CN113150161A

CN113150161A - Anti-idiotype monoclonal antibody of bacillus thuringiensis Cry2Aa toxin and application

Info

Publication number: CN113150161A
Application number: CN202110580591.4A
Authority: CN
Inventors: 刘媛; 林曼曼; 高美静; 卢莉娜; 张霄; 徐重新; 刘贤金
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2021-05-26
Filing date: 2021-05-26
Publication date: 2021-07-23
Anticipated expiration: 2041-05-26
Also published as: CN113150161B

Abstract

The invention relates to a Bacillus thuringiensis Cry2Aa toxin anti-idiotype monoclonal antibody and application, the anti-idiotype monoclonal antibody takes an F (ab)' 2 fragment of a Cry2Aa toxin rabbit polyclonal antibody as an immunogen, and is obtained by screening hybridomas, and the amino acid sequences of a heavy chain and a light chain of the anti-idiotype monoclonal antibody are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; the anti-idiotype monoclonal antibody can compete the combination of Cry2Aa toxin and rabbit polyclonal antibody, can also generate specific combination with a diamond back moth cadherin toxin combination area, and has potential application in identification of potential receptors of Cry2Aa toxin in target insects and research of toxicological mechanism thereof.

Description

Anti-idiotype monoclonal antibody of bacillus thuringiensis Cry2Aa toxin and application

Technical Field

The invention relates to a bacillus thuringiensis Cry2Aa toxin anti-idiotypic monoclonal antibody and application thereof, belonging to the crossing field of immunology technology and plant protection technology.

Background

Yamamoto and McLaughlin were first isolated from parasporal crystals of the Bacillus thuringiensis subspecies kurstaki in 1981 as two insecticidal protein fractions with molecular weights of 130 kDa (P1) and 66 kDa (P2), respectively, of which the P1 protein (Cry 1 toxoid) is toxic to lepidopteran pests and the P2 protein (Cry 2 toxoid) is toxic to both lepidopteran and dipteran pests (Yamamoto T, McLaughlin RE. Isolation of a protein from the paraspore crystal of Bacillus-Thuringiensis var kurstaki toxic to the mosquito larva, Aedes-TaeniorhynchusBiochemical and Biophysical Research communications 1981,103: 414-421). In 1989, Widner et al cloned and sequenced from Bacillus thuringiensis Kurstaki subspeciescry2AaAndcry2Abgene (then namedcryB1AndcryB2genes) encoding 633 amino acids, with 87% sequence similarity, but with a large difference in insecticidal spectra (Widner WR, Whiteley HR. Two high related insecticidal proteins ofBacillus thuringiensis subsp. kurstaki possess different host range specificities. Journal of Bacteriology, 1989;171:965-974）。

Because the Cry2A toxin has weaker toxicity to some target pests than the Cry1 toxoid and the Cry2A toxin is developed later in commercialization, the analysis research on the biological activity of the Cry2A toxin to the target insects is relatively less at present, and the toxicological mechanism is not completely clear. In the receptor study, Qiu et al measured binding proteins of Cry2Aa toxin to Spodoptera exigua using dielectrophoresis and LC-MS, and predicted receptors include polycalin, V-Type ATPase subBunit A, V-Type ATPase subBunit B, actin, 4-hydroxybutyrate coenzyme A-transferase, and activated protein kinase C receptor. After the V-Type ATPase subBunit B gene of the beet armyworm is knocked out by an RNA interference method, the sensitivity of the beet armyworm to Cry2Aa toxin can be obviously reduced, and the fact that the V-Type ATPase subBunit B is a functional receptor of Cry2A toxin (Qiu L, Zhang B, Liu L, Ma W, Wang X, Lei C,et al. Proteomic analysis of Cry2Aa-binding proteins and their receptor function in Spodoptera exiguascientific Reports, 2017;7: 40222.). Onofre et al identified by Western Blot that Cry2Ab bound to tobacco moth BBMV at around 110 kDa, followed by mass spectrometry as aminopeptidase-N2, and confirmed by cloning and expression of this receptor that aminopeptidase-N2 is the binding receptor for Cry2Ab (Onofre J, Gaytan MO, Pena-Cardena A, Garcia-Gomez BI, Pachelo S, Gomez I,et al. Identification of aminopeptidase-N2 as a Cry2Ab binding protein in Manduca sexta. Peptides, 2017.）。

furthermore, although cadherins have been many times shown to be important functional receptors in Cry1A toxoids, there is controversy as to whether cadherins are functional receptors for Cry2Aa toxins in lepidopteran insects. Qiu et al demonstrated that heterologously expressed Cadherin repeats 7 through the membrane proximal extracellular domain (CR 7-MPED) could bind to Cry2Aa and that Cadherin was a functional receptor for Cry2Aa as demonstrated BY RNA interference assays (Qiu L, Hou LL, Zhang BY, Liu L, Li B, et al. 2015. Cadherin in the action of the expression of Cry2AaBacillus thuringiensis toxins Cry1Ac and Cry2Aa in the beet armyworm, Spodoptera exiguaJournal of Invertebrate Pathology 127: 47-53). Zhao et al demonstrated by Western blot analysis and RNA interference experiments that Helicoverpa armigera cadherin, aminopeptidase 4 and alkaline phosphatase are Functional receptors for Cry2Aa toxin (Zhao M, Yuan XD, Wei JZ, Zhang WN, Wang BJ, et al 2017. Functional roles of cadherin, aminopeptidase-N and alkaline phosphatase phosphorus fromHelicoverpa armigera (Hubner) in the action mechanism of Bacillus thuringiensisCry2aa. Scientific Reports 7). However, Gao et al showed that although Cry2Aa has binding ability to heterologously expressed Helicoverpa armigera Cadherin (HaCad) and Plutella xylostella (PxCad), high concentrations of Cry2Aa had no significant lethal effect on sf9 cells expressing fragments of HaCad and Plutella xylostella PxCad, thereby concluding that Cadherin is not a functional receptor for Cry2Aa (Gao MJ, Dong S, Hu XD, Zhang X, Liu Y, et al, 2019. leaves of midgut Cadherin from Motho Mothos in Differencen Bacillus thuringiensis actions: Cortion amond Toxin Binding, Cellular Toxicity, and Synergism. Journal of Agricultural and Food Chemistry 67: 13237-46）。

According to the immune grid theory proposed by Jerne in 1974, after any antigen is used to immunize an animal, it will induce the production of its antibody (Ab 1), the antigen recognition site of Ab1, i.e. Idiotype (Id), in turn induces the production of Anti-Idiotype antibodies (Ab 2, Anti-Idiotype antibodies) by the lymphocytes of the body. Anti-idiotype antibodies can be regarded as "internal images" of the original antigen. Some Anti-idiotypic Antibodies may even have the ability to mimic the structural characteristics of an antigen, even biological activity (including receptor binding capacity) (Stanova, A.K.; Ryabkova, V.A.; Tillib, S.V.; Utekhin, V.J.; Churilov, L.P.; Shoenfeld, Y. Anti-idiotype antigenic Antibodies: candidates for the role of environmental remainders, 2020, 9, 19), or may replace the antigen as a tool for its receptor search (Sege K, Peterson PA. Use of Anti-idiotype Antibodies as reagents for cell-surface receptors, Imandindinavir. Journal of immune, 8: 172-72). The literature, "analysis of pyrethroid pesticide metabolites based on antibodies and Cry2Aa toxin molecule simulation research" (quality of Liu, Nanjing university of agriculture, 2018.) uses screened Cry2Aa anti-idiotypic antibodies B10 and F2 as templates to construct heavy and light chain substitution libraries, and screening is carried out to obtain an anti-idiotypic single-chain antibody MUT10 of Cry2Aa, but the level of affinity of the anti-idiotypic single-chain antibody and the screening antigen is micromolar (1.42 multiplied by 10)⁶mu.M), the affinity is low, and in addition, the test only determines the binding capacity of the anti-idiotype single-chain antibody to the diamond back moth Brush Border Membrane Vesicle (BBMV), and the binding affinity of the anti-idiotype single-chain antibody to a single receptor is not determined. Screening and preparing the Cry2Aa toxin anti-idiotype monoclonal antibody has potential to be used as a new tool for receptor discovery and toxicological research of Cry2Aa toxin in target insects, and the preparation of the Cry2Aa toxin anti-idiotype monoclonal antibody and the receptor binding activity are not reported at present.

Disclosure of Invention

Aiming at the problem that the affinity level of an anti-idiotype antibody obtained by earlier research is low, the Cry2Aa anti-idiotype antibody is prepared by adopting a monoclonal antibody route, the anti-idiotype monoclonal antibody of the Bacillus thuringiensis Cry2Aa toxin is obtained, and the monoclonal antibody has specific binding and extremely high affinity for a diamondback moth cadherin toxin binding region (PxCad-TBR).

The invention firstly provides an anti-idiotype monoclonal antibody of Cry2Aa toxin, wherein the heavy chain and the light chain of the anti-idiotype monoclonal antibody respectively consist of 443 and 216 amino acids, the amino acid sequences of the heavy chain and the light chain are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, the nucleotide sequence of the heavy chain for coding the anti-idiotype monoclonal antibody is shown as SEQ ID NO.5, and the nucleotide sequence of the light chain for coding the anti-idiotype monoclonal antibody is shown as SEQ ID NO. 6. The anti-idiotype monoclonal antibody is obtained by taking an F (ab)' 2 fragment of a Cry2Aa toxin rabbit polyclonal antibody as an immunogen, immunizing a Balb/c series mouse, and then performing hybridoma fusion and screening technology.

Secondly, the application provides the application of the anti-idiotype monoclonal antibody of the Cry2Aa toxin in combination with a Cry2Aa toxin receptor; the Cry2Aa toxin receptor includes a Plutella xylostella cadherin toxin binding region (PxCad-TBR).

Third, the present application also provides the use of an anti-idiotype monoclonal antibody to the Cry2Aa toxin described above to inhibit binding of the Cry2Aa toxin to its polyclonal antibody.

The Cry2Aa anti-idiotype monoclonal antibody obtained by molecular simulation of Cry2Aa toxin by adopting an anti-idiotype antibody technology has competitive inhibition effect on combination of Cry2Aa and a polyclonal antibody thereof; meanwhile, the anti-idiotype antibody 2G4 has high affinity (apparent binding affinity of 37 nM) for the Plutella xylostella cadherin toxin binding region (PxCad-TBR); and through a competitive inhibition test of Cry2Aa on the combination of PxCad-TBR and 2G4, Cry2Aa and 2G4 share a common or partial binding site on the PxCad-TBR, and have wide application prospects in identification of potential receptors of Cry2Aa toxin in target insects and research on toxicological mechanisms of the potential receptors.

Drawings

FIG. 1 is a schematic diagram of the preparation of Cry2Aa toxin anti-idiotype monoclonal antibody;

the method comprises the following steps of (A) determining a standard inhibition curve of a Cry2Aa toxin rabbit polyclonal antibody by an indirect competitive ELISA method, (B) identifying a purified rabbit polyclonal antibody F (ab)' 2 fragment by adopting non-denaturing polyacrylamide gel electrophoresis, and (C) identifying a hybridoma cell culture supernatant by adopting an indirect non-competitive ELISA method and an indirect competitive ELISA method.

FIG. 2 is a graph showing the results of a competitive inhibition assay for Cry2Aa toxin anti-idiotype monoclonal antibody.

FIG. 3 is a schematic representation of the results of the characterization of the binding properties of Cry2Aa toxin anti-idiotype monoclonal antibody to potential receptors; the test method comprises the following steps of (A) testing the binding capacity of 2G4 and prokaryotic expression diamondback moth PxCad-TBR and PxAPN5, (B) identifying the specificity of 2G4 binding with PxCad-TBR, and (C) testing the ELISA binding curve of 2G4 and PxCad-TBR. (D) Analysis for competitive inhibition of 2G4 binding to PxCad-TBR for Cry 2A.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

Reagents, instrument sources referred to in the examples:

new Zealand white rabbits and BALB/c mice were purchased from the center for laboratory animals of the academy of agricultural sciences of Jiangsu province and the center for comparative medicine of Yangzhou university, respectively.

Mouse myeloma cells (Sp 2/0-Ag 14) were maintained by the institute of agricultural product safety and nutrition, national academy of agriculture, Jiangsu province (the cells are commonly used in the literature, "Yuan Liu, Aihua Wu, Jung Hu, Manman Lin, Mengtang Wen, Xiao Zhang, Chongxin Xu, Xiaodan Hu, Jianfeng Zhong, Lingxia Jiano, Yajing Xie, Cunzhen Zhang, Xiangyang Yu, Ying Liung Liu, Xianjin Liu, Detection of 3-phenoxy benzoic acid in river water with a colloidal gold-based cellular flow assay, Analytical Biochemistry, chemistry, 483, 7-11). The diamondback moth cadherin toxin binding domain (PxCad-TBR) and the diamondback moth aminopeptidase 5 (PxAPN 5) were obtained by expression in E.coli in this laboratory (2 receptors see the literature "Gao MJ, Dong S, Hu XD, Zhang X, Liu Y, et al, 2019. circles of Midgut cadherin from two moles in different Bacillus thuringiensis action mechanisms: correction of toxin binding, cellular toxin, and synergy, Journal of Agricultural and Food Chemistry 67: 13237-46 ".

Activated Cry2Aa toxin (molecular weight 68 kDa, purity 94% -96%) was purchased from Envirologix, USA, and the toxin was produced by Bt strain at the university of Case Western Reserve, USA.

Freund's complete adjuvant, Freund's incomplete adjuvant, polyethylene glycol for cell fusion, Hypoxanthine Aminopterin Thymidine (HAT), Hypoxanthine Thymidine (HT) additive components, purchased from Sigma Aldrich, USA.

1640 medium and F (ab)' 2 preparation kit were purchased from Thermo.

Fetal bovine serum was purchased from Tian Hang biological Co., Ltd.

Protein A column, Protein G column and nickel ion affinity chromatography column (His Trap HP) were purchased from GE Health Care.

96-well enzyme-labeled plates were purchased from Corning, USA.

3,3,5, 5-Tetramethylbenzidine (TMB) was purchased from Nanjing Odoforni Biotech Ltd.

Horse Radish Peroxidase (HRP) -labeled goat anti-rabbit IgG and goat anti-mouse IgG were purchased from KPL corporation, usa. HRP-labeled anti-His-tag murine monoclonal antibody was purchased from japan biotechnology limited.

The heavy chain and the light chain of the Cry2Aa toxin anti-idiotype monoclonal antibody are subjected to full-length sequencing and are entrusted to Nanjing Kingsry biotechnology and technology Limited.

12% preformed gel, Tris-MOPS protein electrophoresis buffer solution and Western blot TMB color development solution (chromoSensor)^TMOne-Solution TMB Substrate) was purchased from tsingtaury biotechnology limited, tokyo.

Skimmed milk powder was purchased from Solebao Biotechnology Ltd.

Other chemical reagents and organic solvents used in the following examples are all domestic analytical purifications.

PCR instrument (Takara), metal bath (Hangzhou Europe rice instrument MIULAB), small-sized table-type refrigerated Centrifuge (Eppendoff), nucleic acid electrophoresis instrument (Beijing Liuyi DYY-6C type), vertical plate electrophoresis tank (Beijing Junyi JY-SCZ2 +), electrophoresis instrument power supply (Beijing Junyi JY600E type), Western blot membrane transfer electrophoresis instrument (Beijing Junyi JY-ZY5 type), small-sized table-type Centrifuge (Eppendoff Centrifuge 5424R), ultrasonic cell disruption instrument (Nanjing Yuxu Biotechnology YX-1000H), enzyme labeling instrument (Thermo Multiskan GO), plate washing machine (Thermo Wellwash), autoclave (Sanyo MLS-3750), ultra-low temperature refrigerator (Hal), pure water instrument (Millipore-Q3 UV balance), Liangping instrument FA (Liangping Heng instrument FA), pH meter (Sartorius-10), Sagel shaking table (Scilox-3750), ultra-low temperature refrigerator (Hai Zhao BF-180 instrument), super-BF-1000H, Constant temperature incubator (Jinghong).

Example 1 preparation of Cry2Aa toxin anti-idiotype monoclonal antibody

(1) Preparation of polyclonal antibodies to Cry2Aa toxin and isolation of f (ab)' 2 fragments thereof: after being emulsified by mixing 1 mL of Cry2Aa toxin (dissolved in PBS) containing 250. mu.g/mL with an equal volume of Freund's complete adjuvant, 2 male New Zealand rabbits (2 kg) were immunized and injected subcutaneously in the back at multiple points. The complete Freund adjuvant is replaced by the incomplete Freund adjuvant for boosting immunization, and after 4 times of boosting immunization, the heart collects serum.

The specific recognition capability of the rabbit antiserum on the Cry2Aa is detected by adopting an indirect competitive ELISA method. The method comprises the following specific steps: 1) coating: cry2Aa was diluted to 2. mu.g/mL with CBS buffer, 100. mu.L/well was added to the microplate, overnight at 4 ℃. 2) And (3) sealing: the next day, the plates were washed 3 times with PBST, diluted with 2% skim milk (PBS) at 200. mu.L/well, and washed 3 times with PBST after 1h of incubation at 37 ℃. 3) Adding a primary antibody: mu.L of a mixture of varying concentrations of Cry2Aa and 50. mu.L of rabbit antiserum diluted 5-fold in PBS (3 replicates per concentration) was added to each well and after 1h incubation at 37 ℃ the plates were washed 3 times with PBST. 4) Adding a secondary antibody: mu.L/well, adding HRP-labeled goat anti-rabbit IgG diluted by 1:5000 times of PBS, and after 1h of warm bath at 37 ℃, washing the plate for 3 times by PBST. 5) Color development: add 100. mu.L/well of ready-prepared substrate solution (10 mL CPBS buffer with 100. mu.L 10mg/mL TMB dissolved in dimethyl sulfoxide and 25. mu.L 0.65% H₂O₂) And standing at 37 ℃ for color development for 15 min. 6) TerminateAnd reading: 50 μ L/well 2M H₂SO₄The reaction was stopped and the absorbance was read at 450 nm using a microplate reader.

FIG. 1 (A) shows a standard inhibition curve of a Cry2Aa toxin rabbit polyclonal antibody measured by an indirect competitive ELISA method. The ELISA light absorption value is gradually reduced along with the increase of the concentration of the Cry2Aa toxin, so that the Cry2Aa rabbit polyclonal antibody has a specific recognition effect on Cry2Aa.

The test adopts Protein A column to perform affinity purification on rabbit serum, the purified antibody is separated into F (ab) '2 fragments by using an F (ab)' 2 preparation kit, and the specific test steps refer to the product instruction. FIG. 1 (B) shows the identification of purified rabbit polyclonal antibody F (ab)' 2 fragment by native polyacrylamide gel electrophoresis (J.E. Kolin root et al. eds. protein science, Experimental guidelines. science publishers. Beijing, 2007). The antibody fragment after enzyme digestion (under the non-denaturing condition) has a clear band above 80 kDa, and conforms to the theoretical molecular weight (88 kDa) of the rabbit F (ab) 2 fragment according to the instruction of a F (ab) 2 preparation kit, thereby proving that the F (ab) 2 is successfully prepared.

(2) Animal immunization: 4 female BALB/c-line mice of 6-8 weeks old were selected, and each was emulsified by mixing with 100. mu.L of a mixture containing 20. mu.g/mL of F (ab) '2 fragment and an equal volume of Freund's complete adjuvant, and then injected intraperitoneally. The boosting immunization adopts Freund incomplete adjuvant to replace Freund complete adjuvant, and after 4 times of boosting immunization, the mouse with the highest titer is selected and the spleen of the mouse is taken for hybridoma cell fusion.

(3) Screening and identifying hybridoma by adopting 2.25X 10⁸Spleen cells of an immunized mouse were combined with 4.5X 10⁷Mouse myeloma cells (Sp 2/0-Ag 14) were fused with 50% polyethylene glycol. The fused cells were screened by HAT medium and replaced with HT medium after one week. After one week of further culture, the medium was replaced with complete 1640 medium.

When the hybridoma grows about one tenth of the bottom of the micropore, performing a first round of identification on hybridoma supernatant by adopting indirect non-competitive ELISA, and taking a light absorption value larger than 0.5 as a positive result judgment standard. The method comprises the following specific steps: mu.L/well was added 2. mu.g/mL of CBS diluted F (ab)' 2-coated microplate, overnight at 4 ℃. The next day, the plate was washed 3 times with PBST, the ELISA plate was sealed with 2% skimmed milk powder at 200. mu.L/well, and after 1h of warm bath at 37 ℃, the plate was washed 3 times with PBST. mu.L of hybridoma cell supernatant was added to each well, and after 1h of incubation at 37 ℃, the plates were washed 3 times with PBST. 100 u L/hole added 1:5000 times PBS diluted HRP labeled goat anti mouse IgG, 37 degrees C warm bath for 1h, PBST washing plate 3 times. The remaining color development, termination and reading steps were the same as in step 1.

The positive clones produced in the first round of screening are subjected to a second round of identification by adopting an indirect competitive ELISA method. Indirect competitive ELISA method the same procedure as in step 1 was followed except that in step 3 "Add one antibody" 50. mu.L of hybridoma culture supernatant was used instead of 50. mu.L of a mixture of varying concentrations of Cry2Aa and 50. mu.L of rabbit antiserum diluted 5-fold in PBS.

And (3) sorting Cry2Aa toxin anti-idiotype monoclonal antibodies by taking 50 mu L of hybridoma culture supernatant which can inhibit more than 30 percent of Cry2Aa and rabbit polyclonal antibody binding signals thereof as a positive result judgment standard. And (4) obtaining a pure cultured hybridoma cell strain by adopting a limiting dilution method for the positive clones produced in the second round, and completing cell establishment.

FIG. 1 (C) shows the identification of hybridoma cell culture supernatants by indirect non-competitive ELISA and indirect competitive ELISA. From the left ordinate, it can be seen that the absorbance of 20 clones is greater than 0.5, and the positive result judgment standard is met. From the right ordinate, it can be seen that only 2 of the 20 positive clones (2G 4 and 3C 8) met the positive result criterion with an inhibition rate of more than 30%.

Because the proportion of the anti-idiotypic antibodies capable of simulating the structural characteristics of the antigen is low, and the screening and preparation difficulty is high (Siyue, Liyue, Wang, Hejiang, Liuxianjin. the application of the anti-idiotypic antibodies in the immunodetection of small molecular pesticides and mycotoxins. Zhejiang agriculture newspapers 2010, 23 (3): 398 and 402), in the preparation process of 2G4, the F (ab)' 2 fragment of Cry2Aa rabbit polyclonal antibody is used as immunogen and screening antigen, so that the generation of antibodies for identifying Fc segments (non-antigen binding regions) is avoided, and the screening efficiency of the anti-idiotypic antibodies is improved.

Example 2 analysis of competitive substitutions by anti-idiotype monoclonal antibodies

To further examine the competitive substitution of the two anti-idiotype monoclonal antibodies obtained in example 1 with Cry2Aa toxin, this example 1 prepared these two monoclonal antibodies in large quantities using an in vivo induced ascites method and purified using a Protein G column (see the purification steps: liu yung, antibody-based pyrethroid pesticide metabolite analysis and Cry2Aa toxin molecular simulation studies, nanjing university of agriculture, 2019), and then determined the competitive substitution of the anti-idiotype monoclonal antibodies with Cry2Aa toxin using a competitive ELISA method. The same procedure as in example 1 was followed except that in the third step "Add one antibody" 50. mu.L of 0-200. mu.g/mL anti-idiotype mAb was used instead of varying concentrations of Cry2Aa to mix with 50. mu.L of Cry2Aa rabbit polyclonal antibody diluted 5 ten thousand times in PBS.

As shown in FIG. 2, the inhibition exhibited a clear dose-response relationship with increasing concentrations of anti-idiotypic mab. 200 ug/mL of 2G4 and 3C8 showed 52% and 40% inhibition of Cry2Aa bound to its polyclonal antibody, respectively, and when the two were mixed at equal volumes, the inhibition increased by 90%. This is probably due to the presence of antibody components recognizing different Cry2Aa epitopes in the polyclonal antibodies employed in the test system. 2G4 or 3C8 can only simulate a single epitope of Cry2Aa, so that a single anti-idiotype antibody is adopted to compete with the combination of Cry2Aa and a multi-antibody thereof, and only partial inhibition can be generated. After 2G4 and 3C8 are added in equal amount, the inhibition effect is greatly improved, and the two antibodies are predicted to respectively simulate different Cry2Aa antigen epitopes. The subsequent examples select the anti-idiotype monoclonal antibody 2G4 with stronger competitive inhibition for further study.

Example 3 variable region sequencing and antibody informatics analysis of anti-idiotype monoclonal antibodies

The anti-idiotypic mab 2G4 obtained in example 1 was subjected to full-length sequencing of antibody genes by King-Shirui Biotech Co., Ltd. The heavy chain and light chain amino acids of the anti-idiotype monoclonal antibody 2G4 are shown in sequence tables SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences for coding the heavy chain and the light chain of the anti-idiotype monoclonal antibody 2G4 are respectively shown in SEQ ID NO.5 and SEQ ID NO. 6.

The nucleotide sequences of heavy chain and light chain variable regions of an antibody (the nucleotide sequences of heavy chain and light chain variable regions of 2G4 are respectively shown as SEQ ID NO.7 and SEQ ID NO.8, and the amino acid sequences of heavy chain and light chain variable regions of 2G4 are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4) are submitted by using an online tool (http:// www.imgt.org/IMGT _ vquest/vquest) of an IMGT database of an international immune genetic information system, and the information of alleles, CDR regions and the like of the heavy chain and light chain variable region coding gene segments are analyzed.

The 2G4 variable region gene sequence obtained by sequencing was submitted to IMGT database for analysis. VH 358 bp at 5 'end and VL 327 bp in length at 3' end. Among them, the allele having the highest identity with the V gene fragment of the heavy chain variable region (VH) was IGHV5-17 x 02F (identity = 97.92%). The allele with the highest identity to the V gene segment of the light chain variable region (VL) was Musmus IGLV1 × 01 (identity = 99.65%). IMGT labels 3 antigen Complementarity Determining Regions (CDRs) of VH and VL are 8-8-12 and 9-3-9 in amino acid length, respectively.

By alignment with the most consistent germline genes, the 2G4 monoclonal antibody had a total of 8 mutated amino acids including: VH-CDR2 (S57> N, G59> D and T64> I); VH-FR3 (A68> T and N82> Y), VL-CDR1 (S36> T) and VL-CDR3 (F112> W). These mutant amino acids may be associated with the unique positions of the anti-idiotypic monoclonal recognition of the Cry2Aa polyclonal antibody.

Example 4 binding assay of anti-idiotype monoclonal antibodies to Cry2Aa receptor

Early experiments show that the Cry2Aa toxin has insecticidal activity on diamondback moth (Royal girl, Linjie ru, Linmandan, etc. Cry2Aa toxin has insecticidal mechanism on diamondback moth. Jiangsu agricultural science, 2018,34 (4): 762 one 768.). This example further tests the binding ability of the anti-idiotype mab 2G4 obtained in example 1 to two prokaryotic expression of plutella xylostella midgut proteins, and the specific steps are as follows:

(1) testing the binding capacity of the anti-idiotype monoclonal antibody and the midgut proteins of two plutella xylostella: the diamondback moth cadherin toxin binding domain (PxCad-TBR) and diamondback moth aminopeptidase 5 (PxAPN 5) were diluted to 10. mu.g/mL with CBS buffer, 100. mu.L/well was added to the microplate, and overnight at 4 ℃. The next day, the plates were washed 3 times with PBST, 2% MPBS was added at 200. mu.L/well, incubated for 1h at 37 ℃ and washed 3 times with PBST. 100 u L/hole, adding 15 u G/mL 2G4 monoclonal antibody, 37 ℃ warm bath for 1h, PBST washing plate 3 times.

(2) Specific identification of 2G4 binding to PxCad-TBR: mu.L/well, microplate coated with a gradient concentration of PxCad-TBR diluted in CBS (0.02-2.5. mu.g/mL), overnight at 4 ℃. The next day, the plates were washed 3 times with PBST, 2% MPBS was added at 200. mu.L/well, incubated for 1h at 37 ℃ and washed 3 times with PBST. mu.L/well, 2G4 was added at a constant concentration (7.5. mu.g/mL), incubated at 37 ℃ for 1h, and the plates were washed 3 times with PBST.

(3) ELISA binding curves for 2G4 and PxCad-TBR: 100 μ L/well, plate coated with PxCad-TBR diluted with 10 μ g/mL CBS overnight at 4 ℃. The next day, the plates were washed 3 times with PBST, 2% MPBS was added at 200. mu.L/well, incubated for 1h at 37 ℃ and washed 3 times with PBST. mu.L/well, 0-200 nM (0-30. mu.g/mL) 2G4 was added, incubated at 37 ℃ for 1h, and the plates were washed 3 times with PBST.

(4) Cry2A was analyzed for competitive inhibition of 2G4 binding to PxCad-TBR. CBS dilution PxCad-TBR was diluted to 10. mu.g/mL, 100. mu.L/well was added to the microplate, overnight at 4 ℃. The next day, the plates were washed 3 times with PBST, 2% MPBS was added at 200. mu.L/well, incubated for 1h at 37 ℃ and washed 3 times with PBST. 50 μ L of a mixture of 500 μ G/mL Cry2Aa and 50 μ L of a mixture of 3.75 μ G/mL 2G4 (Cry 2Aa to 2G4 molar ratio 294:1) was added to each well, and the plates were washed 3 times with PBST after 1h of incubation at 37 ℃.

In the above (1) - (4) groups of experiments, 100. mu.L/well of HRP-goat anti-mouse IgG was diluted with 1:5000 times of PBS for detection, and the plate was washed 3 times with PBST after being incubated at 37 ℃ for 1 h. The remaining color development, termination and reading steps were the same as in example 1.

As shown in fig. 3 (a), 2G4 showed a higher binding signal with prokaryotically expressed PxCad-TBR, but no binding was observed with pxapp 5 at the same concentration. FIG. 3 (B) shows that as the concentration of PxCad-TBR coating increases, 2G4 at a constant concentration has an increasing tendency to bind, thus demonstrating that 2G4 specifically binds PxCad-TBR. ELISA binding curves of PxCad-TBR with 2G4 were tested as in FIG. 3 (C), and the apparent binding affinity was calculated to be 37 nM by Scatchard model regression analysis. FIG. 3 (D) then tested the competitive inhibition of 2G4 binding to PxCad-TBR by Cry2Aa toxin, indicating that 44% inhibition of PxCad-TBR binding to 2G4 at 500. mu.g/mL of Cry2Aa predicted that 2G4 and Cry2Aa have the same or partially the same binding site as PxCad-TBR. The antibody has potential application in identification of potential receptors of Cry2Aa toxin in target insects and research on toxicological mechanisms of the potential receptors.

Sequence listing

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<120> anti-idiotype monoclonal antibody of bacillus thuringiensis Cry2Aa toxin and application

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

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Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

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Ala Tyr Ile Asn Ser Asp Ser Ser Ile Ile Tyr Tyr Thr Asp Thr Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Tyr Pro Lys Asn Thr Leu Phe

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Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

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Ala Arg Asp Asp Gly Tyr Ser Ala Trp Phe Thr Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr

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Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu

130 135 140

Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp

145 150 155 160

Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser

180 185 190

Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser

195 200 205

Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys

210 215 220

Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr

245 250 255

Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser

260 265 270

Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile

290 295 300

Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn

305 310 315 320

Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu

340 345 350

Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe

355 360 365

Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala

370 375 380

Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr

385 390 395 400

Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly

405 410 415

Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His

420 425 430

Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440

<210> 2

<211> 216

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Thr

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly

35 40 45

Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn

85 90 95

His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Ala Asp

100 105 110

Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr

115 120 125

Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys

130 135 140

Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly

145 150 155 160

Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn

180 185 190

Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val

195 200 205

Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 3

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Val

35 40 45

Ala Tyr Ile Asn Ser Asp Ser Ser Ile Ile Tyr Tyr Thr Asp Thr Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Tyr Pro Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Asp Asp Gly Tyr Ser Ala Trp Phe Thr Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 4

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr Thr Ser Pro Gly Glu

1 5 10 15

Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Thr

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Thr Gly

35 40 45

Leu Ile Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn

85 90 95

His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105

<210> 5

<211> 1329

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gatgtgcagc tggtggagtc tgggggaggc ttagtgcagc ctggagggtc ccggaaactc 60

tcctgtgcag cctctggatt cactttcagt agctttggaa tgcactgggt tcgtcaggct 120

ccagagaagg gcctggagtg ggtcgcatac attaatagtg acagtagtat catctactat 180

acagacacag tgaagggccg attcaccatc tccagagact atcccaagaa caccctgttc 240

ctgcaaatga ccagtctaag gtctgaggac acggccatgt attactgtgc aagagatgat 300

ggttactcgg cctggtttac ttactggggc caagggactc tggtcactgt ctctgcagcc 360

aaaacgacac ccccatctgt ctatccactg gcccctggat ctgctgccca aactaactcc 420

atggtgaccc tgggatgcct ggtcaagggc tatttccctg agccagtgac agtgacctgg 480

aactctggat ccctgtccag cggtgtgcac accttcccag ctgtcctgca gtctgacctc 540

tacactctga gcagctcagt gactgtcccc tccagcacct ggcccagcga gaccgtcacc 600

tgcaacgttg cccacccggc cagcagcacc aaggtggaca agaaaattgt gcccagggat 660

tgtggttgta agccttgcat atgtacagtc ccagaagtat catctgtctt catcttcccc 720

ccaaagccca aggatgtgct caccattact ctgactccta aggtcacgtg tgttgtggta 780

gacatcagca aggatgatcc cgaggtccag ttcagctggt ttgtagatga tgtggaggtg 840

cacacagctc agacgcaacc ccgggaggag cagttcaaca gcactttccg ctcagtcagt 900

gaacttccca tcatgcacca ggactggctc aatggcaagg agttcaaatg cagggtcaac 960

agtgcagctt tccctgcccc catcgagaaa accatctcca aaaccaaagg cagaccgaag 1020

gctccacagg tgtacaccat tccacctccc aaggagcaga tggccaagga taaagtcagt 1080

ctgacctgca tgataacaga cttcttccct gaagacatta ctgtggagtg gcagtggaat 1140

gggcagccag cggagaacta caagaacact cagcccatca tggacacaga tggctcttac 1200

ttcgtctaca gcaagctcaa tgtgcagaag agcaactggg aggcaggaaa tactttcacc 1260

tgctctgtgt tacatgaggg cctgcacaac caccatactg agaagagcct ctcccactct 1320

cctggtaaa 1329

<210> 6

<211> 648

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

caggctgttg tgactcagga atctgcactc accacatcac ctggtgaaac agtcacactc 60

acttgtcgct caagtactgg ggctgttaca actactaact atgccaactg ggtccaagaa 120

aaaccagatc atttattcac tggtctaata ggtggtacca acaaccgagc tccaggtgtt 180

cctgccagat tctcaggctc cctgattgga gacaaggctg ccctcaccat cacaggggca 240

cagactgagg atgaggcaat atatttctgt gctctatggt acagcaacca ttgggtgttc 300

ggtggaggaa ccaaactgac tgtcctacgg gctgatgctg caccaactgt atccatcttc 360

ccaccatcca gtgagcagtt aacatctgga ggtgcctcag tcgtgtgctt cttgaacaac 420

ttctacccca aagacatcaa tgtcaagtgg aagattgatg gcagtgaacg acaaaatggc 480

gtcctgaaca gttggactga tcaggacagc aaagacagca cctacagcat gagcagcacc 540

ctcacgttga ccaaggacga gtatgaacga cataacagct atacctgtga ggccactcac 600

aagacatcaa cttcacccat tgtcaagagc ttcaacagga atgagtgt 648

<210> 7

<211> 357

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gatgtgcagc tggtggagtc tgggggaggc ttagtgcagc ctggagggtc ccggaaactc 60

tcctgtgcag cctctggatt cactttcagt agctttggaa tgcactgggt tcgtcaggct 120

ccagagaagg gcctggagtg ggtcgcatac attaatagtg acagtagtat catctactat 180

acagacacag tgaagggccg attcaccatc tccagagact atcccaagaa caccctgttc 240

ctgcaaatga ccagtctaag gtctgaggac acggccatgt attactgtgc aagagatgat 300

ggttactcgg cctggtttac ttactggggc caagggactc tggtcactgt ctctgca 357

<210> 8

<211> 327

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caggctgttg tgactcagga atctgcactc accacatcac ctggtgaaac agtcacactc 60

acttgtcgct caagtactgg ggctgttaca actactaact atgccaactg ggtccaagaa 120

aaaccagatc atttattcac tggtctaata ggtggtacca acaaccgagc tccaggtgtt 180

cctgccagat tctcaggctc cctgattgga gacaaggctg ccctcaccat cacaggggca 240

cagactgagg atgaggcaat atatttctgt gctctatggt acagcaacca ttgggtgttc 300

ggtggaggaa ccaaactgac tgtccta 327

Claims

1. An anti-idiotypic monoclonal antibody to the toxin Bacillus thuringiensis Cry2Aa, characterized in that the heavy and light chain amino acid sequences of said anti-idiotypic monoclonal antibody are shown in SEQ ID No.1 and SEQ ID No.2, respectively.

2. Genes encoding the heavy and light chains of an anti-idiotype monoclonal antibody of the Bacillus thuringiensis Cry2Aa toxin according to claim 1, the nucleotide sequences of which are shown in SEQ ID No.5 and SEQ ID No.6, respectively.

3. Use of an anti-idiotype monoclonal antibody of the bacillus thuringiensis Cry2Aa toxin according to claim 1 for binding to a Cry2Aa toxin receptor.

4. Use of an anti-idiotype monoclonal antibody to a bacillus thuringiensis Cry2Aa toxin according to claim 1 for inhibiting binding of a Cry2Aa toxin to its polyclonal antibody.