CN111548414A - Bivalent antibody for broad-spectrum detection of Bt Cry1 toxoid as well as gene sequence and application thereof - Google Patents

Abstract

The invention provides a bivalent antibody for broad-spectrum detection of Bt Cry1 toxoid, a gene sequence and application thereof, wherein the bivalent antibody is a bivalent light chain antibody V_L‑V_L-2D3, diabody V_L‑V_LThe amino acid sequence of-2D 3 is shown in SEQ ID NO. 2. The bivalent antibody provided by the invention has better binding activity with 7 Cry1 toxoids (Cry 1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E and Cry1F), has no cross reaction with Cry2A and Vip3 toxins, can be used for broad-spectrum detection of Bt Cry1 toxoids, is simple and convenient to operate, and is suitable for popularization and application.

Description

Bivalent antibody for broad-spectrum detection of Bt Cry1 toxoid as well as gene sequence and application thereof

Technical Field

The invention relates to a bivalent antibody for broad-spectrum detection of Bt Cry1 toxoid, a gene sequence and application thereof, in particular to a double light chain antibody V derived from an anti-Bt Cry1 toxoid monoclonal cell 2D3 gene_L-V_L-2D3 and its gene sequence and application, belonging to the technical field of genetic engineering antibody and immunological detection.

Background

The Bt Cry toxin is an insecticidal protein produced by Bacillus thuringiensis (Bt for short) in the sporulation stage, and is effective on lepidoptera and coleoptera insect larvae. When ingested by sensitive insect larvae, it is cleaved to an active protein of 65kDa size under the action of alkaline environment and midgut protease, and binds to specific receptors present in intestinal cells, leading to the formation of pores and the lysis of midgut epithelial cells, eventually leading to larval death. Also, because of the lack of receptors for Cry toxin proteins in mammals, Cry toxins are considered safe for humans and livestock. At present, Cry1 toxoid genes are widely introduced into transgenic crops, and the continuous emergence of transgenic insect-resistant crops and the large-area popularization and planting of the transgenic insect-resistant crops generate economic and social benefits, and meanwhile, the risks of ecological safety and potential safety hazards to human beings and other mammals are gradually concerned. At present, the applied Cry1 toxoid gene types are more and more, the technical restrictions of government safety supervision and food processing enterprise raw material control are more and more obvious, and particularly, when the gene detection in some transgenic foods cannot achieve a satisfactory effect, the safety screening and detection of toxin expression products are particularly urgent.

The detection method aiming at toxin protein in a transgenic product mainly adopts an antibody detection technology aiming at single toxin at present, and the preparation process of the antibody is long and high in cost because corresponding antibodies are required to be prepared aiming at different toxins, so that the requirement of broad-spectrum screening detection cannot be met. Therefore, the development of Bt Cry1 toxoid broad-spectrum screening immunoassay technology is a work with great application value.

The current detection methods for toxin proteins in transgenic products are mainly focused on nucleic acid and protein analysis. DNA-PCR based methods have been widely used for the detection of Bt genes, which have a high sensitivity. However, the PCR method requires the operation of a special instrument by experienced personnel, is not only time-consuming but also not suitable for rapid detection in the field. On the other hand, the enzyme-linked immunosorbent assay (ELISA) overcomes the defects of the protein-based detection method, has the advantages of strong specificity, high sensitivity, simple sample pretreatment, low cost, suitability for field batch detection and the like, and is applied to a plurality of fields of food, medical treatment and the like. In the ELISA process, the specificity, sensitivity and the like of the antibody directly influence the detection result, so that a high-quality broad-spectrum antibody for resisting Cry1 toxoid protein must be prepared in order to establish an immunological detection technology for Cry1 toxoid.

Disclosure of Invention

The invention aims to solve the technical problem of providing a bivalent antibody for broad-spectrum detection of Bt Cry1 toxoid, and a gene sequence and application thereof, which overcome the defects of the prior art.

The invention provides a bivalent antibody for broad-spectrum detection of Bt Cry1 toxoid, wherein the bivalent antibody for detection is V_L-V_L-2D3, said diabody V_L-V_LThe amino acid sequence of-2D 3 is shown in SEQ ID NO. 2.

The invention also provides bivalent antibody V_L-V_L-2D3 gene sequence.

Encoding said diabody V_L-V_LThe gene sequence of-2D 3 is shown in SEQ ID NO. 1.

Bivalent antibody V of the present invention_L-V_L2D3 is a double light chain antibody, i.e., the antibody comprises two light chain variable regions (V)_L) Compared with monoclonal antibodies, the double light chain antibody can be prepared by mass expression without an immunization means, is directly synthesized artificially according to a gene sequence, is relatively simpler and more convenient in preparation method and is easy to store for a long time. With single-chain antibodies V_H-V_LCompared with 2D3, the double light chain antibody not only has higher biological activity, but also has better specific binding activity with antigen. Subsequently, in bivalent antibody V_L-V_LOn the basis of-2D 3, a DAS-ELISA detection method capable of simultaneously detecting seven toxins of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E and Cry1F is established.

The bivalent antibody V_L-V_L-2D3 in the detection of Bt Cry1 toxoids, said Bt Cry1 toxoids are Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E and Cry 1F.

Such as bivalent antibody V_L-V_LThe application of the-2D 3 in the detection of the Bt Cry1 toxoid comprises the following specific detection methods:

step 1, coating capture antibody-bivalent anti-Bt Cry1 toxoid antibody V_L-V_LDiluting 2D3 to 1.25. mu.g/mL with PBS buffer, adding to 96-well enzyme label plate, 100. mu.L/well, and coating overnight at 4 ℃;

step 2, after sealing and coating overnight, throwing out liquid in the holes, washing by adopting a PBST solution, then adding MPBS with the mass concentration of 3% into the holes as a sealing solution, performing incubation for 2 hours in an incubator at 37 ℃, wherein 200 mu L/hole is formed in the holes;

step 3, adding a sample, namely washing the ELISA plate by adopting a PBST solution, adding a Bt Cry1 toxoid sample dissolved in a CBS buffer solution into the hole with the concentration of 100 mu L per hole, and then putting the ELISA plate in an incubator at 37 ℃ for incubation for 1 h;

step 4, adding a detection antibody, namely washing the ELISA plate by using a PBST solution, adding an anti-Bt Cry1 toxoid polyclonal antibody with the concentration of 0.2 mu g/mL into the hole, wherein the concentration is 100 mu L/hole, and then putting the ELISA plate in an incubator at 37 ℃ for incubation for 1 h;

step 5, adding an enzyme-labeled antibody, namely washing the ELISA plate by adopting a PBST solution, adding a goat anti-rabbit-HRP antibody into the hole with the concentration of 100 mu L/hole, and placing the ELISA plate in a 37 ℃ incubator for incubation for 1 h;

step 6, developing, namely washing the ELISA plate by adopting a PBST solution, adding a TMB developing solution system into micropores of the ELISA plate at 100 mu L/hole, and placing the ELISA plate in an incubator at 37 ℃ for incubation for 15min in a dark place;

and 7, detection, namely adding the stop solution into the micropores of the ELISA plate, measuring the absorbance value at the condition of 450nm on an ELISA reader at 50 mu L/hole.

In step 3, the Bt Cry1 toxoid sample is at least one of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E and Cry 1F.

The stop solution adopts H with the concentration of 2M₂SO₄。

The bivalent antibody provided by the invention has better binding activity with 7 Bt Cry1 toxoids (namely Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E and Cry1F), has no cross reaction with Cry2A and Vip3 toxins, can be used for broad-spectrum detection of the Bt Cry1 toxoids, is simple and convenient to operate, and is suitable for popularization and application.

Drawings

FIG. 1 is a schematic diagram of scFv-2D3 gene amplification, identification and amino acid sequence thereof.

FIG. 2 is a schematic diagram showing the docking results of scFv-2D3 with Cry1Ac and Cry1B toxin molecules.

FIG. 3 is V_HAnd V_LThe genes are respectively amplified and constructed to the PCR identification and double enzyme digestion identification schematic diagram of an expression vector pET-26 b.

FIG. 4 shows scFv-2D3, V_H、V_LAnd V_L-V_LSchematic representation of the expression identification results of (1).

FIG. 5 is scFv-2D3, V_H、V_LAnd V_L-V_LThe indirect ELISA identification results with seven Cry1 toxoids and the cross-reaction identification results with Cry2A and Vip3 are shown in the figure.

FIG. 6 is scFv-2D3 and V_L-V_LSchematic diagram of standard curve established for seven Cry1 toxoids.

Detailed Description

The technical scheme of the invention is further explained in detail by combining the embodiment as follows: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection authority of the present invention is not limited to the following embodiments.

The reagents and media formulations referred to in the examples are as follows:

(1) PBS buffer: 8.0g NaCl, 0.2g KCl, 2.9g Na₂HPO₄·12H₂O and 0.2g KH₂PO₄Dissolving in double distilled water, diluting to 1L, and adjusting pH to 7.4.

(2) CBS buffer solution: 1.59g Na₂CO₃And 2.93g NaHCO₃Dissolving in double distilled water, diluting to 1L, and adjusting pH to 9.6.

(3) PBST solution: tween-20 was added to the PBS buffer in a volume ratio of 0.05%.

(4) 3% MPBS solution: 3g of skim milk powder was dissolved in 100mL of double distilled water.

(5) CPBS solution: 21g citric acid (C)₆H₇O₈) And 71.6g Na₂HPO₄·12H₂Dissolving O in double distilled water, diluting to 1L, and adjusting pH to 5.5.

(6) TMB mother liquor: 10mg of TMB was dissolved in 1mL of dimethyl sulfoxide and stored at 4 ℃ for a short period.

(7) Color development liquid: TMB mother liquor with concentration of 10mg/mL (100 mu L) and H with mass concentration of 0.65%₂O₂25 μ L of the composition was dissolved in 9.875mL of CPBS solution and was ready for use.

(8) H at a concentration of 2M₂SO₄: 11.8mL of concentrated sulfuric acid (98%) was slowly added to 80mL of distilled water, the mixture was stirred during the addition, and the volume was adjusted to 100mL after cooling.

(9)2 × TY medium: the preparation method comprises the following steps of dissolving 16g of tryptone, 10g of yeast extract and 5g of NaCl in double distilled water, fixing the volume to 1L, subpackaging and then carrying out autoclaving at 121 ℃.

(10)2 × TY-AG medium: ampicillin at a final concentration of 100. mu.g/mL and 1% by mass of glucose were added to 2 XTY medium.

(11) TYE medium: 10g of tryptone, 5g of yeast extract, 8g of NaCl and 15g of agar powder are dissolved in double distilled water, the volume is fixed to 1L, and the mixture is sterilized under high pressure at the temperature of 121 ℃ for later use.

(12) TYE-AG medium: 10g of tryptone, 5g of yeast extract, 8g of NaCl8g and 15g of agar powder are dissolved in double distilled water, the volume is fixed to 1L, and after autoclaving at the temperature of 121 ℃, ampicillin with the final concentration of 100 mu g/mL and glucose with the mass ratio of 1% are added.

(13) IPTG solution at concentration 1M: 2.38g IPTG was dissolved in 10mL of sterile water, which was then sterilized by filtration through a 0.22 μm filter, and stored at-20 ℃ after split charging.

Sources of materials involved in the examples:

the polyclonal antibody against Bt Cry1 toxoid is self-made in the laboratory, and the preparation method is shown in the literature: dong S, Zhang C, Zhang X, Liu Y, Zhong J, Xie Y, Xu C, Ding Y, Zhang L, Liu X (2016) Production and characterization of monoclonal antibody biosynthesis Cry1 toxin using synthesized polypeptide as a host Chem 88:7023-7032 (others may obtain polyclonal antibodies against Bt Cry1 toxoid by self-made or by direct request to the applicant); pET-26b expression vectors, e.coli TG1 and e.coli BL21 were purchased from MRC (Cambridge, England); anti-His-HRP antibody, goat anti-rabbit HRP antibody and His-Trap HP affinity columns were purchased from GE Healthcare (America); cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E, Cry1F, Cry2A and Vip3 are all available from Shanghai Yongong Biotech Limited. All primers involved in sequencing were synthesized by Shanghai Bioengineering, Inc., such as RV-M, M13-47, T7P, T7T.

The preparation of hybridoma 2D3 is described in the literature: dong S, Zhang C, Zhang X, Liu Y, Zhong J, Xie Y, Xu C, Ding Y, Zhang L, Liu X (2016) Production and chromatography of monoclonal antibody hybridization Cry1 toxin using design primer as in inlet Chem 88: 7023-. In addition, hybridoma 2D3 was deposited in 2015 by the agricultural academy of sciences of Jiangsu province in the China center for type culture Collection with the preservation number of CCTCC NO: C2015145. Others may obtain hybridoma cell 2D3 by making a request from home, from the chinese type culture collection, or directly from the applicant.

The remaining reagents and materials involved in this example are commercially available and are not listed here.

Nucleotide and amino acid sequences referred to in the examples:

SEQ ID NO.1：

GACATTGTGTTGACACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAATCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACA GGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCTCCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGAACTGTGGCTGCACCATCTGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATTGTGTTGACACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAATCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCTCCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGAACTGTGGCTGCACCATCTSEQ ID NO.2：

DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPPTFGAGTKLELKRTVAAPSGGGGSGGGGSGGGGSDIVLTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPPTFGAGTKLELKRTVAAPS

SEQ ID NO.3：

RV-M：5’-GAGCGGATAACAATTTCACACAGG-3’

SEQ ID NO.4：

M13-47：5’-CGCCAGGGTTTTCCCAGTCACGAC-3’

SEQ ID NO.5：

T7P：5’-TAATACGACTCACTATAGGG-3’

SEQ ID NO.6：

T7T：5’-TGCTAGTTATTGCTCAGCGG-3’

example 1 amplification and identification of the scFv-2D3 gene.

(1) Antibody V_HAnd V_LAnd (4) amplifying the gene.

Total RNA was extracted from hybridoma cell 2D3 using Trizol Total RNA extraction kit (purchased from Invitrogen) and SuperScript was used^TMKit III (purchased from Invitrogen) reverse transcribes it into cDNA. Using the obtained cDNA as a template, corresponding upstream and downstream degenerate primers (see Table 1) were mixed in equal amounts, respectively, to perform PCR reaction for V amplification_HAnd V_LA gene.

PCR reaction System (20. mu.L) 2 × Pfu MasterMix (available from Kyoto Kogyo Co., Ltd.), 10. mu.L and 10. mu.M of V_H/V_LMixing the upstream primer, 1 mu L; v of 10. mu.M_H/V_LMixing downstream primers, 1 μ L; cDNA template, 1. mu.L; dd H₂O (double distilled water) to 20. mu.L, and carrying out × 30 cycles of PCR reaction at 94 ℃ for 5min, 94 ℃ for 45s, 55 ℃ for 45s, and 72 ℃ for 60s, and 72 ℃ for 10 min.

Get allRunning a PCR amplification product into 1% agarose gel electrophoresis, dyeing by adopting 5% GoldView in volume ratio, observing an image under a gel imaging system, cutting a segment with a target size, and purifying and recovering according to the specification of an Axygen gel recovery kit to obtain V_HPurification of the product and V_LThe product was purified and used directly in subsequent experiments or stored at-20 ℃ for future use.

(2) Antibody V_HAnd V_LCloning and identifying the gene.

Purifying the V_HAnd V_LThe genes were ligated to pMD19-T vectors (purchased from TaKaRa), respectively. The enzyme connecting system is as follows: 1 μ Lvector, 5 μ L buffer and 4 μ L V_HOr V_LThe product was purified, added to a PCR tube, carefully mixed and ligated overnight at 16 ℃. The enzyme linked line was then transformed into Trans1-T1 competent cells (purchased from Kyoto holotype gold, Beijing) by the procedure described in chapter 2, section 1.4.3, of "two Bt toxin single-chain antibody preparations and antibody detection technology research" of King Bo Shi graduation. And (3) coating a proper amount of recovered bacterial liquid on a TYE-AG plate, culturing overnight, and picking single colonies on the plate by using a sterile gun head to perform PCR reaction and sequencing identification.

PCR reaction system (10 μ L) 2 × Taq Mastermix (from Beijing Quanji Co., Ltd.), 5 μ L, 10 μ M upstream primer RV-M (shown as SEQ ID NO. 3), 0.5 μ L, 10 μ M downstream primer M13-47 (shown as SEQ ID NO. 4), 0.5 μ L, bacterial suspension template, 1 μ L, dd H₂Adding O to 10 μ L, reacting at 94 deg.C for 5min, at 94 deg.C for 1min, at 55 deg.C for 45s, and at 72 deg.C for 60s, × 30 cycles, and at 72 deg.C for 10 min.

Taking 5 mu L of PCR amplification product to carry out 1% agarose gel electrophoresis detection, staining 5% GoldView, observing an image under a gel imaging system, and obtaining a clone with an obvious band at the size of a target gene fragment as a positive clone. Sending the positive clone bacterial liquid to Shanghai biological engineering technical service company Limited for bidirectional sequencing identification, after the strain with correct sequencing result is subjected to amplification culture, adding 15% of glycerol by volume (namely the addition of the glycerol is 15% of the total volume) into one part of the strain for storage at-80 ℃, and extracting plasmids from the other part of the strain (the step refers to the specification of the Axygen plasmid extraction kit), thus obtaining V_HRecombinant plasmid and V_LThe recombinant plasmid is directly used for subsequent experiments or stored at the temperature of minus 20 ℃ for later use.

(3) Construction of the scFv-2D3 gene.

Amplification of the full-Length Gene of the Single-chain antibody Using overlap extension PCR, according to V_HAnd V_LAs a result of gene sequencing, specific primers (see Table 2) were designed by connecting peptides (Gly) through intermediate flexibility₄Ser)₃(Linker) reaction of V_HAnd V_LConnection, construction of V_H-Linker-V_LA form of the scFv-2D3 gene sequence.

First, the compound containing V obtained in the step (2) above_HAnd V_LThe plasmids of (1) as templates, respectively, with V_H-NcoI-F、V_H-Linker-R and V_L-Linker-F、V_LNotI-R as amplification V_HAnd V_LAnd connecting the primer of the peptide gene, and performing PCR amplification.

PCR reaction (20. mu.L) 2 × Pfu MasterMix, 10. mu.L, 10. mu.M V_H-NcoI-F/V_L-Linker-F, 1 μ L; v of 10. mu.M_H-Linker-R/V_L-NotI-R，1μL；V_H/V_LRecombinant plasmid, 1 μ L; dd H₂Adding O to 20 μ L, reacting at 94 deg.C for 5min, and (95 deg.C for 45s, 55 deg.C for 45s, and 72 deg.C for 60s) for × 30 cycles at 72 deg.C for 10 min.

Taking the total PCR amplification product, running 1% agarose gel electrophoresis, dyeing 5% GoldView, observing the image under a gel imaging system, and verifying V_HAnd V_LWhether the linker gene was successfully amplified or not, V_HAnd V_LCutting a segment with a target size from the connecting peptide gene, purifying and recovering according to the instructions of a gel recovery kit to obtain NcoI-V_H-Linker and Linker-V_L-NotI purified product.

The purified NcoI-V_H-Linker and Linker-V_Lthe-NotI gene was added in equal amounts to the PCR reaction system, and the amplification of the full-length scFv-2D3 gene sequence was carried out in two steps as follows.

First, PCR reaction system (25. mu.L), 2 × Pfu MasterMix, 12.5. mu.L, NcoI-V_H-Linker，50ng；Linker-V_L-NotI，50ng；dd H₂Adding 25 μ L of O, reacting at 94 deg.C for 1min, 50 deg.C for 1min, and 72 deg.C for 1min, and × 10 cycles.

Second, PCR reaction (25. mu.L), 2 × Pfu Mastermix, 12.5. mu.L, 10. mu.M V_HNcoI-F, 0.5. mu.L; v of 10. mu.M_L-NotI-R, 0.5 μ L; the product of the first step, 2. mu.L, dd H₂Adding O to 25 μ L, reacting at 94 deg.C for 1min, (94 deg.C for 1min, 60 deg.C for 1min, 72 deg.C for 1min) for × 30 cycles, and at 72 deg.C for 10 min.

Taking the full amount of PCR amplification products, running 1% agarose gel electrophoresis, dyeing 5% GoldView, observing images under a gel imaging system, verifying whether the scFv-2D3 gene is successfully amplified, cutting a fragment with a target size from the successfully amplified scFv-2D3 gene, purifying and recovering according to the instructions of a gel recovery kit to obtain a scFv-2D3 purified product, and directly using the purified product in subsequent experiments or storing the purified product at-20 ℃ for later use.

In the embodiment, anti-Cry 1 toxoid monoclonal cell 2D3 is taken as a gene source, and a heavy chain variable region (V) of an antibody is amplified by a universal primer_H) And light chain variable region (V)_L) And from Linker (Gly)₄Ser)₃In series, the single-chain antibody scFv-2D3 is connected.

(4) Cloning and identification of scFv-2D3 gene.

The purified scFv-2D3 gene was ligated into the pMD19-T vector. The enzyme connecting system is as follows: mu.L vector, 5. mu.L buffer and 4. mu.L scFv-2D3 purified product were added to the PCR tube, carefully mixed and ligated overnight at 16 ℃. Then the enzyme linked system is transformed into Trans1-T1 competent cells, and the specific transformation steps refer to the Wangchi graduation paper "two Bt toxin single-chain antibody preparation and antibody detection technology research" chapter 2, section 1.4.3 operation. And (3) coating a proper amount of recovered bacterial liquid on a TYE-AG plate, culturing overnight, and picking single colonies on the plate by using a sterile gun head for PCR and sequencing identification.

PCR reaction system (10 μ L) 2 × Taq Mastermix, 5 μ L, 10 μ M RV-M, 0.5 μ L, 10 μ M13-47, 0.5 μ L, bacterial liquid template, 1 μ L, dd H₂Adding O to 10 μ L, reacting at 94 deg.C for 5min, at 94 deg.C for 1min, at 55 deg.C for 45s, and at 72 deg.C for 60s, × 30 cycles, and at 72 deg.C for 10 min.

Taking 5 mu L of PCR amplification product to carry out 1% agarose gel electrophoresis detection, staining 5% GoldView, observing an image under a gel imaging system, and obtaining a clone with an obvious band at the size of a target gene fragment as a positive clone. And (3) sending the positive clone bacterial liquid to Shanghai biological engineering technical service company Limited for bidirectional sequencing identification, carrying out amplification culture on strains with correct sequencing results, adding 15% of glycerol to one part of the strains for storage at-80 ℃, and extracting plasmids from the other part of the strains (the step refers to the specification of the Axygen plasmid extraction kit) to obtain plasmids containing scFv-2D3 full-length gene sequences, and directly using the plasmids for subsequent tests or storing the plasmids at-20 ℃ for later use.

(5) Construction and identification of scFv-2D3 prokaryotic expression vector

The plasmid containing the full-length gene sequence of scFv-2D3 and the plasmid of expression vector pET-26b were digested with NcoI and NotI in equal amounts, respectively, in the digestion system, NcoI (purchased from NEB) 1. mu.L, NotI (purchased from NEB) 1. mu.L, 10 × NEB Cut smart buffer 5. mu.L, pET-26b plasmid/scFv-2D 3 plasmid 1. mu.g, dd H₂O is complemented to 50 mu L; mixing the above systems, enzyme-cutting at 37 deg.C overnight, and inactivating at 80 deg.C for 20 min. The next day, the enzyme digestion effect was checked by 1% agarose gel electrophoresis, and the digested scFv-2D3 fragment and the enzyme-cleaved linear pET-26b expression vector were recovered by gel cutting, respectively.

Followed by enzyme ligation using 1. mu. L T4 DNA Ligase (from NEB), 2. mu.L 10 × buffer, 10. mu.L scFv-2D3 purified product (recovered from the previous step), 3. mu.L pET-26b linear vector (recovered from the previous step), 4. mu.L ddH₂O, added to the PCR tube, carefully mixed and ligated overnight at 16 ℃ to give the ligation product pET-26b-scFv-2D 3.

The ligation product is transformed into competent cells of a host bacterium E.coli BL21, and a resuscitative bacterium solution is smeared on a TYE-AG plate and placed in an incubator at 37 ℃ for inverted culture for 12-16 h. After the culture, a single colony was picked from the plate, added to 2mL of 2 XTY-AG liquid medium, cultured at 37 ℃ and 250rpm for about 16 hours, and the bacterial solution was subjected to PCR.

PCR reaction system (10 μ L) 2 × Taq Mastermix, 5 μ L, 10 μ M T7P (shown as SEQ ID NO. 5), 0.5 μ L, 10 μ M T7T (shown as SEQ ID NO. 6), 0.5 μ L, bacterial liquid template, 1 μ L, dd H₂Adding O to 10 μ L, reacting at 94 deg.C for 5min, at 94 deg.C for 1min, at 55 deg.C for 1min, and at 72 deg.C for 1min, × 30 cycles, at 72 deg.C for 10 min.

Taking 5 mu L of PCR amplification product to carry out 1% agarose gel electrophoresis detection, staining 5% GoldView, observing an image under a gel imaging system, and obtaining a clone with an obvious band at the size of a target gene fragment as a positive clone. And (3) sending the positive clone bacterial liquid to Shanghai biological engineering technology service company Limited for bidirectional sequencing identification, adding 15% of glycerol into one part of pET-26b-scFv-2D3 strains with correct sequencing results after amplification culture, preserving at-80 ℃ and preserving the other part at 4 ℃ for later use. The scFv-2D3 gene amplification, identification and amino acid sequence are shown in FIG. 1.

Example 2 homologous modeling of scFv-2D3 Gene and docking with Cry1 toxoid molecules

The gene sequence of scFv-2D3 was translated into an amino acid sequence using the existing SWISS-MODEL website (website: SEQ ID NO: SEQhttp://swissmodel.expasy.org/) And carrying out homologous modeling. The three-dimensional structure model of scFv-2D3 and the three-dimensional structure model of seven Cry1 toxoids were then modeled using the existing ZDCK website (website: N.T.)http://zdock.umassmed.edu/) Molecular docking is performed to obtain a structural model of the complex. The three-dimensional structure model is analyzed by adopting the existing SWISS-Pdb Viewer software, and the molecular docking analysis result of scFv-2D3 and Cry1Ab is shown in figure 2.

Example 3V_HAnd V_LConstruction and identification of genes

V_HAnd V_LThe gene was constructed in the same manner as in example 1, and the template was the above-mentioned PET-26b plasmid into which the complete scFv gene sequence was inserted. The primer sequences used are shown in Table 3. Purifying the obtained V with NcoI and NotI enzyme cutting sites_HAnd V_LThe genes are respectively connected to a PMD19-T vector, and the steps of T vector connection, transformation, screening of positive colonies, sequencing identification, plasmid extraction and the like are the same as the example 1; v_HAnd V_LConstruction of prokaryotic expression System for protein pET-26b-V was obtained in the same manner as in example 1_HAnd pET-26b-V_L。V_HAnd V_LPCR identification and double enzyme digestion identification node for gene amplification and construction to expression vector pET-26bThe result is shown in FIG. 3.

Example 4V_L-V_LConstruction and identification of genes

V with NcoI and NotI cleavage sites_L-V_LThe whole gene sequence is synthesized by Nanjing Dingding biotechnology limited company, synthesized V_L-V_LThe whole gene sequence is connected between NcoI and NotI enzyme cutting sites of the PET-26b vector after double enzyme cutting to obtain a recombinant plasmid pET-26b-V_L-V_LAnd the obtained recombinant plasmid pET-26b-V is used_L-V_LColi BL21 strain, and the concrete implementation procedure is the same as example 1.

Example 5scFv-2D3, V_H、V_LAnd V_L-V_LExpression and purification identification of

The correctly sequenced pET-26b-scFv-2D3 and pET-26b-V stored at 4 ℃ were taken_H、pET-26b-V_LAnd pET-26b-V_L-V_LTransferring the recombinant bacteria and the non-loaded bacterial liquid into a 2 × TY-AG liquid culture medium according to the proportion of 1 percent, carrying out shaking culture at 37 ℃ and 250rpm overnight, transferring the overnight bacterial liquid into a 100mL 2 × TY-AG (containing 0.1 percent of glucose) liquid culture medium according to the proportion of 1 percent, carrying out shaking culture at 37 ℃ and 250rpm until OD is obtained₆₀₀About 0.6-0.8, about 3 hours. 1mM IPTG solution was added and expression was induced at 30 ℃ for 10h, with the unloaded strain as a control. After the induction, centrifugation was carried out at 4 ℃ and 10000g for 20min, the supernatant was discarded, and 100mL of PBS buffer was taken to resuspend the cells. And ultrasonically crushing the thalli in an ice bath for 40min, wherein the power is 35%, and the working time is 3s and the stopping time is 4 s. 16000g, centrifuging for 30min, and collecting supernatant to obtain soluble protein. Then, the protein was purified and collected by a His-Trap HP affinity column (the specific purification method is referred to in the article "prokaryotic expression and biological activity assay of humanized anti-Cry 1B toxic protein single-chain antibody", Xuzai et al, proceedings of Nanjing university of agriculture, 3 rd 2013). Finally, SDS-PAGE and WB were performed to analyze the expression of the target protein. FIG. 4 shows scFv-2D3, V_H、V_LAnd V_L-V_LThe expression identification result of (1).

Example 6scFv-2D3, V_H、V_LAnd V_L-V_LIdentification of Activity

Evaluation of scFv-2D3 by indirect ELISA,V_H、V_LAnd V_L-V_LMainly comprising the determination of affinity and specificity. The coating antigens are seven Cry1 toxoids, Cry2A and Vip3 toxins. The method comprises the following specific steps:

(1) coating: the toxins Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E, Cry1F, Cry2A and Vip3 are respectively dissolved in CBS buffer solution to a final concentration of 4 mu g/mL, and then are respectively added into a 96-well enzyme label plate, 100 mu L/well and coated overnight at 4 ℃;

(2) and (3) sealing: after the overnight, the liquid in the holes is thrown out, the holes are washed for 3 times by using a plate washing machine PBST solution, then a 200 mu L/hole 3% MPBS solution is added into the holes as a sealing solution, and the ELISA plate is placed in an incubator at 37 ℃ for incubation for 2h to reduce the nonspecific adsorption of the ELISA plate;

(3) adding a primary antibody: after washing the enzyme label plate for 3 times by the method of the step (2), scFv-2D3, V diluted by PBS buffer solution according to a certain proportion_H、V_LAnd V_L-V_LAdding the antibody into the hole, adding 100 mu L of the antibody into the hole, and placing the ELISA plate in an incubator at 37 ℃ for incubation for 1 h;

(4) adding an enzyme-labeled secondary antibody: after washing the ELISA plate for 3 times by adopting the method in the step (2), adding an ELISA secondary antibody (namely an anti-His-HRP antibody) which is diluted by 4000 times by using a 3% MPBS solution into the hole of the ELISA plate according to 100 mu L/hole, and then putting the ELISA plate in an incubator at 37 ℃ for incubation for 1 h;

(5) color development: and (3) washing the ELISA plate for 4 times by adopting the method in the step (2), adding a developing solution system into the hole of the ELISA plate, performing 100 mu L/hole, and immediately placing the ELISA plate in an incubator at 37 ℃ for incubation for 15min in a dark place.

(6) Reading: then the stop solution 2M H is added₂SO₄Add to the well of the microplate, 50. mu.L/well, and immediately read the absorbance value under 450nm of the microplate reader.

scFv-2D3、V_H、V_LAnd V_L-V_LThe results of indirect ELISA with seven Cry1 toxoids and cross-reaction with Cry2A and vip3 are shown in FIG. 5, showing that V_LThe binding activity to seven Cry1 toxoids is obviously higher than that of V_H，V_L-V_LFor seven Cry1The binding activity of the toxoid is strongest.

Example 7 construction of DAS-ELISA detection method

Step 1, coating capture antibody, namely, anti-Bt Cry1 toxoid single-chain antibody scFv-2D3 or bivalent antibody V_L-V_LDiluting 2D3 to 1.25. mu.g/mL with PBS buffer, adding to 96-well enzyme label plate, 100. mu.L/well, and coating overnight at 4 ℃;

step 2, after sealing and coating overnight, throwing out liquid in the holes, washing the liquid for 3 times by using a plate washing machine and adopting a PBST solution, then adding an MPBS solution with the mass concentration of 3% into the holes as a sealing liquid, wherein the mass concentration of the MPBS solution is 200 mu L/hole, and then putting the ELISA plate in an incubator at 37 ℃ for incubation for 2 hours;

step 3, adding samples, namely washing the ELISA plate for 3 times by using a PBST solution by using a plate washing machine, adding Cry1 toxoid samples (Cry1 toxoid samples are Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E and Cry1F which are dissolved in CBS buffer solution in advance, and using Cry1 toxoid standard samples with the solubility of 0.015, 0.03, 0.06, 0.12, 0.25, 0.5, 1, 2, 4, 8, 16 and 32 mu g/mL into the wells, adding 100 mu L/well, using the CBS buffer solution as a blank control, and then placing the ELISA plate in an incubator at 37 ℃ for incubation for 1 h;

step 4, adding a detection antibody, namely washing the ELISA plate for 3 times by using a PBST solution by using a plate washing machine, adding an anti-Bt Cry1 toxoid polyclonal antibody with the concentration of 0.2 mu g/mL into the hole, wherein the concentration of the anti-Bt Cry1 toxoid polyclonal antibody is 100 mu L/hole, and then putting the ELISA plate into an incubator at 37 ℃ for incubation for 1 h;

step 5, adding an enzyme-labeled antibody, namely washing the enzyme-labeled plate for 3 times by using a PBST solution by using a plate washing machine, adding a goat anti-rabbit-HRP antibody (the volume ratio is 1:5000, and 3% MPBS is used as a solvent) into the holes, incubating for 1h at 100 mu L/hole, and putting the enzyme-labeled plate in an incubator at 37 ℃;

step 6, developing, namely washing the ELISA plate for 4 times by using a PBST solution by using a plate washing machine, adding a developing solution system into micropores of the ELISA plate, wherein each pore is 100 mu L, and immediately placing the ELISA plate in an incubator at 37 ℃ for incubation for 15min in a dark place;

step 7, reading-stop solution (2M H)₂SO₄) Adding into the micro-hole of the enzyme label plate, 50 μ L/hole, immediately reading absorbance value on an enzyme label instrument under the condition of 450nm, and establishing scFv-2D3 and V according to the detection result_L-V_LStandard curves for seven Cry1 toxoids (see fig. 6).

The minimum detection limit, the minimum quantitative limit and the four-parameter equation of the DAS-ELISA detection method for the seven Cry1 toxoids are shown in the table 4. As can be seen from Table 4, based on the single-chain antibody scFv-2D3 and diabody V_L-V_LThe established detection methods have good detection effects on seven Cry1 toxoids, and bivalent antibodies V_L-V_LThe activity is better.

Example 8 additive recovery test

Seven Cry1 toxoid standards were added to rice flour separately, at three levels of addition: 0.2, 0.5 and 1.0. mu.g/g. Three replicates per addition level. The specific operation is as follows: 5g of non-transgenic rice flour is weighed and added with seven Cry1 toxins at corresponding levels respectively. The above samples were vortexed at room temperature for 30min, followed by standing at 4 ℃ overnight. The next day, 5mL of sample extract (0.05% Tween-20 was added to CBS buffer) was added to the sample and extracted at room temperature with gentle shaking for 2 h. Subsequently, the shaken mixture was centrifuged at 3000rpm for 10min, and the supernatant was aspirated and diluted ten-fold with CBS buffer before being used directly in DAS-ELISA assay. Blank is a sample without added toxin. The results are shown in Table 5, which shows that the addition recovery rate of the seven Cry1 toxins in rice flour is 83.7% -93.4%, and the variation coefficient is less than 9.5%, which indicates that the Cry1 toxins are based on V_L-V_LThe established DNA-ELISA has better accuracy for quantitative detection of seven Cry1 toxins in a sample.

TABLE 1 antibody variable regions V_HAnd V_LGeneral primer for gene sequence amplification

Note: y is C/T; b is C/G; w is A/T; G/C; m is A/C; r is A/G; k is G/T; d is A/G/T; v ═ A/C/G

TABLE 2 primers for SOE-PCR amplification of scFv-2D3 full-Length sequence

TABLE 3 primers for amplifying VH and VL sequences with restriction sites NcoI and NotI

Table 4 is based on scFv-2D3 and V, respectively_L-V_LFour-parameter equation, LOD (Lod index) and LOQ (Loq index) values of established DAS-ELISA (detection system-enzyme linked immunosorbent assay) detection method for seven Cry1 toxoids

TABLE 5 bases on V_L-V_LThe established DAS-ELISA detection method is used for determining the addition recovery of seven Cry1 toxoids in rice flour

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can understand that the modifications or substitutions within the technical scope of the present invention are included in the scope of the present invention, and therefore, the scope of the present invention should be subject to the protection scope of the claims.

Sequence listing

<110> Yangzhou university

Bivalent antibody for broad-spectrum detection of <120> Bt Cry1 toxoid as well as gene sequence and application thereof

<141>2020-05-21

<160>6

<170>SIPOSequenceListing 1.0

<210>1

<211>765

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

gacattgtgt tgacacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60

atgagctgca aatccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 120

tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagctat 300

cctcccacgt tcggtgctgg gaccaagctg gagctgaaac gaactgtggc tgcaccatct 360

ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcggacat tgtgttgaca 420

cagtctccat cctccctagc tgtgtcagtt ggagagaagg ttactatgag ctgcaaatcc 480

agtcagagcc ttttatatag tagcaatcaa aagaactact tggcctggta ccagcagaaa 540

ccagggcagt ctcctaaact gctgatttac tgggcatcca ctagggaatc tggggtccct 600

gatcgcttca caggcagtgg atctgggaca gatttcactc tcaccatcag cagtgtgaag 660

gctgaagacc tggcagttta ttactgtcag caatattata gctatcctcc cacgttcggt 720

gctgggacca agctggagct gaaacgaact gtggctgcac catct 765

<210>2

<211>255

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Asp Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ser

130 135 140

Ser Leu Ala Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser

145 150 155 160

Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp

165 170 175

Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala

180 185 190

Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser

195 200 205

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu

210 215 220

Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Pro Thr Phe Gly

225 230 235 240

Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro Ser

245 250 255

<210>3

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

gagcggataa caatttcaca cagg 24

<210>4

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

cgccagggtt ttcccagtca cgac 24

<210>5

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

taatacgact cactataggg 20

<210>6

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

tgctagttat tgctcagcgg 20

Claims (6)

1. A bivalent antibody for broad-spectrum detection of Bt Cry1 toxoid, wherein the bivalent antibody for detection is V_L-V_L-2D3, said diabody V_L-V_LThe amino acid sequence of-2D 3 is shown in SEQ ID NO. 2.
2. 2. The diabody V of claim 1_L-V_L-2D3 gene sequence.
3. 3. The gene sequence of claim 2, wherein the bivalent antibody V is encoded_L-V_LThe gene sequence of-2D 3 is shown in SEQ ID NO. 1.
4. 4. The use of the diabody of claim 1 in the detection of a Bt Cry1 toxoid that is Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E, and Cry 1F.
5. 5. The use according to claim 4, wherein the specific detection method is as follows:

   step 1, coating capture antibody-bivalent anti-Bt Cry1 toxoid antibody V_L-V_LDiluting 2D3 to 1.25. mu.g/mL with PBS buffer, adding to 96-well enzyme label plate, 100. mu.L/well, and coating overnight at 4 ℃;

   step 2, after sealing and coating overnight, throwing out liquid in the holes, washing by adopting a PBST solution, then adding MPBS with the mass concentration of 3% into the holes as a sealing solution, performing incubation for 2 hours in an incubator at 37 ℃, wherein 200 mu L/hole is formed in the holes;

   step 3, adding a sample, namely washing the ELISA plate by adopting a PBST solution, adding a BtCry1 toxoid sample dissolved in a CBS buffer solution into the hole with the concentration of 100 mu L/hole, and then putting the ELISA plate in an incubator at 37 ℃ for incubation for 1 h;

   step 4, adding a detection antibody, namely washing the ELISA plate by using a PBST solution, adding an anti-Bt Cry1 toxoid polyclonal antibody with the concentration of 0.2 mu g/mL into the hole, wherein the concentration is 100 mu L/hole, and then putting the ELISA plate in an incubator at 37 ℃ for incubation for 1 h;

   step 5, adding an enzyme-labeled antibody, namely washing the ELISA plate by adopting a PBST solution, adding a goat anti-rabbit-HRP antibody into the hole with the concentration of 100 mu L/hole, and placing the ELISA plate in a 37 ℃ incubator for incubation for 1 h;

   step 6, developing, namely washing the ELISA plate by adopting a PBST solution, adding a TMB developing solution system into micropores of the ELISA plate at 100 mu L/hole, and placing the ELISA plate in an incubator at 37 ℃ for incubation for 15min in a dark place;

   and 7, detection, namely adding the stop solution into the micropores of the ELISA plate, measuring the absorbance value at the condition of 450nm on an ELISA reader at 50 mu L/hole.
6. 6. The use according to claim 5, wherein said Bt Cry1 toxoid sample is at least one of Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1E and Cry 1F.

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