AI aided design chicken beta-defensin-6 mutant, recombinant expression system and application

Abstract

The invention relates to a chicken beta-defensin-6 mutant based on AI aided design, a recombinant expression system and application, and relates to the technical field of gene engineering and protein engineering. The chicken beta defensin-6 mutant has the following amino acids at the following five sites: lysine at the 33rd site, lysine at the 35th site, asparagine at the 49th site, arginine at the 61st site and proline at the 65th site. The chicken beta-defensin-6 mutant has high stability, activity and solubility, compared with wild chicken beta-defensin-6, the chicken beta-defensin-6 mutant has higher antibacterial activity and expression level, an efficient and safe antibacterial substitute is provided for livestock and poultry breeding, and the chicken beta-defensin-6 mutant can be widely applied to the fields of livestock and poultry breeding, animal health care and biological preservation.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and protein engineering technology, and in particular to AI-assisted design of chicken β-defensin-6 mutants, recombinant expression systems and their applications. Background Technology

[0002] Avian β-defensin-6 (AvBD6) is a natural antimicrobial peptide that exerts broad-spectrum antimicrobial effects through mechanisms such as disrupting pathogen cell membranes and modulating host immune responses. However, the expression level of natural chicken β-defensin-6 is low and its stability is poor, limiting its application in practical production.

[0003] Currently, genetic engineering technology provides an effective means to improve the production efficiency of antimicrobial peptides, but existing recombinant expression systems generally suffer from problems such as low expression levels, poor solubility, and insufficient protein activity. Furthermore, the natural sequence of chicken β-defensin-6 has not been optimized for industrial production, and its antimicrobial activity still has room for improvement.

[0004] Traditional amino acid mutation methods rely on random mutation or empirical design, which are inefficient, costly, and time-consuming. With AI software becoming increasingly integrated into daily life, it can not only solve everyday problems but also be rationally applied to address challenges in bioengineering. Therefore, developing a chicken β-defensin-6 with AI-optimized amino acid mutations and producing it using an efficient recombinant expression system is of great significance to the livestock and poultry farming industry. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides an AI-assisted designed chicken β-defensin-6 mutant. This chicken β-defensin-6 mutant exhibits high stability, activity, and solubility, and compared to wild-type chicken β-defensin-6, it demonstrates higher antibacterial activity and expression levels. It provides a highly efficient and safe antibacterial alternative for livestock and poultry farming and can be widely applied in livestock and poultry farming, animal health, and biological preservation.

[0006] This invention provides an AI-assisted designed chicken β-defensin-6 mutant, which has the following amino acids at the following 5 sites:

[0007] The 33rd position is lysine, the 35th position is lysine, the 49th position is asparagine, the 61st position is arginine, and the 65th position is proline.

[0008] The inventors used the AI ​​software ChatGPT to predict mutation sites of chicken β-defensin-6, optimized its stability, activity and solubility, and improved its antibacterial activity.

[0009] In one embodiment, the remaining amino acid sequence of the chicken β-defensin-6 mutant, excluding the five sites, is identical to the amino acid sequence of wild-type chicken β-defensin-6 after removing the signal peptide sequence. The amino acid sequence of wild-type chicken β-defensin-6 after removing the signal peptide sequence is shown below:

[0010] SPIHACRYQRGVCIPGPCRWPYYRVGSCGSGLKSCCVRNRWA (SEQ ID NO: 1).

[0011] In one embodiment, the amino acid sequence of the chicken β-defensin-6 mutant is as follows: SPIHACRKQKGVCIPGPCRWPYYNVGSCGSGLKSCRVRNPWA (SEQ ID NO: 2).

[0012] The present invention also provides an expression gene encoding the chicken β-defensin-6 mutant, the expression gene comprising several of the chicken β-defensin-6 mutants and a flexible linker peptide.

[0013] In one embodiment, the flexible linker peptide is tandemly spaced with the chicken β-defensin-6 mutant, and the expressed gene further includes a 6×His tag.

[0014] In one embodiment, the sequence of the expressed gene is shown below:

[0015] MSPIHACRKQKGVCIPGPCRWPYYNVGSCGSGLKSCRVRNPWAGGGGSSPIHACRKQKGV

[0016] CIPGPCRWPYYNVGSCGSGLKSCRVRNPWAGGGGSSPIHACRKQKGVCIPGPCRWPYYNV

[0017] GSCGSGLKSCRVRNPWAGGGGSSPIHACRKQKGVCIPGPCRWPYYNVGSCGSGLKSCRVRNPWAHHHHHH (SEQ ID NO: 3).

[0018] The present invention also provides a recombinant expression system, including an expression vector, wherein the expression vector includes the expressed gene.

[0019] In one embodiment, the initial vector for the expression vector includes the PET28a vector.

[0020] The pET-28a high-efficiency expression vector was used, and the promoter and signal peptide sequences were optimized to improve protein expression levels.

[0021] The present invention also provides a recombinant cell, including the aforementioned recombinant expression system.

[0022] In one embodiment, the host cell of the recombinant cells includes Escherichia coli BL21.

[0023] The present invention also provides a method for preparing the chicken β-defensin-6 mutant, comprising the following steps: cloning the expression gene into an initial vector to obtain the recombinant expression system; transferring the expression vector into competent host cells to obtain the recombinant cells; inducing expression to obtain the chicken β-defensin-6 mutant.

[0024] The above preparation method has the advantages of low cost, simple operation, and suitability for large-scale production, and has great market application potential.

[0025] The present invention also provides a feed additive or veterinary drug, wherein the active ingredient of the feed additive or veterinary drug includes the chicken β-defensin-6 mutant.

[0026] The above-mentioned chicken β-defensin-6 mutant can be used as a feed additive or veterinary drug for livestock and poultry to reduce antibiotic use and improve animal health.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention relates to an AI-assisted design of a chicken β-defensin-6 mutant, a recombinant expression system, and its applications. The chicken β-defensin-6 mutant significantly increases the expression level of chicken β-defensin-6, overcoming the problem of low natural expression levels. Compared with wild-type chicken β-defensin-6, it significantly enhances antibacterial activity and improves the inhibitory effect on target pathogens. The recombinant expression system employs an optimized expression strategy, giving the mutant protein higher solubility and stability, thus enhancing its industrial application value. Attached Figure Description

[0029] Figure 1 This is a representation of the PET28A reading frame and its main feature map.

[0030] Figure 2 A schematic diagram illustrating the construction principle of recombinant expression vectors;

[0031] Figure 3 This is a graph showing the results of an SDS-PAGE electrophoresis experiment.

[0032] Figure 4 This is a graph showing the results of a Western blotting experiment.

[0033] Figure 5 The graph shows the results of the minimum inhibitory concentration (MIC) experiment. Detailed Implementation

[0034] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0036] source:

[0037] Unless otherwise specified, all reagents, materials and equipment used in this invention are commercially available; unless otherwise specified, all test methods are conventional test methods in the field.

[0038] Example

[0039] (a) Strains and plasmids.

[0040] The expression strain BL21 and expression vector pet28a were donated by the Microbiology Research Laboratory of Wens Foodstuff Group Co., Ltd.; Staphylococcus aureus, Salmonella, and Escherichia coli K88 were preserved by the Microbiology Research Laboratory of Wens Foodstuff Group Co., Ltd. Figure 1 As shown.

[0041] (II) Mutant design and gene construction.

[0042] The reference sequence (NM_001001193.1) of chicken β-defensin-6 (AvBD6) was obtained from GenBank, and the AvBD6 signal peptide sequence was removed. The mature peptide sequence of chicken β-defensin-6 was input into ChatGPT, and a design mutation command was issued. Based on the ChatGPT output, the mutant gene sequence was designed. The mutation sites were located at positions 33, 35, 49, 61, and 65. AvBD6 was tandemly linked using a flexible linker peptide (G4S) to form AvBD6(G4S)AvBD6(G4S)AvBD6(G4S)AvBD6 (hereinafter referred to as "TB-AvBD6"). The control group was not modified and directly tandemly linked using a flexible linker peptide (G4S) (hereinafter referred to as "AvBD6×4"). Codon optimization was performed based on the codon preference of *E. coli*, and a 6×His tag was added to the end. The gene was synthesized by General Biotechnology and cloned into the PET28a vector to construct the recombinant plasmid PET28a-TB-AvBD6. The construction principle of the recombinant expression vector is as follows: Figure 2 As shown.

[0043] Table 1. Amino acid sequences and molecular weights of AvBD6 and TB-AvBD6

[0044]

[0045]

[0046] (III) The recombinant plasmid was transformed into Escherichia coli BL21.

[0047] Add 10 μL of recombinant plasmid PET28a-TB-AvBD6 to 100 μL of competent BL21(DE3) cells, mix well, and transfer to a 1.5 mL EP tube. Incubate on ice (4 °C) for 30 min. Heat shock the EP tube in a 42 °C water bath for 90 s, then cool on ice for 2-3 min. Add 900 μL of LB broth and gently invert to mix. Incubate at 37 °C, 220 rpm for 1 h. Centrifuge at 4000 rpm for 5 min, collect 100 μL of supernatant, discard the rest, and mix the supernatant with the precipitate. Spread the mixture onto an LB agar plate containing KANA and incubate for 12-16 h. Add 500 μL of LB broth to a 1.5 mL EP tube, pick 10 colonies from the plate, and inoculate them into 10 EP tubes. Incubate at 37 °C, 200-250 rpm for 4 h. Identify the turbid tubes using colony PCR.

[0048] (iv) Identification of recombinant plasmids.

[0049] The above bacterial culture was subjected to PCR identification. The identification system (10 μL) is as follows:

[0050] Table 2 Identification system (10 μL)

[0051] Taq 5μL F 0.2μL R 0.2μL Bacterial solution (sample) 1μL water 3.6μL

[0052] After the reaction was complete, all products were subjected to electrophoresis on a 1% agarose gel and the results were detected and identified using a gel imaging system. Based on the imaging, a portion of the samples were selected and transferred to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0053] (v) Inducible proteins.

[0054] 1. Streak the correctly sequenced strain onto LB agar plates containing KANA and incubate overnight at 37°C;

[0055] 2. Pick a single colony and place it in 1 ml of LB broth containing KANA, and incubate overnight at 37°C and 200 rpm on a shaker.

[0056] 3. Take 200 μL of the above bacterial culture at a 2% ratio and transfer it to 10 ml LB broth containing KANA. Incubate at 37°C and 200 r / min for 4 h in a shaker.

[0057] 4. Take 5 ml of the bacterial culture obtained in step 3 and add it to 100 ml of LB broth containing KANA. Incubate at 37°C and 220 rpm for 3 h in a shaker. Measure the OD value. If the OD value is between 0.6 and 0.8, add 28 μl of IPTG inducer and induce at 37°C for 6 h in a shaker.

[0058] 5. Take about 50ml of bacterial culture, centrifuge at 11000r / 10min, and discard the supernatant;

[0059] 6. Add 5 ml of Tris, resuspend the precipitate under shaking, then centrifuge at 11000 r / 10 min and discard the waste liquid;

[0060] 7. Add 5 ml of Tris, resuspend / pipette the precipitate and mix well, then transfer to a new 10 ml ep tube, add 5 μL of protease inhibitor, and disrupt the cell with a cell disruptor for 5-10 min.

[0061] 8. Aliquot 1ml of the broken sample into 1.5ml EP tubes, centrifuge at 12000r / 10min, and aliquot the supernatant and precipitate into two EP tubes. Mix the precipitate with 1ml of Tris. Mix 20ul of buffer solution with 20ul of sample, centrifuge in a cis-directed manner, and place in a boiling water bath for 10min. After centrifugation to room temperature, analyze the protein expression level by SDS-PAGE.

[0062] according to Figure 3 The band intensity indicates that after induction at 37℃ for 6 hours, the total expression level of TB-AvBD6×4 was significantly higher than that of unmutated AvBD6×4.

[0063] (vi) Protein transfer.

[0064] 1. Cut the separating gel according to the protein size in the SDS-PAGE results, and cut the PVDF membrane to the same size. Gently hold the PVDF membrane with tweezers and place it in anhydrous methanol for 10 seconds to activate it. Then, soak it in an electrophoresis tank containing conversion buffer along with two sponge pads for 10 minutes.

[0065] 2. Installation: With the blackboard at the bottom, place the sponge pad, filter paper, adhesive, PVDF membrane paper, filter paper, and sponge pad in sequence; remove air bubbles during placement to ensure the PVDF membrane is close to the gel and in a consistent position;

[0066] 3. Electrotransfer: Place the device in the electrophoresis tank that has been placed on ice, fill the electrophoresis tank with pre-cooled transfer solution (black to black edge, transparent plate to red edge) 80V, transfer for 45 minutes;

[0067] 4. Sealing: Take out the PVDF membrane, mark the protein side, and use tweezers to hold one corner and transfer it to 5% skim milk powder sealing solution. Incubate overnight at 4°C or shake at room temperature for 1 hour.

[0068] 5. Incubate primary antibody: Remove the blocking solution, add an appropriate amount of mouse 6xHis monoclonal antibody solution diluted with 2% skim milk powder, shake at 37°C for 1 hour at 50 rpm or overnight at 4°C;

[0069] 6. Washing: Remove the antibody, add an appropriate amount of TBST and shake at low speed, wash for 30 minutes, changing the solution every 5 minutes, for a total of 6 times.

[0070] 7. Development: Remove excess TBST, place the membrane on a disposable PE glove with the protein facing up, and evenly drop a 1:1 ECL luminescent detection solution onto the membrane. Incubate at room temperature in the dark for 30 minutes, and then analyze the protein solubility level by Western blotting.

[0071] The results are as follows Figure 4 As shown, soluble proteins are present in the supernatant after lysis, while insoluble proteins are present in the precipitate. Western blotting results indicate that the target protein band intensity in the TB-AvBD6 supernatant is significantly higher than that in the unmutated AvBD6, suggesting that the mutated AvBD6 has improved solubility compared to the unmutated AvBD6.

[0072] (vii) Protein purification.

[0073] Using Ni 2+ The protein was purified by affinity chromatography, and its expression was verified by Western blot. The specific steps are as follows:

[0074] 1. Equilibrium column: Wash with 50-100ml of ultrapure water, then wash the column with 200ml of Tris;

[0075] 2. Sample loading: Load samples in small amounts multiple times, and repeat the loading process after recovering the samples;

[0076] 3. Washing: Wash the column with 50 mm imidazole, 5-10 times the volume of the sample;

[0077] 4. Elution: Elute the column with 25 ml of 500*300 mm imidazole solution, discard the first 2 ml, and collect approximately 23 ml of sample.

[0078] 5. Cleaning: Clean and store with 100ml of Tris solution.

[0079] (viii) Antibacterial activity test.

[0080] The inhibitory effect of the purified recombinant protein on pathogens such as Escherichia coli K88 and Salmonella was evaluated using the minimum inhibitory concentration (MIC) assay.

[0081] The inhibition zone experiment was used to compare the antibacterial activity differences between the mutant and natural chicken β-defensin-6.

[0082] The results are as follows Figure 5 As shown, the red arrow represents the mutant chicken β-defensin 6 (TB-AvBD6×4), and the green arrow represents the unmutated natural chicken β-defensin 6 (AvBD6×4).

[0083] Table 3. Size of the inhibition zone of the recombinant plasmid against the pathogen. The inhibition zone diameter of TB-AvBD6×4 was significantly larger than that of the non-mutated AvBD6×4, indicating that the antibacterial activity of the mutated chicken β-defensin-6 was stronger than that of the natural chicken β-defensin-6.

[0084] (ix) Summary.

[0085] This invention utilizes the AI ​​software ChatGPT to analyze the structure-function relationship of proteins and accurately predict mutation sites. It optimizes chicken β-defensin-6 through amino acid mutations, improving its antibacterial activity and stability. Based on a high-efficiency expression system using recombinant E. coli BL21, it significantly increases protein expression levels. By optimizing induction conditions (such as IPTG concentration, culture temperature, and time), it improves protein solubility and activity, employing Ni... 2+ Affinity chromatography purification strategy improves the purity, recovery rate and bioactivity of target proteins, and validates their in vitro antibacterial activity.

[0086] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0087] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An AI-assisted design of a chicken β-defensin-6 mutant, characterized in that, This chicken β-defensin-6 mutant has the following amino acids at the following 5 sites: The 33rd position is lysine, the 35th position is lysine, the 49th position is asparagine, the 61st position is arginine, and the 65th position is proline.

2. The chicken β-defensin-6 mutant according to claim 1, characterized in that, The remaining amino acid sequence of the chicken β-defensin-6 mutant, excluding the five sites mentioned above, is identical to the amino acid sequence of wild-type chicken β-defensin-6 after removing the signal peptide sequence. The amino acid sequence of wild-type chicken β-defensin-6 after removing the signal peptide sequence is shown below: SPIHACRYQRGVCIPGPCRWPYYRVGSCGSGLKSCCVRNRWA (SEQ ID NO: 1).

3. The chicken β-defensin-6 mutant according to any one of claims 1-2, characterized in that, The amino acid sequence of the chicken β-defensin-6 mutant is shown below: SPIHACRKQKGVCIPGPCRWPYYNVGSCGSGLKSCRVRNPWA (SEQ ID NO: 2).

4. An expression gene encoding the chicken β-defensin-6 mutant according to any one of claims 1-3, characterized in that, The expressed gene includes the chicken β-defensin-6 mutant and the flexible linker peptide as described in any one of claims 1-3.

5. The expressed gene according to claim 4, characterized in that, The flexible linker peptide is tandemly linked to the chicken β-defensin-6 mutant, and the expressed gene also includes a 6×His tag.

6. The expression gene of the chicken β-defensin-6 mutant according to claim 5, characterized in that, The sequence of the expressed gene is shown below: MSPIHACRKQKGVCIPGPCRWPYYNVGSCGSGLKSCRVRNPWAGGGGSSPIHACRKQKGVCIPGPCRWPYYNVGSCGSGLKSCRVRNPWAGGGGSSPIHACRKQKGVCIPGPCRWPYYNVGSCGSGLKSCRVRNPWAGGGGSSPIHACRKQKGVCIPGPCRWPYYNVGSCGSGLKSCRVRNPWAHHHHHH(SEQ ID NO: 3).

7. A recombinant expression system, characterized in that, It includes an expression vector, said expression vector comprising the expression gene of any one of claims 4-6.

8. A recombinant cell, characterized in that, Includes the recombinant expression system as described in claim 7.

9. A method for preparing the chicken β-defensin-6 mutant according to any one of claims 1-3, characterized in that, The method includes the following steps: cloning the expression gene according to any one of claims 4-6 into an initial vector to obtain the recombinant expression system according to claim 7; transferring the expression vector into competent host cells to obtain the recombinant cells according to claim 8; inducing expression to obtain the chicken β-defensin-6 mutant.

10. A feed additive or veterinary drug, characterized in that, The active ingredient of the feed additive or veterinary drug includes the chicken β-defensin-6 mutant as described in any one of claims 1-3.

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