PREPARATION AND APPLICATION OF AN INTACT RECOMBINANT ANTIBODY SPECIFIC TO CLOTHIANIDIN BASED ON THE IDENTIFIED VARIABLE REGION SEQUENCE

Abstract

A variable region sequence of a specific antibody against clothianidin is provided. A gene encoding a heavy chain variable region of the antibody has an amino acid sequence shown in SEQ ID NO: 2. An intact recombinant antibody against clothianidin is provided. The intact recombinant antibody includes 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. An immunostrip containing the antibody for rapid detection of clothianidin residue is also provided. The sequence genes obtained are linked 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 specific antibody against clothianidin, 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 specific antibody against clothianidin according to claim 1, wherein the gene encoding the heavy chain variable region has a nucleotide sequence shown in SEQ ID NO: 1.

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

5. An intact recombinant antibody against clothianidin, 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.

6. The intact recombinant antibody against clothianidin according to claim 5, wherein the gene encoding the heavy chain variable region has a nucleotide sequence shown in SEQ ID NO: 1.

7. The intact recombinant antibody against clothianidin according to claim 6, wherein a gene encoding the light chain variable region has an amino acid sequence shown in SEQ ID NO: 4.

8. The intact recombinant antibody against clothianidin according to claim 7, wherein the gene encoding the light chain variable region has a nucleotide sequence shown in SEQ ID NO: 3.

9. 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 an intact recombinant antibody against clothianidin.

10. An immunostrip for rapid detection of clothianidin residue, comprising the intact recombinant antibody against clothianidin according to claim 5.

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

12. An antibody expression plasmid, wherein a nucleotide sequence comprising the heavy chain variable region according to claim 2 and a mouse IgG1 heavy chain constant region expresses a heavy chain protein of an intact recombinant antibody against clothianidin.

13. An antibody expression plasmid, wherein a nucleotide sequence comprising the light chain variable region according to claim 2 and a mouse kappa light chain constant region expresses a light chain protein of a specific intact recombinant antibody against clothianidin.

14. 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 specific intact recombinant antibody against clothianidin.

15. An immunostrip for rapid detection of clothianidin residue, comprising the intact recombinant antibody against clothianidin according to claim 6.

16. An immunostrip for rapid detection of clothianidin residue, comprising the intact recombinant antibody against clothianidin according to claim 7.

17. An immunostrip for rapid detection of clothianidin residue, comprising the intact recombinant antibody against clothianidin according to claim 8.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] FIG. 2 illustrates the verification of the expression of the intact recombinant antibody against clothianidin in a mammalian cell HEK293F by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the present disclosure.

[0024] FIG. 3 illustrates ELISA competition curves of an intact recombinant antibody against clothianidin (pesticide concentrations, from low to high, are 0.01, 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 ng/mL).

[0025] FIG. 4 illustrates detection of clothianidin standard solutions by gold test strip based on the intact recombinant antibody (pesticide concentrations, from low to high, are CK (blank control), 0.63, 1.25, 2.5, 5, 10, 20, 40 ng/mL).

DESCRIPTION OF THE EMBODIMENTS

[0026] 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.

[0027] An intact recombinant antibody against clothianidin 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. The heavy chain constant region sequence thereof is a mouse IgG1 heavy chain constant region sequence, and the light chain constant region sequence of the antibody is a mouse kappa light chain constant region sequence.

[0028] Preparation of the Intact Recombinant Antibody Against Clothianidin

[0029] 1) Gene Amplification of Variable Region of Monoclonal Antibody Against Clothianidin

[0030] A hybridoma cell-line B2 prepared by mouse immunization, cell fusion and hybridoma cell screening could secrete specific antibody against clothianidin, and the subtype was IgG1/kappa. The total RNA of cell-line B2 was extracted by Trizol method and identified by agar gel electrophoresis. The integrity of RNA could meet the requirements of this experiment. RNA was used as a template to synthesize specific cDNA by reverse transcription using 5′Race technology (SMARTer RACE 5/3′ Kit). 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, and the specific downstream primers for the light chain are CTCATTCCTGTTGAAGCTCTTGACAATGGG and CTCATTCCTGTTGAAGCTCTTGACGACGGG.

[0031] The PCR amplification program was:

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

[0032] 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.

[0033] Monoclonal antibody B2 heavy chain variable region sequence is as follows:

TABLE-US-00002 (SEQ ID NO: 1) GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTC AGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAACTATGTTA TGCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATTT TTTTATCCTTACATTGATTATACTAAATACAATGAGATGTTCAAGGGCAA GGCCACACTGACTTCAGACACATCCTCCAGCACAGCCTACATGGAACTCA GCGGCCTGACCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGATCACAG TTCTTCTATAGATACGACTACTTTGACTACTGGGGCCAAGGCACCGCTCT CACAGTCTCCTCA

[0034] 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 IGHV1-14*01, and the matching rate is 94.9% for the V region and 97.9% for the J region.

[0035] 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 se- 1 to 76 to 100 to 151 to 174 to 289 to 331 to quence 75 99 150 174 288 330 363

[0036] The amino acid sequence of the monoclonal antibody B2 heavy chain variable region is as follows:

TABLE-US-00004 (SEQ ID NO: 2) EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVMHWVKQKPGQGLEWIGF FYPYIDYTKYNEMFKGKATLTSDTSSSTAYMELSGLTSEDSAVYFCARSQ FFYRYDYFDYWGQGTALTVSS

[0037] The amino acid sequence of the monoclonal antibody B2 light chain variable region is as follows:

TABLE-US-00005 (SEQ ID NO: 3) GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGA TCAAGCCTCCATCTCTTGCAGATCCAGTCAGAACATTGTGCACAGTAATG GAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAA GTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGATAGGTT CAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAACAGAGTGG AGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCA TTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA

[0038] 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 is 97.0% for the V region and 96.0% for the J region.

[0039] 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 se- 1 to 79 to 112 to 163 to 172 to 280 to 307 to quence 78 111 162 171 279 306 336

[0040] The amino acid sequence of the monoclonal antibody B2 light chain variable region is as follows:

TABLE-US-00007 (SEQ ID NO: 4) DVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTYLEWYLQKPGQSPK VLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGIYYCFQGSHVP FTFGSGTKLEIK

[0041] 2) Construction of Expression Vector

[0042] After identifying the correct heavy and light chain variable region genes of antibody B2, the correct gene sequence was synthesized. The heavy and light chain variable region genes were connected 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. Therefore, the constructed heavy or light chain recombinant plasmids contain heavy chain variable region gene and constant region gene or light chain variable region gene and constant region gene, respectively. The heavy or light chain expression vector was transformed into Ti competent cells, cultured under shaking, and then sequenced.

[0043] 3) Expression of the Intact Recombinant Antibody

[0044] The bacterial suspension corresponding to the correctly sequenced plasmid was cultured and amplified in a large volume, followed by extracting the plasmid for later use. HEK293F cells were resuscitated and cultured in suspension at 120 rpm, 8% CO.sub.2, and 37° C. for 3 generations. Before transfection, the cells were seeded into a new culture flask with a seeding density of 1.5×10.sup.6 cells/mL. After suspension culture for 2 h, the heavy-chain plasmid, light-chain plasmid and transfection reagent were mixed well into a certain ratio and incubate for 15 min and then transfected into HEK293F cells. The transfected cells were cultured in suspension until the survival rate was less than 70%. After centrifugation at 4000 rpm for 5 min, the supernatant was collected. 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).

[0045] Characterization of the Intact Recombinant Antibody

[0046] 1) Comparison of Anti-Clothianidin Intact Recombinant Antibody and Anti-Clothianidin Natural Mouse Ascitic Monoclonal Antibody by ELISA

[0047] In the early preparation stage of parental ascitic monoclonal antibodies, the assay sensitivity difference was compared between the homologous competitive indirect ELISA (coating antigen was clothianidin-OVA) and the heterologous competitive indirect ELISA (coating antigen was imidaclothiz-OVA). It was found that the sensitivity of the ELISA method was higher when imidaclothiz-OVA was used as a heterologous coating antigen. Therefore, in the present disclosure, the heterologous competitive indirect ELISA was used to evaluate the sensitivity and specificity of the intact recombinant antibody against clothianidin. A 96-well plate was coated with imidaclothiz-OVA as the heterologous competitive antigen; subsequently, ELISA was performed using a rabbit anti-mouse IgG-HRP as a detection antibody, tetramethylbenzidine (TMB) as a reaction substrate, and clothianidin and other neonicotinoid pesticide standards as analytes. The results (FIG. 3) are as follows:

[0048] a) Detection Range

[0049] The detection range of the intact recombinant antibody against clothianidin for ELISA (IC.sub.20-IC.sub.80) was 0.92-23.04 ng/mL.

[0050] The detection range of the ascitic monoclonal antibody against clothianidin for ELISA (IC.sub.20-IC.sub.80) was 1.45-20.01 ng/mL.

[0051] b) Sensitivity (IC.sub.50)

[0052] The IC.sub.50 of the intact recombinant antibody against clothianidin was 4.62 ng/mL.

[0053] The IC.sub.50 of the ascitic monoclonal antibody against clothianidin was 5.39 ng/mL.

[0054] c) Specificity by ELISA

[0055] In the present disclosure, the specificity of the antibody was evaluated by detecting the cross-reactivity with seven common neonicotinoid insecticides. The data of ELISA showed that the intact recombinant antibody and mouse monoclonal antibody prepared in the present disclosure had no cross-reactivity with other seven common neonicotinoid insecticides (cross-reactivity <0.1%), and could be used for specific analysis of clothianidin. The results also showed that the intact recombinant antibody had highly consistent selectivity with the parental monoclonal antibody.

[0056] 2) Characterization of Antibody Binding Kinetics by Surface Plasmon Resonance (SPR)

[0057] The CM7 chip was used in this invention, and the optimal pH for both antibodies was 5.0. The final signal values of the antibodies coupled on the chip were 38663 RU and 39854 RU, respectively. Kinetic experiments were performed to determine the affinity of both the parental ascitic monoclonal antibody and the intact recombinant antibody to clothianidin at a range of analyte concentrations (50, 25, 12.5, 6.25, 3.12, 1.623, and 0.78 nM). The KD were 5.05×10.sup.−9 M and 3.24×10.sup.−9 M, respectively, indicating that the affinity of the intact recombinant antibody to clothianidin was very close to that of the ascitic antibody.

Example 3: Application of the Intact Recombinant Antibody on Gold Test Strips

[0058] As verified by ELISA and SPR method, the intact recombinant antibody against clothianidin prepared by this method can replace the ascitic antibody. In order to achieve the rapid detection of clothianidin, this invention prepares a competitive immunochromatographic test strip for clothianidin detection.

[0059] 1) Preparation of Gold-Labelled Antibody

[0060] The optimal buffer for labeling in this method was determined to be 0.01 M PBS, the optimal pH was 6.5, and the optimal amount of labelled antibody was 75 μg/mL. An appropriate amount of antibody was added into 10 mL of colloidal gold solution and mixed well. After incubation at room temperature for 1 h, the gold-labelled antibody solution was blocked by 10% BSA and 1% PEG 20000 (the final concentrations were 1% and 0.1%, respectively), and then incubated at room temperature for 1 h. After centrifuging at 13500 g, 4° C. for 30 min, the collected sediment was redissolved with the buffer containing 1% BSA and 0.1% PEG20000 pH6.5, and centrifuged again to remove the unbound antibody. Then, the sediment was resuspended with 1 mL of the buffer (containing 5% sucrose and 1% BSA) for later use.

[0061] 2) Assembly of Test Strips

[0062] Immunochromatographic test strips were made of plastic liner, absorbent pad, NC membrane, sample pad (pre-treated with 1% BSA, 0.1% PEG20000, 0.25% PVP), gold-labelled conjugate pad, test line (0.15 mg/mL of imidaclothiz-OVA) and control line (0.09 mg/mL of goat anti-mouse IgG), assembled according to the conventional test strip assembly method.

[0063] 3) Sensitivity Experiment of Test Strip

[0064] Clothianidin stock solution of 100 μg/mL was prepared with pure methanol, and a series of standard working solutions at 0.63, 1.25, 2.5, 5, 10, 20, and 40 ng/mL were prepared with 0.01M PBS buffer to test the same batch of strips. The test results are shown in FIG. 4. Judging by naked eyes, when the concentration of clothianidin was above 2.5 ng/mL, the color of T line was obviously weakened. When the concentration of clothianidin was above 10 ng/mL, the T line disappeared. And other seven neonicotinoid pesticide solutions were used to test the specificity of the test strip. The standard solution was diluted with 0.01M PBS to 1000 ng/mL for testing. After 15 minutes, compared the color of the test strip T line with the results of clothianidin, and the specificity of the method could be determined according to the color of the T line. The results showed that compared with the color of T line from the sample CK (blank control) without any pesticide, the color of T lines from the seven structural analogs of clothianidin was not significantly weakened, so the results were negative.

[0065] In short, the gold test strips based on the recombinant antibody against clothianidin in the present disclosure has strong specificity and high sensitivity. The visual LOD of clothianidin is 2.5 ng/mL, which can be used for rapid detection of clothianidin residues in environment and agricultural products.

[0066] The above method is used to explain the invention, not to limit. Any modifications and changes made to the invention within the spirit of the invention and the protection scope of the claims fall within the protection range of the invention.