High-activity vip3c mutant protein and application thereof

Abstract

The application discloses a high-activity Vip3C mutant protein and application, wherein the Vip3C mutant protein is Vip3Ca Q198VKK / G302K , Vip3Ca Q198VKK / N390K , Vip3Ca QGI198VKK / G302K or Vip3Ca QGI198VKK / N390K . Compared with a wild-type protein, the Vip3Ca mutant protein has obviously improved insecticidal activity on multiple lepidopteran pests such as Spodoptera frugiperda, Helicoverpa armigera, Spodoptera litura, Spodoptera exigua and Ostrinia furnacalis. The application further discloses a nucleic acid sequence coding the mutant protein and application of the nucleic acid sequence in preparation of a biological insecticide for preventing and treating lepidopteran pests.

Description

Technical Field

[0001] This invention belongs to the fields of biological control and biotechnology, specifically relating to a highly active Vip3C mutant protein and its applications. Background Technology

[0002] Fall armyworm, beet armyworm, and Asian corn borer, among other lepidopteran pests, severely impact agricultural production and threaten national food security. Effective pest control is crucial for achieving food security, directly impacting the stability of grain production and the safety of agricultural products. However, traditional chemical pesticides pose problems such as non-target damage, environmental pollution, pesticide residues, and soil degradation. Furthermore, pesticide resistance in pests is becoming increasingly prominent, necessitating innovative green pest control technologies.

[0003] Bacillus thuringiensis (Bt) is an insect pathogen. Expressing Bt proteins in crops can achieve green control of target pests. Bt proteins are divided into insecticidal crystal proteins (ICPs) and vegetative insecticidal proteins (Vips). Among them, Vip3 protein differs from Cry proteins in structure and mechanism of action and can serve as an important protein resource for controlling lepidopteran pests and delaying the development of resistance.

[0004] Vip3 protein consists of 786-803 amino acids with an average molecular weight of approximately 89 kDa. Vip3 protein can be hydrolyzed by insect midgut fluid into two still-linked fragments of 22 kDa and 67 kDa, both existing as tetramers before and after hydrolysis. The efficiency of Vip3 protein activation by protease cleavage and the receptor-interacting regions in domains II-III after activation can both affect its insecticidal activity. Therefore, adding more cleavage sites between domains I and II of the Vip3Ca protein, or improving the receptor affinity of the Vip3Ca protein by modifying domains II-III, may enhance its insecticidal activity and expand the agricultural applications of Vip3-like proteins.

[0005] Currently, there have been attempts to improve the insecticidal activity of Vip3 protein through site-directed mutagenesis. For example, patent publication CN119775374A (publication date: April 8, 2025) discloses an activity-enhanced Vip3 protein. This is achieved by making a single-point mutation at the Y19 site of domain I or the V383 site of domain III of the Vip3Aa protein, or at the K383 site of domain III of the Vip3Ca protein, to reduce the hydrogen bond interaction between the N-terminus of domain III and domain I, thereby accelerating protein activation and improving insecticidal activity. However, the above-mentioned prior art only involves single-point mutation, and the improvement in the insecticidal activity of Vip3Ca protein is limited. Furthermore, this technology does not involve modification of the restriction enzyme cleavage site region between domains I and II of the Vip3Ca protein, nor does it involve combined mutations of receptor-binding related sites in domains II and III. Its insecticidal activity assay is only targeted at four pests: fall armyworm, beet armyworm, cotton bollworm, and cotton bollworm. It does not involve Asian corn borer, which is naturally insensitive to many Vip3 proteins and belongs to the non-susceptible pests of Vip3 proteins. Summary of the Invention

[0006] The purpose of this invention is to design a highly active Vip3C mutant protein and its applications.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A highly active Vip3C mutant protein, wherein the Vip3C mutant protein uses the wild-type Vip3Ca protein shown in SEQ ID NO.1 as a backbone and includes the following combination of mutations: a first mutation is introduced in the protease cleavage site region between domain I and domain II, and a second mutation is introduced in the receptor binding-related region between domain II and domain III; the first mutation is located at amino acid residue 198 or its adjacent region corresponding to SEQ ID NO.1, and the second mutation is located at amino acid residue 302 or 390 corresponding to SEQ ID NO.1.

[0009] Furthermore, the first mutation in the protease cleavage site region between domain I and domain II includes any of the following mutations:

[0010] Replace the amino acid Q corresponding to the 198th position of SEQ ID NO.1 with K; or replace the amino acids QGI corresponding to the 198th to 200th positions of SEQ ID NO.1 with VKK; or replace the amino acid Q corresponding to the 198th position of SEQ ID NO.1 with VKK.

[0011] Furthermore, the second mutation in the receptor-binding-related region between domain II and domain III includes any of the following mutations:

[0012] Furthermore, replace amino acid G at position 302 of SEQ ID NO.1 with K; or replace amino acid N at position 390 of SEQ ID NO.1 with K; or replace amino acid Q at position 468 of SEQ ID NO.1 with K.

[0013] Furthermore, the Vip3C mutant protein comprises any one of the following combinations of mutations:

[0014] (1) Replace amino acid Q at position 198 of SEQ ID NO.1 with VKK, and replace amino acid G at position 302 of SEQ ID NO.1 with K, i.e., the sequence is Vip3Ca as shown in SEQ ID NO.2. Q198VKK / G302K ;

[0015] (2) Replace the 198th amino acid Q in SEQ ID NO.1 with VKK, and replace the 390th amino acid N in SEQ ID NO.1 with K, that is, the sequence is Vip3Ca as shown in SEQ ID NO.3. Q198VKK / N390K ;

[0016] (3) Replace amino acids QGI corresponding to positions 198 to 200 of SEQ ID NO.1 with VKK, and replace amino acid G corresponding to position 302 of SEQ ID NO.1 with K, i.e., the sequence is Vip3Ca as shown in SEQ ID NO.4. QGI198VKK / G302K ;

[0017] (4) Replace amino acids QGI corresponding to positions 198 to 200 of SEQ ID NO.1 with VKK, and replace amino acid N corresponding to position 390 of SEQ ID NO.1 with K, i.e., the sequence is Vip3Ca as shown in SEQ ID NO.5. QGI198VKK / N390K .

[0018] In one embodiment, the Vip3C protein is a Vip3Ca insecticidal protein.

[0019] A nucleic acid molecule that encodes the Vip3C mutant protein.

[0020] The sequence of the nucleic acid molecule is shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.

[0021] A recombinant expression vector containing the aforementioned nucleic acid molecule.

[0022] Application of Vip3C mutant protein in the preparation of biological insecticides for controlling lepidopteran pests; the lepidopteran pests are selected from one or more of the following: fall armyworm (Spodoptera frugiperda), cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), and Asian corn borer (Ostrinia furnacalis).

[0023] Biological pesticides contain Vip3Ca mutant protein, nucleic acid molecules, and recombinant expression vectors.

[0024] The above technical solution can achieve the following beneficial effects:

[0025] Mutations (such as Q198VKK and QGI198VKK) were introduced into the protease cleavage site region between domain I and domain II, increasing or replacing protease recognition and cleavage sites. This significantly improved the efficiency of Vip3Ca protein activation by hydrolysis in insect midgut fluid. Mutations of specific residues (G302 and N390) to lysine in the receptor-binding related regions of domains II-III enhanced the binding affinity to BBMV (brush border membrane vesicles) in the insect midgut. The synergistic effect of the two enhanced the overall effect.

[0026] The mutant protein of this invention significantly improves the insecticidal activity against fall armyworm, cotton bollworm, beet armyworm, and Asian corn borer. It significantly reduces the amount of protein used in actual control operations, which is beneficial for biological control of fall armyworm, cotton bollworm, beet armyworm, and Asian corn borer at a lower cost. It covers major pests of various crops such as corn, cotton, and vegetables, thereby enhancing the application of Vip3 proteins in the control of lepidopteran pests. Attached Figure Description

[0027] Figure 1 The amino acid sequence alignment results of Vip3Ca protein and its mutant protein between domain I and domain II. In the figure, Vip3Ca2 is the Vip3C protein specifically used in the example.

[0028] Figure 2 Figure showing the purification results of the Vip3C protease cleavage site replacement mutant protein;

[0029] Figure 3The image shows the SDS-PAGE electrophoresis results of Vip3C protein and its mutant proteins after digestion with enzymes in the midgut fluid of fall armyworm. C represents the undigested control protein; 0.5, 1, 1.5, and 2 represent the electrophoresis results of protein samples digested with enzymes for 0.5, 1, 1.5, and 2 hours, respectively.

[0030] Figure 4 Figure showing the purification results of the Vip3C protein alanine mutant protein;

[0031] Figure 5 Insecticidal activity results of Vip3C alanine mutant protein;

[0032] Figure 6 Figure showing the purification results of the Vip3C protein receptor binding site mutant protein;

[0033] Figure 7 Binding saturation curves of Vip3C protein and its mutants with BBMV;

[0034] Figure 8 Figure showing the purification results of the Vip3C protein combinatorial mutant;

[0035] Figure 9 The image shows the SDS-PAGE electrophoresis results of Vip3C protein and its mutant proteins after digestion with enzymes in the midgut fluid of Asian corn borer. C represents the control protein without enzyme digestion; 10, 20, 30, and 60 represent the electrophoresis results of protein samples digested for 10, 20, 30, and 60 minutes, respectively. Detailed Implementation

[0036] Terminology Explanation: Amino acids are also represented by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C), glutamine (Gln; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile; 1), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0037] The following is in conjunction with the appendix Figure 1-9 The present invention will be further illustrated by the following examples:

[0038] Example 1: Construction and protein purification of Vip3Ca protease cleavage site replacement mutant

[0039] The Vip3Ca protein is mainly composed of 803 amino acids, with a molecular weight of approximately 89 kDa. After being produced and secreted by Bacillus thuringiensis, Vip3Ca exists as a protoxin. Upon entering the midgut of insect pests, it is cleaved by midgut proteases between its domain I and domain II, transforming into an insecticidal activator. Studies have shown that the efficiency of Vip3Ca cleavage by midgut proteases is directly proportional to its insecticidal activity. This invention used the Exapasy website to analyze the protease cleavage sites of the Vip3Ca protein, finding them to be arginine at position 195 and lysine at position 197. Therefore, we hypothesize that inserting or replacing other highly efficient cleavage sites near the protease cleavage sites between domain I and domain II of the Vip3Ca protein can increase its cleavage activation efficiency. In this embodiment, the amino acid sequence of the wild-type Vip3Ca used is shown in SEQ ID No. 1, which is as follows:

[0040] MNMNNTKLNARALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDEILKNQQLLNEISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVLNDVNNKLDAINTMLNIYLPKITSMLSDVMKQNYALSLQIEYLSRQLQEISDKLDVINLNVLINSTLTEITPSYQRIKYVNEKFDKLTFATESTLRAKQGIFNEDSFDNNTLENLTDLAELAKSITKNDVDSFEFYLHTFHDVLIGNNLFGRSALKTASELITKDEIKTSGSEIGKVYSFLIVLTSLQAKAFLTLTTCRKLLGLSDIDYTSIMNEHLNNEKNEFRDNILPALSNKFSNPSYAKTIGSDNYAKVILESEPGYALVGFEIINDPIPVLKAYKAKLKQNYQVDNQSLSEIVYLDIDKLFCPENSEQKYYTKNLTFPDGYVITKITFEKKLNNLIYEATANFYDPSTGDIDLNKKQVESTFPQTDYITMDIGDDDGIYMPLGVISETFLTPINSFGLEVDAKSKTLTLKCKSYLREYLLESDLKNKETGLIAPPNVFISNVVKNWDIEEDSLEPWVANNKNAYVDNTGGIERSKALFTQGDGEFSQFIGDKLKPNTDYIIQYTVKGKPAIYLKNKSTGYITYEDTNGNSEEFQTIAVKFTSETDLSQTHLVFKSQNGYEAWGDNFIILEAKLFETPESPELIKFNDWERFGTTYITGNELRIDHSRGGYFRQSLNIDSYSTYDLSFSFSGLWAKVIVKNSRGVVLFEKVKNNGSSYEDISESFTTASNKDGFFIELTAERTSSTFHSFRDISIKEKIE.

[0041] Referring to Table 1, the region from position 192 to position 202 of Vip3Ca protein is selected (S 192 T 193 L 194 R 195 A 196 K 197 Q 19 8G 199 I 200 F 201N 202 Enzyme cleavage sites were added or improved. First, amino acids 192 to 198 of the Vip3Ca protein were replaced with amino acids 192 to 198 of the VipAa protein, constructing the M1 mutant in Table 1. In addition, glutamine at position 198 of the Vip3Ca protein was directly replaced with lysine, constructing the M2 mutant in Table 1. Further, glutamine, glycine, and isoleucine at positions 198, 199, and 200 of the Vip3Ca protein were directly replaced with VKK, constructing the M3 mutant in Table 1. Glutamine, glycine, isoleucine, and phenylalanine at positions 198, 199, 200, and 201 of the Vip3Ca protein were directly replaced with DEKK, constructing the M4 mutant in Table 1. The glutamine at position 198 of the Vip3Ca protein was directly replaced with valine, and KK was inserted between the mutated amino acids 198 and 199 to construct the M5 mutant shown in Table 1.

[0042] Table 1: List of mutation sites in Vip3Ca mutant protein relative to Vip3Ca

[0043]

[0044] The Vip3Ca shown in SEQ ID No. 1 was used to add or replace the above-mentioned restriction enzyme sites. The activation efficiency of insecticidal proteins is related to their insecticidal activity; therefore, this invention uses the protease cleavage site between domains I and II of Vip3Aa as a reference, and performs amino acid sequence alignment on mutant proteins that replace Vip3Ca and its restriction enzyme sites. Figure 1 As shown, five Vip3Ca protease cleavage sites were replaced with mutant proteins.

[0045] The M1, M4, and M5 mutants of the Vip3Ca protein in Table 1 were all constructed using similar methods. Specifically, the mutation site was designed into the primers for PCR of the Vip3Ca protein gene fragment. PCR was performed on the Vip3Ca gene to obtain two DNA fragments overlapping at their mutation sites. Based on the principle of homologous recombination, the Vip3Ca DNA fragment containing the mutation site was homologously recombinated into the pET28a vector using the Beijing TransGen pEASYR-UniSeamless Cloning and Assembly Kit. The reaction mixture was 10 μL (1 μL of pET28a-His vector linearized by PCR, 2 μL of the PCR fragment before the mutation site, 2 μL of the PCR fragment after the mutation site, and 5 μL of 2×Assembly Mix). After mixing by pipetting, the mixture was incubated at 50°C for 30 min, and the recombination product was cooled on ice. The recombinant product was mixed with competent *E. coli* BL21(DE3) cells, incubated on ice for 30 min, heat-shocked at 42℃ for 45 s, and then incubated on ice for 2 min. 600 μL of LB medium was added, and the mixture was incubated at 37℃ and 180 rpm for 1 h. After centrifugation, the bacterial cells were plated on 50 μg / mL kanamycin-resistant LB agar plates. The plates were incubated overnight at 37℃, and single colonies were picked for PCR verification. Transformants with correct PCR results were amplified, the bacterial culture was preserved, and plasmids were extracted. The plasmids were sent to a sequencing company for sequencing verification. Plasmids with correct sequencing results and bacterial strains were stored at -80℃. The M2 and M3 mutants of the Vip3Ca protein in Table 1 were constructed using site-directed mutagenesis PCR. PCR was performed using the Vip3Ca protein-encoding gene as a template. The Vip3Ca protein-encoding gene sequence is shown in SEQ ID No. 1. The obtained PCR product was digested with DMT enzyme for 1 h. 5 μL of the digested product was added to 50 μL of DMT competent cells. After incubating on ice for 30 min, the cells were heat-shocked in a 42℃ water bath for 45 s. Immediately after heat shock, the cells were incubated on ice for 2 min. 600 μL of antibiotic-free LB medium was added, and the cells were cultured at 37℃ and 180 rpm for 1 h. After centrifugation and discarding 450 μL of supernatant, 150 μL of the bacterial culture was plated on Kan-resistant LB agar plates and incubated upside down at 37℃ overnight. Single colonies were picked and placed in 5 mL LB tubes (Kan-resistant) and cultured at 37℃ for 8-10 h. The bacterial culture was then preserved and sequenced. Plasmids were extracted from the correctly sequenced bacterial cultures and transformed into E. coli BL21(DE3) to prepare glycerol-containing bacteria, which were then stored at -80℃.

[0046] Expression of mutant proteins

[0047] (1) Streak the preserved glycerol tube strain on LB (containing Kan resistance) plates and incubate overnight at 37°C with the plates upside down;

[0048] (2) Pick a single colony and inoculate it into 5 mL of LB (containing Kan resistance) liquid medium, and incubate overnight at 37°C and 180 rpm;

[0049] (3) Transfer 10 mL of the overnight culture to 1000 mL of LB (containing Kan resistance) liquid medium and incubate at 37°C and 180 rpm until the OD600 is approximately 0.8. Add IPTG to a final concentration of 0.5 mmol / L to induce Vip3Ca protein expression and incubate overnight at 16°C and 180 rpm for 15 h.

[0050] (4) Collect bacterial cells by centrifugation at 8000 rpm for 5 min at 4℃, and suspend them in Lysis buffer (containing PMSF with a final concentration of 1 mmol / L) and sonicate to disrupt them;

[0051] (5) Centrifuge at 4℃ and 18000 rpm for 40 min to collect the supernatant for purification of Vip3Ca protein;

[0052] (6) Add the supernatant to the nickel column of the affinity chromatography column at a flow rate of 0.8 mL / min, allow it to adhere to the column twice, and wash the column with 15 column volumes of washing buffer at a flow rate of 1.0 mL / min;

[0053] (7) Add 5 mL of Elution buffer to the nickel column, let stand for 10 min, control the flow rate at 0.5 mL / min, and collect the eluent;

[0054] (8) Aspirate the eluent into the dialysis bag, place it in the dialysis buffer and dialyze 3 to 4 times. Change the dialysis buffer every 3 hours. The Vip3Ca protein used in the experiment is obtained after dialysis.

[0055] (9) The purified protein was aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C;

[0056] (10) The Vip3Ca protein and mutants purified in this invention were subjected to SDS-PAGE analysis. The results are shown in the figure. Figure 2 .

[0057] Example 2: Determination of hydrolytic activation efficiency of Vip3Ca mutant protein

[0058] Extraction of midgut fluid from fall armyworm:

[0059] (1) Larval treatment: Select normal-growing fall armyworm larvae (4th-5th instar), place them on ice for 15-30 minutes to induce rigor mortis, which will facilitate subsequent operations;

[0060] (2) Midgut tissue separation: Use sterile scissors to remove the head and tail of the larva, and then use pointed forceps to gently pull out the midgut tissue containing the contents. Rinse the separated midgut tissue repeatedly with distilled water until clean.

[0061] (3) Homogenize the midgut tissue: Transfer the rinsed midgut tissue to a pre-cooled homogenizer and grind it on ice for 10 minutes. A small amount of sterile saline or PBS buffer can be added.

[0062] (4) Centrifugation: The homogenate was centrifuged at 10,000 rpm for 25 min at 4°C to precipitate tissue fragments and cell residues;

[0063] (5) Collection and preservation of supernatant: After centrifugation, the supernatant is taken as midgut fluid. It is dispensed into sterilized EP tubes, quickly frozen with liquid nitrogen, and then transferred to a -80℃ refrigerator for long-term storage.

[0064] Hydrolysis activation efficiency assay of Vip3Ca mutant protein: The same hydrolysis activation efficiency assay was performed on Vip3Ca protein and all its mutants. Vip3Ca protein was divided into 5 equal portions, each containing 20 μg of protein. Four portions were mixed with midgut fluid at a ratio of 100:1, and the remaining portion was used as a control without midgut fluid. After mixing, the mixture was placed at 27℃ for hydrolysis activation. At 0.5 h, 1 h, 1.5 h, and 2 h, one portion of the mixture was taken out, and a final concentration of 1 mM protease inhibitor PMSF was added and mixed. 5 μL of 5× loading buffer was added to each sample, and the samples were boiled in boiling water for 10 min. SDS-PAGE was then used to detect the hydrolysis activation of each protein. Each protein was analyzed three times consecutively to compare the efficiency of midgut fluid hydrolysis activation of different Vip3Ca mutant proteins. The midgut fluid hydrolysis activation results showed ( Figure 3 The efficiency of the activation of Vip3Ca mutants, except for M1, by midgut fluid hydrolysis by fall armyworm was significantly increased compared to Vip3Ca, indicating that their activation efficiency was significantly increased.

[0065] Example 3: Indoor bioactivity assay of Vip3Ca mutant protein against fall armyworm

[0066] Indoor bioactivity assay of fall armyworm (24-well plate feed method) was conducted by preparing insect feeds containing different concentrations of insecticidal proteins. Blank feed and feed supplemented with 20 mmol / L Tris-HCl buffer were used as the blank control and negative control, respectively. Newly hatched fall armyworm larvae were selected as the test insects.

[0067] (1) Weigh 20 g of artificial feed and place it in a sterile petri dish. Press the feed until there are no lumps and spread it evenly in a 9 cm sterile petri dish.

[0068] (2) Use a pipette to draw 3 mL of the prepared mixed solution of the test sample, mix the protein sample with the feed thoroughly, and evenly dispense it into 24 wells. Let it air dry at room temperature for 3 h to evaporate excess water from the feed.

[0069] (3) Use a spatula to evenly distribute the feed into the 24-well culture plate and make the feed stick to one side;

[0070] (4) First, shake the larvae onto A4 paper, and use a brush to pick out one-day-old larvae that are pulling silk, crawling fast, and active, and inoculate them into a 24-well culture plate, one larvae per well;

[0071] (5) After the larvae are attached, cover them with the top cover of the 24-hole board (with a blown plastic board), and then fix them with two rubber bands to prevent the larvae from escaping.

[0072] (6) Place the 24-well culture plate in an insect incubator at 28°C and RH 65%;

[0073] (7) Regularly observe the dryness and wetness of the feed, whether the photoperiod of the incubator is normal, and whether the conditions are suitable. After 7 days of incubation, count the survival and mortality of the test insects. Use the compiled data to calculate the mortality rate and calculate LC50 using SPSS software.

[0074] Table 2: Insecticidal activity of Vip3Ca protein and mutants against fall armyworm

[0075]

[0076] As shown in Table 2, among the mutant proteins with five replaced restriction enzyme sites, the insecticidal activity of the mutants with the remaining restriction enzyme sites, except for the M1 mutant, was significantly enhanced, increasing to 3.24 times, 3.58 times, 3.09 times, and 4.25 times that of the Vip3Ca protein, respectively. This may be related to the increased efficiency of its hydrolysis by midgut proteases. However, the insecticidal activity of the M1 mutant was significantly reduced, and the reason for this decrease needs further investigation. In summary, this invention, based on inserting or replacing more restriction enzyme sites between domain I and domain II of the Vip3Ca protein, can significantly improve its efficiency of hydrolysis by midgut proteases, which helps to obtain mutant proteins with significantly enhanced insecticidal activity.

[0077] Example 4: Construction and insecticidal activity assay of Vip3Ca protein domain II and III alanine mutants.

[0078] Studies have shown that Vip3 tetramer protoxin is activated by larval intestinal proteases, promoting the formation of dissolution pores, damaging midgut epithelial cells, and killing susceptible larvae. This invention, by analyzing the residues of domains II and III exposed after Vip3Ca is activated by protease hydrolysis, identified a binding site that plays an important role in receptor binding and toxicity. In this embodiment, the amino acid sequence of wild-type Vip3Ca used is shown in SEQ ID No. 1.

[0079] This invention utilizes GETAREA and PDBePISA programs to analyze the exposure of residues in domains II and III of Vip3Ca before and after activation. Seven residues located in the Loop region (I266, K267, G302, N390, Q468, N530, G534) were selected for alanine scanning mutagenesis to clarify the effect of these residues on the insecticidal activity of Vip3Ca. (Table 3)

[0080] Table 3: Amino acid residues exposed after Vip3Ca protein activation

[0081]

[0082] PCR amplification was performed using the Vip3Ca protein expression plasmid (Vip3Ca gene sequence shown in SEQ ID No. 1) as a template. Primers are shown in Table 4. The PCR product was treated with DMT enzyme, transformed with E. coli, and verified by DNA sequencing to obtain the corresponding mutant. The protein expression of the mutant was the same as in Example 1. The Vip3Ca protein and its mutant were detected by SDS-PAGE, and the results are shown in Table 4. Figure 4 .

[0083] Table 4: Primers for constructing Vip3Ca protein alanine mutants

[0084]

[0085] The insecticidal activity was determined in the same manner as in Example 3. Newly hatched larvae of the fall armyworm were selected as the test insects. After 7 days of cultivation, the survival and mortality of the test insects were recorded, and the data were compiled to calculate the mortality rate. The results are as follows: Figure 5 As shown.

[0086] Compared to the Vip3Ca protein, the Vip3Ca alanine mutant protein exhibited significantly reduced insecticidal activity. These results suggest that residues I266, K267, G302, N390, Q468, N530, and G534 of Vip3Ca may play an important role in its toxicity.

[0087] Example 5: Construction and insecticidal activity assay of Vip3Ca protein domain II-III mutants

[0088] As demonstrated in Example 4, residues I266, K267, G302, N390, Q468, N530, and G534 in domains II and III of the Vip3Ca protein may play an important role in the toxicity of the Vip3Ca protein. Therefore, we mutated isoleucine at position 266 of the Vip3Ca protein to aspartic acid; and mutated glycine, asparagine, glutamine, and glycine at positions 302, 390, 468, and 534 to lysine, respectively, obtaining a total of five domain II-III mutant proteins.

[0089] PCR amplification was performed using the Vip3Ca protein expression plasmid (Vip3Ca gene sequence shown in SEQ ID No. 1) as a template. Primers are shown in Table 6. The PCR product was treated with DMT enzyme, transformed with E. coli, and verified by DNA sequencing to obtain the corresponding mutant. The protein expression of the mutant was the same as in Example 1. The Vip3Ca protein and its mutant were analyzed by SDS-PAGE, and the results are shown in Table 6. Figure 6 Vip3Ca G534K Soluble expression levels decreased.

[0090] Table 6: Primers for constructing Vip3Ca protein domain II-III mutants

[0091]

[0092] The insecticidal activity was determined in the same manner as in Example 3. Newly hatched larvae of the fall armyworm were selected as the test insects. After culturing for 7 days, the survival and mortality of the test insects were recorded. The data were compiled to calculate the mortality rate and LC50. The results are shown in Table 7.

[0093] Table 7: Insecticidal activity of Vip3Ca protein domain II-III mutants against fall armyworm

[0094]

[0095] As shown in Table 7, among the four domain II-III mutant proteins, the insecticidal activity of the domain II-III mutants was significantly enhanced, increasing to 1.58 times, 2.06 times, 3.78 times, and 2.06 times that of Vip3Ca, respectively. In summary, this invention, by mutating specific lysine residues in domains II-III of the Vip3Ca protein, helps to obtain mutant proteins with significantly enhanced insecticidal activity.

[0096] Example 6: Affinity analysis of Vip3Ca protein domain II-III mutants with fall armyworm BBMV

[0097] To investigate the reasons for the increased insecticidal activity of the Vip3Ca domain II-III mutant, this invention analyzed the binding affinity between the highly active mutant and fall armyworm BBMV.

[0098] Extraction of BBMV from Fall Armyworm

[0099] (1) Sample preparation: The fifth instar larvae of fall armyworm were starved for 2 h, then placed on ice to stiffen. The stiffened larvae were dissected in a petri dish using sterile tweezers and scissors. The midgut tissue was rinsed with PBS buffer until clean and then transferred to a sterile container for later use.

[0100] (2) Tissue homogenization: Take an appropriate amount of midgut tissue and place it in a glass homogenizer, and add 9 times the volume of solution A. Place the homogenizer in an ice box, and let it stand for 1 minute after every 1 minute of grinding. Repeat this process 5 times to ensure thorough homogenization;

[0101] (3) Initial centrifugation treatment: Add an equal volume of 24 mmol / L MgCl2 solution to the homogenate, let it stand on ice for 15 min, transfer the mixture to a centrifuge tube pre-cooled to 4℃, centrifuge at 5000 rpm for 15 min, discard the precipitate, and keep the supernatant;

[0102] (4) Resuspension of precipitate and second centrifugation: Resuspend the precipitate from the previous step with 1 / 2 volume of solution A from step (2), add an equal volume of 24 mmol / L MgCl2 solution, let stand on ice for 15 min, transfer the mixture to a centrifuge tube that has been pre-cooled to 4°C, centrifuge at 5000 rpm for 15 min, discard the precipitate and keep the supernatant.

[0103] (5) Supernatant mixing and ultra-high speed centrifugation: Combine the supernatants obtained in steps (3) and (4), transfer them to an ultra-high speed centrifuge tube pre-cooled to 4°C, centrifuge at 18000 rpm for 30 min, discard the supernatant, and collect the precipitate;

[0104] (6) Resuspension of precipitate and high-speed centrifugation: Resuspend the precipitate from the previous step with an appropriate amount of solution B, transfer the resuspension to a centrifuge tube that has been pre-cooled to 4°C, centrifuge at 15000 rpm for 30 min, discard the supernatant and collect the precipitate;

[0105] (7) Final resuspension and storage: Resuspend the precipitate with an appropriate amount of solution C, dispense it into 1 mL EP tubes, quickly place the EP tubes into liquid nitrogen for quick freezing, and then transfer them to a -80℃ freezer for long-term storage.

[0106] (8) Protein quantification: The total protein of the prepared BBMV was quantified using the BCA protein concentration assay kit. The specific operation steps are described in the kit instructions.

[0107] Biotin labeling of Vip3Ca protein and its mutants

[0108] Biotin labeling was performed according to the instructions of the Frdbio HRP rapid labeling kit. The specific steps are as follows:

[0109] (1) Antibody pretreatment: Take 1 mg of the antibody to be labeled precisely and transfer it into an ultrafiltration tube. Add an appropriate amount of labeling buffer according to the antibody mass. Calculate to ensure that the final concentration of the antibody in the mixture is precisely 2 mg / mL. After the addition is completed, put the ultrafiltration tube into a centrifuge and set the centrifugation force to 12000×g for 10 min to pre-treat the antibody solution.

[0110] (2) Biotin labeling reaction: Add an appropriate amount of biotin solution to the ultrafiltration tube after the first step of centrifugation, and add a certain amount of labeling buffer. Then use a pipette to gently blow the liquid in the tube to promote the biotin solution, labeling buffer and antibody to mix thoroughly. After mixing, carefully place the ultrafiltration tube in a 37°C incubator and place it in a dark place for 30 min of incubation to allow biotin and antibody to undergo labeling reaction.

[0111] (3) Centrifugation after the first reaction: After the incubation reaction is completed, put the ultrafiltration tube back into the centrifuge, set the centrifugal force to 12000×g, and perform a centrifugation operation for 10 min.

[0112] ELISA experiment

[0113] (1) Coating: Prepare 20 mL of PBS buffer containing 1 μg fall armyworm BBMV, add 0.1 mL of the solution to each reaction well in a 96-well microplate, cover the 96-well plate, seal the edges with sealing film, place in a 4°C refrigerator overnight, discard the solution in the well, and wash each reaction well 6 times with PBS buffer.

[0114] (2) Blocking: Weigh 0.3 g bovine serum albumin (BSA) and add it to 30 mL PBS buffer to prepare a PBS buffer solution containing 2% BSA. Add 0.1 mL of this solution to each reaction well in the 96-well microplate and block at 37°C for 2 h. Then wash each reaction well 6 times with PBS buffer solution for 5 min each time.

[0115] (3) Adding samples: Add 0.1 mL of serially diluted protein sample to the reaction wells of fall armyworm BBMV that have been coated in the 96-well microplate, block at 37℃ for 2 h, and then wash the reaction wells 6 times with PBST buffer solution for 5 min each time.

[0116] (4) Add 0.1 mL of PBST buffer solution containing 1 / 40000 Streptavidin-Horseradish Peroxidase (HRP) to each well of the 96-well microplate and incubate at 37°C for 1 h;

[0117] (5) Color development: Add 0.1 mL of TMB substrate to each well of the 96-well microplate, and then place the microplate in a 37°C environment and incubate in the dark for 10 min.

[0118] (6) Termination of reaction: Add 0.1 mL of 2 mol / L HCl solution to the reaction well;

[0119] (7) OD value measurement: The OD value was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0120] (8) Unlabeled Vip3Ca protein at a concentration of 90 μM was used to competitively bind to BBMV against the mutant;

[0121] (9) Using Sigma-Plot software, the measured data of each group were analyzed and the equilibrium dissociation constant (Kd) was calculated to determine the affinity of Vip3Ca protein and its mutants for BBMV. The results are as follows: Figure 7 As shown.

[0122] Example 7: Construction and Insecticidal Activity Determination of Vip3Ca Combination Mutants

[0123] In Example 3, four mutants with significantly enhanced insecticidal activity were obtained through targeted mutation of the restriction enzyme sites. The M3 and M5 mutants, exhibiting the most significant insecticidal effects, were selected for further research after activity assays. Based on this, in Example 5, residues in domains II-III that may be involved in receptor binding were mutated, successfully obtaining four mutants with even higher insecticidal activity. The M7 and M8 mutants, showing the greatest increase in activity, were preferred. These were then combined with the aforementioned restriction enzyme site substitution mutants M3 and M5 to construct novel mutant proteins with synergistic effects.

[0124] PCR amplification was performed using Vip3Ca mutant expression plasmids M3 and M5 as templates, with primers shown in Table 6. The PCR products were then treated with DMT enzyme, transformed with *E. coli*, and verified by DNA sequencing to obtain the corresponding mutants. Protein expression of the mutants was performed as in Example 1. The Vip3Ca protein and its mutants were analyzed by SDS-PAGE, and the results are shown in Table 6. Figure 8 .

[0125] The insecticidal activity was determined in the same manner as in Example 3. Newly hatched fall armyworm larvae were selected as test insects. After culturing for 7 days, the survival and mortality of the test insects were recorded. The data were compiled to calculate the mortality rate and LC50. The results are shown in Table 8.

[0126] Table 8: Insecticidal activity of Vip3Ca hybrid mutant against fall armyworm

[0127]

[0128] As shown in Table 8, compared with Vip3Ca protein, the four combined mutants showed significantly enhanced insecticidal activity against fall armyworm, increasing to 2.72 times, 5.23 times, 2.95 times, and 4.53 times that of Vip3Ca protein, respectively.

[0129] Example 8: Insecticidal activity of enhanced Vip3Ca protein against other lepidopteran pests

[0130] This invention detected the Vip3Ca protein (Vip3Ca) with increased insecticidal activity against fall armyworm. Q198VKK / G302K and Vip3Ca Q198VKK / N390K The insecticidal activity against cotton bollworm, beet armyworm, sugar beet armyworm, and Asian corn borer was determined using the same method as in Example 3. Newly hatched larvae of cotton bollworm, beet armyworm, and Asian corn borer were selected as the test insects. After 7 days of cultivation, the survival and mortality of the test insects were recorded, and the data were compiled to calculate the mortality rate and LC50. The results are shown in Table 9.

[0131] Table 9: Insecticidal activity of the enhanced Vip3Ca protein against cotton bollworm, beet armyworm, corn borer, and Asian corn borer.

[0132]

[0133] Table 9 shows that the Vip3Ca hybrid mutant exhibits enhanced insecticidal activity against cotton bollworm, beet armyworm, corn borer, and Asian corn borer. Compared to the Vip3Ca protein, Vip3Ca... Q198VKK / G302K The insecticidal activity against cotton bollworm, beet armyworm, sugar beet armyworm, and Asian corn borer was increased by 2.10 times, 3.65 times, 6.00 times, and 12.30 times, respectively; Vip3Ca Q198VKK / N390K The insecticidal activity against cotton bollworm, beet armyworm, sugar beet armyworm and Asian corn borer was increased to 4.06 times, 2.61 times, 5.20 times and 10.67 times that of Vip3Ca protein, respectively.

[0134] In summary, this invention significantly enhances the activation efficiency and receptor binding affinity of the Vip3Ca protein by adding an enzyme cleavage site and altering the receptor binding site through a combined mutation, thereby increasing its insecticidal activity.

[0135] Example 9: Insecticidal activity assay of Vip3Ca protein lysine mutant against non-susceptible pests

[0136] To investigate the combinatorial mutant protein (Vip3Ca) Q198VKK / G302K and Vip3Ca Q198VKK / N390K The reason for the significantly increased insecticidal activity against non-susceptible pests (Asian corn borer) is that the hydrolysis activation efficiency of the mutant protein was tested using the same method as in Example 2. The results showed that... Figure 9 Neither of the two mutant protein combinations showed a significant increase in hydrolytic activation efficiency, suggesting that the increased insecticidal activity may be related to the lysine mutation. Therefore, the inventors modified the Vip3Ca mutant protein from Example 5... G302K and Vip3Ca N390K Insecticidal activity against non-susceptible pests was determined using the same method as in Example 3. Newly hatched larvae of the Asian corn borer were selected as test insects. After 7 days of cultivation, the survival and mortality of the test insects were recorded, and the data were compiled to calculate the mortality rate and LC50. The results are shown in Table 10.

[0137] Table 10: Vip3Ca protein Vip3Ca G302K and Vip3Ca N390K Insecticidal activity of mutant against Asian corn borer

[0138]

[0139] The results are shown in Table 10. Compared with Vip3Ca protein, Vip3Ca... G302K and Vip3Ca N390K The insecticidal activity was significantly enhanced, increasing by 10.03 and 3.26 times respectively.

[0140] Mutant protein name: Vip3Ca Q198VKK / G302K The sequence of SEQ ID NO.2 is as follows:

[0141]

[0142] Mutant protein name: Vip3Ca Q198VKK / N390K The sequence of SEQ ID NO.3 is as follows:

[0143]

[0144] Mutant protein name: Vip3Ca QGI198VKK / G302K The sequence of SEQ ID NO.4 is as follows:

[0145]

[0146] Mutant protein name: Vip3Ca QGI198VKK / N390K The sequence of SEQ ID NO.5 is as follows:

[0147]

[0148] The above descriptions are all preferred embodiments of the present invention. For those skilled in the art, any modifications to the present invention in various equivalent forms without departing from the principle of the present invention shall fall within the protection scope of the appended claims.

Claims

1. A highly active Vip3C mutant protein, characterized in that: The Vip3C mutant protein uses the wild-type Vip3Ca protein shown in SEQ ID NO.1 as its backbone and contains the following combination of mutations: a first mutation is introduced in the protease cleavage site region between domain I and domain II, and a second mutation is introduced in the receptor binding-related region between domain II and domain III; the first mutation is located at amino acid residue 198 or its adjacent region corresponding to SEQ ID NO.1, and the second mutation is located at amino acid residue 302 or 390 corresponding to SEQ ID NO.

1.

2. The highly active Vip3C mutant protein according to claim 1, characterized in that: The first mutation in the protease cleavage site region between domain I and domain II includes any of the following mutations: Replace the amino acid Q corresponding to the 198th position of SEQ ID NO.1 with K; or replace the amino acids QGI corresponding to the 198th to 200th positions of SEQ ID NO.1 with VKK; or replace the amino acid Q corresponding to the 198th position of SEQ ID NO.1 with VKK.

3. The highly active Vip3C mutant protein according to claim 1, characterized in that: The second mutation in the receptor-binding region between domain II and domain III includes any of the following mutations: Replace amino acid G at position 302 of SEQ ID NO.1 with K; or replace amino acid N at position 390 of SEQ ID NO.1 with K; or replace amino acid Q at position 468 of SEQ ID NO.1 with K.

4. The highly active Vip3C mutant protein according to any one of claims 1-3, characterized in that: The Vip3C mutant protein comprises any of the following combinations of mutations: (1) Replace amino acid Q at position 198 of SEQ ID NO.1 with VKK, and replace amino acid G at position 302 of SEQ ID NO.1 with K, i.e., the sequence is Vip3Ca as shown in SEQ ID NO.

2. Q198VKK / G302K ; (2) Replace amino acid Q at position 198 of SEQ ID NO.1 with VKK, and replace amino acid N at position 390 of SEQ ID NO.1 with K, i.e., the sequence is Vip3Ca as shown in SEQ ID NO.

3. Q198VKK / N390K ; (3) Replace amino acids QGI corresponding to positions 198 to 200 of SEQ ID NO.1 with VKK, and replace amino acid G corresponding to position 302 of SEQ ID NO.1 with K, i.e., the sequence is Vip3Ca as shown in SEQ ID NO.

4. QGI198VKK / G302K ; (4) Replace amino acids QGI corresponding to positions 198 to 200 of SEQ ID NO.1 with VKK, and replace amino acid N corresponding to position 390 of SEQ ID NO.1 with K, i.e., the sequence is Vip3Ca as shown in SEQ ID NO.

5. QGI198VKK / N390K .

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the Vip3C mutant protein as described in any one of claims 1 to 4.

6. The nucleic acid molecule as described in claim 5, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.

5.

7. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid molecule as described in claim 5 or 6.

8. The use of the Vip3C mutant protein as described in any one of claims 1 to 4 in the preparation of a biological insecticide for controlling lepidopteran pests.

9. The application as described in any one of claims 8, characterized in that... Lepidoptera pests are selected from one or more of the following: fall armyworm (Spodoptera frugiperda), bollworm (Helicoverpa armigera), beet armyworm (Spodoptera litura), beet armyworm (Spodoptera exigua), and Asian corn borer (Ostrinia furnacalis).

10. The application as described in any one of claims 8, characterized in that: The biopesticide contains the Vip3C mutant protein as described in any one of claims 1 to 4, the nucleic acid molecule as described in claim 5 or 6, and the recombinant expression vector as described in claim 7.

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