CN114106133A

CN114106133A - Gene editing housefly antibacterial peptide Sarcotoxin-1B nucleotide sequence and method for preparing antibacterial peptide

Info

Publication number: CN114106133A
Application number: CN202111450960.4A
Authority: CN
Inventors: 徐国防; 白杰; 李文嘉; 卢敏; 王改利; 席燕燕; 盛卫东; 柴磊; 魏凤仙; 李绍钰
Original assignee: Peoples Hospital of Zhengzhou
Current assignee: Peoples Hospital of Zhengzhou
Priority date: 2021-12-01
Filing date: 2021-12-01
Publication date: 2022-03-01

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a gene editing housefly antibacterial peptide Sarcotoxin-1B nucleotide sequence and a method for preparing the antibacterial peptide. According to the invention, a nucleotide sequence capable of expressing the antibacterial peptide Sarcotoxin-1B gene is obtained by editing the Sarcotoxin-1B gene secreted and expressed in vitro of a yeast system, the nucleotide sequence is synthesized and then cloned into a Pichia pastoris expression vector pPIC9K, the vector is transformed into competent cells, plasmids are extracted after bacteria expansion, enzyme digestion identification and sequencing verification are carried out, the correct vector is verified to be transformed into the competent yeast through electric transformation, further, the yeast strain GS115-pPIC9K-Sarcotoxin-1B containing the antibacterial peptide Sarcotoxin-1B mature peptide is obtained through screening and verification, and the strain is enabled to secrete and express the housefly antibacterial peptide Sarcotoxin-1B under the action of an inducer.

Description

Gene editing housefly antibacterial peptide Sarcotoxin-1B nucleotide sequence and method for preparing antibacterial peptide

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a gene editing housefly antibacterial peptide Sarcotoxin-1B nucleotide sequence and a method for preparing the antibacterial peptide.

Background

The wide and long-term use of antibiotics in the field of medicine and health leads to the generation of drug-resistant bacteria, so that the search for a substitute/substitutes for antibiotics becomes a imperative development trend. The antibacterial peptide, also called host defense peptide, is an important component of the body's innate immune system, can cooperate with other proteins of the body's natural immunity to jointly protect the host, and has a wide spectrum of biological functions of inhibiting or eliminating bacteria, fungi, viruses, parasites and the like. Many documents indicate that the addition of antibiotics generally causes the generation of drug-resistant bacteria in livestock and poultry. Therefore, the antibacterial function of antibacterial peptides is one of the alternatives to antibiotics. In the research for searching for highly active antibacterial peptides, we know that the antibacterial effect of the antibacterial peptides in vivo should theoretically be stronger because the houseflies live in the environment where bacteria grow. Therefore, the invention selects the housefly Sarcotoxin-1B for research (NCBI, amino acid sequence number: XP _ 005179717.1), and finds that the mature peptide sequence thereof is as follows: GWLKKIGKKIERIGQHTRDATIQAIGVAQQAANVAATLKGK, has strong antibacterial ability to staphylococcus aureus, escherichia coli and salmonella typhimurium, but because the housefly is difficult to purify in vivo and the chemical synthesis cost is too high, the application prospect of the housefly as an antibiotic substitute in the industries of medicine, livestock raising, fresh-keeping products and the like is seriously restricted.

Disclosure of Invention

Aiming at the defects and problems of difficult in-vivo purification and higher chemical synthesis cost of the mature peptide sequence of the housefly Sarcotoxin-1B at present, the invention designs a new nucleotide sequence (SEQ S1 Bl) of the housefly antibacterial peptide Sarcotoxin-1B subjected to gene editing and provides a new method for preparing the antibacterial peptide by in-vitro fermentation of pichia pastoris, thereby providing a new thought and solution method for the wide application of the antibacterial peptide in animal production.

The technical scheme adopted by the invention for solving the technical problems is as follows: provides a nucleotide sequence for preparing housefly antibacterial peptide Sarcotoxin-1B, wherein the nucleotide sequence is shown as SEQ S1 Bl.

The invention also provides a preparation method of the housefly antibacterial peptide Sarcotoxin-1B, which comprises the following steps:

step one, manually editing a nucleotide sequence corresponding to an amino acid sequence of the housefly antibacterial peptide Sarcotoxin-1B, and designing and synthesizing a nucleotide sequence shown as SEQ S1 Bl;

synthesizing a promoter sequence, a restriction enzyme EcoR I, a 6-his tag sequence, a restriction enzyme Not I and a terminator sequence, and then cloning into a vector to construct a recombinant vector;

and step three, transferring the recombinant vector into a yeast strain after sequencing verification, and obtaining the housefly antibacterial peptide Sarcotoxin-1B after screening, induced expression and purification.

The preparation method of the housefly antibacterial peptide Sarcotoxin-1B comprises the second step of inserting a restriction enzyme EcoR I in front of an initiation codon, inserting a 6-his tag sequence in front of a termination codon, inserting a restriction enzyme Not I synthetic sequence behind the termination codon, constructing a protein translation reading frame by a yeast secretion expression signal peptide and a SEQ S1Bl sequence, and cloning into a pPIC9K vector to construct a recombinant vector pPIC 9K-Sarcotoxin-1B.

The preparation method of the housefly antibacterial peptide Sarcotoxin-1B specifically comprises the following steps:

s1, transforming the constructed recombinant expression vector pPIC9K-Sarcotoxin-1B into competent cells of Pichia pastoris GS 115;

s2, screening high-copy transformants by YPD plates containing G418 with different concentrations, and screening Mut + type strains of methanol rapid-use transformants by MD and MM plates;

s3, selecting the screened high-copy transformant, inoculating the high-copy transformant on a YPD culture medium for culture, collecting and cleaning a thallus precipitate, and performing induced expression by using 1% methanol;

s4, after induction expression, carrying out separation and purification to obtain the Sarcotoxin-1B antibacterial peptide.

The preparation method of the housefly antibacterial peptide Sarcotoxin-1B comprises the following specific steps of S1: by usingSac I, linearizing a recombinant expression vector pPIC 9K-Sarcotoxin-1B; then the linearized recombinant expression vector pPIC9K-Sarcotoxin-1B gene is purified by an agarose gel DNA recovery kit, and a purified product is electrically transferred into Pichia pastoris GS 115.

In the preparation method of the housefly antibacterial peptide Sarcotoxin-1B, the step S2 further comprises extracting a Mut + -type strain to identify the strain on a YPD plate, specifically: firstly, extracting the genome DNA of a His + Mut + high-copy yeast transformant by using a yeast genome DNA extraction kit, and carrying out PCR by using a yeast genome as a template and using a universal primer of a pPIC9K eukaryotic expression vector, wherein the universal primer is as follows:

forward primer 5'AOX1 （5’-GACTGGTTCCAATTGACAAGC-3’），

Reverse primer3’AOX1 （5’-GGCAAATGGCATTCTGACATCCT -3’）

And sequencing the strains successfully identified by the PCR to obtain the Pichia pastoris GPS-I capable of producing the housefly Sarcotoxin-1B antibacterial peptide in high volume.

The preparation method of the housefly antibacterial peptide Sarcotoxin-1B comprises the following specific steps of S3:

(1) selecting the screened high-copy yeast transformant, inoculating the high-copy yeast transformant into 5 mL of YPD medium, and culturing overnight;

(2) inoculating 1mL of bacterial liquid into 50mL of BMGY medium, and performing shaking culture at 30 ℃ and 250rpm until OD600= 2-6; centrifuging at 4 deg.C and 6000rpm for 5min, collecting thallus, and removing supernatant; washing the thallus precipitate with sterilized water, centrifuging, and discarding the supernatant; repeating;

(3) washing the thallus precipitate with BMMY culture medium, centrifuging, and discarding the supernatant;

(4) suspending the thallus precipitate with 50mLBMMY medium until OD600=1.0, transferring into 250mL conical flask, shaking culturing at 30 deg.C and 250rpm, inducing expression with 1% methanol, and adding methanol every 24h until the final concentration is still 1%; the expression is induced for 72 h.

The preparation method of the housefly antibacterial peptide Sarcotoxin-1B comprises the following steps of separation and purification in step S4: taking fermentation supernatant for inducing expression for 72h, filtering fine solid impurities through a 0.22 mu m filter membrane, then placing the filtered supernatant on a nickel column balanced by a binding buffer, purifying, and balancing the column by the binding buffer after all filtrate is purified to enable the column to be in an activated state; and then adding an elution buffer to obtain an eluent, namely the antibacterial peptide purification solution.

The invention has the beneficial effects that: the invention edits the Sarcotoxin-1B gene (the protein sequence number in NCBI is XP _ 005179717.1) expressed by the in vitro secretion of the yeast system to obtain the nucleotide sequence capable of expressing the antibacterial peptide Sarcotoxin-1B gene, and the expressed protein sequence only expresses the mature peptide sequence of 41 amino acid residues at the carbon end of the protein

GWLKKIGKKIERIGQHTRDATIQAIGVAQQAANVAATLKGK, the signal peptide sequence consisting of the 23 amino acid residues at the nitrogen end is not expressed.

The prepared housefly Sarcotoxin-1B protein has strong antibacterial capacity on staphylococcus aureus, escherichia coli and salmonella typhimurium.

The method effectively reduces the production cost of the housefly antibacterial peptide Sarcotoxin-1B, realizes the large-scale efficient and rapid expression of the housefly antibacterial peptide Sarcotoxin-1B, can improve the purification efficiency of the housefly Sarcotoxin-1B mature peptide, reduces the detection cost, and solves the problems of difficult purification from the housefly body and overhigh chemical synthesis cost; the invention also establishes a purification system with simple steps, rapidness and reliability, and can realize large-scale production.

The housefly antibacterial peptide Sarcotoxin-1B related by the invention is not only limited to the field of antibacterial drugs, but also can be applied to the fields of research and development of feed additives in animal husbandry, food preservatives and the like.

The method provided by the invention is efficient, cheap and rapid, is beneficial to promoting the marketization and scale production of the housefly antibacterial peptide Sarcotoxin-1B, and provides a new idea and solution for the wide application of the housefly antibacterial peptide in animal production.

Drawings

FIG. 1 is a schematic diagram of the nucleotide sequence of the housefly antibacterial mature peptide expressed by the invention.

FIG. 2 is the agarose gel electrophoresis picture after double digestion of recombinant expression vector and target gene.

FIG. 3 is an agarose electrophoresis picture of the sequence of SEQ S1Bl amplified using the GPS-I yeast genome as a template; in the figure,

lanes

1, 2 and 3 show the amplification products of the GPS-I yeast gene as a template; lane 4 is a 150bp molecular weight standard.

FIG. 4 is a sequence diagram of Musca domestica SEQ S1Bl gene mature peptide constructed into pPIC9K vector.

FIG. 5 is a schematic diagram of the construction of Musca domestica SEQ S1Bl gene mature peptide into pPIC9K vector.

FIG. 6 is a graph showing the results of western blot analysis after purification of fermentation supernatants using 6-his as the tag protein.

FIG. 7 is a graph showing the bacteriostatic effect of housefly antibacterial peptide SEQ S1Bl on Staphylococcus aureus; in the figure, 1 is virginiamycin control; 2 is antibacterial peptide Sarcotoxin-1B of 1080 mug/ml; 3 is a PBS blank.

FIG. 8 is a graph showing the bacteriostatic effect of housefly antibacterial peptide SEQ S1Bl on Escherichia coli; in the figure, 1 is virginiamycin control; 2 is antibacterial peptide Sarcotoxin-1B of 1080 mug/ml; 3 is 540 mug/ml antibacterial peptide Sarcotoxin-1B.

FIG. 9 is a graph showing the bacteriostatic effect of the housefly antibacterial peptide Sarcotoxin-1B on Salmonella typhimurium; in the figure, 1 is an antibacterial peptide Sarcotoxin-1B with the concentration of 1080 mug/ml; 2 is virginiamycin control; 3 is a PBS blank.

Detailed Description

The invention is further illustrated with reference to the following figures and examples.

Example 1: this example provides a method for preparing Sarcotoxin-1B, which includes the following steps:

1 Gene Synthesis and construction of expression plasmid

According to codon preference of pichia pastoris, artificially editing a nucleotide sequence corresponding to an amino acid sequence of the housefly antimicrobial peptide Sarcotoxin-1B, and designing a nucleotide sequence shown as SEQ S1 Bl;

wherein the amino acid sequence of the housefly antibacterial peptide Sarcotoxin-1B is as follows:

GWLKKIGKKIERIGQHTRDATIQAIGVAQQAANVAATLKGK；

the designed nucleotide sequence is as follows:

GGTTGGTTGAAGAAGATTGGTAAAAAGATTGAAAGAATTGGTCAACATACTAGAGATGCTACTATTCAAGCTATTGGTGTTGCTCAACAAGCTGCTAACGTTGCTGCTACTTTGAAGGGTAAA（SEQ S1Bl）。

for ease of detection, nucleic acid sequence CATCATCATCATCATCAC was added 6 × his after the 3' end of the sequence and before the stop codon, and the sequence was as follows:

GGTTGGTTGAAGAAGATTGGTAAAAAGATTGAAAGAATTGGTCAACATACTAGAGATGCTACTATTCAAGCTATTGGTGTTGCTCAACAAGCTGCTAACGTTGCTGCTACTTTGAAGGGTAAACATCATCATCATCATCACTAA；

the recognition sequence of restriction enzyme EcoR I is linked at the 5 'end of the gene segment, the recognition sequence of restriction enzyme Not I is linked at the 3' end of the gene segment, and yeast secretion expression signal peptide and the SEQ S1Bl sequence are constructed in an Open Reading Frame (ORF) for protein translation, as shown in figure 1, the constructed expression vector is named as pPIC9K-Sarcotoxin-1B, as shown in figure 5. The procedures of synthesizing genes, cloning and constructing to an expression vector are carried out by the biological engineering (Shanghai) Co., Ltd, and the synthesized gene sequence is subjected to enzyme digestion verification, as shown in FIG. 2.

2 fermentation expression of housefly antibacterial peptide SEQ S1Bl Pichia pastoris

2.1 pPIC9K vector transfection into Pichia pastoris GS115

The successfully constructed recombinant expression vector pPIC9K-Sarcotoxin-1B is transformed into competent cells of Pichia pastoris GS115, and the specific steps are as follows:

by usingSac I, the recombinant expression vector pPIC9K-Sarcotoxin-1B is linearized, and a single enzyme digestion reaction system is shown in the following table 1.

Purifying the linearized recombinant expression vector pPIC9K-Sarcotoxin-1B gene by using an agarose gel DNA recovery kit, and electrically transferring a purified product into Pichia pastoris GS 115:

(1) mixing 80. mu.L of yeast competent cell bacterium solution with 10. mu.L of linear DNA;

(2) adding the mixture into precooled electric rotor ice, standing for 5min (the electric rotor needs to be treated in advance: soaking overnight in 75% alcohol, ultrasonically washing for 30min, drying, standing upside down, and irradiating overnight with ultraviolet light);

(3) and (3) electrotransfer conditions: 1.5kV, 25 muF, 200 omega, 5 ms;

(4) immediately adding 600 mu L of precooled 1M sorbitol after electrotransfer, uniformly mixing, transferring to a sterilized centrifugal tube, and standing for 1-2h at 30 ℃;

(5) the cells were divided into 200. mu.L aliquots, plated on MD plates, and incubated at 30 ℃ until colonies grew out.

2.2 screening and identification of high copy transformants

After washing with sterile water, high-copy transformants were selected on YPD plates containing G418 at various concentrations, and methanol rapid-use transformants (Mut + type) were selected on MD and MM plates.

The specific steps of screening high-copy transformants by G418 with different concentrations are as follows:

(1) 1-2ml of sterile water is sucked on all MD plates, and His + transformants are resuspended;

(2) transferring the cell suspension into a 50mL sterilized centrifuge tube, and performing vortex oscillation for 5-10 s;

(3) concentration was determined with a spectrophotometer (1OD600=5 × 10)⁷Cells/ml);

(4) the bacterial suspension was applied to YPD plates with G418 concentrations of 1.0, 2.0, 3.0 and 4.0G/L in this order, and approximately 10G/L was applied to each YPD plate containing G418⁵(ii) individual cells;

(5) the plates were incubated at 30 ℃ until single colonies were produced.

Clones on YPD plates containing G418 took 2-5 days to grow.

The method comprises the following specific steps of screening methanol rapid utilization type transformants:

(1) picking a monoclonal by using a sterilized toothpick, dotting the monoclonal on an MM plate, and dotting the same colony at a corresponding position on an MD plate; toothpicks are needed to be replaced once for 1 clone at each point, and the plates are numbered in sequence;

(2) incubating at 30 ℃ for 2 days;

(3) after two days, the plates were observed, and the strains that grew normally on the MD plate but little or no growth on the MM plate were of the methanol slow-use type (Muts type), while the strains that grew normally on both the MD and MM plates were of the methanol fast-use type (Mut + type);

(4) selecting a Mut + type strain on a YPD plate for subsequent identification:

extracting yeast genome and identifying: extracting the genome DNA of His + Mut + high copy yeast transformant by a yeast genome DNA extraction kit, carrying out PCR by taking a yeast genome as a template and a universal primer of a pPIC9K eukaryotic expression vector, wherein,

a forward primer: 5'AOX1 （5’-GACTGGTTCCAATTGACAAGC-3’），

Reverse primer: 3'AOX1 （5’-GGCAAATGGCATTCTGACATCCT -3’）；

2% agarose gel electrophoresis test is carried out, as shown in figure 3, then the strains successfully identified by PCR are sequenced, as shown in figure 4, the sequencing result is completely consistent with the design scheme through comparison, and therefore, the Pichia pastoris strain GS115-pPIC9K-Sarcotoxin-1B with high housefly Sarcotoxin-1B antibacterial peptide yield is obtained, and GPS-I is shortened.

2.3 fermentation expression of housefly antibacterial peptide Sarcotoxin-1B in Pichia pastoris by SEQ S1Bl

(2) inoculating 1mL of bacterial liquid into 50mL of BMGY medium, and performing shaking culture at 30 ℃ and 250rpm until OD600= 2-6;

(3) centrifuging at 4 deg.C and 6000rpm for 5min, collecting thallus, and removing supernatant;

(4) washing the thallus precipitate with sterilized water, centrifuging, and discarding the supernatant;

(5) repeating the above steps;

(6) washing the thallus precipitate with BMMY culture medium, centrifuging as above, and discarding the supernatant;

(7) suspending the thallus precipitate with 50mLBMMY medium until OD600=1.0, transferring into 250mL conical flask, shaking culturing at 30 deg.C and 250rpm, inducing expression with 1% methanol, and adding methanol every 24h until the final concentration is still 1%;

(8) induced expression for 72h 50mL was sampled for analysis in subsequent experiments.

3 separating and purifying the housefly antibacterial peptide Sarcotoxin-1B expressed by SEQ S1Bl

Taking fermentation supernatant for inducing expression for 72h, filtering through a 0.22 mu m filter membrane to remove some fine solid impurities, then placing the filtered supernatant on a nickel column balanced by a binding buffer, purifying, after the filtrate is completely purified, balancing the column by the binding buffer to enable the column to be in an activated state, then adding an elution buffer, and obtaining eluent, namely the antibacterial peptide purification solution.

After purification, the column was washed twice with ultrapure water, and then the nickel column was preserved by adding about 4ml of 20% ethanol. And then the purified solution and the unpurified antibacterial peptide solution are subpackaged in freeze-drying bottles and placed in a freeze dryer to prepare freeze-dried powder for later concentration detection and bacteriostasis experiments.

4 identifying the expressed antibacterial peptide and measuring the concentration thereof

The BCA method is used for detecting the concentration of the antibacterial peptide in the purified liquid, and the step can be used for detecting the concentration of the antibacterial peptide and preliminarily judging whether the target antibacterial peptide exists in the purified liquid. The principle is as follows:

under the alkaline condition, divalent Cu ions are reduced into monovalent Cu ions by the protein, the monovalent Cu ions and the BCA reagent form a purple complex, a maximum absorption peak is formed at 562nm, and the concentration of the protein to be detected can be calculated by measuring the absorbance according to the principle that the absorbance is in direct proportion to the concentration. Calculating the formula: total protein concentration (μ g/ml) = [ (measured OD value-blank OD value)/(standard OD value-blank OD value) ] × [ standard/563 (μ g/ml) ].

Sequentially adding 10 mul of distilled water +250 mulBCA reagent (namely a blank control group), 10 mul of standard protein solution +250 mulBCA reagent, 10 mul of distilled water +250 mulBCA reagent, purified antibacterial peptide Sarcotoxin-1B 10 mul +250 mulBCA reagent, 10 mul of distilled water +250 mulBCA reagent and purified antibacterial peptide Sarcotoxin-1B 10 mulBCA reagent into a 96-well plate A-H well, wherein each group is subjected to 2 repetitions, and the protein concentration is shown in Table 2.

Western Blot detection of target protein

Respectively collecting 200 mul of each of two antibacterial peptide solutions before and after purification and mixing with a loading buffer, placing the mixture in a water bath pot for boiling and high-temperature denaturation, taking 20 mul of protein samples for SDS-PAGE electrophoresis, transferring the protein samples to a nitrocellulose membrane (NC membrane), sealing the NC membrane by a confining liquid for 2h, transferring the protein samples into an Anti-His antibody of a mouse source, incubating the protein samples for 1-2h at room temperature, labeling a second antibody with fluorescein to obtain goat Anti-mouse IgG, incubating the protein solutions for 1 h at room temperature, and scanning, photographing and storing the antibody solutions. The result is shown in FIG. 3, and it can be seen that the specific band of the target protein is between 4.6-10kDa standard molecular weight, indicating that the antibacterial peptide is stably expressed in Pichia pastoris cells.

Example 2: musca domestica antibacterial peptide Sarcotoxin-1B bacteriostasis experiment expressed by SEQ S1Bl

Adding 20 μ l of Staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli) and Salmonella typhimurium (Salmonella typhimurium) which are cultured to logarithmic growth phase into different culture dishes respectively, pouring 20ml of sterilized LB solid culture medium into the culture dishes, cooling and solidifying the culture medium, punching 3 holes with the same distance on the culture medium by a puncher, wherein the number of the holes is 1, 2 and 3, and 70uL is added into each hole. Wherein 1 in the figure 7 is a control of 20 mug/ml virginiamycin, 2 is 1080 mug/ml antibacterial peptide Sarcotoxin-1B, and 3 is a PBS blank control; in the figure 8, 1 is a control of 20 mug/ml virginiamycin, 2 is 1080 mug/ml antibacterial peptide Sarcotoxin-1B, and 3 is 540 mug/ml antibacterial peptide Sarcotoxin-1B; in FIG. 9, 1 is 1080 mug/ml antimicrobial peptide Sarcotoxin-1B, 2 is 20 mug/ml virginiamycin control, and 3 is PBS blank control; the cells were incubated overnight at 37 ℃ in a constant temperature incubator.

The result shows that the in vitro expressed housefly antibacterial peptide Sarcotoxin-1B has very obvious inhibition zones for three different types of strains, namely staphylococcus aureus, escherichia coli and salmonella typhimurium, and the virginiamycin has resistance only to the staphylococcus aureus, which indicates that the Sarcotoxin-1B protein prepared by the invention has wider antibacterial spectrum. As shown in FIG. 7, FIG. 8 and FIG. 9, it is shown that the in vitro recombinant housefly Sarcotoxin-1B protein has strong antibacterial ability against the above pathogenic bacteria.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and scope of the present invention are intended to be covered thereby.

Sequence listing

<110> Zhengzhou people hospital (Zhengzhou people hospital medical management center)

<120> gene editing housefly antibacterial peptide Sarcotoxin-1B nucleotide sequence and method for preparing antibacterial peptide

<141> 2021-12-01

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 123

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggttggttga agaagattgg taaaaagatt gaaagaattg gtcaacatac tagagatgct 60

actattcaag ctattggtgt tgctcaacaa gctgctaacg ttgctgctac tttgaagggt 120

aaa 123

Claims

1. a nucleotide sequence for preparing the housefly antibacterial peptide Sarcotoxin-1B, is characterized in that: described nucleotide sequence is as shown in SEQ S1B1.

2. a preparation method of housefly antibacterial peptide Sarcotoxin-1B, is characterized in that: may further comprise the steps:

Step 1, manually edit the nucleotide sequence corresponding to the amino acid sequence of the housefly antibacterial peptide Sarcotoxin-1B, design and synthesize the nucleotide sequence shown in SEQ S1B1;

Step 2, synthesizing the promoter sequence, restriction endonuclease EcoR I, 6*his tag sequence, restriction endonuclease Not I and terminator sequence, and then cloned into the vector to construct the recombinant vector;

Step 3: The recombinant vector is sequenced and verified, and then transferred into yeast strains. After screening, inducing expression, and purification, the housefly antibacterial peptide Sarcotoxin-1B is obtained.

3. the preparation method of housefly antibacterial peptide Sarcotoxin-1B according to claim 2, is characterized in that: step 2 is to insert restriction endonuclease EcoR I before start codon, insert 6* before stop codon his tag sequence, insert the restriction endonuclease Not I synthetic sequence after the stop codon, construct a protein translation reading frame with the yeast secretory expression signal peptide and the SEQ S1B1 sequence, and then clone into the pPIC9K vector to construct the recombinant vector pPIC9K- Sarcotoxin-1B.

4. the preparation method of housefly antibacterial peptide Sarcotoxin-1B according to claim 2, is characterized in that: step 3 specifically comprises the following steps:

S1. Transform the constructed recombinant expression vector pPIC9K-Sarcotoxin-1B into competent cells of Pichia pastoris GS115;

S2. Screen the high-copy transformants through YPD plates containing different concentrations of G418, and then screen the methanol fast-utilizing transformants Mut+ strains through MD and MM plates;

S3. Pick the screened high-copy transformants and inoculate them on YPD medium for culture, collect and wash the cell pellet, and then induce expression with 1% methanol;

S4. After induction and expression, separation and purification are carried out to obtain Sarcotoxin-1B antimicrobial peptide.

5. the preparation method of housefly antibacterial peptide Sarcotoxin-1B according to claim 3, is characterized in that: step S1 is specially: with Sac I, the recombinant expression vector pPIC9K-Sarcotoxin-1B is linearized; The recombinant expression vector pPIC9K-Sarcotoxin-1B gene was purified by agarose gel DNA recovery kit, and the purified product was electroporated into Pichia GS115.

6. the preparation method of housefly antibacterial peptide Sarcotoxin-1B according to claim 4, is characterized in that: step S2 also comprises extracting Mut+ type bacterial strain to identify on YPD flat plate, specifically: first by yeast genomic DNA extraction kit Extract the genomic DNA of His+Mut+ high-copy yeast transformants, use the yeast genome as a template, and perform PCR with the universal primers of the pPIC9K eukaryotic expression vector, wherein the universal primers are:

Forward primer 5' AOX1 (5'-GACTGGTTCCAATTGACAAGC-3'),

Reverse primer 3' AOX1 (5'-GGCAAATGGCATTCTGACATCCT -3');

Then, the strains identified by PCR were sequenced to obtain the Pichia species GPS-I, which can produce the Sarcotoxin-1B antimicrobial peptide of housefly in high mass.

7. the preparation method of housefly antibacterial peptide Sarcotoxin-1B according to claim 4, is characterized in that: step S3 is specially:

(1) Pick the screened high-copy yeast transformants, inoculate them in 5 mL of YPD medium, and cultivate overnight;

(2) Inoculate 1 mL of bacterial liquid into 50 mL of BMGY medium, 30 °C, 250 rpm shaking culture to OD600 = 2-6; 4 °C, 6000 rpm centrifugation for 5 min, collect the bacterial cells, discard the supernatant; wash the bacterial cells with sterilized water Precipitate, centrifuge as above, discard supernatant; repeat;

(3) Wash the bacterial pellet with BMMY medium, centrifuge as above, and discard the supernatant;

(4) Resuspend the bacterial cell pellet with 50mL BMMY medium to OD600=1.0, transfer to 250mL conical flask, 30℃, 250rpm shaking culture, induce expression with 1% methanol, add methanol every 24h until the final concentration is still 1 %; induced expression for 72h.

8. the preparation method of housefly antibacterial peptide Sarcotoxin-1B according to claim 4, is characterized in that: in step S4, the separation and purification method is: take the fermentation supernatant of induced expression 72h, filter through 0.22 μm membrane to remove fine To remove solid impurities, place the filtered supernatant on a nickel column that has been equilibrated with binding buffer for purification. After the filtrate is completely purified, equilibrate the column with binding buffer to activate the column; then add elution buffer , the obtained eluate is the purified solution of antimicrobial peptides.