Camel source insect-resistant protein as well as coding gene and application thereof

Abstract

The invention provides a camel source insect-resistant protein as well as a coding gene and application thereof, and belongs to the field of genetic engineering and biological control. The nucleotide sequence and the amino acid sequence of the insect-resistant protein are respectively shown as SEQ IN NO.1 and SEQ IN NO.2, and the insect-resistant protein is a single-domain antibody which is screened from a camel source nano antibody library, has insecticidal activity, is small in molecular weight and simple to prepare, is derived from alpaca, is used for agricultural insect pest control, and has important scientific and practical significance for expanding and enriching novel insecticidal gene resources.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering and biological control, and in particular to a camel-derived insect-resistant protein F8 and its encoding gene and application. Background Art

[0002] Crop diseases and insect pests are one of the main agricultural disasters, and their outbreaks often cause significant losses to agricultural production. At present, the main means of crop pest control include chemical pesticide control and biological control. The long-term use of chemical pesticides will seriously pollute the environment and destroy the ecological balance while causing insects to develop drug resistance. At the same time, the residues of chemical pesticides also 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 great technical success of Bt protein in controlling a variety of important pests has brought great inspiration and demonstration effects to the application of protein biological insecticides, making the screening and creation of new protein insecticides an important exploration hotspot for the development of new technologies for pest control. Summary of the invention

[0003] In view of the above problems, the purpose of the present invention is to provide a camel-derived insect-resistant protein and its encoding gene and application. Specifically, the present application provides the following technical solutions:

[0004] First, the present application discloses a camel-derived protein, which the applicant named F8, with its amino acid sequence as shown in SEQ IN NO.2 and its coding gene sequence as shown in SEQ IN NO.1.

[0005] Secondly, the present invention provides a recombinant vector for expressing camel-derived insect-resistant gene F8, comprising an initial vector and a camel-derived insect-resistant gene F8 having a nucleotide sequence as shown in SEQ IN NO. 1. The present application has no limitation on the initial vector, such as conventional vectors such as pcomb3XSS and pET series vectors; at the same time, there is no special limitation on the position where the camel-derived insect-resistant gene F8 is cloned into the initial vector, as long as the expression of the camel-derived insect-resistant gene F8 can be achieved.

[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 nucleotide sequence of the camel-derived insect-resistant gene F8 as shown in SEQ IN NO. 1. Preferably, the host strain includes Escherichia coli.

[0007] Fourthly, the present invention provides the use of camel-derived insect-resistant protein F8 having an amino acid sequence as shown in SEQ IN NO. 2 in the prevention and treatment of lepidopteran pests. Preferably, the lepidopteran pests include Plutella xylostella.

[0008] Fifth, the present invention provides an insecticide, which comprises a camel-derived insecticide protein F8 with an amino acid sequence as shown in SEQ IN NO.2, an excipient (such as PBS), and the like.

[0009] The present application first discovered the application of camel-derived insect-resistant protein F8 with an amino acid sequence as shown in SEQ IN NO.2 in killing lepidopteran pests. The insect-resistant protein F8 encoded by the camel-derived insect-resistant gene F8 can specifically bind to the cadherin-like protein in the midgut of beet armyworm. The results of the indoor insecticidal biological activity assay of diamondback moth showed that the insect-resistant protein F8 concentration was 40 μg / cm 2 The 3-day mortality rate of diamondback moth was 35±4.7%. It can be seen that the insect-resistant protein can be used as a new source of insect-resistant protein to enrich the types of biological pest control products.

[0010] In addition, camel-derived nanoantibodies are superior to 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, the preparation cost of nanoantibodies is lower, and nanoantibodies with insecticidal activity have excellent conditions for the development of potential biological pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is the result of expression and identification of the toxin binding region fragment of Spodoptera exigua cadherin (SeCad-TBR);

[0012] Among them, A is the SDS-PAGE detection result of the toxin binding region fragment of Spodoptera exigua (SeCad-TBR) after prokaryotic expression, lane M is the protein molecular weight Maker, and lanes 1-4 are the purified toxin binding region fragment of Spodoptera exigua (SeCad-TBR); B is the Western blot identification result, lane M is the protein molecular weight Maker, and lane 1 is the purified toxin binding region fragment of Spodoptera exigua (SeCad-TBR).

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

[0014] Figure 3 It is the result of monoclonal phage competition ELISA identification.

[0015] Figure 4 It is the result of prokaryotic expression and identification of nanobodies;

[0016] Among them, A is the SDS-PAGE detection result, lane M is the protein molecular weight Maker, 1-5 is the purified camel-derived nanoantibody F8, and B is the Western blot identification result of F8 protein.

[0017] Figure 5 These are the results of indoor bioassay of Plutella xylostella. DETAILED DESCRIPTION

[0018] SEQ IN NO.1:

[0019] GAGGTGCAGCTGGTGGAGTCTGGAGGAGGTTCGGTACAAGATGGAGGGTCTCTGAGACTCGCCTGTGGC

[0020] GCCTCTGGACGCATCAGTTCACACTGTATGGCCTGGTTCCGCCAGGCTCCAGGGGCGGAGCGCGAGTGG

[0021] CTCGCAGGTTTGATATAGATCTCAGGACAAGTTATGCAGATTCCGTGAAGGGCCGATTCACCATCTCC

[0022] CAAGATCGCGTCAAGAATACTCTGTCTCTGCAGATGAACAGCCTGAAGCCTGAGGACACTGCCATTTAC

[0023] TACTGTGTGGCCGCGAGTGCGGGCTATTGCGGTCGTTCATTGAGTTACCGCATTTGGGCCAGTAGTTTTTGGGGCCAGGGGGACCCAGGTTACCGTCTCCTCAGCGCACCACAGCGAAGACCCCGGCCAGGCCGGCCAG.

[0024] SEQ IN NO.2:

[0025] EVQLVESGGGSVQDGGSLLACGRISSHCMAWFRQAPGAEREWLAGFDIDLRTSYADSVKGRFTISQDRVKNTLSLQMNSLKPEDTAIYYCVAASAGYCGRSLSYRIWASSFWGQGTQVTVSSAHHSEDPGQAGQ

[0026] Sources of materials and instruments in the examples:

[0027] The insect-resistant protein encoded by the camel-derived insect-resistant gene F8 was obtained by screening in a phage-displayed antibody library, which is a camel-derived nanoantibody library in the form of a KM13-assisted phage-rescued TG1 host bacteria (purchased from China NBBiolab Company).

[0028] The initial vector in the examples was pET-28a vector, purchased from GenScript Biotech Co., Ltd. (China).

[0029] Other chemical reagents and organic solvents used in the following examples were all of domestic analytical grade.

[0030] Small desktop refrigerated centrifuge (Eppendoff), vertical plate electrophoresis tank (Beijing Junyi JY-SCZ2+), electrophoresis power supply (Beijing Junyi JY600E), Western blot transfer electrophoresis instrument (Beijing Junyi JY-ZY5), small desktop centrifuge (Eppendorff Centrifuge 5424R), microplate reader (Thermo Multiskan GO), plate washer (ThermoWellwash), ultra-low temperature refrigerator (Haier), water purifier (Millipore Direct-Q 3UV), balance (Ryohei Instrument FA2004), pH meter (Sartorius PB-10), decolorization shaker (Scilogex SF-O180-E).

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

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

[0033] 1. Preparation of Spodoptera exigua cadherin (SeCad-TBR)

[0034] In this example, the preparation and detection methods of Spodoptera exigua cadherin (SeCad-TBR) are conventional methods in the art. In the example, the protein was prepared and prokaryotic expression and detection were performed according to the method disclosed in the document "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". The results are shown in FIG. Figure 1As shown, A is the SDS-PAGE detection result of the toxin binding region fragment of Spodoptera exigua (SeCad-TBR) after prokaryotic expression, wherein lanes 1-4 are the purified toxin binding region fragment of Spodoptera exigua (SeCad-TBR); B is the Western blot detection result of the toxin binding region fragment of SeCad-TBR. Figure 1 The test results showed that Secad-TBR was successfully expressed and could be used as a screening target molecule for subsequent screening.

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

[0036] a. Coat the 6-well plate with 3 mL of 5% skim milk powder dissolved in PBS buffer (MPBS) at 4°C overnight, wash three times with PBS, and place 5×10 12 The phages displaying antibodies were mixed with 1 mL of 5% MPBS and added to the wells and incubated at 37°C for 1 h.

[0037] b. 800 μL 10 μg / mL SeCadCR9-CR11 protein (the detailed preparation method of the protein is referred 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 Cadherinsand Mimicking Domain II of Cry2Aa Toxin" (DOI: 10.1021 / acs.jafc.3c07295; Journal of Agricultural and Food Chemistry 202472(3), 1582-1591) was used to coat the 96-well plate overnight at 4°C, washed three times with PBST, and blocked with 3 mL 5% MPBS at 37°C for 2h. After washing three times with PBST, the library reacted in step a was added to the wells and incubated at 37°C for 1h;

[0038] c. After the reaction, the wells were 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 treated at 37°C for 10 min. Subsequently, 24 μL of Tris-HCl neutralization buffer was added for neutralization. The eluate at this time was the product of the first round of selection.

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

[0040] 3. Monoclonal phage ELISA identification.

[0041] The third round of panning products were infected into E. coli TG1 competent cells and single clones were picked. After rescue by KM13 auxiliary phage, monoclonal phage ELISA identification was performed. The specific process is as follows: 100 μL 2.5 μg / mL SeCadCR9-CR11 protein was added to each well of the 96-well plate for overnight coating at 4°C, and washed 3 times with PBST; 300 μL 5% MPBS was added for blocking at 37°C for 1.5h, and washed 3 times with PBST; 100 μL KM13 auxiliary phage rescued monoclonal supernatant was added for incubation at 37°C for 1h, and washed 3 times with PBST; 100 μL (1:3000 dilution) anti-M13-HRP antibody was added for incubation at 37°C for 1h, and washed 3 times with PBST; TMB single-component colorimetric solution was added for color development at 37°C for 10min; 50 μL 2mol / L H2SO4 was added to terminate and read. Each clone was coated with 5% MPBS well as a negative control. After sequencing analysis, monoclonal clones with the same antibody sequence were excluded. A total of 5 monoclonal clones with complete and different antibody sequences were obtained and can be used for subsequent phage competition ELISA tests. The applicant named these 5 monoclonal antibodies B8, C8, F8, H8, and F10. The ELISA results of their corresponding phage display antibodies and SeCad CR9-CR11 are as follows: Figure 2 , Figure 3 Select OD 450 Greater than 1.0 and corresponding to the negative well OD 450 The monoclonal clone with a p-value less than 0.4 was selected as the target clone, and the monoclonal antibody F8 was finally obtained, whose 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] Qingke Biotechnology Co., Ltd. was commissioned to synthesize and construct the antibody F8 gene sequence (SEQ ID No. 1) into a pET-28a vector after fusing the HA tag sequence to the C-terminus, and the recombinant plasmid was transformed into the strain Escherichia coli BL21 (DE3) (conventional methods in the art, such as disclosed in the document "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] The single colony verified by sequencing was inoculated into 10mL of 2×TY liquid medium containing 50μg / mL kanamycin, and cultured overnight at 37℃ to obtain seed liquid. The next day, the seed liquid was inoculated into 1000mL of 2×TY liquid medium containing kanamycin at a ratio of 1:100, and cultured at 37℃, 250rpm to OD600 of 0.5, and IPTG was added to a final concentration of 0.5mM, 25℃200rpm, and cultured for 16h to induce the expression of the target protein. Centrifuge at 4℃5000rpm for 10min, collect 1000mL of induced bacteria, add 30mL of resuspension solution to resuspend, mix well and then ultrasonically break, take ultrasonic every 2s for 3s, 60w, ultrasonic for 30min, so that the bacteria are fully broken, and the ultrasonic process is carried out on ice throughout. The expressed protein was purified by GE's His-Trap affinity column. The purified protein was desalted by GE's Hi-Trap Desalting column. The purified and desalted protein was quantified using a Coomassie Brilliant Blue Protein Quantification Kit to obtain an insecticide protein F8 with a concentration of 1.02 mg / mL. Anti-HA tag antibodies were used for SDS-PAGE and Western blot verification, and the results were as follows: Figure 4 As shown, it indicates that the insect-resistant protein F8 was successfully prepared.

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

[0046] Spread the artificial feed in a 6 cm flat dish, dilute the F8 protein with PBS, spread 1 mL on the surface of the feed in each dish, and let it dry. In this test, an equal volume of PBS was used as a negative control. Take different plates, inoculate 20 2-year-old diamondback moths in each dish, and place them in an incubator with a temperature of 28°C ± 1°C, a relative humidity of 80 ± 5%, and a photoperiod (L:D) of 16h:8h. After 3 days, observe and record the number of deaths. Inoculate 60 larvae in each treatment, and repeat 3 times. After 3 days, the mortality rate of the diamondback moth was counted, and the results are as follows: Figure 5 The results showed that the concentration of F8 protein was 40 μg / cm 2 The mortality rate was 35.0±4.7%, and the mortality rate of the negative treatment diamondback moth was 0.5±0.5%.

[0047] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as 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 coding gene of the insect-resistant protein as claimed in claim 1, wherein the nucleotide sequence thereof is shown in SEQ IN NO.

1.

3. Use of the insect-resistant protein as claimed in claim 1 in controlling lepidopteran pests.

4. The use according to claim 3, characterized in that The lepidopteran pest to be controlled is Plutella xylostella.

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

1.

6. An insecticide containing a protein having an amino acid sequence as shown in SEQ IN NO. 2.

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