Humanized single-domain antibody insect-resistant gene C4D, protein humanized single-domain antibody C4D coded by humanized single-domain antibody insect-resistant gene C4D and application of humanized single-domain antibody insect-resistant gene C4D

Abstract

The invention belongs to the technical field of gene engineering and biological prevention and control, and particularly relates to a human single-domain antibody insect-resistant gene C4D, a protein human single-domain antibody C4D coded by the same and application of the human single-domain antibody insect-resistant gene C4D. The humanized single-domain antibody C4D disclosed by the invention can be specifically combined with the BBMV of the middle intestine of the plutella xylostella. The determination result of the indoor insecticidal biological activity of the plutella xylostella shows that the death rate within 5 days is 47.78 + / -4.16% under the concentration of the specific humanized single-domain antibody C4D. Meanwhile, the human single-domain antibody C4D is obtained by screening from a human single-domain antibody library, has the characteristics of small molecular weight and simple preparation, has lower heterology compared with antibodies from other sources, causes small immune response in a human body, is beneficial for improving safety as an insect-resistant material, and has a broad application prospect. The method has important scientific and practical significance for reducing various safety risks caused by wide use of the existing Bt toxin and expanding and enriching novel insecticidal gene resources.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering and biological control, and particularly relates to a human single-domain antibody anti-insect gene C4D, a protein human single-domain antibody C4D encoded by the human single-domain antibody anti-insect gene C4D, and application thereof. BACKGROUND

[0002] Lepidoptera pests are widely distributed, with the most abundant species in the tropics. The larvae of most species damage various cultivated plants. These pests feed on leaves, flower buds, or fruits with chewing mouthparts. The larvae often feed on leaves by rolling or tying them, and some species hide their bodies by spinning silk sheaths. Larger larvae can eat leaves or bore into branches, causing crop yield reduction or even death.

[0003] The control of Lepidoptera pests includes agricultural control, physical control, and chemical pesticide control. Chemical pesticide control is the most common method, and common Lepidoptera chemical control agents include indoxacarb, lufenuron, abamectin, and spinosad. However, long-term use of chemical pesticides can lead to the development of pesticide resistance in pests, and in some planting areas, the pesticides have even completely lost their effectiveness. The increased resistance forces farmers to increase the frequency and concentration of pesticide use, which exacerbates the risk of pesticide residues and ecological pollution.

[0004] Green insecticides, such as protein insecticides, are an important way to achieve sustainable agricultural development. Protein insecticides against Lepidoptera pests are most represented by Bt toxins (Bacillus thuringiensis crystal proteins). Bt toxins have strong toxic effects on Lepidoptera pests and mainly achieve insecticidal effects by damaging their digestive systems. However, the existing commercial insecticidal proteins are limited, and the development of new insect-resistant protein materials has become an important research focus in the field of Lepidoptera pest control. SUMMARY

[0005] The present application aims to provide a human single-domain antibody anti-insect gene C4D and a protein human single-domain antibody C4D encoded by the human single-domain antibody anti-insect gene C4D, and application thereof. The human single-domain antibody C4D can be used for the control of Lepidoptera pests and provides technical support for the opening of Lepidoptera pest control products.

[0006] The present application provides a human single-domain antibody C4D, which comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3. The amino acid sequence of the HCDR1 is VKVSAKNMA, the amino acid sequence of the HCDR2 is SINNRD, and the amino acid sequence of the HCDR3 is GRRRRTANFRY.

[0007] Preferably, the amino acid sequence of the human single-domain antibody C4D is shown in SEQ ID NO: 2.

[0008] The present invention also provides a human single-domain antibody anti-insect gene C4D, wherein the human single-domain antibody anti-insect gene C4D is a gene encoding any one of the following proteins (a) to (c):

[0009] (a) A protein comprising the amino acid sequence of the human single-domain antibody C4D of claim 1;

[0010] (b) A protein consisting of the amino acid sequence shown in SEQ ID NO:2;

[0011] (c) A protein derived from (b) with one or more amino acids substituted, deleted or added to the amino acid sequence shown in SEQ ID NO:2 and having the same function.

[0012] Preferably, the nucleotide sequence of the human single-domain antibody anti-insect gene C4D is shown in SEQ ID NO:1.

[0013] The present invention also provides a recombinant expression vector, comprising a basic vector and the human single-domain antibody anti-insect gene C4D described in the above technical solution.

[0014] Preferably, the base vector includes a plasmid vector.

[0015] The present invention also provides an engineered bacterium, comprising the human single-domain antibody anti-insect gene C4D described in the above technical solution or the recombinant expression vector described in the above technical solution.

[0016] The present invention also provides the application of the human single-domain antibody C4D described in the above technical solution, or the human single-domain antibody anti-insect gene C4D described in the above technical solution, or the recombinant expression vector described in the above technical solution, or the engineered bacteria described in the above technical solution in the control of lepidopteran pests and / or the preparation of insecticides.

[0017] Preferably, the lepidopteran pests include the diamondback moth.

[0018] The present invention also provides an insecticide, the active ingredient of which includes the human single-domain antibody C4D described in the above technical solution.

[0019] Beneficial effects:

[0020] This invention provides a human single-domain antibody anti-insect gene C4D, its encoded protein, and its applications. In this invention, the human single-domain antibody C4D specifically binds to the brush border membrane (BBMV) of the midgut of the diamondback moth. Indoor insecticidal bioactivity assays of the diamondback moth showed that at a specific concentration of human single-domain antibody C4D, the 5-day mortality rate was 47.78 ± 4.16%. This demonstrates that human single-domain antibody C4D can serve as a novel source of anti-insect proteins, enriching the variety of biological pest control products. Furthermore, the human single-domain antibody C4D described in this invention is obtained through screening from a human single-domain antibody library, possessing the advantages of small molecular weight, simple preparation, and low heterologity compared to antibodies from other sources. It also induces a small immune response in the human body, which is beneficial for improving safety as an anti-insect material. This has significant scientific and practical implications for reducing the various safety risks associated with the widespread use of existing Bt toxins and for expanding and enriching novel insecticidal gene resources. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0022] Figure 1 The data are ELISA data of monoclonal phage binding in Example 1, where different letters indicate significant differences.

[0023] Figure 2 The data are competitive ELISA identification data of monoclonal phages in Example 1, where different letters indicate significant differences;

[0024] Figure 3 The change in ELISA absorbance of human single-domain antibody C4D bound to diamondback moth BBMV in Example 2;

[0025] Figure 4 The data represents the results of the indoor activity assay of the C4D human single-domain antibody C4D in Example 3, which is used to kill diamondback moth. Detailed Implementation

[0026] This invention provides a human single-domain antibody C4D, which includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is VKVSAKNMA (SEQ ID NO:4), the amino acid sequence of HCDR2 is SINNRD (SEQ ID NO:5), and the amino acid sequence of HCDR3 is GRRRRTANFRY (SEQ ID NO:6).

[0027] As one embodiment, the amino acid sequence of the human single-domain antibody C4D is shown in SEQ ID NO:2.

[0028] The present invention also provides a human single-domain antibody anti-insect gene C4D, wherein the human single-domain antibody anti-insect gene C4D is a gene encoding any one of the following proteins (a) to (c):

[0029] (a) A protein comprising the amino acid sequence of the human single-domain antibody C4D of claim 1;

[0030] (b) A protein consisting of the amino acid sequence shown in SEQ ID NO:2;

[0031] (c) A protein derived from (b) with one or more amino acids substituted, deleted or added to the amino acid sequence shown in SEQ ID NO:2 and having the same function.

[0032] As one embodiment, the nucleotide sequence of the human single-domain antibody anti-insect gene C4D described in this invention is shown in SEQ ID NO:1.

[0033] This invention also provides a recombinant expression vector, comprising a base vector and the human single-domain antibody anti-insect gene C4D described in the above-described technical solution. In one embodiment, the base vector is a plasmid vector; in another embodiment, the plasmid vector is a pET-26b vector. This invention does not specifically limit the insertion position of the human single-domain antibody anti-insect gene C4D into the pET-26b vector, as long as the expression of the human single-domain antibody anti-insect gene C4D can be achieved. This invention does not specifically limit the construction method of the recombinant expression vector; conventional methods for preparing recombinant expression vectors in the art can be used.

[0034] This invention also provides an engineered bacterium, comprising the human single-domain antibody anti-insect gene C4D described in the above-described technical solutions or the recombinant expression vector described in the above-described technical solutions. As one embodiment, the engineered bacterium of this invention is obtained by introducing the recombinant expression vector into a host strain. As one embodiment, the host strain is *Escherichia coli*; as another embodiment, the *Escherichia coli* is *Escherichia coli* BL21(DE3). This invention does not specifically limit the specific process of introducing the recombinant expression vector into the host strain; conventional methods in the art can be used.

[0035] The present invention also provides the application of the human single-domain antibody C4D described in the above technical solution, or the human single-domain antibody anti-insect gene C4D described in the above technical solution, or the recombinant expression vector described in the above technical solution, or the engineered bacteria described in the above technical solution in the control of lepidopteran pests and / or the preparation of insecticides.

[0036] In one implementation, the lepidopteran pest is the diamondback moth.

[0037] This invention also provides an insecticide whose active ingredient includes the human single-domain antibody C4D described in the above-described technical solution. As one embodiment, the insecticide of this invention also includes pesticide-acceptable excipients; the type and amount of the excipients are not particularly limited and can be conventionally selected according to preparation needs.

[0038] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0039] Unless otherwise specified, the following examples are performed under standard experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Manual, or according to the manufacturer's instructions.

[0040] Example 1

[0041] The screening steps for anti-insect human single-domain antibodies are as follows:

[0042] Human single-domain antibody libraries purchased from Source BioScience in the UK were rescued using KM13 helper phages; single-domain antibodies with binding activity against diamondback moth BBMV were screened from the rescued phage display antibody library (for specific methods, please refer to the following reference [Lee, CM, Iorno, N., Sierro, F. and Christ, D. (2007) "Selection of human antibody fragments by phage display", Nat. Protoc., 2(11): 3001-3008]).

[0043] The screening process is as follows:

[0044] a. Coat the 6-well plate wells with 4 mL of 5% phosphate-buffered saline (MPBS) overnight at 4°C. The next day, wash the coated wells three times with PBS.

[0045] b. Take 6 × 10 12 After the rescue, the phage display antibody library was added to the well with 1 mL of 5% MPBS and vortexed. The mixture was then incubated at 25°C and 150 rpm for 1 h.

[0046] c. Transfer the phage display library from step b into 1 mL of 300 μg / mL diamondback moth BBMV (extracted from the midgut of laboratory-raised diamondback moths; extraction method as described in the following literature [M. Wolfersberger, P. Luethy, A. Maurer, P. Parentia, F. V. Sacchi, B. Giordana, G. M. Hanozet, Preparation and partial characterization of amino acid transporting brush border membra ne vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae), Comp. Biochem. Physiol. A Physiol.86(1987)301-308, https: / / doi.org / 10.1016 / 0300-9629(87)90334-3】) 6-well plates coated overnight at 4℃ and washed 3 times with PBS were slowly shaken at 150 rpm for 1 h at 25℃ and then allowed to stand at 25℃ for 1 h.

[0047] d. After the reaction is complete, wash the wells coated with diamondback moth BBMV 7 times with PBST and 3 times with PBS, then add 1 mL of 1 mg / mL trypsin and treat with gentle shaking at 25°C and 100 rpm for 1 h; the eluent at this time is the first round of screening product.

[0048] e. The products from each round of screening were amplified and rescued by KM13 helper phage before being used in the next round of screening. The second and third rounds of screening were the same as the first round, but the elution buffer was changed from 1 mL of 1 mg / mL trypsin to 1 mL of 100 μg / mL Cry1Ac toxin. In the second round of screening, the BBMV coating concentration of *Plutella xylostella* was reduced to 90 μg / mL, and the number of PBST washes was increased to 15. In the third round of screening, the BBMV coating concentration of *Plutella xylostella* was 45 μg / mL, and the number of PBST washes was increased to 25.

[0049] Positive clone ELISA identification:

[0050] After the third round of screening products were infected with the logarithmic TG1 phase, they were plated to obtain single clones. After rescue by KM13 helper phage, single clone phage ELISA was performed for identification.

[0051] The specific procedure is as follows: Add 100 μL of 10 μg / mL Diamondback mitochondritis BBMV to each well of a 96-well plate and incubate overnight at 4°C, then wash three times with PBST; add 300 μL of 5% MPBS to each well and incubate at 37°C for 1.5 h, then wash three times with PBST; add 100 μL of monoclonal rescue supernatant to each well and incubate at 37°C for 1 h, then wash three times with PBST; add 100 μL (1:5000 dilution) of anti-M13-HRP secondary antibody to each well and incubate at 37°C for 30 min, then wash six times with PBST; add TMB single-component chromogenic solution to each well and incubate at 37°C for 10 min; add 50 μL of 2M H2SO4 to each well to stop the chromogenic process and read the OD. 450 Absorbance value; simultaneously, each single clone was used as a negative control with wells coated with 5% MPBS, and the OD value of the group coated with Diamondback moth BBMV was selected. 450 With the corresponding negative OD 450比 Single clones with an OD value greater than 2.5 are considered positive clones and proceed to the next step of competitive ELISA identification to screen for positive clones. 450 Absorbance values ​​such as Figure 1 As shown.

[0052] Competitive ELISA identification:

[0053] Positive clones were subjected to competitive ELISA detection. The specific procedure was as follows: 100 μL of 10 μg / mL diamondback moth BBMV was added to each well of a 96-well plate and incubated overnight at 4°C, followed by washing three times with PBST; 300 μL of 5% MPBS was added to each well and the plate was blocked at 37°C for 1.5 h, followed by washing three times with PBST; 100 μL of monoclonal rescue supernatant containing 100 μg / mL Cry1Ac toxin was added to each well and incubated at 37°C for 1 h, followed by washing three times with PBST; 100 μL (1:5000 dilution) of anti-M13-HRP secondary antibody was added to each well and incubated at 37°C for 30 min, followed by washing six times with PBST; TMB single-component chromogenic solution was added to each well and chromogenic reaction was performed at 37°C for 10 min; 50 μL of 2M H2SO4 was added to each well to stop the chromogenic reaction and the OD was read. 450 Absorbance; simultaneously, 100 μL of monoclonal rescue supernatant was added to each well as a control, using the same coating of diamondback moth BBMV.

[0054] Competitive ELISA testing showed that the Cry1Ac toxin could competitively bind to the diamondback moth BBMV with a single-domain antibody named C4D, while the binding of the other five single-domain antibodies to BBMV was unaffected by Cry1Ac (e.g., ...). Figure 2As shown), the C4D single domain antibody nucleotide sequencing result SEQ ID NO.1 is as follows: CAGGTGCAGCTG TTGGAGGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGAGTTAAGGTTAGCGCTAAGAATATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGTCTAGAGTGGGTATCAAGCATTAATAACCGAGACGGTAGCACATACTACGCAGA CTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACCGCGGTATATTATTGCGCGGGTAGGAGGAGGCGTACCGCCAACTTCCGCTATTGGGGTCAGGGAACCCTGGTCACCGTCTCGAGC;

[0055] The corresponding amino acid sequence SEQ ID NO.2 is as follows:

[0056] QVQLLESGGGLVQPGGSLRLSCAASG VKVSAKNMA WVRQAPGKGLEW VS SINNRD GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA GR RRRTANFRY WGQGTLVTVSS; The bold italicized and underlined parts, from left to right, are the heavy chain complementarity determination regions HCDR1, HCDR2, and HCDR3.

[0057] Example 2

[0058] Prokaryotic expression and purification of C4D human single-domain antibody

[0059] The C4D (SEQ ID NO:1) gene sequence was amplified by PCR and constructed into the pET-26b vector. The recombinant plasmid was then transformed into the expression strain E. coli BL21(DE3). Single colonies with confirmed sequence were inoculated into 10 mL of 2×TY liquid medium containing Kan and cultured overnight at 37°C with shaking at 250 rpm. The next day, 2 mL of seed culture was added to 200 mL of fresh 2×TY liquid medium containing Kan and cultured at 37°C with shaking at 250 rpm. When OD... 600IPTG was added to a final concentration of 0.5 mM, and the mixture was incubated at 16°C with shaking at 200 rpm for 18 h. The bacterial culture was then centrifuged at 4°C and 5000 rpm for 10 min to collect the bacterial cells. 30 mL of PBS buffer was added to the bacterial cells for resuspending and mixing. The mixture was then sonicated at a 60 W ultrasonic sonicator for 30 min, alternating between 2 s and 3 s intervals, to thoroughly disrupt the bacterial cells. The disrupted product was centrifuged at 4°C and 10000 rpm for 30 min. The supernatant was filtered through a membrane and purified using a His-Trap affinity column, followed by desalting using a Hi-Trap Desalting column. The protein concentration was determined using a Coomassie Brilliant Blue protein quantification kit, yielding C4D at a concentration of 1.2 mg / mL. An ELISA assay was prepared to determine the binding of C4D protein to diamondback moth BBMV. Figure 3 As shown.

[0060] Example 3

[0061] Indoor insecticidal test of C4D protein

[0062] Artificial feed was spread in 6cm petri dishes. Purified C4D protein was diluted with PBS to different concentrations (100μg / mL, 200μg / mL, and 300μg / mL). 1mL of C4D was added to each petri dish and spread on the surface of the feed. After drying, 20 second-instar diamondback moths were inoculated into each dish. The single-domain antibody ZA7, which does not bind to the diamondback moth BBMV, was used as a negative control. The dishes were reared in an incubator at 26℃±1℃ with a photoperiod (L:D) of 14h:10h. Mortality was observed and recorded after 5 days. Each treatment was inoculated with 60 larvae, and the experiment was repeated three times. Mortality rates were recorded after 5 days. Figure 4 As shown.

[0063] The amino acid sequence of the single-domain antibody ZA7 is: QVQLLESGGGLVQPGGSLRLSCAAS GDMFNSQNMGWVRQAPGKGLEWVSTITGGSGSTYYADSVKGRFTISRDNSK NTLYLQMNSLRAEDAAVYYCAGHHLGETSNYSTLESWGQGTLVTVSS (SEQ ID NO:3).

[0064] The results showed that when the C4D concentration was 300 μg / mL, the mortality rate of diamondback moth was 14.44 ± 4.91% at 3 days and 47.78 ± 4.16% at 3 days, while the mortality rate of ZA7 at 300 μg / mL was 0 at both 3 days and 5 days.

[0065] From the above embodiments, it can be concluded that the human single-domain antibody C4D can be used for the control of lepidopteran pests, providing technical support for the development of lepidopteran pest control products.

[0066] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A human single-domain antibody C4D, characterized in that, The human single-domain antibody C4D includes heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is VKVSAKNMA, the amino acid sequence of HCDR2 is SINNRD, and the amino acid sequence of HCDR3 is GRRRRTANFRY.

2. The human single-domain antibody C4D according to claim 1, characterized in that, The amino acid sequence of the human single-domain antibody C4D is shown in SEQ ID NO:

2.

3. A human single-domain antibody anti-insect gene C4D, characterized in that, The human single-domain antibody anti-insect gene C4D is a gene encoding any one of the following proteins (a) to (c): (a) A protein comprising the amino acid sequence of the human single-domain antibody C4D of claim 1; (b) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; (c) A protein derived from (b) with one or more amino acids substituted, deleted or added to the amino acid sequence shown in SEQ ID NO:2 and having the same function.

4. The human single-domain antibody insect-resistant gene C4D according to claim 3, characterized in that, The nucleotide sequence of the human single-domain antibody anti-insect gene C4D is shown in SEQ ID NO:

1.

5. A recombinant expression vector, characterized in that, It includes the basic vector and the human single-domain antibody anti-insect gene C4D as described in claim 3 or 4.

6. The recombinant expression vector according to claim 5, characterized in that, The basic vector includes plasmid vectors.

7. An engineered bacterium, characterized in that, Includes the human single-domain antibody anti-insect gene C4D as described in claim 3 or 4, or the recombinant expression vector as described in claim 5 or 6.

8. The use of the human single-domain antibody C4D of claim 1 or 2, the human single-domain antibody anti-insect gene C4D of claim 3 or 4, the recombinant expression vector of claim 5 or 6, or the engineered bacteria of claim 7 in the control of lepidopteran pests and / or the preparation of insecticides.

9. The application according to claim 8, characterized in that, The Lepidoptera pests mentioned include the diamondback moth.

10. An insecticide, characterized in that, The active ingredient includes the human single-domain antibody C4D as described in claim 1 or 2.

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