VARIABLE REGION SEQUENCE OF BROAD-SPECTRUM ANTIBODY AGAINST CLOTHIANIDIN AND DINOTEFURAN AND PREPARATION OF INTACT RECOMBINANT ANTIBODY THEREOF

Abstract

The present disclosure provides a variable region sequence of a broad-spectrum antibody against clothianidin and dinotefuran, where a gene encoding a heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 2. The present disclosure further discloses a broad-spectrum intact recombinant antibody against clothianidin and dinotefuran, including a heavy chain constant region, a heavy chain variable region, a light chain constant region, and a light chain variable region, where a gene encoding the heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 2. The sequence genes obtained by the present disclosure are ligated to an expression vector containing a heavy chain constant region gene and a light chain constant region gene, respectively, and an intact recombinant antibody is expressed and obtained by using mammalian cells with a double-plasmid system.

Claims

1. A variable region sequence of a broad-spectrum antibody against clothianidin and dinotefuran, wherein a gene encoding a heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 2.

2. The variable region sequence of a broad-spectrum antibody against clothianidin and dinotefuran according to claim 1, wherein the gene encoding a heavy chain variable region has a nucleotide sequence shown in SEQ ID NO: 1.

3. The variable region sequence of a broad-spectrum antibody against clothianidin and dinotefuran according to claim 1, wherein a gene encoding a light chain variable region has an amino acid sequence shown in SEQ ID NO: 4.

4. The variable region sequence of a broad-spectrum antibody against clothianidin and dinotefuran according to claim 2, wherein a gene encoding a light chain variable region has an amino acid sequence shown in SEQ ID NO: 4.

5. The variable region sequence of a broad-spectrum antibody against clothianidin and dinotefuran according to claim 3, wherein the gene encoding a light chain variable region has a nucleotide sequence shown in SEQ ID NO: 3.

6. The variable region sequence of a broad-spectrum antibody against clothianidin and dinotefuran according to claim 4, wherein the gene encoding a light chain variable region has a nucleotide sequence shown in SEQ ID NO: 3.

7. A broad-spectrum intact recombinant antibody against clothianidin and dinotefuran, comprising a heavy chain constant region, a heavy chain variable region, a light chain constant region, and a light chain variable region, wherein a gene encoding the heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 2.

8. The broad-spectrum intact recombinant antibody against clothianidin and dinotefuran according to claim 7, wherein the gene encoding the heavy chain variable region has a nucleotide sequence shown in SEQ ID NO: 1.

9. The broad-spectrum intact recombinant antibody against clothianidin and dinotefuran according to claim 8, wherein a gene encoding the light chain variable region has an amino acid sequence shown in SEQ ID NO: 4.

10. The broad-spectrum intact recombinant antibody against clothianidin and dinotefuran according to claim 9, wherein the gene encoding the light chain variable region has a nucleotide sequence shown in SEQ ID NO: 3.

11. An antibody expression plasmid, wherein a nucleotide sequence comprising the heavy chain variable region according to claim 1 and a mouse IgG1 heavy chain constant region expresses a heavy chain protein of a broad-spectrum intact recombinant antibody against clothianidin and dinotefuran.

12. The antibody expression plasmid of claim 11, wherein the gene encoding a heavy chain variable region has a nucleotide sequence shown in SEQ ID NO: 1.

13. An antibody expression plasmid, wherein a nucleotide sequence comprising the light chain variable region according to claim 3 and a mouse kappa light chain constant region expresses a light chain protein of a broad-spectrum intact recombinant antibody against clothianidin and dinotefuran.

14. The antibody expression plasmid of claim 13, wherein the gene encoding a light chain variable region has a nucleotide sequence shown in SEQ ID NO: 3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 illustrates the agarose gel electrophoresis results of PCR amplification of a heavy chain variable region gene and a light chain variable region gene using cDNA as a template obtained by reverse transcription based on 5′RACE technology in the present disclosure.

[0022] FIG. 2 illustrates the verification of the expression of an intact recombinant antibody in a mammalian cell HEK293F by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the present disclosure.

[0023] FIG. 3 illustrates ELISA competition curves of an intact recombinant antibody against clothianidin and dinotefuran (pesticide concentrations, from low to high, are 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 ng/mL).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] To enable those skilled in the art to better understand the solutions of the present disclosure, the technical solutions in the examples of the present disclosure will be clearly and completely described below in conjunction with the accompanying drawings in the examples of the present disclosure. Obviously, the described examples are only a part of, not all of, the examples of the present disclosure. Based on the examples of the present disclosure, all other examples obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present disclosure. Unless otherwise specified, all experimental methods in the examples are conventional.

[0025] A broad-spectrum intact recombinant antibody against clothianidin and dinotefuran provided by the present disclosure includes a heavy chain constant region, a heavy chain variable region, a light chain constant region, and a light chain variable region. A heavy chain variable region sequence thereof is a mouse IgG1 heavy chain constant region sequence, and a light chain constant region sequence of the antibody is a mouse kappa light chain constant region sequence.

Preparation of the Intact Recombinant Antibody Against Clothianidin and Dinotefuran

1) Gene Amplification of Variable Region of Monoclonal Antibody Against Clothianidin and Dinotefuran

[0026] A hybridoma cell line G4 that could secrete antibodies that recognize both clothianidin and dinotefuran was used as a material, and the antibodies secreted thereby were identified as an IgG1 heavy chain and a kappa light chain. Total RNA was extracted from the hybridoma cell line by the Trizol method, and the 5′-end cDNA was amplified by 5′RACE technology. Adapter primers and subtype-specific primers were used to obtain heavy and light chain variable region genes of the antibody. Herein, upstream primers for the heavy and light chains were the adapter primers included in the kit; the specific downstream primer for the heavy chain is CTCAATTTTCTTGTCCACCTTGGT (SEQ ID NO: 5), and the specific downstream primers for the light chain are CTCATTCCTGTTGAAGCTCTTGACAATGGG (SEQ ID NO: 6) and CTCATTCCTGTTGAAGCTCTTGACGACGGG (SEQ ID NO: 7).

[0027] The PCR amplification program was:

TABLE-US-00001 95° C. for 45 s {close oversize brace} 68° C. for 45 s 25 cycles 72° C. for 3 min

[0028] The agarose gel electrophoresis results of PCR amplified products are shown in FIG. 1. Bands containing VH and VL gene fragments were amplified. Purified by a gel extraction kit, purified products were cloned into a pEASY-Blunt vector, transformed, and sequenced. After sequences were aligned to the NCBI database by Blast, VH and VL genes with intact sequences, consistent subtypes, and correct expression cassettes were identified.

[0029] G4 heavy chain variable region sequence is as follows:

TABLE-US-00002 (SEQ ID NO: 1) CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAG AGCCTGTCCATCACTTGCACTATCTCTGGGTTTTCATTAACCAACTAT GGTGTTCACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTG GGAGTGATATGGGCTGGTGGAAACACAAATTATAATTCGGCTCTCATG TCCAGACTGAGCATCAGCAAAGACAACTCCAAGACCCAAGTTTTCTTG AGAATGAACAGTCTGCAAACTGATGATACAGCCATGTACTACTGTGCC AGCCCTTTACGGCCGCCGCGATATTACTATGGTTTGGACTACTGGGGT CAAGGAACCTCAGTCACCGTGTCCTCA

[0030] The functional heavy chain variable region has a full length of 363 bases. Starting from base 1, the domain encodes 121 amino acids. The functional heavy chain belongs to IGHV2-9*02, and the matching rate of the V region is 96.9%.

[0031] The domain is defined by IMGT method, and the specific domain is divided into the following:

TABLE-US-00003 Domain FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Base 1 to 76 to 100 to 151 to 172 to 286 to 331 to sequence 75 99 150 171 285 330 363

[0032] The amino acid sequence of the G4 heavy chain variable region is as follows:

TABLE-US-00004 (SEQ ID NO: 2) QVQLKESGPGLVAPSQSLSITCTISGFSLTNYGVHWVRQPPGKGLEWL GVIWAGGNTNYNSALMSRLSISKDNSKTQVFLRWINSLQTDDTAMYYC ASPLRPPRYYYGLDYWGQGTSVTVSS

[0033] The amino acid sequence of the G4 light chain variable region is as follows:

TABLE-US-00005 (SEQ ID NO: 3) GATGTTTTGATGACCCAAAGTCCACTCTCCCTGCCTGTCAGTCTTGGA GATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTTCATAGT AATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCT CCAAAGCTCCTGATCTATAAAGTTTCCAACCGATTTTCTGGGGTCCCA GACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCATACTCAAGATC AGTAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGC TCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0034] The functional light chain variable region has a full length of 336 bases. Starting from base 1, the domain encodes 112 amino acids. The functional light chain belongs to IGKV1-117*01, and the matching rate of the V region is 98.0%.

[0035] The domain is defined by IMGT method, and the specific domain is divided into the following:

TABLE-US-00006 Domain FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 Base 1 to 79 to 112 to 163 to 172 to 280 to 307 to sequence 78 111 162 171 279 306 336

[0036] The amino acid sequence of the G4 light chain variable region is as follows:

TABLE-US-00007 (SEQ ID NO: 4) DVLMTQSPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQS PKLLIYKVSNRFSGVPDRFSGSGSGTDFILKISRVEAEDLGVYYCFQG SHVPYTFGGGTKLEIK

2) Construction of Expression Vector

[0037] The heavy and light chain variable region genes were ligated to linearized expression vectors pCDNA3.4-Mouse-IgG1 and pCDNA3.4-Mouse-Kappa by homologous recombination, respectively. Herein, the pCDNA3.4-Mouse-IgG1 contained a mouse IgG1 heavy chain constant region gene, and the pCDNA3.4-Mouse-Kappa contained a mouse kappa light chain constant region gene. The heavy/light chain expression vector was transformed into T1 competent cells, cultured under shaking, and sequenced.

3) Expression of the Intact Recombinant Antibody

[0038] The bacterial suspension corresponding to the correctly sequenced plasmid was cultured and amplified in a large volume, and an EndoFree Plasmid Maxi Kit was used to extract the heavy/light chain expressing plasmid, respectively. In the early stage of transfection, HEK293F cells were resuscitated and cultured in suspension at 120 rpm, 8% CO.sub.2, and 37° C. to a density of 3×10.sup.6 cells/mL; a first passage was carried out with a density of 0.3×10.sup.6 cells/mL. When the cell density during the first passage was proliferated to 3×10.sup.6 cells/mL, a second passage was carried out with a density of 0.3×10.sup.6 cells/mL. When the cell density reached about 3×10.sup.6 cells/mL, it was ready for transfection and expression. Before transfection, the cells were seeded into a new culture flask with a seeding density of 1.5×10.sup.6 cells/mL. A heavy chain plasmid, a light chain plasmid, and a liposome transfection reagent were mixed well with culture medium in advance, and allowed to stand at 37° C. for 15 min. The above transfection buffer was added dropwise to the suspension cell culture medium, and shaken well while adding. The transfected cells were cultured in suspension for five days to collect a supernatant. The protein A affinity chromatography column The intact recombinant antibody in the cell supernatant was purified by Protein A affinity chromatography, and the liquid flow rate during the purification was 1 mL/min. The purified antibody was dialyzed overnight with 0.01 M PBS solution. After dialysis, the antibody concentration was determined to be 3 mg/mL, which was verified by SDS-PAGE (FIG. 2).

Characterization of the Intact Recombinant Antibody

1) Establishment of Heterologous Indirect Competitive ELISA for the Intact Recombinant Antibody

[0039] In the early stage of preparation of parental ascitic monoclonal antibodies, a difference in sensitivity was compared between the homologous indirect competitive ELISA (coating antigen was clothianidin-OVA) and the heterologous indirect ELISA (coating antigens were thiacloprid-OVA and imidaclothiz-OVA). It was found that the sensitivity of the ELISA method was the highest when the thiacloprid-OVA was used as a heterologous coating antigen. Therefore, in the present disclosure, the heterologous indirect competitive ELISA using thiacloprid-OVA as the coating antigen was used to evaluate the sensitivity and specificity of the intact recombinant antibody against clothianidin and dinotefuran. A 96-well plate was coated with the thiacloprid-OVA as the heterologous coating antigen; subsequently, ELISA was performed using a rabbit anti-mouse IgG-HRP as a detection antibody, tetramethylbenzidine (TMB) as a reaction substrate, and clothianidin, dinotefuran, and other structural analog pesticide standards as analytes.

1) Sensitivity of Intact Recombinant Antibody (IC.SUB.50.)

[0040] According to the inhibitory rate and the concentrations of clothianidin and dinotefuran, standard curves were plotted and established (FIG. 3), and the median inhibitory concentrations (IC.sub.50) and linear ranges (IC.sub.20 to IC.sub.80) of clothianidin and dinotefuran were calculated, respectively. Herein, the IC.sub.50 of clothianidin toward the intact recombinant antibody was 5.22 ng/mL, with a linear range of 1.08-15.06 ng/mL; the IC.sub.50 of dinotefuran toward the intact recombinant antibody was 10.66 ng/mL, with a linear range of 3.78-30.03 ng/mL.

2) Specificity of the Intact Recombinant Antibody

[0041] In the present disclosure, the specificity of the antibody was evaluated by detecting the cross-reactivity with eight common neonicotinoid insecticides. The data of heterologous indirect competitive ELISA showed that the intact recombinant antibody prepared in the present disclosure could specifically recognize clothianidin and dinotefuran, but had no apparent cross-reactivity with imidacloprid, thiacloprid, acetamiprid, imidaclothiz, thiamethoxam, and nitenpyram (IC.sub.50>1,000 ng/mL, cross-reactivity <0.5%); the prepared intact recombinant antibody could be used for specific analysis of clothianidin and dinotefuran.

[0042] 3) Kinetic Surface Plasmon Resonance (SPR) Characterization of the Intact Recombinant Antibody

[0043] In the present disclosure, the affinity of the intact recombinant antibody to a target analyte was detected by Biacore T200.

[0044] Kinetic determination analyzed the affinity of the intact recombinant antibody to clothianidin or dinotefuran at a series of analyte concentrations. The results showed that the affinity of the intact recombinant antibody to clothianidin was 7.96×10.sup.−9 M and the affinity thereof to dinotefuran was 7.56×10.sup.−9 M. Therefore, it was found that the intact recombinant antibody prepared in the present disclosure had a high affinity to both clothianidin and dinotefuran.