CN114457102B - Gene expression cassette for encoding secreted Mersacidin and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of genetic engineering, and in particular relates to a gene expression cassette for encoding secreted Mersacidin and a preparation method thereof. The invention discloses a gene expression cassette for encoding secreted Mersacidin, the nucleotide sequence of which is shown as SEQ ID NO. 1. The invention utilizes high-fidelity enzyme to amplify the mrsK2R2FGE, mrAR 1D and mrMT of Mersacidin gene cluster genes mrsK2R2FGEAR1DMT, and utilizes Gibson cloning technology to connect the 3 segments of Mersacidin gene cluster genes to construct a Mersacidin expression cassette, the expression cassette is placed at the downstream of a strong promoter, and the Mersacidin expression cassette is introduced into genetic engineering bacteria Bacillus subtilisSCK to realize the efficient secretion expression of Mersacidin, thereby providing a cheap biological antibacterial agent and preservative for feed additives, veterinary drugs, foods and medicines.

Description

Gene expression cassette for encoding secreted Mersacidin and preparation method thereof

Technical Field

The invention belongs to the field of genetic engineering, and in particular relates to a gene expression cassette for encoding secreted Mersacidin and a preparation method thereof.

Technical Field

Bacteriocins are polypeptides or precursor polypeptides synthesized by the bacteria through ribosomes in the metabolic process, have ideal probiotic characteristics, have the functions of effectively inhibiting pathogens, regulating inflammation, promoting wound healing, protecting mucous membrane and the like, and are not easy to generate drug resistance.

Mersacidin was produced by Bacillus HIL Y-8554728, 20aa long, belonging to class Ib bacteriocins. The Mersacidin is pure white amorphous powder, stable at the ambient temperature, stable at pH 5.0-7.0 and insoluble in water; the metal salt is easily soluble in water and can enhance its bactericidal activity. Mersacidin inhibits the transglycosylation reaction of peptidoglycan biosynthesis by forming a complex with the glycophosphoric acid group of peptidoglycan precursor lipid II, thereby inhibiting cell wall synthesis and killing microorganisms. Merdacidin has an MIC of 1-32 mug/mL for methicillin-resistant staphylococcus aureus (MRSA), 0.5-16 mug/mL for methicillin-resistant staphylococcus epidermidis, 0.5-8 mug/mL for streptococcus, and 0.5-8 mug/mL for clostridium perfringens. Although Mersacidin had a MIC for MRSA greater than vancomycin, mice challenged with MRSA (st. Aureus E710) were treated subcutaneously with 25mg/kg Mersacidin with a cure rate of 100% and the same dose of vancomycin had only 67% survival. Furthermore, the intraperitoneal administration of Mersacidin eliminates nasal inoculated MRSA strains in the rhinitis model of mice. The evidence proves that although Mersacidin has higher MIC in vitro, the Mersacidin has good in vivo effect and has the potential of developing into antibacterial medicines.

Mersacidin was synthesized from a gene cluster AJ250862.2 consisting of multiple related genes including transcription, translation, transport and modification. The gene cluster is 12324bp long, consists of 10 ORFs, encodes a 68-residue propeptide from mrsA, which is decarboxylated and oxidized by mrsD to produce 2-thioethylamine function at the C-terminal cysteine. The modified mrsA is used as a substrate for producing a four-ring structure of mrsM, and is secreted into a final bioactive compound after mrsT processing. One of the regulatory genes mrsR1 is responsible for controlling the synthesis of mrsA; there is also a two-component system, mrsR2/mrsK2, encoding sensors and kinases involved in the induction of immune genes mrsE, mrsF and mrsG. Bacillus amyloliquefaciens FZB42 exogenous expression of Mersacidin by host bacteria has been successful, but the expression level is low. This demonstrates that exogenous expression of Mersacidin is possible, with the problem of how to increase the expression level, mainly how to increase the tolerance of the expression strain to Mersacidin.

Disclosure of Invention

In order to solve the problems, the invention utilizes high-fidelity enzyme to amplify the mrsK2R2FGE, mrAR 1D and mrMT of Mersacidin gene cluster genes mrsK2R2FGEAR1DMT, and utilizes Gibson cloning technology to connect the 3 segments of Mersacidin gene cluster genes to construct a Mersacidin expression cassette, the expression cassette is placed at the downstream of a strong promoter, and the expression cassette is introduced into genetic engineering bacteria Bacillus subtilis SCK to realize the efficient secretion expression of Mersacidin, thereby providing a cheap biological antibacterial agent and preservative for feed additives, veterinary drugs, foods and medicines.

It is an object of the present invention to provide a gene expression cassette for encoding secreted Mersacidin.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the gene expression cassette adopts a cloning amplification technology to obtain three cloning fragments: gene mrsK2R2FGE, gene mrsAR1D, and gene mrmt; the gene msK 2R2FGE is a fragment 1, and consists of a gene msK 2 encoded by an msK 2 protein, a gene msR 2 encoded by an msR 2 protein, a gene msF encoded by an msF protein, a gene msG encoded by an msG protein and a gene msE encoded by an msE protein, wherein the gene msAR 1D is a fragment 2, and consists of a gene msA encoded by a pro-peptide msA, a gene msR 1 encoded by an msR 1 protein and a gene msD encoded by an msD protein, and the gene msMT is a fragment 3, and consists of a gene msM encoded by an msM protein and a gene msT encoded by an msT protein.

Further, the three cloning fragments are respectively selected from a primer G1 cloning fragment 1 and a primer G2 cloning fragment 2, a primer G3 cloning fragment 2 and a primer G5 cloning fragment 3; the nucleotide sequence of the primer G1 is shown as SEQ ID NO.2, the nucleotide sequence of the primer G2 is shown as SEQ ID NO.3, the nucleotide sequence of the primer G3 is shown as SEQ ID NO.4, the nucleotide sequence of the primer G4 is shown as SEQ ID NO.5, the nucleotide sequence of the primer G5 is shown as SEQ ID NO.6, and the nucleotide sequence of the primer G6 is shown as SEQ ID NO. 7.

Further, the nucleotide sequence of the gene expression cassette is shown as SEQ ID NO. 1.

Further, the gene expression cassette is sequentially from the 5 'end to the 3' end: the genes mrK 2 encoded by the mrK 2 protein, the genes mrR 2 encoded by the mrR 2 protein, the genes mrF encoded by the mrF protein, the genes mrG encoded by the mrG protein, the genes mrE encoded by the mrE protein, the genes mrA encoded by the pro-peptide mrA, the genes mrR 1 encoded by the mrR 1 protein, the genes mrD encoded by the mrD protein, the genes mrM encoded by the mrM protein, and the genes mrT encoded by the mrST protein.

Further, the total sequence of the expression cassette genes is 12324bp:1 to 1440 are the genes mrsK2;1437 to 2159 gene mrsR2;2402 to 3313 gene mrsF;3310 to 4068 gene mrsG;4085 to 4819 gene mrsE;5104 to 5310 gene mrsA;5406 to 6047 gene mrsR1;6096 to 6680 gene mrsD;6892 to 10080 gene mrsM;10132 to 12324 gene mrsT.

Further, the gene expression cassette comprises a promoter, and the 3' -end of the promoter is connected with the pro-peptide coding gene mrsA.

The second object of the present invention is to provide a recombinant expression vector which can realize the expression of secreted Mersacidin.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the recombinant expression vector comprises the gene expression cassette.

Further, the recombinant expression vector is a plasmid vector or a viral vector.

Further, the viral vector is a lentiviral vector, an adeno-associated viral vector or an adenovirus vector.

Further, as a preferred embodiment, the plasmid vector is a pP43NMK vector.

Further, the recombinant expression vector includes, but is not limited to, a pP43NMK vector, other bacillus expression vectors with promoters and signal peptides, and vectors containing a secreted Mersacidin expression cassette "promoter-Mersacidin" can also be constructed.

The invention aims at providing a genetically engineered bacterium, which has higher expression level than wild bacterium after culture, and has low cost of fermentation medium, short fermentation period and easy industrialization.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the genetically engineered bacterium comprises the gene expression cassette or the recombinant expression vector.

Further, as a preferable example, the genetically engineered bacterium is Bacillus subtilis SCK.

Further, the medium composition of Bacillus subtilis SCK and 168 comprises: 10+/-5 g/L of soybean meal powder, 10+/-5 g/L of yeast powder, 10+/-5 g/L of peptone, 10+/-5 g/L of glucose and 5+/-2 g/L of sodium chloride.

Further, the genetically engineered bacteria include, but are not limited to, bacillus subtilis, and other genetically engineered bacteria such as bacillus licheniformis and bacillus pumilus can also realize transformation and expression of vectors.

The invention aims at providing a preparation method of secreted Mersacidin, which has low culture cost and high yield and provides a new direction and thought for realizing industrial production of Mersacidin.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the preparation method based on the secreted Mersacidin specifically comprises the following steps:

1) Constructing the recombinant expression vector;

2) Transferring the recombinant expression vector into the genetically engineered bacterium;

3) Screening positive recombinants, and fermenting and culturing in a culture medium.

Further, the preparation method of the secreted Mersacidin can further comprise the following steps:

4) Taking fermentation liquor to measure the antibacterial activity and content of the fermentation liquor;

5) Determining the antibacterial effect of the fermentation broth, and detecting antibacterial activity;

6) Filtering the fermentation liquor, and measuring the content thereof by a high performance liquid phase method.

The fifth object of the present invention is to obtain a secreted Mersacidin obtained by the above preparation method.

The invention has the advantages that:

1. the secreted Mersacidin obtained by the invention has a random structure, good activity, strong biological stability, good tolerance to heat, gastric juice, intestinal juice and the like, and remarkable antibacterial effect.

2. The Mersacidin expression cassette vector obtained by the invention is a promoter-mrsK 2R2FGE-mrsa 1D-mrmt gene, and realizes the synthesis and transportation of Mersacidin to extracellular secretion expression by combining Mersacidin gene cluster coding genes.

3. The invention discloses a preparation method of secreted Mersacidin, which has low culture cost and high yield and provides a new direction and thought for realizing industrial production of Mersacidin.

4. The invention uses Gibson cloning technology to connect the gene fragments of mrSK2R2FGE, mrAR 1D and mrMT to the expression vector with promoter to construct the Merdacidin expression cassette vector promoter-mrSK 2R2 FGE-mrAR 1D-mrMT gene. Because the carrier has mrsR1DMT which can process, modify and transport the propeptide, the synthesis and transport of Mersacidin to extracellular secretion expression are realized.

5. The invention converts a recombinant expression vector containing a Merdacidin expression cassette into genetically engineered bacteria to construct a strain. The strain is cultivated in a relevant culture medium, the activity of the strain is judged by measuring a bacteriostasis circle, and the expression quantity of Mersacidin in Bacillus subtilis is measured by adopting HPLC. The expression system has the advantages that the Mersacidin expression level is higher than that of wild bacteria, the cost of a fermentation medium is low, the fermentation period is short, and the industrialization is easy.

6. The invention ferments and expresses Mersacidin by genetic engineering and fermentation engineering for the first time, uses a safe strain Bacillus subtilis SCK168 as an expression host bacterium, uses a pP43NMK expression vector with a promoter P43, carries out recombination on a Mersacidin gene cluster AJ250862.2, and constructs an expression vector by using an expression cassette Mersacidin and the pP43 NMK. The Mersacidin expressed by the invention has high activity, improves the secretion expression level of Mersacidin in Bacillus subtilis, and is favorable for application and popularization.

7. The expression level of the secreted Mersacidin produced by the preparation method disclosed by the invention is up to 1.26g/L, and is higher than that of Mersacidin in all the current reports.

Drawings

FIG. 1 is a diagram of a recombinant expression vector construction process;

FIG. 2 shows the bacteriostatic activity of the broth against Clostridium perfringens (A: bacillus subtilis SCK: 168 broth supernatant; B: bacillus subtilis MRS: 08 broth supernatant; C: bacillus subtilis MRS: 24 broth supernatant; D:100ng vancomycin);

FIG. 3 shows the high performance liquid spectra of Mersacidin pure (A) and broth (B).

Detailed Description

The technical scheme of the present invention will be further clearly and completely described in connection with specific embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. Therefore, all other embodiments obtained by those skilled in the art without undue burden are within the scope of the invention based on the embodiments of the present invention.

EXAMPLE 1 construction of recombinant vector

1) The vector pP43NMK (BioVector NTCC inc.) was double digested with HindIII and PstI.

The enzyme digestion reaction system is as follows: 10. Mu.L 10X FastDigest buffer, 10. Mu.L vector (100 ng/. Mu.L), 5. Mu.L HindIII and 5. Mu.L PstI, ddH ₂ After mixing 70. Mu.L of O, the linearized carrier is recovered from the digested product in a water bath at 37℃for 60 min.

2) Using Bacillus HIL Y-8554728 genome as template, the mrsK2R2FGE, mrsAR1D and mrsMT gene fragments were amplified using G1/G2, G3/G4 and G5/G6 as primers, respectively. Primers G1 and G6 have homologous sequences to the vector, and G2 and G3, and G4 and G5 also have homologous sequences (as shown in FIG. 1, regions of the same color are homologous sequences).

The amplification system was 50 μl: 2.5U/. Mu.L of high-fidelity enzyme, 1. Mu.L; 2 Xreaction buffer, 25. Mu.L; primers, 1 μl each; template, 1 μl; ddH ₂ O, 21. Mu.L; the amplification conditions were: denaturation, 98 ℃,0.5min; annealing at 55 ℃ for 0.5min; extension at 68℃for 1.5min for fragment 1, 3min for fragment 2 and 2min for fragment 3.

3) The three fragments are mixed with a linearization pP43NMK vector and then connected by adopting a recombinant cloning kit method.

The reaction system is as follows: mu.L of 2 XGibson mixture, 1. Mu.L of linearized vector (50 ng/. Mu.L), 0.9. Mu.L of recovered and purified DNA fragment 1 (50 ng/. Mu.L), 1.8. Mu.L of recovered and purified DNA fragment 2 (50 ng/. Mu.L), 1.3. Mu.L of recovered and purified DNA fragment 3 (50 ng/. Mu.L), were mixed, placed in a water bath at 25℃for 30min and an ice bath at 0℃for 15min, and the reaction product was transferred into E.coli competent DH 5. Alpha. To screen positive recombinants (kanamycin resistance), thus constructing vector pP43NMK-mersacidin (FIG. 1).

The total gene sequence of the expression cassette is 12324bp:1 to 1440 are the genes mrsK2;1437 to 2159 gene mrsR2;2402 to 3313 gene mrsF;3310 to 4068 gene mrsG;4085 to 4819 gene mrsE;5104 to 5310 gene mrsA;5406 to 6047 gene mrsR1;6096 to 6680 gene mrsD;6892 to 10080 gene mrsM;10132 to 12324 gene mrsT.

Such vectors include, but are not limited to, pP43NMK, other Bacillus expression vectors with promoters and signal peptides, and vectors containing the Mersacidin expression cassette "promoter-Mersacidin" may also be constructed.

EXAMPLE 2 transformation of recombinant vectors

The vector pP43NMK-mersacidin was transferred to Bacillus subtilis SCK168 by electrotransformation under the following conditions: c=50 μf; pc=200 ohm; v=1.0 kV. Positive recombinants were screened and designated MRS08 and MRS24.

The host bacteria include, but are not limited to Bacillus subtilis, other genetically engineered bacteria such as bacillus licheniformis and bacillus pumilus, and transformation and expression of vectors can be realized.

Example 3 detection of bacteriostatic Activity of fermentation supernatant on Staphylococcus aureus

After the MRS08 and MRS24 strains obtained by screening were cultured for 16-22 hours under the conditions of 37 ℃ and 200rpm/min of culture medium (10 g/L of soybean meal powder, 10g/L of yeast powder, 10g/L of peptone, 10g/L of glucose and 5g/L of sodium chloride), the bacteriostatic activity of the fermentation supernatant on clostridium perfringens was measured in different time periods.

The antibacterial activity is measured by the following method:

1) Bacterial recovery: clostridium perfringens ATCC13124 plates were streaked and incubated at 37℃for 20h; single colonies are picked up and cultured in a corresponding liquid culture medium at 37 ℃ and 200rpm/min for about 12-16 hours. Diluting the bacterial solution to OD with physiological saline ₆₀₀ Absorbance of 0.1 (about 10 ⁸ CFU/mL), ready for use;

2) Taking 10mL of fermentation liquor sample 8000g, centrifuging at 4 ℃, and collecting supernatant for preservation at-20 ℃ for later use;

3) In an ultra-clean workbench, 100 mu L of indicator bacteria liquid is absorbed and added into 100mL of corresponding agar culture medium with constant temperature of 50+/-5 ℃ to be gently shaken to avoid foaming, and the content of bacteria in the culture medium is 10 ⁶ CFU/mL; pour 15-20mL into each accompanying dish, spread evenly, and let it cool and solidify. Uniformly punching by using a sterile puncher, sucking 150-200 mu L of fermentation liquor sample, injecting into the corresponding hole, setting Bacillus subtilis SCK fermentation liquor as negative control, vancomycin (1 mu g/mL) as positive control, marking on a culture dish, and placing the culture dish in a refrigerator at 2-8 ℃ for 1-2h for sample diffusion. The dishes were transferred to a 37℃incubator for cultivation.

The growth condition and the antibacterial effect of the thalli are observed in the following 6-12h, the antibacterial circle can be observed in 8-12h generally, the diameter of the antibacterial circle of the strain fermentation liquor of Bacillus subtilis MRS (figure 2B) and Bacillus subtilis MRS (figure 2C) is equivalent to that of vancomycin (figure 2D), and the antibacterial effect is strong.

Example 4 determination of stability of Mersacidin in fermentation broth

1) Respectively taking 5mL of fermentation liquor supernatant, and regulating the pH value of the supernatant to 2.0, 7.3 and 12.0 by using 5mol/L HCl or 5mol/L NaOH, and carrying out water bath at 37 ℃ for 3 hours;

2) Taking 5mL of fermentation broth supernatant, and treating at 100 ℃ for 1 hour; adding 0.2mL trypsin and proteinase K solution into 1.5mL fermentation supernatant with pH value adjusted to 7.3 respectively to make the final concentration of enzyme be l mg/mL, carrying out water bath at 37 ℃ for 4 hours, and carrying out boiling water bath treatment on the reaction system of the control group and all enzymes for 5 minutes to inactivate the enzyme;

3) The antibacterial activity of the treated fermentation broth against clostridium perfringens was tested according to the test procedure of example 3; mersacidin was found to be resistant to acid and base, pancreatin and proteinase K, and not durable in high temperature tolerance (table 2).

TABLE 2 stability of the antibacterial Activity of fermentation broths against Clostridium perfringens

O: 1.25 times dilution of distilled water, 5000rpm/min, centrifuging for 10 min;

a: diluting the centrifugal supernatant with distilled water by 1.25 times, and adjusting the pH to 7.3;

t: diluting the centrifugal supernatant with distilled water by 1.25 times, adjusting the pH to 7.3, and heating at 80 ℃ for 2h;

2: pepsin blank control;

7.3: trypsin, proteinase K, catalase blank;

p: pepsin treatment;

tr: trypsin treatment;

k: proteinase K treatment;

h: and (5) catalase treatment.

Example 5 determination of Merdacidin content in fermentation broth

Culturing the MRS08 strain and MRS24 strain obtained by screening under the conditions of 37 ℃ and 200rpm/min of culture medium (10 g/L of soybean meal powder, 10g/L of yeast powder, 10g/L of peptone, 10g/L of glucose and 5g/L of sodium chloride), and measuring the content of Mersacidin in fermentation supernatant in different time periods; the method comprises the following steps:

the method is characterized in that Mersacidin (99% of Nanjing Jinsri) synthesized by biological company is used as a standard, and high performance liquid chromatography is adopted for detection, wherein the chromatographic conditions are as follows:

reversed phase chromatographic column: c18 (4.6X1250 mm or 150 mm);

eluent: mobile phase a (0.1% trifluoroacetic acid), mobile phase B (90% acetonitrile);

column temperature: 25 ℃ (or room temperature);

detection wavelength: 260nm;

maximum pressure: 200bar;

gradient elution was performed as in table 3:

TABLE 3 gradient elution procedure for mobile phases

By adopting the detection method, the highest content of the active ingredients is 1.26g/L.

The pure product is dissolved in water, the peak time of the pure product is measured by loading, and then the fermentation liquor is loaded.

The external standard method is calculated in proportion to the peak area and the concentration (consistent loading amount), and the content is calculated by adopting the following formula:

wherein AX is the peak area of the fermentation broth Mersacidin; CX is the content of Mersacidin in the fermentation broth (μg/g); AR is peak area of control: CR is the concentration of control (. Mu.g/mL). The pure product and fermentation broth patterns are respectively shown in fig. 3A and 3B.

Sequence listing

<110> Chongqing City academy of livestock sciences

<120> Gene expression cassette for encoding secreted Mersacidin and method for preparing the same

<130> 2022.1.14

<141> 2022-02-24

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12324

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

ctaattgaca ttgttatact tagatggcgg actcggaaag gtaattttaa atcttgtgcc 60

aattcccggc tgactatata tatcaatatt tcccccatgt gccttgataa tttgttgagc 120

aatagccatt cctaatccag tccctgagtt atttgacgta gcgcttgtcc ctctgaaata 180

ccgatcaaat aaacattcga ccgtctcttc gtccattcct atcccatcgt cttctattat 240

aatttgtatt tcattgtcat gtaacacgtc ctgatataat atgaccttta ttttagtgcc 300

cggaggatta tgtttaatac aattagcaat caagttttca actgcacgct ttaggtacct 360

ttcatccata ttaaaaagta tcttattgtg actggattct aaagcgaatc tcttattttc 420

tgactccggt agcagcttca ttttttctaa agtgtcatga attactttca ctacgttttt 480

ttcttcaaga tggattggaa gcgactcatt tttcaattga aaagttaagt tgaaatcttc 540

aatcagcttt tccatatatt cgacacgttc ttccattatt aaggaaaaat cacgcacttg 600

ctgattttcc cactcatatt catttgaaga taaaagaacc gtatatccct taataaccga 660

aagaggggtt tttaaatcat gtgagacacc ggccatccac tcttctcgtg tcttttccaa 720

taattctcgc tcagccttat tcctttgaag cgttgaggta agttcagaca gagcctgcat 780

taattcttga taggtcttat atccagattt attatgtcta tatttgcttg aatgaaactt 840

ccaatcataa ggttcttcat atttttcttt agacaaattc tcaatccaag atacaataaa 900

cagcagggga gcacctagtt tttttccgta aaatattgca atgacaatcc ctgccataat 960

aatagaaaca atccacaagt tattcaccca atagaaaaaa gggttatttt tttccatcgg 1020

ctttcccaat acccatgtta aatcctgatt gtttttcgtt tcataccacg tagataattg 1080

ataacctttt ttggcaggat acatgtaatc tgatattaac tctccaagaa tataatgctt 1140

aggtatattt tgaggtttat taaaagaaaa aacctcatct ccttgttcat ctaaaatttg 1200

aatccacatc cggttctcta ataaatcttc ttttgctttc gtatttattg aaaattgttt 1260

atctgcaaca actgtttggt caacaatttt ttttaaggat aagtgcggcg tttccatttt 1320

attctgacct aagacgaggc tgaaaaatat gatactgccc acaatgagca caccccaaat 1380

aaacatgagt aaaaaaagtc tcgatacgaa gtaaaatgct atcctatttc tcagcttcaa 1440

aataaatcct catctatagg ctctgcttca aatatatagc cctgtcctcg aaccgtttta 1500

atccatctag gtctgctcgg gtcttcctct aatttttcac gtaatcgtct tacatgtacc 1560

attacagtac tatctccgcc ataaaactcc ccccacacat ctttataaat ttggtgttta 1620

cttaaaattt gattgggatg ttcacagaaa taaatcagaa gtttcatttc ctgagcaggg 1680

caatctattc ttgtcccgtt aacaacaagt cttccggtat tgggatctat tttaaaatat 1740

tcataatcgt atacctgttt tagagggtaa gaagaagaga gttgagtcgt tctctttaat 1800

tgggctttta cacgagctac aatttctagt ggattaaatg gtttagtaat atagtcatct 1860

cccccaaaac taaacccttg caatttatct aaatctgttg tttttgccgt taagaagagt 1920

atcggcacat ttgtatgcga gcgtattttg ctgcataagg tgaagccatc agtgtccgga 1980

agcatgacat ctaataaaat aatatttgga agttcctgat cgattttcag aagggtttcg 2040

ctccctgtca tagctgtgga aatattgctg aaaccttcct tttcaaagca agttattaat 2100

aagtttaaga tatgttcgtc atcatccacc atgaggattt tgttctcttc taacgtcata 2160

agttaccctc ctttttattt cttattttat atgagatcaa tgacataaag gtcagaaaag 2220

gaaagaattt aactttcgtt aagggttcag aatattcttt cgaattttta accttgatat 2280

tcaggtatca atataactac atacagaaaa taaaccaggg atgtcgttaa gtgaatgttt 2340

aggaagagtt tcgtttgtgt ttaaagaaca tcgcttatga taaaagtgag gtgagagaca 2400

aatgaatgat caaatagtgg taacacacga tctgactaaa aagtataaaa agcatacttc 2460

tgttgatgga ttaaacttaa ggattaggcg tggcgaaatt tatggttttc ttggaccgaa 2520

tggtgctggt aaaacaacaa ccatccgaat gttattaggt ttaattaaac caacaaaagg 2580

aaacatcgaa atctttggcc aaaacctaaa caagaatcgt ttgcagatat tgcaaagaat 2640

cgggtcactg gtagaatctc caacttatta tggtaattta acaggttacg aaaacttaga 2700

agctgttagg aggttacgag gacttccgga acaacaggtc aatgaagtat tggaaactgt 2760

aaggttatct aaagtagcaa atcgactgac taaggagtat tcccttggaa tgaaacaacg 2820

tttgggaatt gcagttgccc tgttaagcag tcctgattta ttaattctgg atgaaccgac 2880

aaatggttta gatccatccg gtatccaaga aataagagag ctaattaaag aattgcctaa 2940

atcgggaatg agtgttattg tatccagcca cttattgagt gaaatagatc aaatggctac 3000

tcaagtggga attatcaaca atggaaaaat gatttttcag gactcgattg caagtttaca 3060

tcaaaaaaga aaaccactat taaaagtcgg tgttagtgac gtaatcgaag caaaaacaat 3120

attaaacagg aaaggattaa aggttgattt acaaaaaaat tatttgtggc tgtctcaaac 3180

agaaccggaa ttcgtttcag aaatcaattc catacttctt cattcagggc tgtctgtatt 3240

tcgacttgaa gaaaagacac gatcacttga ggatattttt ttagaattaa ccggtacaga 3300

gggaagtcta tgaaaaagtt attatgggca gatcaattaa aactgaaacg ttcgtcgtta 3360

ttgattgtag tcttattggt tcccttactc attatagcat atgagttggt aaatcttact 3420

tatcgatctg aatacgtgga aaaacaagct gaaatgttcc atgctggatc aatgtggatg 3480

tatttactgt atgataacag tttgttattt ggtctgggtt ttccattagc cgccacactt 3540

tctgcgtcaa taatagcaaa tatagagcat caagcaaacg gatggaagca aaccctttct 3600

tttcctgtat ctagaatgcg aatttatgta agtaaattta tttgcttagt agtcagttta 3660

tttatttcgt caacaatctt cttgctcggc atggtactgc tgggcaaact ggtaggattc 3720

gaaggtagtg taccttgggg acttttgttt ggagatagtt acagcatgtt agttacagta 3780

ctgcctataa tggcattcca gatatggtta tctatggtgt ttagcaacca agcattctca 3840

attcttgtag gatcagtatc atctataatg ggtttgtttt tggcagctgc tcaatcaacg 3900

agatggtttc cgctggctta tcctagtcaa tcttcgacag tcattctgca gtatgaaggt 3960

ataggatata acccagatct ttcatcttac ctttgcatta gccttttctt agggataatt 4020

atattatttc taggctctat tcattttgct aaacgggatg ctttgtaaaa aggaggcgaa 4080

atacttgaaa aacattttat ttgtagaacg tttaaagctt aagcgatcaa agttatggat 4140

catctactta ttagggcctt tgttaggtgt atccctggca tacactaact ttattaaaaa 4200

ctataacctt tttatgaatc ccggagacaa tccctgggta gaggcttgga cacaagttgc 4260

cttatttatg ggcccttttg tattgcctat cgtagtggga attttcgccg cccttgtttg 4320

caggggggaa catgtaggag gcggttggaa acagcttcta gccttaccag tcaaacactc 4380

agatatcttt ctgggaaagt ttctaacagt ggtccgcatg atattcatta gtatgtccat 4440

tttaatcctc ttatttattg gattcggtta tatgttaggc ataagcggga gtcttccttt 4500

acttacaatt ctaggttatg gaattagagg gatcttagct tgtttaccat taattttatt 4560

gcagcttatt gtttcaatca ggtctaaaac atttggcata ccactcgctg ttagcattgt 4620

ttttacatta ccggccatta ttattgccag tacaccatta ggtcaagtat atccatggac 4680

acagcctatg ttagcaatgt cacccgaaga tgaatcaccg attcaatcaa atttcctgtt 4740

ttactcaatt atggtaataa cgtgtctcgg tctcctggtt tacggaataa gaagttttac 4800

taaacgagac cttacgtagg gtatatgcgg tataaactta tgagaattcg agacaaggta 4860

aactaatttg actagcgcta tgtaaaacag aaatttatat gaaggtaaga ctaaatcttt 4920

ttggttgtat tctttgaaac taaacgacta atggaaaagg tgatgtttca aagcataaga 4980

cataagaaat ataaagatat cttaagactc tttatttaaa cattttttac atttaataat 5040

atctcttcca tttttttgat tatgagttaa tataatacta tacttaataa gggggtgaat 5100

acaatgagtc aagaagctat cattcgttca tggaaagatc ctttttcccg tgaaaattct 5160

acacaaaatc cagctggtaa cccattcagt gagctgaaag aagcacaaat ggataagtta 5220

gtaggtgcgg gagacatgga agcagcatgt acttttacat tgcctggtgg cggcggtgtt 5280

tgtactctaa cttctgaatg tatttgttaa tttgatttat ataggctgtt tcccttcaga 5340

aggaacagcc tatattttat tatataaact attagaaaat tcttaaaaaa caggagggta 5400

aatcattggg gaaaactctc gtttgttcag aaaatagcta tttccaagca tatatgaatc 5460

atttattaac gccagatagt aatgaaataa taaatgttaa cacattggat gaactaaaac 5520

aaataatatc caaagaaaat ttttcctctg taatcatcga tacttgccac cctaatgact 5580

tagtgttgca actgataaaa tcgatatctt gcccagtcat aatacttaac tccttagaaa 5640

ctaacgtttc agattataac cctggtccaa tattaaatca aataaataat gtttcaaccc 5700

taaaacataa tgtttttcaa ttgtctttca caactttttt cgacgttgga aagcattgta 5760

tttggaaaaa gaatgaatat atccctctag caatacaaga atttaaaatt ttatatttgc 5820

tttatttaaa ttcaaataaa atagtctgtt ctgaagaact tattgaatat gctgacctta 5880

ctggtaggtc aagtctttat gtacacatta gttctcttag agaaaaagta gaggataacc 5940

ctggagatcc aaagattctt caaacaaagt ttggaaaagg gtacctctta tctgacagta 6000

catatatttg tcttgaaaaa aaagcagact caaaaaatgt cgtgtaaaaa ggatgaccca 6060

aaaatcattt taaaatgact tttgagtcat cccatttatg ttagtgaggg gtgttttgtt 6120

ctttctttaa aacctttttc tattgctaat aaagctttgt cgggtgtaat tagaccacga 6180

ttaggttttc ttgtacccgt cgcaatctca aacgccataa tttcaactgg ttcaataaca 6240

atatgcccat cttttcttaa ttgttcaata ttcctggaaa caactgtttt attccacatt 6300

aaatcgttca tattaggaaa gaaaatagtg ttatgtggat gagctaatac agtagttgca 6360

actaaattca ttgctacacc attagcagtt tgtcctaaga tgttcgctgt agctggaata 6420

atgcaatata tgtccgccca gcggccaatt tctacgtggc tatgtctttt tccgttttcc 6480

ccatgttctg agtatacatg gtcacaaaaa taagaaacag tatgagctgg aataaggtct 6540

tccgctgttt ttgtcataac aacccttatt tccttaaaga agcttttaaa atataagaga 6600

tacgaggaga tgccgacaga tgagattgat ccgcaaatcc cgatcaataa ctttttatct 6660

tttaatattg aaatactcat ttttttctcc ctctgtaaat tttaaacaat catactacac 6720

aattattctt tttgtggtgt ttattccaaa tattaaatat tcttaataaa cacatataaa 6780

ataaacctaa acttctttta aaattctttt aacttttaaa tggctctgct atatttatgg 6840

ataattaagg aatattaatt gttcaaattt attaaaaatg aggtgttcaa tatgcataca 6900

aaattcaaac ggaattcggt atggaatagg tcttcttcga taagtgaaag aaaagtaagg 6960

cgttcactta atactaattg ggatgaacta acaaacagac gcttcgaaag atggaaaagc 7020

cttgttgaaa gtgatgaggg gattcgaatt gaggatgtat tagccaccca aaatattgac 7080

gaagaaaccc taaagcatac tatcaatgca aaagaagtgg aatttattaa tgaaggtgat 7140

catcaaggat ggcttgaaat tattcagcta gttgatgaac aatcctataa aaatgtaaat 7200

atagaagtta gaaaagatat tctcttcttt agttttataa aaccattttt gaaaatagca 7260

agaggcaaat tagaggaagt attatattct cactctacta aatctctgat aaaggaagaa 7320

cttagtccgt cggtaattga tgatttactt aataatcttg gtgaaacttt atcagctata 7380

agtagtcgga tattaatatt ggagttaaat gtagcaaggg tatcaggaaa gctgcggggt 7440

gaaacttctg aagaacgagc ctcttatttc aatcaagccc ttttaaatga cccggcatat 7500

gttcgatcaa ttcgagaaga atacatagtt ttaacaaggc tgttagcaac aaaaacaatg 7560

tattggattc aaaatacttc tgatctcctt gtaagatttc atcaggataa agggatatta 7620

gaatctgagt ttagtaatgg tcaaaaatta gggaaaatta tttccatcga tacaggaagt 7680

ggcgtctcag atactcataa caaaggaaaa acagtcgcca ttttaaactt tgaaactgga 7740

attaaaattg tgtataagcc tcgttcatta gaaatagacg taaaatttaa taaatttgta 7800

aattatctaa atggtaaaaa tttaagtttt gacttaaaaa ctgtacacac acttaacaaa 7860

aaatcttatg gatggacgca gtttatctct tataaagaat gtcaggaaga actgcaaatt 7920

ggaaagtttt attggcgtat tggaagttat ttggctatct tatatgcaat gaatgcggta 7980

gattttcata tgcaaaattt aattgctgat ggagaatatc ctattttagt tgatttagaa 8040

tctttatttc ataataattc tacgtataca gataccagtg cttttagtcg cgcacaagaa 8100

catattgaac ggtcagtttt acgaattggg ttattaccga gaaaaataaa tagtaaagct 8160

ggatttgaag gaattgacct tagtgcatta ggtgcacaag aaggacaagt atctccgcat 8220

aaaaccagta caatagttga tcgagataaa gatacagtca ggatagaaga aaaaaacttt 8280

ccaattccag ttagtcagca tagacctatg ctgcatggac aaatcattaa tactgttgct 8340

tacgaaggaa atattataaa aggatttgaa gaaacctact tccttttcat gaaatataaa 8400

caagacatgc tcgaacaaat agattctttt aaaggggtta ctgtaagaca aatattgcgt 8460

ggtacatctc gctatgcaaa ccttctgaaa ataagtttac atccagattt catgagagac 8520

ggcttagatc gtgaaatgat tttagataaa ttgtggctag atacaaaatt gaatccacga 8580

ttgaaccaag ttgtaaatag tgaaaaggaa ggcttattcc ttggggatat cccatatttc 8640

acaagcaagc ctgaatctac aaatatgtgg gattccagcg gtcgaaaaat caacaatttt 8700

tttaaaacaa gtgccttaaa tgagacaaag gaaaaaataa atgaaatgaa tgaaagtgac 8760

tgtcatgaac aagtaagctt tataaaaaca gctattttag taattaagga ttcttatcgt 8820

aaacataaag tatttgatat aaatcctcga ttacacgttt ttaacccaaa agattttttt 8880

caagaagcta ttaaaatcgg agactttctt gctagtaggg cgattgaagg tgaacagtta 8940

gatggacaag aggatgtatc ttggatcgga tcgtttgttg acaatcaacg agaggatcaa 9000

ttcaagattt cagctgcaaa tagttcatta tacgagggtg taggtggtat ttctctcttc 9060

cttgcttatt taggtcgcct ctctaacaat gaaaaataca ccaaactatc taaaaaagct 9120

ttagtggcag tacacaaaaa tatgtctgca tctagtgatt taggtgcctt tgggggaatt 9180

gcttcttacc tttatttgtt ggatcattta tcaaaattat ggaatgatga acaactattg 9240

aaaaacgaac tttattctgc cctcaacaaa ttagattctt tgatcgaaag ggatgaaaac 9300

aacgatattt taacaggggt ggctgggaca gcagttattt taataaattt gtttaaacgg 9360

tataaagaag aacaaatctt aaacttgatt acaaaatgcg gaaatcgtct tattcaaaat 9420

ataaacgtta tggaaaaggg agttggatgg aaagtcccgg caaacccaac gccagcctcc 9480

ggatttgctc atggtgcctc aggaattata tgggctcttt atgagattta cgcaattact 9540

aagcaaactg tatttaaaga ggtagctgaa aaagcgttag aatttgaaag aactttgttt 9600

attccggaaa aaaacaattg ggcagatatt aaacttgaaa acggacagtt tcgaaatgat 9660

aattttgttg cttggtgtaa tggcgcagca ggcataggat taagtaggat attgatcctg 9720

ccacacaatc aaaatgaatt gataaaagat gaagcacatg tcgcaattaa tacaacccta 9780

aaatatggtt ttgaacatga tcaatcttta tgccatggtg atttaggtaa tctggacatc 9840

cttatgtacg cagcggaaaa ctttaataaa aagttaagcg taaatgtaac agaactaagc 9900

cataaaattt taaatgatat aaagctcaga ggatggttaa ctggatttga aaaaaataac 9960

gaatccccat ccttaatgat ggggtatgca ggtataggac ttggattgct taagattttt 10020

gcaccagtcg aagtgccatc agttttgaga ctccaatcac ctttagaact aaaattgtaa 10080

aattaaaatg agccttactt tgttaaggct ctatttctta gaggtgatcc aatgagaaga 10140

agagttcctc tagtaaggca aatgggacaa tatgagtgtg ggcccgcctg tctcaccatg 10200

attttaagtt attatgggag tactatatct ctaaataaaa taagtgaaca atgtgatgcc 10260

caaagaaatg gcgtatctgt atccatttta aaatcagtat cagaatatta tgggcttaac 10320

tgcaaagtat accaagtttc gtttaaagat ttgaaaaaat atatcaattc atatcttccc 10380

tgcatcatat tttgggatga acggcacttt gtggttttag aacagattaa aaaaggtctt 10440

tttcatataa ttgaccccaa cagaggaaaa ttgaaattat ccgaagaaga atttaaaaga 10500

cattatagca aggtaatctt gacttttaag aagtcagata aatttaaaga gatgatgcca 10560

tcacccgcag caaagtatta tttacgttat atagttaaaa gccgtactat tgtttcactt 10620

atcattctct tttcactgat cacacaagta gtcttcttag ccgttccttt tttaattaaa 10680

tatttagtgg atcattcttt aataagtaaa tcaacaaatt cttttctatt tttaggaatt 10740

gtagtcatca tagcagtttt tatattggga cttgttatgt ttatccgcaa ttatttcaca 10800

attaaacttc aagctattat aagtaaaagc atctcaaacg attttgtgga acatttatta 10860

aaattacctc tcaattttta tgaaaatcga acaaccggtg atattgcaat gagagtaagt 10920

aacatttcca tgataaggga gattatagct aagaatggag caacaatagt actagatatt 10980

attactttga tttcattttt tattgctatg ttaactcaat catttaaact tgcatttttt 11040

gctattggat tggcaattat tcaattctta ctaatgatga ttttgattcc aagaataaaa 11100

aatttaatcc ataatgactt atcaattcaa acaaccacac aaagtttttt agtggaagct 11160

ctaagagcta tcacttttat taaatcaaat ggtttagatc attctatcct aacaaagtgg 11220

tcaaattact atgataaaca aatcgaagca ttttcacagc gataccactt agacgctata 11280

atggatagca taagtgtaag catccgatat tgtgcacccc ttctcttatt atggtttgga 11340

tcaaaagaag ttatcaccgg aaatttaaca ctcggaggct tattagggtt tagcagctta 11400

ggaacatctt tcttgttgcc gattgcttct ttaataacag gcatgcaaca atttcaattg 11460

gtcggtgaca cttttgaacg aatgcaagat gttatggaaa ctgagcccga acaaatcaac 11520

caaacttcaa taattgaaac tgaactatca aaacaagata taaaacttga aaatgtcaca 11580

tttacacatc atagcttaca tattcttaaa gaagtttcat taaacattaa gtcaggtact 11640

aaattagctc tcgtaggccg tacaggtagt ggaaagacaa ctttatcaag aattatactg 11700

gggttataca aacccactaa gggaaaagta ttttatggtg aacaggactt gaagaattta 11760

aacctttacg agctccgaaa acaaatgggg gtcgtattac aagaaagctt tcttttcaat 11820

gatactattg caaacaatat agctggattt aaaagcttat ctcaggataa aattgaacaa 11880

gcagcaaaga gagtgcagtt acatgaagat ataataagaa tgccaatggg atataacaca 11940

attatcgggg aaaatggcag tatgttatca ggagggcagc gtcagcgcat agcaatagca 12000

agagctatcg tcgataaccc ttctgtagtc attttagatg aaataacaag taacttggat 12060

acattaacag agcatgaaat tgacgaatac tttgcaaaat caaatattac ccgtattgta 12120

ataactcatc gtcttttaag tagccaagac tctgatttaa tagtggtatt agatcaagga 12180

aaaatagtcg aaaaaggaaa acatcaagag ttattagaga aaaagggata ctattataac 12240

ttatggataa aacaagtagg ggatagacag aaagctacgc tgcaaaagcc ctttttcctc 12300

aatgagcaaa ctggcaaaac ttga 12324

<210> 2

<211> 48

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

taacacatgc ctcagctgcc taattgacat tgttatactt agatggcg 48

<210> 3

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

taagtttata ccgcatatac cctacgtaag gtctcgttta 40

<210> 4

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ggtatatgcg gtataaactt atgagaattc gagacaaggt 40

<210> 5

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atttacagag ggagaaaaaa atgagtattt caatattaaa 40

<210> 6

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ttttttctcc ctctgtaaat tttaaacaat catactacac 40

<210> 7

<211> 43

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tctaaccgca ttagtaccag ttcaagtttt gccagtttgc tca 43

Claims (4)

1. The preparation method of the secreted Mersacidin is characterized by comprising the following steps of:

1) Constructing a recombinant expression vector containing a gene expression cassette with a nucleotide sequence shown as SEQ ID NO. 1;

2) Transferring the recombinant expression vector obtained in the step 1) into genetically engineered bacteria; the genetically engineered bacterium isBacillus subtilis SCK168；

3) Screening positive recombinants, and fermenting and culturing in a culture medium to obtain the secreted Mersacidin;

in step 1), the vector is a pP43NMK vector.

2. The method of claim 1, wherein the gene expression cassette employs a clonal amplification technique to obtain three cloned fragments: gene mrsK2R2FGE, gene mrsAR1D, and gene mrmt; the gene mrK 2R2FGE is a fragment 1, and consists of a gene mrK 2 encoded by an mrK 2 protein, a gene mrR 2 encoded by an mrR 2 protein, a gene mrSF encoded by an mrF protein, a gene mrG encoded by an mrG protein and a gene mrE encoded by an mrE protein; the gene mrsAR1D is a fragment 2, and consists of a gene mrsA encoded by a propeptide mrsA, a gene mrsR1 encoded by an mrsR1 protein and a gene mrsD encoded by an mrsD protein; the gene mrsMT is a fragment 3, and consists of a gene mrsM encoded by an mrsM protein and a gene mrsT encoded by an mrsT protein.

3. The preparation method of claim 2, wherein the three cloning fragments are selected from the group consisting of a primer G1 and a primer G2 cloning fragment 1, a primer G3 and a primer G4 cloning fragment 2, and a primer G5 and a primer G6 cloning fragment 3; the nucleotide sequence of the primer G1 is shown as SEQ ID NO.2, the nucleotide sequence of the primer G2 is shown as SEQ ID NO.3, the nucleotide sequence of the primer G3 is shown as SEQ ID NO.4, the nucleotide sequence of the primer G4 is shown as SEQ ID NO.5, the nucleotide sequence of the primer G5 is shown as SEQ ID NO.6, and the nucleotide sequence of the primer G6 is shown as SEQ ID NO. 7.

4. The method of claim 1, wherein the medium composition comprises: 10+/-5 g/L of soybean meal powder, 10+/-5 g/L of yeast powder, 10+/-5 g/L of peptone, 10+/-5 g/L of glucose and 5+/-2 g/L of sodium chloride.