CN112940116B

CN112940116B - Single domain antibody, recombinant antibody, coding gene, preparation method and application

Info

Publication number: CN112940116B
Application number: CN202110280916.7A
Authority: CN
Inventors: 高美静; 刘贤金; 卢莉娜; 胡晓丹; 仲建锋; 张霄; 徐重新; 谢雅晶; 朱庆; 林曼曼; 陈蔚
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2021-03-16
Filing date: 2021-03-16
Publication date: 2022-07-12
Anticipated expiration: 2041-03-16
Also published as: CN112940116A

Abstract

The invention provides a single domain antibody specifically combined with insect BBMV, a recombinant antibody, a coding gene, a preparation method and application, belonging to the fields of genetic engineering and pest control, wherein the amino acid sequence of the single domain antibody is shown as SEQ ID No.1, and the recombinant antibody with synergistic effect on Bt toxin comprises the single domain antibody, a connecting peptide and an antibody light chain variable region for identifying Bt toxin which are connected in sequence; the recombinant antibody has high specific binding activity with diamondback moth BBMV and four Bt toxins, can improve the lethality of the four toxins to diamondback moth larvae from 23.3-38.3% to 63.3-88.3%, and has remarkable synergistic effect on the Bt toxins; the application of the recombinant antibody can effectively improve the control effect of the Bt preparation or the Bt crops on pests and effectively delay the evolution of the resistance of the target pests Bt.

Description

Single domain antibody, recombinant antibody, coding gene, preparation method and application

Technical Field

The invention belongs to the field of genetic engineering and pest control, and particularly relates to a single domain antibody specifically combined with insect BBMV, a recombinant antibody, a coding gene, a preparation method and application.

Background

Bacillus thuringiensis (Bt) is one of the most successful biopesticides applied at present, and a transgenic biotechnology is used for transferring Bt insecticidal genes into plants to stably express the Bt insecticidal genes to generate insect-resistant crops, so that a new chapter for applying Bt is opened. The method is popularized and used since 1996, farmers grow the transgenic crops in an accumulated way for more than 930 million hectares, the population quantity of target pests is effectively inhibited, the dependence on conventional pesticides is reduced, and the income of the farmers is increased. However, the long-term intensive planting of transgenic Bt crops places tremendous selection pressure on the target pests and has led to the evolution of their resistance. In view of the fact that the evolution of insect Bt resistance becomes an important factor restricting the long-term application of Bt toxin, how to effectively delay Bt resistance becomes a problem to be solved at present.

The Bt preparation and the synergistic factor are mixed for use to improve the insecticidal activity and the field control stability of the Bt preparation, which is one of the most rapid and effective ways in production and application and is also an effective means for effectively delaying the resistance of the Bt of pests. There are many types of Bt toxin potentiators. Among them, Bt toxin receptor protein fragments (particularly cadherin toxin binding domain fragments) have been studied most extensively. However, the preparation cost of the receptor protein fragment is high, the effect is unstable, and the aimed toxin type is relatively single, which is not beneficial to popularization and application. In addition, the development of potentiators of novel receptor protein fragments is heavily dependent on the discovery of novel Bt receptor proteins, and thus studies on the potentiation of receptor protein fragments have been suspended in recent years.

Disclosure of Invention

Aiming at the problems of drug resistance widely existing in current rotary Bt crops and Bt preparations, limitation of the existing Bt synergist and the like, the invention provides a recombinant antibody with a synergistic effect on Bt toxin, a gene for coding the recombinant antibody, a preparation method and application thereof.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a single domain antibody specifically bound with insect BBMV, and the amino acid sequence of the single domain antibody is shown as SEQ ID No. 1.

The invention provides a gene for coding the single domain antibody, and the nucleotide sequence of the gene is shown as SEQ ID No. 2.

The invention provides a recombinant antibody with a synergistic effect on Bt toxin, which comprises the single domain antibody, a connecting peptide and an antibody light chain variable region for recognizing Bt toxin which are sequentially connected.

Preferably, the amino acid sequence of the variable region of the antibody light chain for identifying the Bt toxin is shown in SEQ ID No.3, and the amino acid sequence of the connecting peptide is shown in SEQ ID No. 4.

Preferably, the amino acid sequence of the recombinant antibody is shown as SEQ ID No. 5.

The invention provides a gene for coding the recombinant antibody, and the nucleotide sequence of the gene is shown as SEQ ID No. 6.

The invention provides a Bt toxin synergist, which comprises the recombinant antibody and a solvent.

Preferably, the solvent comprises a PBS solution; the Bt toxins include Cry1Ac, Cry1B, Cry1C, and Cry 1F.

The invention provides application of the recombinant antibody and the Bt toxin synergist in pest control.

Preferably, the pests include diamondback moth.

The invention has the beneficial effects that: the single domain antibody provided by the invention can be specifically combined with insect BBMV, the recombinant antibody comprises the single domain antibody, a connecting peptide and an antibody light chain variable region for identifying Bt toxin which are sequentially connected, the recombinant antibody has high specific binding activity with diamondback moth BBMV and four Bt toxins (Cry1Ac, Cry1B, Cry1C and Cry1F), the lethality rate of the four toxins to diamondback moth larvae can be improved from 23.3-38.3% to 63.3-88.3%, and the synergistic effect on the Bt toxin is obvious. The recombinant antibody is a single-chain antibody, has short preparation period and small molecular weight, is suitable for in vitro large-scale production and can be co-expressed with Bt toxin in plants by utilizing a transgenic technology; the application of the recombinant antibody can effectively improve the control effect of Bt preparations or Bt crops on pests and effectively delay the evolution of the resistance of target pests Bt.

Drawings

FIG. 1 is a schematic representation of the bispecific binding of recombinant antibodies of the invention to toxins and insect BBMVs;

FIG. 2 shows a recombinant single-chain antibody (4C 1V) of the present invention_H-2D3V_L) The result of electrophoresis after prokaryotic expression of (1), wherein M: protein molecular weight standards; 1, crushing thalli; 2, purifying the fragment; 3, a WesternBlot detection result of the broken thallus;

FIG. 3 shows a recombinant single-chain antibody (4C 1V)_H-2D3V_L) Results of ELISA binding assays to Bt toxin;

FIG. 4 shows a recombinant single-chain antibody (4C 1V)_H-2D3V_L) Combining the analysis result with ELISA of diamondback moth BBMV;

FIG. 5 shows a recombinant single-chain antibody (4C 1V)_H-2D3V_L) And detecting the result of the synergistic effect of the Bt toxin in the plutella xylostella.

Detailed Description

The invention provides a single domain antibody specifically bound with insect BBMV, wherein the amino acid sequence of the single domain antibody is shown as SEQ ID No.1, and specifically comprises the following steps:

QVQLLESGGGLVQPGGSLRLSCAASGYIFSYYTMAWVRQAPGKGLEWVSSIAEASGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATALDIIRLDRCCTKEDLYFWGQGTLVTVSS。

the invention also provides a gene for coding the single domain antibody, and the nucleotide sequence of the gene is shown as SEQ ID No.2, and specifically comprises the following steps:

CAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATATATCTTTAGCTATTATACTATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAAGCATTGCTGAGGCTAGCGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGACAGCGCTGGATATTATTCGGTTGGATCGTTGTTGTACGAAGGAGGACCTCTACTTTTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC。

in the invention, the single-domain antibody specifically binding to the insect BBMV is preferably obtained by performing targeted screening on the diamondback moth BBMV through a phage display library. In the present invention, the phage display library is preferably obtained using a KM 13-assisted phage rescue TG1 host bacteria form human-derived single domain antibody library; the method for rescuing the human-derived single-domain antibody library in the form of TG1 host bacteria by using KM13 as an auxiliary phage is not particularly limited, and the method can be carried out according to the descriptions of the Selection of human antibody fragments by phase display, NATURE PROTOCOLS | VOL.2NO.11|200, doi: 10.1038/nprot.2007.448.

In the invention, the amino acid sequence of the variable region of the antibody light chain for recognizing the Bt toxin is preferably shown as SEQ ID No.3, and the amino acid sequence is as follows:

DIVLTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPPTFGAGTKLELKRTVAAPS；

the preferred amino acid sequence of the connecting peptide is shown as SEQ ID No.4, and specifically comprises the following steps:

GGGGSGGGGSGGGGS；

in the present invention, the amino acid sequence of the recombinant antibody is preferably as shown in SEQ ID No.5, specifically as follows:

QVQLLESGGGLVQPGGSLRLSCAASGYIFSYYTMAWVRQAPGKGLEWVSSIAEASGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATALDIIRLDRCCTKEDLYFWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPPTFGAGTKLELKRTVAAPS。

the invention also provides a gene for coding the recombinant antibody, and the nucleotide sequence of the gene is shown as SEQ ID No.6, and specifically comprises the following steps:

CAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATATATCTTTAGCTATTATACTATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAAGCATTGCTGAGGCTAGCGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGACAGCGCTGGATATTATTCGGTTGGATCGTTGTTGTACGAAGGAGGACCTCTACTTTTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATTGTGTTGACACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAATCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCTCCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGAACTGTGGCTGCACCATCT。

in the present invention, the recombinant antibody is preferably prepared by prokaryotic expression. In the present invention, the specific preparation method preferably comprises the steps of: cloning the gene of the coding recombinant antibody to an expression vector to obtain a recombinant vector, then transferring the recombinant vector to host bacteria to obtain a recombinant strain, and inducing the recombinant strain to express to obtain the recombinant antibody.

In the present invention, the expression vector is preferably a pET-26b (+) vector; the host bacterium is preferably E.coli BL21(DE 3). In the present invention, the synthesis of the gene encoding the recombinant antibody, the preparation of the recombinant vector, and the transfer of the recombinant vector into the host cell are preferably performed by a biological company. According to the invention, after the recombinant strain is obtained, sequencing verification is preferably carried out, and the method for sequencing verification is not particularly limited, and can be carried out by adopting a conventional sequencing verification method in the field.

In the invention, the recombinant strain after sequencing verification is preferably cultured and the expression of the recombinant antibody is preferably induced. In the present invention, the recombinant strain is preferably subjected to activation culture in a kanamycin-resistant LB liquid medium to obtain a seed solution; the seed solution was then inoculated into a kanamycin-resistant LB liquid medium to induce expression of recombinant antibodies. In the invention, the temperature of the activation culture is preferably 36-38 ℃, more preferably 37 ℃, and the time of the activation culture is preferably 10-14 h, more preferably 12 h. In thatIn the present invention, the inoculation volume of the seed liquid is preferably 0.5% to 2%, more preferably 1% of the kanamycin-resistant LB liquid medium. In the present invention, when the recombinant strain is cultured to OD₆₀₀When the value is 0.4-0.6, adding IPTG (isopropyl-beta-D-thiogalactoside) for induction expression; the final concentration of the IPTG is preferably 0.4-0.6 mmol/L, and more preferably 0.5 mmol/L; the temperature for inducing expression is preferably 15-18 ℃, more preferably 16 ℃, and the time for inducing expression is preferably 18-22 h, more preferably 20 h. After the induction expression, the recombinant antibody is obtained by preferably collecting and crushing thallus, purifying thallus protein and desalting and purifying the purified protein. In the present invention, the disruption of the bacterial cells is preferably performed by ultrasonic waves, the purification of the bacterial proteins is preferably performed by a GE His-Trap affinity column, and the Desalting is preferably performed by a GE desaling Desalting column.

The invention also provides a Bt toxin synergist which comprises the recombinant antibody and a solvent. In the present invention, the solvent preferably comprises a PBS solution, the concentration of the PBS solution is preferably 0.01 mol/L; the concentration of the recombinant antibody in the Bt toxin synergist is preferably 0.1-1.5 mg/mL, and more preferably 1 mg/mL; in the present invention, the Bt toxins include Cry1Ac, Cry1B, Cry1C, and Cry 1F. The preparation method of the Bt toxin synergist is not particularly limited, and the recombinant antibody is dissolved to a limited concentration by adopting a PBS (phosphate buffer solution).

The invention also provides application of the recombinant antibody and the Bt toxin synergist in pest control. In the present invention, the pests include diamondback moth. In the invention, the application is specifically that the recombinant antibody, the Bt toxin synergist and the Bt toxin act together to control pests. In the present invention, it is preferable to administer the recombinant antibody or the Bt toxin potentiator first, and then to use the Bt toxin; or administering the recombinant antibody, the Bt toxin potentiator, and the Bt toxin simultaneously.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

A human-derived single-domain antibody library in the form of TG1 host bacteria purchased from Source Bioscience, UK was rescued as a phage display antibody library using KM13 helper phage (for concrete operations, see the literature (Selection of human antibodies fragments by phase display, NATURE PROTOCOLS. vol.2NO. 11. 200, doi: 10.1038/nprot.2007.448)); and (3) performing targeted screening on the diamondback moth BBMV by taking a phage display library.

The preparation process of the diamondback moth BBMV comprises the following steps: and (3) selecting diamondback moth larvae of the next day of 4 th instar for dissection, cleaning dissected midgut tissues by using 0.15M sodium chloride, putting the dissected midgut tissues into an ep tube placed on ice, and placing the dissected samples into a refrigerator at the temperature of-80 ℃ for later use. BBMV is obtained by a Mg-EGTA sedimentation method, and the specific operation steps are described in the reference literature: wolfersberger M, Luethyl P, Maurer A, et al, preparation and partial catalysis of amino acid transport fiber membrane catalysts from the large middle of the carbon fiber (Pieris fiber) [ J ] comprehensive Biochemistry and Physiology Part A. Physiology,1987,86(2): 301-. The prepared and dispensed BBMV was stored in a-80 ℃ freezer. Protein concentration was determined using the Bradford method with Bovine Serum Albumin (BSA) as a standard.

The screening process is as follows: the same amount (2. mu.g) of the diamondback moth BBMV was coated in each round, 3 rounds of panning were performed, and a total of 384 clones were used for monoclonal ELISA identification when the OD of the coated wells was₄₅₀Value and negative well OD₄₅₀And (3) judging the clone as a positive clone when the value ratio is more than 2, wherein the clone with the number of 4C1 has the highest binding capacity with the diamondback moth BBMV, performing DNA sequencing on the positive clone, and obtaining the nucleotide sequencing result as follows:

CAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATATATCTTTAGCTATTATACTATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAAGCATTGCTGAGGCTAGCGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGACAGCGCTGGATATTATTCGGTTGGATCGTTGTTGTACGAAGGAGGACCTCTACTTTTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC(SEQ ID No.2)。

the corresponding amino acid sequences are as follows:

QVQLLESGGGLVQPGGSLRLSCAASGYIFSYYTMAWVRQAPGKGLEWVSSIAEASGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATALDIIRLDRCCTKEDLYFWGQGTLVTVSS(SEQ ID No.1)。

example 2

Bispecific single chain antibody 4C1V_H-2D3V_LProkaryotic expression of (recombinant antibody)

2D3 is a monoclonal antibody capable of broad-spectrum recognition of Cry1A toxoid (see the literature references: conformation, Expression, and Identification of Double Light Chain (V)_L-V_L) The light chain variable region V of 2D3 was modified by the following formula, Antibody from a Unique Bt Cry1-Specific Monoclonal Antibody, Food Antibody methods (2020)13: 1570-1582, https:// doi. org/10.1007/s12161-020-01754-y)_LThe sequence is connected with the sequence of 4C1 by a 45bp linker, and a 786bp single-chain antibody 4C1V is obtained by splicing_H-2D3V_L. The nucleotide sequence of linker is shown below: GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCG (SEQ ID No. 7).

The corresponding amino acid sequences are as follows: GGGGSGGGGSGGGGS (SEQ ID No. 4).

Single chain antibody 4C1V_H-2D3V_LThe nucleotide sequencing results of (a) were as follows: CAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATATATCTTTAGCTATTATACTATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAAGCATTGCTGAGGCTAGCGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGACAGCGCTGGATATTATTCGGTTGGATCGTTGTTGTACGAAGGAGGACCTCTACTTTTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATTGTGTTGACACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAATCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCTCCCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGAACTGTGGCTGCACCATCT (SEQ ID No.6)

The corresponding amino acid sequences are as follows:

QVQLLESGGGLVQPGGSLRLSCAASGYIFSYYTMAWVRQAPGKGLEWVSSIAEASGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATALDIIRLDRCCTKEDLYFWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPPTFGAGTKLELKRTVAAPS(SEQ ID No.5)。

the gene sequence of the single-chain antibody is entrusted to general biology company of Anhui and is synthesized and constructed on a pET-26b (+) vector, and the recombinant plasmid is transferred into a strain E.coli BL21(DE 3). Single colonies with correct sequencing were inoculated into 10mL of LB liquid medium with kanamycin resistance and shake-cultured overnight at 37 ℃ to obtain a seed solution. The next day, the seed solution was inoculated into 200mL LB liquid medium containing kanamycin at a ratio of 1:100, cultured with shaking at 37 ℃ and 250rpm to OD₆₀₀When the concentration is 0.5, IPTG is added to the final concentration of 0.5mmol/L, the mixture is cultured at the temperature of 16 ℃ and 200rpm for 20 hours, and the expression of the target protein is induced. Centrifuging the induced expression bacterial liquid at 4 ℃ and 5000rpm for 10min, collecting the thalli, adding 20mL of resuspension solution (the composition of the resuspension solution is 25mmol/L Tris-HCl, 150mmol/L NaCl and 15mol/L imidazole), mixing uniformly, and carrying out ultrasonic crushing in an ice bath, wherein ultrasonic crushing is carried out every 2s for 3s, 60w and 30min, so that the thalli are crushed fully. The supernatant of the disrupted cells was purified by GE His-Trap affinity column. And (4) Desalting the purified protein by using a GE desaling Desalting column. The protein concentration was measured by Coomassie Brilliant blue at 1.5mg/mL and 10mL volume.

Example 3

Bispecific single chain antibody 4C1V_H-2D3V_L(recombinant antibody) binding Activity identification

Identification of the Single chain antibody 4C1V by ELISA_H-2D3V_LBinding activity of the recombinant antibodies to 4 Bt toxins (Cry1Ac, Cry1B, Cry1C, Cry1F) and diamondback moth BBMV. Each well was coated with CBS solubilized 2 μ g Bt toxin (Cry1Ac, Cry1B, Cry1C, Cry1F) or diamondback moth BBMV protein in a 96 well microplate. Incubate with 1% BSA in PBST for 2h at 37 ℃ and wash PBST 5 times for 3min each.

Biotin marker 4C1V_H-2D3V_LThe recombinant antibody and the biotin labeling method are referred to the EZLink-Sulfo-NHS-SS-Biotinylation Kit specification, and the specific labeling process is as follows: 4C1V was washed with PBS buffer (pH 7.4)_H-2D3V_LDissolving to 1 mg/ml; 4C1V at 1mg/mL per mL_H-2D3V_L14 mu.L of 10mmol/L Sulfo-NHS-SS-Biotin is added; reacting at room temperature for 1h, and uniformly mixing every 15 min; the reaction solution was transferred to a dialysis bag, and about 5L of 0.01mol/L PBS buffer (pH 7.4) was put into a large number of cartridges, dialyzed overnight in a refrigerator at 4 ℃ to remove free biotin molecules, and stored in a refrigerator at-80 ℃ for further use.

Adding biotin-labeled 4C1V with corresponding concentration into an enzyme-labeled hole coated with Bt toxin or diamondback moth BBMV protein_H-2D3V_L(determination of binding to different Bt toxins, Biotin marker 4C1V_H-2D3V_LThe concentration of (A) is 100 nmol/L; determination of binding to the Plutella xylostella BBMV selected Biotin marker 4C1V_H-2D3V_LThe concentration series of (b) are respectively 200nmol/L, 100nmol/L, 50nmol/L, 25nmol/L and 12.5nmol/L) non-specific binding determination, 100 times of concentration of unlabeled protein 4C1V is required_H-2D3V_LIncubate at 37 ℃ for 1h, wash 5 times with PBST, 3min each time. Diluted HRP-labeled avidin (1: 3000) was added, incubated at 37 ℃ for 1h, and PBST washed 5 times for 3min each. Adding TMB single-component developing solution, incubating at 37 deg.C for 10min, adding 1mol/L H₂SO₄The reaction was stopped and the absorbance at 450nm was measured. The absorbance of specific binding was subtracted from the absorbance of non-specific binding, each experiment was repeated 3 times independently, and the results are shown in tables 1 and 2, and the data obtained were analyzed using GraphPad Prism 5 software, and it was shown that the single-chain antibody 4C1V was obtained using a prokaryotic system_H-2D3V_LThe recombinant antibody has better specific binding activity with Bt toxin or diamondback moth BBMV.

TABLE 1 Single chain antibody 4C1V_H-2D3V_LDetermination of Bt toxin-specific binding Activity

TABLE 2 Single chain antibody 4C1V_H-2D3V_LDetermination result of specific binding activity with diamondback moth BBMV

Example 4

Bispecific single chain antibody 4C1V_H-2D3V_L(recombinant antibody) synergistic effect on 4 Bt toxins in plutella xylostella

Evaluation of bispecific Single chain antibody 4C1V Using bioassay_H-2D3V_LSynergistic effect on 4 Bt toxins in plutella xylostella. Bt toxin was diluted to corresponding concentrations with 0.01mol/L PBS, with working concentrations of Cry1Ac, Cry1B, Cry1C, Cry1F toxins of 5ng/cm each²、80ng/cm²、20ng/cm²And 50ng/cm²。

And adding the corresponding toxin diluted solution into a culture dish with the artificial feed for the plutella xylostella, and naturally drying the plutella xylostella. Then add 4C1V at 100 times toxin concentration_H-2D3V_LRecombining antibodies, namely inoculating 20 heads of plutella xylostella larvae of 2 years old into each culture dish after airing, wherein the concentration of each toxin is 3 times; mortality was recorded after 5 days and was considered dead or not as long as 4 years old. The results are shown in Table 3, PBS alone or 4C1V alone_H-2D3V_LThe mortality rate of the diamondback moth larvae treated by the recombinant antibody is less than 10 percent, which indicates that 4C1V_H-2D3V_LThe recombinant antibody has no toxic effect on diamondback moth. 5ng/cm²The death rate of the diamondback moth after the Cry1Ac toxin treatment is 25.0 +/-2.9 percent, and 4C1V is added_H-2D3V_LThe larval mortality rate after the recombinant antibody can be improved to 80.0 +/-5.8%; 80ng/cm²The death rate of the diamondback moth after the Cry1B toxin treatment is 23.3 +/-1.7 percent, and 4C1V is added_H-2D3V_LThe larval mortality rate after the recombinant antibody can be improved to 71.7 +/-1.7%; 20ng/cm²The death rate of the diamondback moth treated by the Cry1C toxin is 26.7 +/-3.3 percent, and 4C1V is added_H-2D3V_LThe larval mortality rate after the recombinant antibody can be improved to 88.3 +/-3.5%; 50ng/cm²Cry1F toxin treatment ofThe death rate of the plutella xylostella is 38.3 + -4.4%, and 4C1V is added_H-2D3V_LThe larval mortality rate can be improved to 63.3 +/-3.3% after the recombinant antibody.

TABLE 34C 1V_H-2D3V_LThe result of the synergistic effect of the recombinant antibody on 4 Bt toxins

From the above examples, it can be seen that recombinant antibody 4C1V_H-2D3V_LHas obvious synergistic effect on Cry1Ac, Cry1B, Cry1C and Cry1F toxins in the process of preventing and controlling diamondback moths.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> agricultural science and academy of Jiangsu province

<120> single domain antibody specifically bound with insect BBMV, recombinant antibody, encoding gene, preparation method and application

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

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ile Phe Ser Tyr Tyr

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

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Ser Ser Ile Ala Glu Ala Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

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

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

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Ala Thr Ala Leu Asp Ile Ile Arg Leu Asp Arg Cys Cys Thr Lys Glu

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Asp Leu Tyr Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

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caggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60

tcctgtgcag cctccggata tatctttagc tattatacta tggcctgggt ccgccaggct 120

ccagggaagg gtctagagtg ggtatcaagc attgctgagg ctagcggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggac accgcggtat attattgcgc gacagcgctg 300

gatattattc ggttggatcg ttgttgtacg aaggaggacc tctacttttg gggtcaggga 360

accctggtca ccgtctcgag c 381

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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

115 120

<210> 4

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 4

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 5

<211> 262

<212> PRT

<213> Artificial Sequence

<400> 5

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ile Phe Ser Tyr Tyr

20 25 30

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

35 40 45

Ser Ser Ile Ala Glu Ala Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Thr Ala Leu Asp Ile Ile Arg Leu Asp Arg Cys Cys Thr Lys Glu

100 105 110

Asp Leu Tyr Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

210 215 220

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

225 230 235 240

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

245 250 255

Thr Val Ala Ala Pro Ser

260

<210> 6

<211> 786

<212> DNA

<213> Artificial Sequence

<400> 6

caggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60

tcctgtgcag cctccggata tatctttagc tattatacta tggcctgggt ccgccaggct 120

ccagggaagg gtctagagtg ggtatcaagc attgctgagg ctagcggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggac accgcggtat attattgcgc gacagcgctg 300

gatattattc ggttggatcg ttgttgtacg aaggaggacc tctacttttg gggtcaggga 360

accctggtca ccgtctcgag cggtggaggc ggttcaggcg gaggtggctc tggcggtggc 420

ggatcggaca ttgtgttgac acagtctcca tcctccctag ctgtgtcagt tggagagaag 480

gttactatga gctgcaaatc cagtcagagc cttttatata gtagcaatca aaagaactac 540

ttggcctggt accagcagaa accagggcag tctcctaaac tgctgattta ctgggcatcc 600

actagggaat ctggggtccc tgatcgcttc acaggcagtg gatctgggac agatttcact 660

ctcaccatca gcagtgtgaa ggctgaagac ctggcagttt attactgtca gcaatattat 720

agctatcctc ccacgttcgg tgctgggacc aagctggagc tgaaacgaac tgtggctgca 780

ccatct 786

<210> 7

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 7

ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcg 45

Claims

1. A recombinant antibody with synergistic effect on Bt toxin is characterized by consisting of a single domain antibody, a connecting peptide and an antibody light chain variable region for recognizing Bt toxin which are connected in sequence;

the amino acid sequence of the single domain antibody is shown as SEQ ID No. 1;

the amino acid sequence of the antibody light chain variable region for identifying the Bt toxin is shown as SEQ ID No.3, and the amino acid sequence of the connecting peptide is shown as SEQ ID No. 4;

the amino acid sequence of the recombinant antibody is shown in SEQ ID No. 5.

2. The gene encoding the recombinant antibody of claim 1, wherein the nucleotide sequence is represented by SEQ ID No. 6.

3. A Bt toxin potentiator comprising the recombinant antibody of claim 1 and a solvent;

the solvent comprises a PBS solution; the Bt toxins include Cry1Ac, Cry1B, Cry1C, and Cry 1F.

4. Use of the recombinant antibody of claim 1, the Bt toxin potentiator of claim 3 for controlling pests;

the pests include diamondback moths.