Construction method of Serpin5 chimeric mutation cricket cricket capable of producing high-activity antibacterial peptide

Abstract

The invention discloses a construction method of Serpin5 chimeric mutant cricket cricket capable of producing high-activity antibacterial peptide, and belongs to the technical field of gene engineering. The invention discloses sgRNA with a Serpin5 gene knocked out. The nucleotide sequence of the sgRNA is as shown in SEQ ID NO. 25. The CRISPR / Cas9 gene editing technology is utilized to knock out the specific site of the Serpin5 gene of the cricket bimaculata, the antibacterial activity of the antibacterial peptide in the cricket bimaculata is improved, and a theoretical support and a technical thought are provided for improving the quality of the antibacterial peptide and promoting the industrialization process of the high-quality antibacterial peptide.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a method for constructing a Serpin5 chimeric mutant two-spotted cricket that produces highly active antimicrobial peptides. Background Art

[0002] In some countries and regions (especially some developing countries), due to the lack of effective medical supervision, patients often use antibiotics incorrectly or excessively. Similarly, in animal husbandry, antibiotics are often used as growth promoters and preventive drugs in feed, which not only leads to the proliferation of drug-resistant bacteria in animals, but may also affect human health through the food chain. The above abuse of antibiotics not only promotes bacterial resistance, but also seriously threatens public health. The World Health Organization (WHO) has listed antibiotic resistance as one of the most pressing public health threats in the world, and it is estimated that by 2050, 10 million people will die each year from drug-resistant infections. Therefore, strengthening the research and development of alternative therapies to antibiotics is one of the important measures to deal with antibiotic resistance.

[0003] As a new type of antimicrobial agent, antimicrobial peptides (AMPs) are gradually becoming an effective alternative to antibiotics due to their unique mechanism of action and spectrum of antimicrobial activity. Antimicrobial peptides show effective antimicrobial activity against a variety of pathogenic microorganisms (including bacteria, fungi and some viruses), which makes them have a wider application potential than traditional antibiotics. In the medical field, antimicrobial peptides can be used to treat various infections, such as skin infections, respiratory infections, intestinal infections, etc.; in the food industry, as natural preservatives, antimicrobial peptides can extend the shelf life of food, prevent the growth of pathogens in food, and meet consumers' demand for healthy and safe food; in agriculture, antimicrobial peptides are used as plant protection agents to help crops stay below pathogens, reduce dependence on chemical pesticides, and improve agricultural sustainability; in animal husbandry, antimicrobial peptides can not only be used as feed additives to prevent and control infections in animal populations, but also have immunomodulatory effects, which help improve the overall immunity of animals, enhance their resistance, and fight various diseases.

[0004] Antimicrobial peptides are a class of short peptides with antimicrobial activity that are widely found in nature, such as animals, plants, insects, amphibians, and microorganisms. As a traditional insect, the two-spotted cricket has an increasingly high potential to become a new type of insect resource due to its high nutritional value, strong growth and reproduction ability, high environmental adaptability, and eco-friendly nature. Modifying the synthesis pathway of two-spotted cricket antimicrobial peptides through genetic engineering methods will greatly promote the industrialization of antimicrobial peptides. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing a chimeric mutant Serpin5 of the two-spotted cricket that produces highly active antimicrobial peptides, so as to solve the problems existing in the above-mentioned prior art. The present invention uses CRISPR / Cas9 gene editing technology to knock out specific sites of the Serpin5 gene of the two-spotted cricket, thereby improving the antimicrobial activity of the antimicrobial peptides in the two-spotted cricket, and providing theoretical support and technical ideas for improving the quality of antimicrobial peptides and promoting the industrialization process of high-quality antimicrobial peptides.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an sgRNA for knocking out the Serpin5 gene, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.25.

[0008] Preferably, the method for preparing the sgRNA comprises the following steps:

[0009] (1) Using the PAC-sgRNA-Cas9 plasmid as a template, amplification was performed using the primer pair shown in SEQ ID NO. 22-23 to obtain crRNA / tracrRNA fragments;

[0010] (2) using the crRNA / tracrRNA fragment as a template, amplifying using the primer pair described in SEQ ID NO.23-24 and the primer pair shown in SEQ ID NO.16-17 to obtain a DNA fragment of gRNA;

[0011] (3) Using the DNA fragment of the gRNA as a template, performing in vitro transcription to obtain the sgRNA.

[0012] The present invention also provides the use of the above-mentioned sgRNA in preparing a reagent for knocking out the Serpin5 gene or reducing the expression amount of the Serpin5 gene.

[0013] The present invention also provides a reagent for knocking out the Serpin5 gene, wherein the reagent comprises the above-mentioned sgNRA and Cas9 protein.

[0014] Preferably, the mass ratio of the sgNRA and Cas9 proteins is 2:3.

[0015] The present invention also provides the use of the above reagent in improving the antibacterial activity of antimicrobial peptides in two-spot crickets, or in constructing a Serpin5 chimeric mutant two-spot cricket that produces highly active antimicrobial peptides.

[0016] The present invention also provides a method for improving the antibacterial activity of antimicrobial peptides in the body of two-spotted crickets, comprising the following steps:

[0017] (a) injecting the above reagent into two-spotted cricket eggs, incubating and culturing, and obtaining two-spotted cricket mutants of the G0 generation;

[0018] (b) The two-spot cricket mutant G0 generation is mated with the wild-type two-spot cricket, and the two-spot cricket chimeric mutant G1 generation is selected after incubation, culture and identification. The antimicrobial activity of the antimicrobial peptide in the two-spot cricket chimeric mutant G1 generation is higher than that in the wild-type two-spot cricket.

[0019] The present invention also provides a method for constructing a Serpin5 chimeric mutant cricket that produces highly active antimicrobial peptides, comprising the following steps:

[0020] (i) injecting the above reagent into two-spotted cricket eggs, incubating and culturing, and obtaining two-spotted cricket mutants of the G0 generation;

[0021] (ii) The G0 generation of the two-spotted cricket mutant is mated with the wild type, and after incubation, culture and identification, a Serpin5 chimeric mutant two-spotted cricket that produces highly active antimicrobial peptides is obtained.

[0022] The present invention discloses the following technical effects:

[0023] The production of antimicrobial peptides is regulated by the Toll signaling pathway of insect humoral immunity. The Toll pathway is activated by the serine protease cascade. The serine protease inhibitor family (Serpins) has an inhibitory effect on serine proteases, so Serpins negatively regulate the Toll signaling pathway and inhibit the production of antimicrobial peptides. Serpin5, as a member of the Serpin family, is also involved in negatively regulating the production of antimicrobial peptides. The present invention uses CRISPR / Cas9 gene editing technology to knock out specific sites of the Serpin5 gene of the two-spotted cricket, and the knockout efficiency can reach 75%. It can produce mutant chimeric two-spotted crickets, improve the antibacterial activity of antimicrobial peptides in the two-spotted cricket, and provide theoretical support and technical ideas for improving the quality of antimicrobial peptides and promoting the industrialization process of high-quality antimicrobial peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The exon structure of Serpin5 gene and the distribution of sgRNA-014 sites;

[0026] Figure 2 The results of in vitro site gene editing efficiency verification are shown, where A is a schematic diagram of the sgRNA-014 site gene editing process, and B is the in vitro enzyme cutting efficiency test results of each site;

[0027] Figure 3 The results are the optimization results of microinjection parameters for C. bimaculata.

[0028] Figure 4 Verify the gene editing efficiency at different sites in vivo and provide sequencing peak diagrams;

[0029] Figure 5 The flowchart for the construction and screening of chimeric knockout mutants of Serpin5 in two-spotted crickets;

[0030] Figure 6 are the inhibitory effects of different two-spot cricket enzymatic peptides on Escherichia coli and Staphylococcus aureus, wherein A is the inhibition zone of different two-spot cricket enzymatic peptides on Escherichia coli, 1-6 are pH=3, 1 mol / L sodium chloride solution, 0.05 mg / mL ampicillin solution, wild-type two-spot cricket female enzymatic peptide solution, wild-type two-spot cricket male enzymatic peptide solution, Serpin5 knockout chimeric mutant two-spot cricket female enzymatic peptide solution, Serpin5 knockout chimeric mutant two-spot cricket male enzymatic peptide solution; B is The inhibition zones of different two-spot cricket enzymatic peptides against Staphylococcus aureus, 1-6 are pH=3, 1 mol / L sodium chloride solution, 0.1 mg / mL ampicillin solution, wild-type two-spot cricket female enzymatic peptide solution, wild-type two-spot cricket male enzymatic peptide solution, Serpin5 knockout chimeric mutant two-spot cricket female enzymatic peptide solution, Serpin5 knockout chimeric mutant two-spot cricket male enzymatic peptide solution; C is the statistical results of the diameters of the inhibition zones of different two-spot cricket enzymatic peptides against Escherichia coli and Staphylococcus aureus. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1

[0037] 1. sgRNA synthesis and verification of in vitro enzyme cleavage activity

[0038] (1) sgRNA site design

[0039] The mRNA sequence and whole genome sequence of Serpin5 of two-spotted cricket were obtained by using the Blast tool on the official website of NCBI, and the sequence was compared and analyzed using SnapGene to mark the exons and introns. The genome sequence of GbSerpin5 was analyzed and it was found that the full length of the gene was 11388930 bp, containing 7 exons (see Figure 1 ). Log in to the E-CRISP website, use the 1-3 exon sequences of the Serpin5 gene as the design range, screen the sgRNA target sites online, and select the top three sequences with high scores and few off-target sites on the third exon as target sequences based on the comprehensive score. The target sequence information is shown in Table 1. Taking the sgRNA-014 site as an example, its distribution position is shown in Figure 1 .

[0040] Table 1 GbSerpin5 gene target sequence

[0041]

[0042] (2) Design of specific genomic amplification primers containing target sequences

[0043] According to the location of each sgRNA site in the genome, SnapGene analysis was used on the genomic sequence of about 400 bp containing the sgRNA target sequence to design specific nested PCR amplification primers (primer sequence information is shown in Table 2) to amplify the genomic sequence containing the target sequence.

[0044] Table 2 Primers for nested PCR amplification of GbSerpin5 target gene sequence

[0045]

[0046]

[0047] (3) Design of sgRNA synthesis primers

[0048] Forward and reverse oligonucleotides were designed and assembled by PCR with 80 bp crRNA / tracrRNA fragments to generate the DNA template of gRNA. The sequence information of sgRNA synthesis primers is shown in Table 3.

[0049] Table 3 sgRNA synthesis primers

[0050]

[0051] (4) Cas9 / sgRNA in vitro enzyme digestion verification

[0052] a. PCR amplification to obtain specific genomic fragments containing target sequences

[0053] The genome of the two-spotted cricket was extracted using a genome extraction kit. The genome was used as a template and the specific nested first-round amplification primers F1 / R1 in Table 2 were used to amplify the target gene fragment (the first-round PCR system is shown in Table 4, and the PCR program is shown in Table 5); the first-round amplification product was used as a template and the second-round amplification primers F2 / R2 in Table 2 were used to amplify the target gene fragment (the second-round PCR system is shown in Table 6, and the PCR program is shown in Table 7). The PCR product was detected by 1% agarose gel to observe the size of the amplified band. After the band size met expectations, the target gene fragment amplified in the second round was recovered using a Gel Extraction Kit agarose gel DNA recovery kit to obtain the target sequences of GbSerpin5-014, GbSerpin5-193, and GbSerpin5-261.

[0054] Table 4 PCR system for the first amplification of target gene sequence

[0055]

[0056] Table 5 Target gene amplification first amplification procedure

[0057]

[0058] Table 6 Target gene sequence second amplification PCR system

[0059]

[0060] Table 7 Target gene amplification second amplification program

[0061]

[0062] b. Amplification of 80 bp crRNA / tracrRNA fragment

[0063] Using the plasmid (PAC-sgRNA-Cas9) as a template, the upstream primer crRNA / tracrRNA-F and the downstream primer Universal-R shown in Table 8 were used for amplification to obtain an 80 bp fragment, which was recovered using a gel recovery kit and diluted to 50 ng / μL after quantification for standby use. The PCR amplification system and procedure are shown in Tables 9 and 10, respectively.

[0064] Table 8 Primer sequences

[0065]

[0066] Table 9 PCR amplification system

[0067]

[0068] Table 10 PCR amplification program

[0069]

[0070] c. gRNA DNA fragment amplification

[0071] Using the 80bp crRNA / tracrRNA fragment as a template, amplification was performed using the upstream primer Universal-F and the downstream primer Universal-R shown in Table 11 and the sgRNA synthetic primers shown in Table 3. The sgRNA primer was diluted to 0.3 μM before use, and a 50 μL reaction system (Table 12) was established in a 200 μL Ep tube. The amplification program in Table 10 was followed to obtain the DNA fragments of the gRNAs at the sgRNA-014 site, the sgRNA-193 site, and the sgRNA-261 site, respectively. The DNA fragments were purified by a gel recovery kit, and the band size was detected by electrophoresis after quantification.

[0072] Table 11 Primer sequences

[0073]

[0074] Table 12 PCR amplification system

[0075]

[0076] d. In vitro transcription and purification of sgRNA

[0077] Using T7 RiboMAX TM The Express RNAi System kit was used to operate, and the DNA fragments of the gRNAs at the sgRNA-014 site, sgRNA-193 site, and sgRNA-261 site were transcribed in vitro, quantified after transcription, and 2% agarose gel was prepared. 0.5 μL was taken for electrophoresis to detect whether the band was single. The GeneJET RNA purification and concentration microextraction kit was used for purification to obtain sgRNA-014, sgRNA-193, and sgRNA-261. The sequences are shown in Table 13.

[0078] Table 13 sgRNA sequences

[0079]

[0080] e. In vitro enzyme digestion efficiency detection

[0081] The target sequences of GbSerpin5-014, GbSerpin5-193, and GbSerpin5-261 obtained in step (4) were used as DNA templates to verify the efficiency of in vitro enzyme cleavage. The enzyme cleavage reaction system is shown in Table 14. During the experiment, one experimental group (adding sgRNA and Cas9) and two control groups (adding sgRNA without adding Cas9 and adding Cas9 without adding sgRNA) need to be set up; incubate at 37°C in a PCR instrument for 2 hours; and detect the enzyme cleavage efficiency with 2% agarose gel. Taking the sgRNA-014 site as an example, the schematic diagram of the gene editing process is as follows Figure 2 The results of in vitro enzyme digestion efficiency test at each site are shown in Figure 2 As shown in B, through grayscale value calculation, it was found that the in vitro cutting efficiency of the sgRNA-014 site was 0.99, the in vitro cutting efficiency of the sgRNA-193 site was 0.99, and the in vitro cutting efficiency of the sgRNA-261 site was 0.99.

[0082] Table 14 Cas9 / sgRNA in vitro enzyme digestion system

[0083]

[0084]

[0085] 2. Verification of the in vivo knockout efficiency of Serpin5 in two-spotted crickets

[0086] (1) Optimization of microinjection parameters in C. bimaculata

[0087] The injection pressure was set to 200, 300 and 400 hPa (pi), the injection time was 0.5 and 1 s (ti), and the return pressure was 10 and 20 hPa (pc) for microinjection. 100 eggs were injected for each parameter combination, and the number of eggs hatched into adults was counted to calculate the survival rate. Figure 3 As shown (the horizontal axis is "injection pressure, injection time, and reflux pressure"), the injection pressure of 300 hPa, the injection time of 0.5 s, and the reflux pressure of 10 hPa were finally selected for microinjection of two-spotted crickets.

[0088] (2) Acquisition of RNP complex

[0089] Mix 3 μL of Cas9 protein (300 ng / μL) with 2 μL of sgRNA (300 ng / μL) obtained in step (1) to obtain 5 μL of RNP complex for injection.

[0090] (3) Detection of in ovo mutation efficiency

[0091] The optimal injection parameters were selected to inject the RNP complex into the eggs of two-spot crickets, and 90 two-spot cricket eggs were injected for each sgRNA site. On the 5th day after injection, 20 samples were selected from each site and divided into 4 groups, each containing 5 samples. The genome of the egg to be tested was extracted, and the extracted genome was used as a template. The target gene fragment was amplified by nested PCR according to the primers in Table 2 and the systems and procedures in Tables 4-7. The mutation efficiency was analyzed by electrophoresis detection and sequencing. The results showed that 3 groups had overlapping peaks after injection of the sgRNA-014 site, 1 group had no impurity peaks, and the mutation efficiency was 75%; 2 groups had overlapping peaks after injection of the sgRNA-193 site, 2 groups had no impurity peaks, and the mutation efficiency was 50%; 1 group had overlapping peaks after injection of the sgRNA-261 site, 3 groups had no impurity peaks, and the mutation efficiency was 25% (such as Figure 4 Therefore, the sgRNA-014 site was selected for subsequent experiments.

[0092] 3. Obtaining a chimeric mutant of Serpin5 knockout in two-spotted cricket

[0093] The remaining eggs injected with the sgRNA-014 site group were cultured until the first-instar nymphs were hatched, and the hatched first-instar nymphs were collected and raised in disposable plastic boxes. When the insect body developed to the 6th instar, 3 were raised separately in a culture cup, and the antennae were cut off about 1 cm in length using dissecting scissors and placed in a 1.5mL centrifuge tube. The genome was obtained using the alkaline lysis method, that is, 45 μL 50mMNaOH was added and lysed at 95°C for 5min; then 5 μL 1M Tris-HCl (pH=9.5) was added. Take 1 μL of the above template, perform nested PCR on the target gene fragment, and sequence the PCR product. The sequencing company is Sangon Biotech (Beijing) Co., Ltd., and the sequencing primer sequence is 014-Serpin5-F2 in Table 2.

[0094] According to the analysis of the sequencing results, it was found that there were mutants of the G0 generation with base mutations and base deletions, and there were hybrid peaks at the target site. The G0 generation mutants were mated with the wild type, and the chimeric mutants of the G1 generation were identified and screened from their offspring, which were the two-spotted cricket Serpin5 knockout chimeric mutants. The process of screening the two-spotted cricket Serpin5 knockout chimeric mutants is shown in Figure 5 .

[0095] 4. Antibacterial activity detection of Serpin5 knockout chimeric mutant peptides of two-spotted cricket

[0096] (1) The peptide extraction process is as follows:

[0097] a. Sample pretreatment: Separate the male and female G1 adults of the Serpin5 knockout chimeric mutant of the two-spotted cricket and freeze them in a -80°C refrigerator for 24 hours, then freeze-dry them in a vacuum freeze dryer for 24 hours, grind the freeze-dried insects in a grinder, and the obtained insect powder can be sealed and stored in a -20°C refrigerator;

[0098] b. Degreasing: weigh 20g of insect powder in a beaker, add 400mL of anhydrous ethanol, seal with plastic wrap and poke holes, stir at room temperature for 1h using a rotor and magnetic stirrer, then centrifuge at room temperature at 4000rpm in a 50mL centrifuge tube for 30min, discard the supernatant, repeat 3 times, take out all the precipitate in the centrifuge tube with a spoon, flatten it in a petri dish, place it in a fume hood for 12h, take it out after drying, and store it in a -20℃ refrigerator;

[0099] c. Protein extraction: Take 1g of defatted powder, add 20mL of 1mol / L NaCl solution, stir in a 50℃ water bath for 1h, centrifuge at 4000rpm for 20min, filter the supernatant to obtain a protein solution, which can be stored in a 4℃ refrigerator for a short period of time;

[0100] d. Use 1 mol / L hydrochloric acid to adjust the pH of the protein solution to the optimal enzymatic hydrolysis pH = 3, add 5000U of acidic protease, and enzymatically hydrolyze at the optimal temperature of 48°C for 1 hour. Then inactivate the enzyme in a boiling water bath for 10 minutes, cool to room temperature, centrifuge at 4000rpm for 20 minutes, take the supernatant and filter it through a 0.45μm filter membrane to obtain the enzymatic polypeptide solution.

[0101] e. Prepare wild-type (WT) two-spotted cricket enzymatic polypeptide solution as a control group according to steps ad.

[0102] (2) The detection process of peptide yield is as follows:

[0103] Take 2mL of enzymatic polypeptide solution, add an equal volume of 20% trichloroacetic acid solution, mix well and let stand for 10 minutes. After precipitation, add 4mL of biuret reagent, mix well and let stand at room temperature for 30 minutes. Replace the supernatant with 1mL of single distilled water and treat in the same way as a blank control. Measure the absorbance at 540nm. According to the standard curve, substitute △A determination (y, △A determination) into the formula to calculate the polypeptide concentration (x, mg / mL).

[0104] The peptide yield calculation formula is:

[0105]

[0106] Where:

[0107] W: polypeptide yield, in %; C1: polypeptide concentration, in mg / mL; V1: volume of the obtained enzymatic polypeptide solution, in mL; m: mass of defatted powder raw material, in g; C2: protein concentration, in mg / mL; V2: volume of anhydrous ethanol added to the defatted powder raw material at a material-liquid ratio of 1:20, in mL; 100: conversion factor.

[0108] The results of peptide yield are shown in Table 15. There is no significant difference in the peptide yield of each sample.

[0109] Table 15 Yield of polypeptides from chimeric mutant crickets of the G1 generation

[0110]

[0111]

[0112] (3) The detection process of polypeptide activity is as follows:

[0113] The antibacterial activity of the four enzymatic peptide solutions was tested, with Escherichia coli and Staphylococcus aureus as the test bacteria, and the concentration of each was 5×10 6CFU / mL; 11 mg / mL of four kinds of enzymatic polypeptide liquids were used as drug liquids; 1 mol / L sodium chloride solution with the same pH (pH=3) was used as a negative control; 0.05 mg / mL of ampicillin was used as a positive control for Escherichia coli, and 0.1 mg / mL of ampicillin was used as a positive control for Staphylococcus aureus, and then the antibacterial effect of the Serpin5 chimeric mutant two-spotted cricket enzymatic polypeptide liquid was reflected by measuring the size of the inhibition zone and calculating the inhibition rate.

[0114] The results are shown in Table 16 and Figure 6 The four enzymatic hydrolyzed polypeptide liquids of two-spot crickets had significant inhibitory effects on Escherichia coli and Staphylococcus aureus. Among them, the inhibitory effect of Serpin5 female two-spot cricket enzymatic polypeptide on Escherichia coli was significantly better than that of the control group, and the inhibitory effect of Serpin5 two-spot cricket enzymatic polypeptide on Staphylococcus aureus was significantly better than that of the control group, indicating that these enzymatic polypeptide liquids have good inhibitory effects on Escherichia coli and Staphylococcus aureus.

[0115] Table 16 Inhibition zone diameter and inhibition rate of different enzymatic peptides against Escherichia coli and Staphylococcus aureus

[0116]

[0117] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A sgRNA for knocking out the Serpin5 gene, characterized in that: The nucleotide sequence of the sgRNA is shown in SEQ ID NO.

25.

2. The sgRNA according to claim 1, characterized in that The method for preparing the sgRNA comprises the following steps: (1) Using the PAC-sgRNA-Cas9 plasmid as a template, amplification was performed using the primer pair shown in SEQ ID NO. 22-23 to obtain crRNA / tracrRNA fragments; (2) using the crRNA / tracrRNA fragment as a template, amplifying using the primer pair described in SEQ ID NO.23-24 and the primer pair shown in SEQ ID NO.16-17 to obtain a DNA fragment of gRNA; (3) Using the DNA fragment of the gRNA as a template, performing in vitro transcription to obtain the sgRNA.

3. Use of the sgRNA according to claim 1 or 2 in preparing a reagent for knocking out the Serpin5 gene or reducing the expression amount of the Serpin5 gene.

4. A reagent for knocking out the Serpin5 gene, characterized in that: The reagent comprises the sgNRA and Cas9 protein according to claim 1 or 2.

5. The reagent according to claim 4, characterized in that The mass ratio of the sgNRA and Cas9 proteins is 2:

3.

6. Use of the reagent according to claim 4 or 5 in improving the antimicrobial activity of antimicrobial peptides in two-spot crickets, or in constructing a Serpin5 chimeric mutant two-spot cricket that produces highly active antimicrobial peptides.

7. A method for improving the antibacterial activity of antimicrobial peptides in two-spotted crickets, characterized in that: The steps include: (a) injecting the reagent described in claim 4 or 5 into two-spotted cricket eggs, incubating and culturing, and obtaining two-spotted cricket mutants of the G0 generation; (b) The two-spot cricket mutant G0 generation is mated with the wild-type two-spot cricket, and the two-spot cricket chimeric mutant G1 generation is selected after incubation, culture and identification. The antimicrobial activity of the antimicrobial peptide in the two-spot cricket chimeric mutant G1 generation is higher than that in the wild-type two-spot cricket.

8. A method for constructing a chimeric mutant Serpin5 of a two-spotted cricket that produces highly active antimicrobial peptides, characterized in that: The steps include: (i) injecting the reagent described in claim 4 or 5 into two-spotted cricket eggs, incubating and culturing, and obtaining two-spotted cricket mutants of the G0 generation; (ii) The G0 generation of the two-spotted cricket mutant is mated with the wild type, and after incubation, culture and identification, a Serpin5 chimeric mutant two-spotted cricket that produces highly active antimicrobial peptides is obtained.

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