Camelid anti-insect protein and its coding gene and application

Abstract

The present application provides a camel-derived anti-insect protein and its coding gene and application, and belongs to the field of genetic engineering and biological control; the nucleotide and amino acid sequences of the anti-insect protein are shown in SEQ IN NO. 1 and SEQ IN NO. 2 respectively, the anti-insect protein is a single-domain antibody with insecticidal activity screened from a camel-derived nanobody library, has small molecular weight, is simple to prepare, and is derived from a llama, is used for agricultural pest control, and has important scientific and practical significance for expanding new insecticidal gene resources.

Description

Technical Field

[0001] This application relates to the fields of genetic engineering and biological control, and in particular to a camel-derived insect-resistant protein F8, its encoding gene, and its applications. Background Technology

[0002] Crop diseases and pests are among the major agricultural disasters, and outbreaks often cause significant losses to agricultural production. Currently, the main methods of crop pest control include chemical pesticides and biological control. The long-term use of chemical pesticides not only leads to insect resistance but also severely pollutes the environment and disrupts the ecological balance. Furthermore, pesticide residues pose a serious threat to human health. Therefore, finding safe, reliable, and environmentally friendly biological control methods has become a major scientific issue in current agricultural research and production. The significant technological success of Bt protein in controlling various important pests has provided substantial inspiration and a demonstration effect for the application of protein-based biological insecticides, making the screening and creation of novel protein insecticides a key research focus in the development of new pest control technologies. Summary of the Invention

[0003] To address the aforementioned problems, the present invention aims to provide a camel-derived insect-resistant protein, its encoding gene, and its applications. Specifically, this application provides the following technical solutions:

[0004] First, this application discloses a camel-derived protein, which the applicant has named F8. Its amino acid sequence is shown in SEQ IN NO.2, and its encoding gene sequence is shown in SEQ IN NO.1.

[0005] Secondly, this invention provides a recombinant vector for expressing the camel-derived insect-resistant gene F8, comprising an initial vector and the camel-derived insect-resistant gene F8 with the nucleotide sequence shown in SEQ IN NO. 1. This application does not limit the initial vector, such as conventional vectors like pcomb3XSS or pET series vectors; furthermore, there are no special limitations on the location where the camel-derived insect-resistant gene F8 is cloned into the initial vector, as long as it enables the expression of the camel-derived insect-resistant gene F8.

[0006] Third, the present invention provides a recombinant bacterium expressing the camel-derived insect-resistant gene F8, which is obtained by transfecting a host strain with a recombinant vector containing the camel-derived insect-resistant gene F8 with the nucleotide sequence shown in SEQ IN NO. 1. Preferably, the host strain includes Escherichia coli.

[0007] Fourth, this invention provides the application of camel-derived insect-resistant protein F8, with the amino acid sequence shown in SEQ IN NO.2, in the control of lepidopteran pests. Preferably, the lepidopteran pest includes the diamondback moth.

[0008] Fifth, the present invention provides an insecticide containing camel-derived insecticidal protein F8 with the amino acid sequence shown in SEQ IN NO.2, excipients (such as PBS), etc.

[0009] This application is the first to discover the application of camel-derived insect-resistant protein F8, with the amino acid sequence shown in SEQ IN NO.2, in the control of lepidopteran pests. The insect-resistant protein F8 encoded by this camel-derived insect-resistant gene F8 can specifically bind to the midgut cadherin of the beet armyworm. Indoor insecticidal bioactivity assays for the diamondback moth showed that at a concentration of 40 μg / cm³, the insect-resistant protein F8 exhibited significant insecticidal activity. 2 At that time, the mortality rate of diamondback moth was 35±4.7% after 3 days. It can be seen that this insect-resistant protein can serve as a new source of insect-resistant proteins, enriching the variety of biological control products for pests.

[0010] Furthermore, camel-derived nanobodies possess advantages over traditional antibodies and other types of genetically engineered antibodies in terms of thermal stability and solubility due to their small size, large specific surface area, high surface energy, and macroscopic quantum tunneling effect. At the same time, nanobodies are more cost-effective to prepare, and nanobodies with insecticidal activity have excellent conditions for developing potential biopesticides. Attached Figure Description

[0011] Figure 1 These are the results of expression and identification of the toxin-binding region fragment of the beet armyworm cadherin (SeCad-TBR);

[0012] In this table, A represents the SDS-PAGE results of prokaryotic expression of the beet armyworm cadherin (SeCad-TBR) toxin-binding region fragment, with lane M representing the protein molecular weight marker and lanes 1-4 representing the purified beet armyworm cadherin (SeCad-TBR) toxin-binding region fragment; B represents the Western blot results, with lane M representing the protein molecular weight marker and lane 1 representing the purified beet armyworm cadherin (SeCad-TBR) toxin-binding region fragment.

[0013] Figure 2 This is the result of monoclonal phage binding ELISA identification.

[0014] Figure 3 This is the result of a monoclonal phage competitive ELISA identification.

[0015] Figure 4 These are the results of prokaryotic expression and identification of nanobodies;

[0016] In this table, A represents the SDS-PAGE detection results, lane M represents the protein molecular weight marker, lanes 1-5 represent the purified camel-derived nanobody F8, and lane B represents the Western blot identification results of the F8 protein.

[0017] Figure 5 These are the results of indoor biological assays for the diamondback moth. Detailed Implementation

[0018] SEQ IN NO.1:

[0019] GAGGTGCAGCTGGTGGAGTCTGGAGGAGGTTCGGTACAAGATGGAGGGTCTCTGAGACTCGCCTGTGGC

[0020] GCCTCTGGACGCATCAGTTCACACTGTATGGCCTGGTTCCGCCAGGCTCCAGGGGCGGAGCGCGAGTGG

[0021] CTCGCAGGTTTGATATAGATCTCAGGACAAGTTATGCAGATTCCGTGAAGGGCCGATTCACCATCTCC

[0022] CAAGATCGCGTCAAGAATACTCTGTCTCTGCAGATGAACAGCCTGAAGCCTGAGGACACTGCCATTTAC

[0023] TACTGTGTGGCCGCGAGTGCGGGCTATTGCGGTCGTTCATTGAGTTACCGCATTTGGGCCAGTAGTTTTTGGGGCCAGGGGGACCCAGGTTACCGTCTCCTCAGCGCACCACAGCGAAGACCCCGGCCAGGCCGGCCAG.

[0024] SEQ IN NO.2:

[0025] EVQLVESGGGSVQDGGSLLACGRISSHCMAWFRQAPGAEREWLAGFDIDLRTSYADSVKGRFTISQDRVKNTLSLQMNSLKPEDTAIYYCVAASAGYCGRSLSYRIWASSFWGQGTQVTVSSAHHSEDPGQAGQ

[0026] Source of materials and instruments used in the examples:

[0027] The insect-resistant protein encoded by the camel-derived insect-resistant gene F8 was obtained by screening a phage display antibody library, which was a camel-derived nanobody library in the form of KM13 helper phage rescue TG1 host bacteria (purchased from NBBiolab, China).

[0028] In this embodiment, the initial vector was pET-28a, which was purchased from Genscript Biotech Ltd. (China).

[0029] All other chemical reagents and organic solvents used in the following examples are domestically produced analytical grade.

[0030] Small benchtop refrigerated centrifuge (Eppendoff), vertical plate electrophoresis tank (Beijing Junyi JY-SCZ2+), electrophoresis power supply (Beijing Junyi JY600E), Western blot transfer electrophoresis system (Beijing Junyi JY-ZY5), small benchtop centrifuge (Eppendorff Centrifuge 5424R), microplate reader (Thermo Multiskan GO), plate washer (Thermo Wellwash), ultra-low temperature freezer (Haier), pure water system (Millipore Direct-Q 3UV), balance (Liangping Instruments FA2004), pH meter (Sartorius PB-10), decolorizing shaker (Scilogex SF-O180-E).

[0031] In the following examples, the term "screening" refers to the targeted screening of the beet armyworm midgut cadherin toxin-binding region protein (SeCad CR9-CR11) by a phage display antibody library.

[0032] Example 1: Screening of anti-insect camel-derived nanobodies

[0033] 1. Preparation of beet armyworm cadherin (SeCad-TBR)

[0034] In this embodiment, the preparation and detection methods of beet armyworm cadherin (SeCad-TBR) are conventional methods in the art. In this embodiment, the protein was prepared and expressed in prokaryotes according to the method disclosed in the literature "Screening and Identification of Anti-Idiotypic Nanobody Capable of Broad-Spectrum Recognition of the Toxin Binding Region of Lepidopteran Cadherins and Mimicking Domain II of Cry2Aa Toxin," and the results are as follows: Figure 1As shown, A represents the SDS-PAGE detection results of the toxin-binding region fragment of the beet armyworm cadherin (SeCad-TBR) after prokaryotic expression, where lanes 1-4 represent the purified toxin-binding region fragment of the beet armyworm cadherin (SeCad-TBR); B represents the Western blot detection results of the toxin-binding region fragment of SeCad-TBR. Figure 1 The test results indicate that Secad-TBR was successfully expressed and can be used as a target molecule for subsequent screening.

[0035] 2. The screening process is as follows:

[0036] a. The 6-well plates coated with 3 mL of skim milk powder (MPBS) dissolved in PBS buffer (5% by volume) and incubated overnight at 4°C were washed 3 times with PBS. 5 × 10⁻⁶ ppm of the solution was added. 12 One phage displaying antibodies was mixed with 1 mL of 5% MPBS and added to the well, then incubated at 37°C for 1 h.

[0037] b. Coat the 96-well plate with 800 μL of 10 μg / mL SeCadCR9-CR11 protein (the detailed preparation method of this protein is as disclosed in the literature "Screening and Identification of Anti-Idiotypic Nanobody Capable of Broad-Spectrum Recognition of the Toxin Binding Region of Lepidopteran Cadherins and Mimicking Domain II of Cry2Aa Toxin" (DOI:10.1021 / acs.jafc.3c07295; Journal of Agricultural and Food Chemistry 202472(3),1582-1591) overnight at 4℃, wash the plate 3 times with PBST, and block it with 3 mL of 5% MPBS at 37℃ for 2 h. After washing 3 times with PBST, add the reaction solution from step a to the well and incubate at 37℃ for 1 h.

[0038] c. After the reaction, the well was washed 5 times with PBST and 3 times with PBS. 100 μL of Gly-HCl (0.1 M, pH 2.2-2.5) elution buffer was added to each well and the mixture was treated at 37 °C for 10 min. Then, 24 μL of Tris-HCl neutralization buffer was added for neutralization. The eluent at this point is the first round of panning product.

[0039] The products from each round of panning were amplified and used in the next round of panning. The process for the next two rounds of panning was the same as that for the first round. In the second and third rounds of panning, the SeCadCR9-CR11 protein coating concentration was reduced to 5 μg / mL and 2.5 μg / mL, respectively, and the number of PBST washings in step c was increased to 10 and 15 times, respectively.

[0040] 3. Identification of monoclonal phages by ELISA.

[0041] After infecting *E. coli* TG1 competent cells with the third round of panning products, single clones were picked and identified by ELISA using KM13 helper phage rescue. The specific procedure is as follows: 100 μL of 2.5 μg / mL SeCadCR9-CR11 protein 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 and the plate was blocked at 37°C for 1.5 h, followed by washing three times with PBST; 100 μL of KM13 helper phage rescued single clone supernatant was added and the plate was incubated at 37°C for 1 h, followed by washing three times with PBST; 100 μL (1:3000 dilution) of anti-M13-HRP antibody was added and the plate was incubated at 37°C for 1 h, followed by washing three times with PBST; TMB single-component chromogenic solution was added and the plate was developed at 37°C for 10 min; 50 μL of 2 mol / L H2SO4 was added to stop the reaction and the readings were taken. Each clone used a 5% MPBS-coated well as a negative control. Sequencing analysis excluded monoclonal antibodies with identical sequences, resulting in five monoclonal antibodies with complete and distinct sequences for subsequent phage competition ELISA assays. The applicant named these five monoclonal antibodies B8, C8, F8, H8, and F10. The ELISA results of their corresponding phage display antibodies against SeCad CR9-CR11 are as follows: Figure 2 , Figure 3 As shown. Select OD 450 Greater than 1.0 and corresponding negative well OD 450 Monoclonal antibodies with a molecular weight of less than 0.4 were used as target clones, and the monoclonal antibody F8 was finally obtained. Its nucleotide sequence and amino acid sequence are shown in SEQ IN NO.1 and SEQ IN NO.2, respectively.

[0042] Example 2: Prokaryotic expression and purification of F8 camel-derived nanobodies

[0043] The pET-28a vector was constructed by fusing the HA tag sequence to the C-terminus of the F8 gene sequence (SEQ ID No. 1) with that of Qingke Biotechnology Co., Ltd., and the recombinant plasmid was then transformed into Escherichia coli BL21(DE3) (a conventional method in the field, as disclosed in the literature "Screening and Identification of Anti-Idiotypic Nanobody Capable of Broad-Spectrum Recognition of the Toxin Binding Region of Lepidopteran Cadherins and Mimicking Domain II of Cry2Aa Toxin" (DOI:10.1021 / acs.jafc.3c07295; Journal of Agricultural and Food Chemistry 202472(3),1582-1591").

[0044] Single colonies verified by sequencing were inoculated into 10 mL of 2×TY liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking to obtain a seed culture. The next day, the seed culture was inoculated into 1000 mL of 2×TY liquid medium containing kanamycin at a ratio of 1:100 and cultured at 37°C with shaking at 250 rpm until the OD600 reached 0.5. IPTG was added to a final concentration of 0.5 mM and cultured at 25°C with shaking at 200 rpm for 16 h to induce the expression of the target protein. The cells were centrifuged at 5000 rpm for 10 min at 4°C, and 1000 mL of the induced cells were collected. The cells were resuspended in 30 mL of resuspension solution, mixed, and then sonicated to disrupt the protein expression. The sonication was performed at 60 W for 30 min every 2 s to ensure complete disruption of the cells. The entire sonication process was performed on ice. The expressed protein was purified using a GE His-Trap affinity column. The purified protein was then desalted using a GE Hi-Trap Desalting column. The purified and desalted protein was quantified using a Coomassie Brilliant Blue protein quantification kit, yielding an insect-resistant protein F8 with a concentration of 1.02 mg / mL. The results were validated by SDS-PAGE and Western blot using an anti-HA tag antibody. Figure 4 As shown, this indicates that the insect-resistant protein F8 was successfully prepared.

[0045] Example 3: Indoor insecticidal test of insect-resistant protein F8

[0046] Artificial feed was spread in 6cm petri dishes. F8 protein was diluted with PBS, and 1mL was spread on the surface of each dish, then allowed to air dry. An equal volume of PBS was used as a negative control. Twenty second-instar diamondback moths were inoculated into each dish on different plates and placed in an incubator at 28℃±1℃, relative humidity 80±5%, and a photoperiod (L:D) of 16h:8h. The number of deaths was observed and recorded after 3 days. Each treatment was inoculated with 60 larvae, and the experiment was repeated three times. The mortality rate of the diamondback moths was calculated after 3 days. The results are shown below. Figure 5 As shown in the figure. The results indicate that the F8 protein concentration is 40 μg / cm³. 2 The mortality rate was 35.0±4.7%, while the mortality rate of the diamondback moth treated with negative treatment was 0.5±0.5%.

[0047] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A camel-derived insect-resistant protein, the amino acid sequence of which is shown in SEQ IN NO.

2.

2. The gene encoding the insect-resistant protein as described in claim 1, the nucleotide sequence of which is shown in SEQ IN NO.

1.

3. The application of the insect-resistant protein as described in claim 1 in the control of lepidopteran pests; wherein the lepidopteran pest is the diamondback moth.

4. A recombinant vector or recombinant bacterium containing a gene with a nucleotide sequence as shown in SEQ IN NO.

1.

5. An insecticide containing a protein with the amino acid sequence shown in SEQ IN NO.2.

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