtained based on mammalian cell expression and purification. Through indirect competitive ELISA analysis, it is clear that both have detection activities similar to those of the parental ascites antibody. The above variable region sequence can be applied to the stable production of multiple batches of recombinant antibodies against chlorpyrifos, providing a reliable and stable core reagent for the construction of immunoassay methods for chlorpyrifos residues in the environment and food and the development of accurate and rapid detection products.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The present invention discloses the variable region sequences of two monoclonal antibodies (mAb) against chlorpyrifos, including the heavy chain variable region and the light chain variable region sequences. By constructing a heavy chain gene expression vector (containing the heavy chain variable region gene and the heavy chain constant region gene) and a light chain gene expression vector (containing the light chain variable region gene and the light chain constant region gene), based on a dual plasmid transfection system, the reliability and accuracy of the above variable region sequences were verified using a mammalian cell HEK293(F) recombinant expression system. Verified by ELISA, the two recombinant full-length antibodies (rAb-13C7 and rAb-14G4) involved in the present invention have sensitivities similar to those of the corresponding ascites monoclonal antibodies, and the cross-reactivity rate of rAb-13C7 to methylchlorpyrifos is less than 5%, belonging to highly specific antibodies. The variable region sequences of the present invention can be applied to the stable expression among multiple batches of specific recombinant full-length antibodies against chlorpyrifos, which is conducive to promoting the construction of immunoassay methods for chlorpyrifos residues and the development of their precise and rapid detection products. Brief Description of the Drawings

[0018] Figure 1 This is the result of agarose gel electrophoresis for PCR amplification of the antibody variable region using the cDNA obtained by reverse transcription as a template in the present invention (electrophoresis diagram of the fragment sizes of the heavy and light chain variable regions).

[0019] Figure 2 This is the specific recognition ability of two ascites mAbs, mAb-13C7 (A) and mAb-14G4 (B), against eight organophosphorus pesticides (1 mg / L) analyzed by ic-ELISA in the present invention.

[0020] Figure 3 This is the standard curve for detecting chlorpyrifos and methylchlorpyrifos constructed by ic-ELISA using two recombinant full-length antibodies in the present invention. (A) Recombinant antibody rAb-13C7 and ascites mAb-13C7; (B) Recombinant antibody rAb-14G4 and ascites mAb-14G4.

[0021] Explanation of the Appendix

[0022] Table 1 shows the detection sensitivity of the developed ic-ELISAs for chlorpyrifos and their cross-reactivity to methylchlorpyrifos. Detailed Description of the Invention

[0023] To enable those skilled in the art to better understand the solution of the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the embodiments are all conventional methods.

[0024] The present invention relates to two novel specific monoclonal antibodies against chlorpyrifos. The amino acid sequences of the heavy chain variable region encoding genes are shown as SEQ ID NO:2 and SEQ ID NO:6 respectively, and the amino acid sequences of the light chain variable region encoding genes are shown as SEQ ID NO:4 and SEQ ID NO:8 respectively. The variable region sequences of the monoclonal antibodies can be applied to the large-scale and standardized production of recombinant antibodies, providing reliable and stable core reagents for the accurate and rapid detection of chlorpyrifos residues in the environment and food.

[0025] 1. Preparation and performance characterization of monoclonal antibodies against chlorpyrifos

[0026] Based on the traditional hybridoma technology, the present invention uses the artificial antigen CHBu-BSA prepared in the early stage of the laboratory. Through steps such as artificial antigen for mice, titer test, cell fusion, and limited dilution, two hybridoma cell lines CHBu-13C7 and CHBu-14G4 secreting monoclonal antibodies are screened. The cell lines are inoculated into the abdominal cavity of F1 mice injected with pristane. After ascites purification by the caprylic acid-ammonium sulfate precipitation method, monoclonal antibodies mAb-13C7 and mAb-14G4 are obtained. Identified by the subtype kit, the heavy and light chain subtypes of the above two monoclonal antibodies are both IgG1 and Kappa. The ic-ELISA method is used to characterize the sensitivity and specificity of the two antibodies, and the SPR technology is used to characterize the recognition selectivity and binding affinity of the mAb-14G4 antibody.

[0027] (1) Sensitivity: The coating antigen CHHe-OVA (3 mg / L) is coated onto a 96-well enzyme-linked immunosorbent assay (ELISA) plate, incubated, and blocked. 50 μL of chlorpyrifos standard solution diluted in a concentration gradient and 50 μL of the antibody solution at the optimal working concentration are added to each well respectively. After the competitive reaction is completed, an enzyme-labeled rabbit anti-mouse secondary antibody is used to amplify the detection signal, and 3,3',5,5'-tetramethylbenzidine (TMB) is used as the reaction substrate. After the reaction is terminated, the absorbance value of each well at 450 nm is measured. With the logarithmic concentration as the abscissa and the corresponding inhibition rate as the ordinate, a standard curve is constructed using GraphPad software according to the four-parameter fitting method. As Figure 2 shown, the ic-ELISA standard curve established by mAb-13C7 has a sensitivity to chlorpyrifos (IC50 ) was 2.26 μg / L, and the recognition linear range was 0.17 - 29.48 μg / L; the sensitivity (IC 50 ) of the ic-ELISA standard curve established by mAb-14G4 for chlorpyrifos was 44.21 μg / L, and the recognition linear range was 7.32 - 266.92 μg / L.

[0028] (2) Specificity: The present invention further evaluated the specificity of ascites monoclonal antibodies based on the ic-ELISA single-point inhibition method. Eight common organophosphorus pesticides including chlorpyrifos, methylchlorpyrifos, parathion, methyl parathion, triazophos, phoxim, isocarbophos, and fenitrothion were selected as target analytes, and the analysis concentration was 1 mg / L. The results were as Figure 2 shown. The ascites monoclonal antibodies mAb-13C7 and mAb-14G4 prepared by the present invention specifically recognized only chlorpyrifos and methylchlorpyrifos. Subsequently, an ELISA standard curve for methylchlorpyrifos was established, and the cross-reactivity rates of other compounds were calculated with reference to the sensitivity of chlorpyrifos. The calculation formula was CR(%) = IC 50 (chlorpyrifos) / IC 50 (other analytes) × 100%. The results were as Figure 3 , using the monoclonal antibodies developed by the present invention to establish the ic-ELISA method, the sensitivity IC 50 of mAb-13C7 for detecting methylchlorpyrifos was 88.83 ng / mL respectively, and the cross-reactivity rate was 2.5%. The sensitivity IC 50 of mAb-14G4 for detecting methylchlorpyrifos was 255.57 ng / mL respectively, and the cross-reactivity rate was 17%.

[0029] (3) Recognition selectivity test: The present invention was based on non-competitive direct binding SPR technology, and evaluated the antibody specificity by testing the binding-dissociation curves of eight common organophosphorus pesticides (concentration 100 nM). The results showed that the RU value of the antibody for binding to chlorpyrifos was 14, and the dissociation rate was slow; the RU value for methylchlorpyrifos was 9, and the dissociation rate was fast. The binding RU values of other organophosphorus pesticides (parathion, methyl parathion, triazophos, phoxim, isocarbophos, fenitrothion) were about 2, similar to the negative control. Although it seemed to be a "fast up and fast down" binding mode, there was no effective binding. This indicated that mAb-14G4 had high specificity and strong binding activity for chlorpyrifos, also had medium binding reaction to the structural analog methylchlorpyrifos, and did not recognize the remaining six organophosphorus pesticides.

[0030] (4) Affinity test: Based on SPR technology, the standard samples of chlorpyrifos and methylchlorpyrifos were serially diluted with PBS-P + running buffer, and the automatic injection program was executed successively from low concentration to high concentration. The samples flowed through Chip Flowcell 1 and 2 at a flow rate of 30 μL / min. Each sample was injected for 180 s and accompanied by a natural dissociation reaction for 600 s. A 10 mM glycine-HCl solution (pH = 2.0) was used to flow through the chip for 30 s for regeneration. The results showed that the binding of chlorpyrifos mAb-14G4 to chlorpyrifos belonged to the "fast binding - slow dissociation" mode, and its K a value was 2.17×10 5 (mol / L·s) -1 , and its K d value was 5.40×10 -4 (s -1 ), and its K D value was 2.49×10 -9 (mol / L). The rapid rise of the curve in the binding stage indicated rapid binding, and the slow decline of the curve in the dissociation stage indicated slow dissociation. The K D value at the 10 -9 level indicated that the affinity of mAb for chlorpyrifos was at the nanomolar level, belonging to high affinity. The binding of chlorpyrifos mAb-14G4 to methylchlorpyrifos belonged to the "fast binding - fast dissociation" mode, and its K a value was 2.02×10 5 (mol / L·s) -1 , and its K d value was 3.89×10 -3 (s -1 ), and its K D value was 1.93×10 -8 (mol / L). The rapid rise of the curve in the binding stage was similar to that of chlorpyrifos, and the rapid decline of the curve in the dissociation stage indicated a faster dissociation rate. The K D value at the 10 -8 level indicated that the affinity of mAb for methylchlorpyrifos was lower than the nanomolar level, one order of magnitude lower than that for chlorpyrifos.

[0031] 2. Amplification and identification of antibody variable region genes

[0032] Total RNA in hybridoma cell lines CHBu-13C7 and CHBu-14G4 was extracted by the one-step method using Trizol reagent, and the extraction process was referred to the instruction manual of the Takara kit. After identification by 1% agarose gel electrophoresis, the purity and integrity of the extracted total RNA samples were good, meeting the requirements of subsequent experiments. Using the prepared total RNA as a template, PrimeScript 1 stStrand cDNA Synthesis Kit, for reverse transcription to synthesize cDNA. Using antibody subtype-specific primers, after PCR amplification, the variable region of the heavy chain (VH) and the variable region of the light chain (VL) of the above-mentioned hybridoma monoclonal antibody were successfully obtained.

[0033] The procedure for PCR amplification was as follows:

[0034]

[0035] The results of agarose gel electrophoresis of the amplification products are shown in Figure 1 . The DNA target fragment was purified using a gel extraction kit, and the purified product was cloned into the pUC19-T vector containing an ampicillin resistance tag. After transformation with Escherichia coli, Sanger sequencing was performed. Using the NCBI database igBLAST for alignment and analysis, VH and VL genes with complete sequences, matching subtypes, and correct expression frames were identified.

[0036] The screened functional heavy and light chain variable region DNAs and amino acid sequences are as follows:

[0037] 1) The DNA sequence of the variable region of the heavy chain of chlorpyrifos mAb-13C7 was:

[0038] CAGGTCCAACTGCAGCAGTCTGGACCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAG

[0039] ATATCCTGCAAGGCTTCTGGCTACAGCTTCACTAACCAGTGGCTAGGTTGGGTTAAGG

[0040] AGAGGCCTGGACAAGGACTTGAGTGGATTGGAGATATGTACCCTAGAGGTGGTCATG

[0041] TTAATTACAATGAGAAGTTCAAGAATAAGGCCACACTGACTGCAGACACATCCTCCA

[0042] ACAGTGTCTACATACAACTCAGGAGCCTGACACCCGAGGACTCTGCTGTCTATTTCTG

[0043] CTATGACAACGGTAGGGGCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA(SEQ ID NO:1)

[0044] The full-length functional heavy-chain variable region is 342 bases, and the domain starts from the 1st base, encoding 114 amino acids.

[0045] The domain is defined by the IMGT method, and the specific domain division is as follows:

[0046] Domain FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Base sequence 1-75 76-99 100-150 151-174 175-288 289-309 310-342 Length 75 24 51 24 114 21 33

[0047] 2) The amino acid sequence of the heavy-chain variable region of chlorpyrifos mAb-13C7 is:

[0048] QVQLQQSGPELVRPGTSVKISCKASGYSFTNQWLGWVKERPGQGLEWIGDMYPRGGHV NYNEKFKNKATLTADTSSNSVYIQLRSLTPEDSAVYFCYDNGRGYWGQGTTLTVSS(SEQ ID NO:2)

[0049] 3) The DNA sequence of the light-chain variable region of chlorpyrifos mAb-13C7 is:

[0050] GACGTCAAGATGACCCAGTCTCCATCTTCCATGTTTGTATCTCTAGGAGAGAGAGTCACTCTCACTTGCAAGGCGAGTCAGGACATTAGTAGCCATTTGGCCTGGTTCCAGCAGAAACCAGGAAAGTCTCCTAAGACCCTGATCTATCATACAAGCAGATTGGTGGCTGGGGTCCCATCAAGGTTCAGTGGCCGTGGATCTGGGCAAGATTATTCTCTCACCATTAGCAGCCTGGAATATGAGGATATGAAAATTTATTATTGTCTACAGTATGATGAACTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAGA(SEQ ID NO:3)

[0051] The full-length functional light-chain variable region is 321 bases, and the domain starts from the 1st base, encoding 107 amino acids.

[0052] The domain is defined by the IMGT method, and the specific domain division is as follows:

[0053] Domain FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Base sequence 1-78 79-96 97-147 148-156 157-264 265-291 292-321 Length 78 18 51 9 108 27 30

[0054] 4) The amino acid sequence of the light chain variable region of chlorpyrifos mAb-13C7 is as follows:

[0055] DVKMTQSPSSMFVSLGERVTLTCKASQDISSHLAWFQQKPGKSPKTLIYHTSRLVAGVPS RFSGRGSGQDYSLTISSLEYEDMKIYYCLQYDELPYTFGGGTKLEIR (SEQ ID NO:4)

[0056] 5) The DNA sequence of the heavy chain variable region of chlorpyrifos mAb-14G4 is as follows:

[0057] GAGTGAAACTTGAGGAGTCTGGAGGAGGCTTGGTGCACTCTGGAGGATCCATGAAAC

[0058] TCTCCTGCGTTGACTCTGGATTCTCTTTCAGTAACTACTGGATGAACTGGGTCCGCCA

[0059] GTCTCCAGACAAGGGGCTTGAGTGGGTTGCTGAAATGAGATTGAAGTCTAATAATTA

[0060] TGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCCAGAGATGATTC

[0061] CAAAAGTAGTGTCTATCTGCAAATGAACAATCTAAGAACTGAAGACACTGGCATTTA

[0062] TTATTGTAATTGGCCCCTTACTATGGCATGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ IDNO:5)

[0063] The full length of the functional heavy chain variable region is 348 bases, and the domain starts from the first base and encodes 116 amino acids.

[0064] The domain is defined by the IMGT method, and the specific domain division is as follows:

[0065] Domain FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Base sequence 1-75 76-99 100-150 151-180 181-294 295-315 316-348 Length 75 24 51 30 114 21 33

[0066] 6) The amino acid sequence of the heavy chain variable region of chlorpyrifos mAb-14G4 is as follows:

[0067] EVKLEESGGGLVHSGGSMKLSCVDSGFSFSNYWMNWVRQSPDKGLEWVAEMRLKSNN

[0068] YATHYAESVKGRFTISRDDSKSSVYLQMNNLRTEDTGIYYCNWPLTMAWGQGTSVTVSS(SEQ IDNO:6)

[0069] 7) The DNA sequence of the light chain variable region of chlorpyrifos mAb-14G4 is:

[0070] GACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCA

[0071] CTATCACTTGCAAGGCGACTCAGGACATTAATAGGTATTTAGGCTGGTTCCAGCAGA

[0072] AACCAGGGAAATCTCCGAAGACCCTGATCTATCGTGCAGACAGAATGGTCGCTGGGG

[0073] TCCCATCAAGGTTCAGTGGGCGTGGATCTGGGCAAGATTATTCTCTGACCATCAGCAG

[0074] CCTGGAGTATGAAGATTTGGGAATTTATTATTGTCTACAGTATGATGGATTGCCGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAAA(SEQ ID NO:7)

[0075] The full length of the functional light chain variable region is 321 bases, and the domain starts from the 1st base, encoding 107 amino acids.

[0076] The domain is defined by the IMGT method, and the specific domain division is:

[0077] Domain FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Base sequence 1-78 79-96 97-147 148-156 157-264 265-291 292-321 Length 78 18 51 9 108 27 30

[0078] 8) The amino acid sequence of the light chain variable region of chlorpyrifos mAb-14G4 monoclonal antibody is:

[0079] DIKMTQSPSSMYASLGERVTITCKATQDINRYLGWFQQKPGKSPKTLIYRADRMVAGVPS RFSGRGSGQDYSLTISSLEYEDLGIYYCLQYDGLPYTFGGGTKLEIK(SEQ ID NO:8)

[0080] 3. Construction of recombinant full-length antibody expression plasmid

[0081] Recombinant plasmids of heavy and light chains containing variable regions and constant regions were constructed using homologous recombination technology. VH and VL were cloned into the expression vectors pCDNA3.4-Mouse-IgG1-CH and pCDNA3.4-Mouse-Cκ that had been linearized by double digestion, respectively. Among them, pCDNA3.4-Mouse-IgG1-CH (HindIII / EcoRI) contains the gene of the constant region of mouse IgG1 heavy chain, and pCDNA3.4-Mouse-Cκ (Hind III / BamHI) contains the gene of the constant region of mouse Kappa light chain. The artificially constructed expression plasmid was transformed into competent Escherichia coli cells. After shaking culture, resistance screening was carried out, and recombinants were selected for sequencing verification.

[0082] 4. Expression and activity evaluation of recombinant full-length antibody

[0083] In the present invention, HEK293(F) cells were selected as the expression system to prepare recombinant full-length antibodies. Based on the ic-ELISA method, the detection performances of two recombinant full-length antibodies and two ascites monoclonal antibodies were analyzed to evaluate the effectiveness and authenticity of the antibody variable region sequences in this invention. The steps are as follows: HEK293(F) cells with good growth state and having been passaged twice were inoculated into a culture flask at an inoculation density of 1.5×10 6 cells / mL. Before transfection, suspension shaking culture was carried out for 2 h (37 °C, 120 rpm, 5% CO 2 2); according to the correct plasmid corresponding to the sequencing result, the volume of the bacterial liquid was enlarged, and the culture was continued. The endotoxin-free plasmid was extracted using a kit. The heavy chain expression plasmid and the light chain expression plasmid were added to the cell culture medium containing transfection reagent according to a mass ratio of 2:3 and left standing at 37 °C for 15 min. Subsequently, the plasmid mixture was added dropwise to the cell culture medium, and the cells were cultured with shaking at 120 rpm for 5 d, and then the supernatant was collected. The protein was eluted using a Protein A affinity chromatography column, and the recombinant full-length antibody product was obtained by dialysis with 0.01 M PBS.

[0084] The ic-ELISA test results showed that the IC 50They were 3.01 and 42.84 ng / mL respectively, which were similar to the sensitivity of the monoclonal antibody in ascites (IC 50 They were 2.26 and 44.21 ng / mL respectively). This indicates that the antibody variable region sequences obtained by sequencing are accurate and have recognition activity, and can be applied to the large-scale stable production of recombinant full-length antibodies, and are suitable for the construction of immunoassay methods for chlorpyrifos and the development of precise rapid detection products. Comparing with the anti-chlorpyrifos monoclonal antibodies previously reported in Table 1, which were also obtained using the chlorpyrifos structural analog (CHBu) as the immunogenic hapten, they all had relatively high cross-reactivity rates (12.5% - 125%) with the chlorpyrifos structural analog methyl chlorpyrifos. However, the chlorpyrifos antibody mAb-13C7 and its rAb-13C7 developed in this invention not only have high affinity but also strong specificity (the cross-reactivity rate with methyl chlorpyrifos is less than 5%), and are very suitable for the highly sensitive, precise and rapid detection of chlorpyrifos residues.

[0085] The above specific embodiments are used to explain and illustrate the present invention, rather than to limit the present invention. Any modifications and changes made within the spirit and scope of the claims of the present invention fall within the protection scope of the present invention.

[0086] Table 1 Detection sensitivities of developed ic-ELISAs for chlorpyrifos and their cross-reactivities with methyl chlorpyrifos

[0087]

Claims

1. A specific monoclonal antibody against chlorpyrifos, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

4.

2. A gene encoding the specific monoclonal antibody against chlorpyrifos as claimed in claim 1, characterized in that: The nucleotide sequence of the heavy chain variable region of the gene encoding the specific monoclonal antibody against chlorpyrifos is shown in SEQ ID NO: 1, and the nucleotide sequence of the light chain variable region of the gene encoding the specific monoclonal antibody against chlorpyrifos is shown in SEQ ID NO:

3.

3. The specific monoclonal antibody against chlorpyrifos according to claim 1, characterized in that: The heavy chain subtype is IgG1, and the light chain subtype is Kappa .

4. An antibody expression plasmid, characterized in that: Contains a nucleotide sequence encoding a mouse heavy chain IgG1 constant region and a nucleotide sequence encoding a heavy chain variable region as claimed in claim 1, contains a nucleotide sequence encoding a mouse light chain Kappa The nucleotide sequence of the constant region and the nucleotide sequence encoding the light chain variable region as claimed in claim 1.

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