CN114457102A - Gene expression cassette for encoding secretory Mersacidin and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of genetic engineering, and particularly relates to a gene expression cassette for coding secretory Mersacidin and a preparation method thereof. The invention discloses a gene expression cassette for coding secretory Mersacidin, which has a nucleotide sequence shown in SEQ ID No. 1. The invention utilizes high fidelity enzyme to amplify mrsK2R2FGE, mrsK 1D and mrsm MT of Mersacidin gene cluster gene mrsK2R2FGEAR1DMT, and utilizes Gibson cloning technology to connect 3 segments of genes of Mersacidin gene cluster to construct a Mersacidin expression cassette, the expression cassette is arranged at the downstream of a strong promoter, and the expression cassette is introduced into a genetically engineered bacterium Bacillus subtilis SCK168 to realize the high-efficiency secretory 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, in particular to a gene expression cassette for encoding secreted Mersacidin and a preparation method thereof.

technical background

Bacteriocins are a class of polypeptides or precursor polypeptides with bacteriostatic activity that are synthesized by bacteria through ribosomes during metabolism. They have ideal probiotic properties and can effectively inhibit pathogens, regulate inflammation, and promote wound healing and mucosal defense. , and is not easy to develop drug resistance characteristics.

Mersacidin is produced by Bacillus HIL Y-8554728, is 20aa long, and belongs to class Ib bacteriocin. Pure Mersacidin is white amorphous powder, stable at ambient temperature, stable at pH 5.0-7.0, insoluble in water; metal salts are easily soluble in water and can enhance its bactericidal activity. Mersacidin inhibits the transglycosylation reaction of peptidoglycan biosynthesis by forming a complex with the sugar phosphate group of peptidoglycan precursor lipid II, thereby inhibiting cell wall synthesis and killing microorganisms. The MIC of Mersacidin against methicillin-resistant Staphylococcus aureus (MRSA) is 1-32 μg/mL, the MIC against methicillin-resistant Staphylococcus epidermidis is 0.5-16 μg/mL, and the MIC against Streptococcus is 0.5-8 μg/mL , the MIC to Clostridium perfringens is 0.5～8μg/mL. Although the MIC of mersacidin against MRSA is greater than that of vancomycin, subcutaneous injection of 25 mg/kg mersacidin in MRSA (St. aureus E710)-challenged mice resulted in a 100% cure rate, while the survival rate of the same dose of vancomycin treatment group Only 67%. In addition, intraperitoneal administration of Mersacidin can eliminate nasally inoculated MRSA strains in a mouse model of rhinitis. The above evidence confirms that Mersacidin is effective in vivo despite its high MIC in vitro and has the potential to be developed into an antibacterial drug.

Mersacidin is synthesized from a gene cluster AJ250862.2, which includes transcription, translation, transport and modification genes. The gene cluster is 12324 bp long and consists of 10 ORFs, which encode a 68-residue propeptide by mrsA, which is decarboxylated and oxidized by mrsD to generate a 2-thioethylamine function at the C-terminal cysteine. The modified mrsA serves as a substrate for the formation of a tetracyclic structure by mrsM, which is processed by mrsT and secreted into the final biologically active compound. Among them is a regulatory gene, mrsR1, responsible for the synthesis of mrsA; and a two-component system, mrsR2/mrsK2, which encodes sensors and kinases involved in the induction of immune genes mrsE, mrsF, and mrsG. Bacillus amyloliquefaciens FZB42 has been successfully used as a host to express Mersacidin, but the expression level is low. This shows that exogenous expression of Mersacidin is feasible. The problem is how to increase the expression level, mainly how to improve the tolerance of expression strains to Mersacidin.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention uses high-fidelity enzymes to amplify mrsK2R2FGE, mrsAR1D, and mrsMT of the Mersacidin gene cluster gene mrsK2R2FGEAR1DMT, and then uses Gibson cloning technology to connect the three segments of the Mersacidin gene cluster to construct a Mersacidin expression cassette. In the downstream of the strong promoter, it was introduced into the genetically engineered bacteria Bacillus subtilis SCK168 to realize the efficient secretion and expression of Mersacidin, which provides a cheap biological antibacterial agent and preservative for feed additives, veterinary drugs, food and medicine.

One of the objects of the present invention is to provide a gene expression cassette for encoding secreted Mersacidin.

To achieve the above object, the present invention adopts the following technical solutions:

The gene expression cassette adopts cloning amplification technology to obtain three cloned fragments: gene mrsK2R2FGE, gene mrsAR1D and gene mrsMT; the gene mrsK2R2FGE is fragment 1, and the genes mrsR2 and mrsF proteins encoded by mrsK2 protein and mrsR2 protein are encoded by mrsK2 protein. The encoded gene mrsF, the mrsG protein encoded gene mrsG and the mrsE protein encoded gene mrsE are composed, the gene mrsAR1D is fragment 2, the gene mrsA encoded by the propeptide mrsA, the mrsR1 protein encoded gene mrsR1 and the mrsD protein encoded gene mrsD Composition, the gene mrsMT is segment 3, which is composed of the gene mrsM encoded by the mrsM protein and the gene mrsT encoded by the mrsT protein.

Further, the three cloned fragments select primer G1 and primer G2 clone fragment 1 respectively, select primer G3 and primer G4 clone fragment 2, select primer G5 and primer G6 clone fragment 3; the nucleotide sequence of described primer G1 is as shown in SEQ ID NO.2, the nucleotide sequence of the primer G2 is shown in SEQ ID NO.3, the nucleotide sequence of the primer G3 is shown in SEQ ID NO.4, and the nucleotide sequence of the primer G4 The sequence is shown in SEQ ID NO.5, the nucleotide sequence of the primer G5 is shown in SEQ ID NO.6, and the nucleotide sequence of the primer G6 is shown in SEQ ID NO.7.

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

Further, the gene expression cassettes are sequentially from the 5' end to the 3' end: the gene mrsK2 encoded by the mrsK2 protein, the gene mrsR2 encoded by the mrsR2 protein, the gene mrsF encoded by the mrsF protein, and the gene encoded by the mrsG protein are encoded by the mrsG and mrsE proteins. The gene mrsE, the gene mrsA encoded by the propeptide mrsA, the gene mrsR1 encoded by the mrsR1 protein mrsR1, the mrsD protein encoded gene mrsD, the mrsM protein encoded gene mrsM, and the mrsT protein encoded gene mrsT.

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

Further, the gene expression cassette includes a promoter, and the 3' end of the promoter is connected to the propeptide-encoding gene mrsA.

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

To achieve the above object, the present invention adopts the following technical solutions:

The recombinant expression vector comprises the above-mentioned 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 adenoviral vector.

Further, as a preference, the plasmid vector is pP43NMK vector.

Further, this recombinant expression vector includes but is not limited to pP43NMK vector, other Bacillus expression vectors with promoter and signal peptide, and a vector containing secreted Mersacidin expression cassette "promoter-mersacidin" can also be constructed.

The third object of the present invention is to provide a genetically engineered bacterium, the genetically engineered bacterium has a higher expression level than wild bacterium after culture, and the fermentation medium cost is low, the fermentation period is short, and the industrialization is easy.

To achieve the above object, the present invention adopts the following technical solutions:

The genetically engineered bacteria comprise the above-mentioned gene expression cassette or the above-mentioned recombinant expression vector.

Further, as a preference, the genetically engineered bacteria is Bacillus subtilis SCK168.

Further, the medium components of Bacillus subtilis SCK168 include: soybean meal powder 10±5g/L, yeast powder 10±5g/L, peptone 10±5g/L, glucose 10±5g/L, sodium chloride 5±2g /L.

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 achieve vector transformation and expression.

The fourth purpose of the present invention is to provide a method for preparing secreted Mersacidin, which has low culture cost and high yield, and provides a new direction and idea for realizing the industrialized production of Mersacidin.

To achieve the above object, the present invention adopts the following technical solutions:

Based on a preparation method of secreted Mersacidin, it specifically includes the following steps:

1) construct the above-mentioned recombinant expression vector;

2) the above-mentioned recombinant expression vector is transferred into the above-mentioned genetically engineered bacteria;

3) Screen the positive recombinants and ferment them in the medium.

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

4) get the fermentation broth to measure its antibacterial activity and content;

5) Measure the bacteriostatic effect of the fermentation broth, and detect the bacteriostatic activity;

6) Filter the fermentation broth and measure its content by 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 benefits of the present invention are:

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

2. The Mersacidin expression cassette vector "promoter-mrsK2R2FGE-mrsAR1D-mrsMT gene" obtained in the present invention realizes the synthesis of Mersacidin and transports it to extracellular secretion and expression by combining the genes encoding the Mersacidin gene cluster.

3. The present invention discloses a method for preparing secreted Mersacidin, which has low culture cost and high yield, and provides a new direction and idea for realizing the industrialized production of Mersacidin.

4. The present invention uses Gibson cloning technology to connect mrsK2R2FGE, mrsAR1D and mrsMT gene fragments to an expression vector with a promoter to construct a Mersacidin expression cassette vector "promoter-mrsK2R2FGE-mrsAR1D-mrsMT gene". Because the mrsR1DMT on the carrier can process, modify and transport the propeptide, the synthesis and transport of Mersacidin to extracellular secretion and expression are realized.

5. The present invention transforms the recombinant expression vector containing the Mersacidin expression cassette into genetically engineered bacteria to construct a strain. The strains were cultured in the relevant medium, and their activity was judged by measuring the inhibition zone. The expression of Mersacidin in Bacillus subtilis was determined by HPLC. The expression level of Mersacidin of the expression system is higher than that of wild bacteria, and the fermentation medium cost is low, the fermentation period is short, and the industrialization is easy.

6. The present invention utilizes genetic engineering and fermentation engineering to ferment and express Mersacidin for the first time. The safe strain Bacillus subtilis SCK168 is used as the expression host, the pP43NMK expression vector with promoter P43 is used to recombine the Mersacidin gene cluster AJ250862.2, and the expression cassette mersacidin is recombined. An expression vector was constructed with pP43NMK. The Mersacidin expressed in the invention has high activity, improves the secretion and expression level of Mersacidin in Bacillus subtilis, and is beneficial to application and popularization.

7. The expression level of secreted Mersacidin produced by the preparation method of the present invention is as high as 1.26 g/L, which is higher than the expression level of Mersacidin in all current reports.

Description of drawings

Fig. 1 is the construction process of recombinant expression vector;

Figure 2 shows the bacteriostatic activity of the fermentation broth against Clostridium perfringens (A: Bacillus subtilis SCK168 fermentation broth supernatant; B: Bacillus subtilis MRS08 fermentation broth supernatant; C: Bacillus subtilis MRS24 fermentation broth supernatant; D: 100ng Vancomycin);

Figure 3 is the high performance liquid phase diagram of pure Mersacidin (A) and fermentation broth (B).

Detailed ways

The technical solutions of the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope 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 digestion reaction system was as follows: 10 μL of 10×FastDigest buffer, 10 μL of vector (100 ng/μL), 5 μL of HindIII and 5 μL of PstI, 70 μL of ddH ₂ O were mixed in a water bath at 37°C for 60 min, and the linearized vector was recovered from the digestion product.

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

The amplification system was 50 μL: 2.5U/μL high-fidelity enzyme, 1 μL; 2× reaction buffer, 25 μL; primers, 1 μL each; template, 1 μL; ddH ₂ O, 21 μL; amplification conditions: denaturation, 98°C, 0.5 min; annealing at 55°C for 0.5min; extension at 68°C, fragment 1 is extended for 1.5 min, fragment 2 is extended for 3 min, and fragment 3 is extended for 2 min.

3) The above three fragments are mixed with the linearized pP43NMK vector and ligated by the method of recombinant cloning kit.

The reaction system is as follows: 5 μL of 2×Gibson mixture, 1 μL of linearized vector (50 ng/μL), 0.9 μL of recovered and purified DNA fragment 1 (50 ng/μL), 1.8 μL of recovered and purified DNA fragment 2 (50 ng/μL), 1.3 The purified DNA fragment 3 (50ng/μL) was recovered by μL, mixed in a water bath at 25°C for 30 minutes, and an ice bath at 0°C for 15 minutes. The reaction product was transferred into E. coli competent DH5α, and positive recombinants (kanamycin resistance) were screened. ), which was constructed into the vector pP43NMK-mersacidin (Fig. 1).

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

This vector includes but is not limited to pP43NMK, other Bacillus expression vectors with promoter and signal peptide, and a vector containing the Mersacidin expression cassette "promoter-mersacidin" can also be constructed.

Example 2. Transformation of recombinant vectors

The vector pP43NMK-mersacidin was transferred into Bacillus subtilis SCK168 by electroporation, and the electrotransfer conditions were as follows: C=50μF; PC=200ohm; V=1.0kV. The positive recombinants were screened and named as MRS08 and MRS24.

The host bacteria include but are not limited to Bacillus subtilis, other genetically engineered bacteria Bacillus licheniformis, Bacillus pumilus, and the transformation and expression of the vector can also be achieved.

Embodiment 3. Detection of the antibacterial activity of fermentation supernatant to Staphylococcus aureus

The MRS08 and MRS24 strains obtained by screening were placed in the culture medium (soybean meal powder 10g/L, yeast powder 10g/L, peptone 10g/L, glucose 10g/L, sodium chloride 5g/L) under the conditions of 37°C and 200rpm/min After culturing for 16-22 hours, the antibacterial activity of the fermentation supernatant against Clostridium perfringens was determined at different time periods.

The method for determining the antibacterial activity is as follows:

1) Bacterial recovery: Clostridium perfringens ATCC13124 was streaked on the plate and cultured at 37°C for 20h; single colonies were picked and placed in the corresponding liquid medium, and cultured at 37°C and 200rpm/min for about 12-16h. Dilute the bacterial solution with physiological saline to an OD ₆₀₀ absorbance value of 0.1 (about 10 ⁸ CFU/mL), for subsequent use;

2) Take 8000g of 10mL fermentation broth sample, centrifuge at 4°C, collect the supernatant and store it at -20°C for later use;

3) In the ultra-clean workbench, draw 100 μL of the indicator bacteria solution and add it to 100 mL of the corresponding agar medium at a constant temperature of 50±5 °C, shake gently to avoid foaming, and the content of bacteria in the medium at this time. 10 ⁶ CFU/mL; pour 15-20 mL into each petri dish, spread evenly, and wait for it to cool and solidify. Punch holes evenly with a sterile hole punch, draw 150-200 μL of fermentation broth samples and inject them into the corresponding wells. Set up Bacillus subtilis SCK168 fermentation broth as a negative control and vancomycin (1 μg/mL) as a positive control. Make a mark on it, and put the petri dish in a refrigerator at 2-8°C for 1-2h to diffuse the sample. Transfer the petri dish to a 37°C incubator.

In the next 6-12h, observe the growth of the bacteria and the bacteriostatic effect. Generally, the bacteriostatic zone can be observed in 8-12h. The diameter of the inhibition zone was comparable to that of vancomycin (Fig. 2D), and the bacteriostatic effect was strong.

Example 4. Stability determination of Mersacidin in fermentation broth

1) Take 5 mL of the supernatant of the fermentation broth respectively, adjust the pH of the supernatant to 2.0, 7.3 and 12.0 with 5 mol/L HCl or 5 mol/L NaOH, and take a water bath at 37°C for 3 hours;

2) Take 5 mL of the fermentation broth supernatant and treat at 100°C for 1 hour; add 0.2 mL of trypsin and proteinase K solutions to 1.5 mL of the fermentation supernatant whose pH is adjusted to 7.3, so that the final concentration of the enzyme is 1 mg/mL, The control group and all enzyme reaction systems were treated in a boiling water bath for 5 minutes to inactivate the enzymes;

3) The bacteriostatic activity of the treated fermentation broth against Clostridium perfringens was detected according to the test procedure of Example 3; it was found that Mersacidin was resistant to acid and alkali, trypsin and proteinase K, and the high temperature tolerance was not durable (Table 2).

The stability of the bacteriostatic activity of table 2 fermentation broth to Clostridium perfringens

O: the centrifugation supernatant diluted 1.25 times with distilled water and centrifuged at 5000rpm/min for 10min;

A: Dilute the centrifugation supernatant 1.25 times with distilled water, and adjust the pH to 7.3;

T: Dilute the centrifugation supernatant 1.25 times with distilled water, adjust the pH to 7.3, and heat at 80°C for 2h;

2: pepsin blank control;

7.3: Blank control of trypsin, proteinase K, catalase;

P: pepsin treatment;

Tr: trypsin treatment;

K: proteinase K treatment;

H: Catalase treatment.

Example 5. Determination of the content of Mersacidin in fermentation broth

The MRS08 and MRS24 strains obtained by screening were placed in the culture medium (soybean meal powder 10g/L, yeast powder 10g/L, peptone 10g/L, glucose 10g/L, sodium chloride 5g/L) under the conditions of 37°C and 200rpm/min Culture, and measure the content of Mersacidin in the fermentation supernatant at different time periods; the method is as follows:

Taking the pure Mersacidin (Nanjing GenScript) synthesized by Bio-Company as the standard (content is 99%), it was detected by high performance liquid chromatography. The chromatographic conditions are as follows:

Reversed-phase column: C18 (4.6×250mm or 150mm);

Eluent: mobile phase A (0.1% trifluoroacetic acid), mobile phase B (90% acetonitrile);

Column temperature: 25°C (or room temperature);

Detection wavelength: 260nm;

Maximum pressure: 200bar;

Carry out gradient elution according to Table 3:

Table 3 Mobile phase gradient elution procedure

Using this detection method, the maximum content of its active ingredients is 1.26g/L.

The pure product was dissolved in water, and the sample was loaded to measure the peak time of the pure product, and then the fermentation broth was loaded.

The external standard method is calculated in proportion to the peak area and concentration (consistent sample loading), and the following formula is used to calculate the content:

where AX is the peak area of Mersacidin in the fermentation broth; CX is the content of Mersacidin in the fermentation broth (μg/g); AR is the peak area of the reference substance; CR is the concentration of the reference substance (μg/mL). The pure and fermentation broth profiles are shown in Figures 3A and 3B, respectively.

sequence listing

<110> Chongqing Academy of Animal Husbandry

<120> Gene expression cassette for encoding secreted Mersacidin and preparation method thereof

<130> 2022.1.14

<141> 2022-02-24

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<170> SIPOSequenceListing 1.0

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agaaccggaa ttcgtttcag aaatcaattc catacttctt cattcagggc tgtctgtatt 3240

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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

cagggggggaa catgtaggag gcggttggaa acagcttcta gccttaccag tcaaacactc 4380

agatatcttt ctgggaaagt ttctaacagt ggtccgcatg atattcatta gtatgtccat 4440

tttaatcctc ttatttattg gattcggtta tatgtttaggc 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 ttttttttgat 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 aaccttttttc 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 attattattct 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 agatttttttt 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 ttatttgtt 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 tagcagttttt 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

<400> 2

taacacatgc ctcagctgcc taattgacat tgttatactt agatggcg 48

<210> 3

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 3

taagtttata ccgcatatac cctacgtaag gtctcgttta 40

<210> 4

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 4

ggtatatgcg gtataaactt atgagaattc gagacaaggt 40

<210> 5

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 5

atttacagag ggagaaaaaa atgagtattt caatattaaa 40

<210> 6

<211> 40

<212> DNA

<213> Artificial Sequence

<400> 6

ttttttctcc ctctgtaaat tttaaacaat catactacac 40

<210> 7

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 7

tctaaccgca ttagtaccag ttcaagtttt gccagttttgc tca 43

Claims (10)

1. A gene expression cassette for encoding secreted Mersacidin, characterized in that it employs clonal amplification techniques to obtain three cloned fragments: gene mrsK2R2FGE, gene mrsAR1D, and gene mrsMT; the gene mrsK2R2FGE is fragment 1 and consists of a gene mrsK2 coded by an mrsK2 protein, a gene mrsR2 coded by an mrsR2 protein, a gene mrsF coded by an mrsF protein, a gene mrsG coded by an mrsG protein and a gene mrsE coded by an mrsE protein; the gene mrsAR1D is a segment 2 and consists of a gene mrsSA coded by a propeptide mrsA, a gene mrsR1 coded by an mrsR1 protein and a gene mrsD coded by an mrsD protein; the gene mrsMT is a fragment 3 and consists of a gene mrsM coded by an mrsM protein and a gene mrsT coded by an mrsT protein.

2. The gene expression cassette of claim 1, wherein the three cloned fragments are cloned fragment 1 with primer G1 and primer G2, cloned fragment 2 with primer G3 and primer G4, and cloned fragment 3 with primer G5 and primer G6, respectively; 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.

3. The gene expression cassette of claim 1, wherein the nucleotide sequence of the gene expression cassette is shown in SEQ ID No. 1.

4. A recombinant expression vector comprising the gene expression cassette according to any one of claims 1 to 3.

5. The recombinant expression vector of claim 4, wherein the recombinant expression vector is a plasmid vector or a viral vector.

6. A genetically engineered bacterium comprising the gene expression cassette according to any one of claims 1 to 3 or the recombinant expression vector according to claim 4 to 5.

7. The genetically engineered bacterium of claim 6, wherein the genetically engineered bacterium is Bacillus subtilis SCK 168.

8. The genetically engineered bacterium of claim 7, wherein the culture medium of the Bacillus subtilis SCK168 comprises: 10 plus or minus 5g/L of soybean meal, 10 plus or minus 5g/L of yeast powder, 10 plus or minus 5g/L of peptone, 10 plus or minus 5g/L of glucose and 5 plus or minus 2g/L of sodium chloride.

9. The preparation method of the secretory Mersacidin is characterized by comprising the following steps:

1) constructing the recombinant expression vector of claim 4;

2) transferring the recombinant expression vector of claim 4 into the genetically engineered bacterium of claim 6;

3) screening positive recombinants to carry out fermentation culture in a culture medium.

10. The secreted Mersacidin obtained by the preparation process according to claim 9.

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