Antimicrobial peptide fusion protein and its preparation method and application

Abstract

The present invention discloses an antimicrobial peptide fusion protein and a preparation method and application thereof, and relates to the field of biopharmaceutical engineering technology. The amino acid sequence of the antimicrobial peptide fusion protein is shown in SEQ ID NO.7. The present invention fuses the two ends of human zinc-copper superoxide dismutase with the antimicrobial peptide CecropinB through a flexible peptide chain, and designs an antimicrobial peptide fusion protein Cb-SOD-Cb. The antimicrobial peptide fusion protein has both antioxidant activity and antibacterial activity, and can be used in the preparation of antimicrobial drugs and beauty skin care products. The present invention uses inclusion bodies to express the antimicrobial peptide fusion protein Cb-SOD-Cb in host cells, thereby avoiding the damage caused to the host by the recombinant protein produced by soluble expression, and uses inclusion body washing, impurity removal and renaturation technology to prepare a recombinant protein with both antimicrobial activity and antioxidant properties.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical engineering, and in particular to an antimicrobial peptide fusion protein and a preparation method and application thereof. Background Art

[0002] In the metabolic process of aerobic organisms, incomplete reduction of molecular oxygen leads to the continuous generation of reactive oxygen species (ROS), including hydrogen peroxide (H2O2), hydroxyl radicals (HO - ) and superoxide anion radicals (O2 - ) etc. Excessive accumulation of ROS can lead to oxidative stress, damage DNA, inactivate proteins, and cause peroxidation of cell membrane lipids. In severe cases, it can lead to cell dysfunction and even cancer and apoptosis. Superoxide dismutase (SOD, EC 1.15.1.1) is a widely existing natural antioxidant. As the most critical component of the antioxidant defense system, it is the first line of defense to eliminate ROS. SOD can promote the dismutation reaction of superoxide anion free radicals, converting O2 - Degrades to hydrogen peroxide (H2O2) and oxygen (O2), ascorbic acid and catalase then decompose H2O2 into H2O and O2, thus removing O2 - , maintaining the dynamic balance of ROS production and elimination in the body, and plays an important role in maintaining the balance of cellular oxidation and reduction. According to the different auxiliary factors, superoxide dismutase is mainly divided into copper-zinc superoxide dismutase (Cu / Zn-SOD), iron superoxide dismutase (Fe-SOD), manganese superoxide dismutase (Mn-SOD) and nickel superoxide dismutase (Ni-SOD), but mammals only contain Cu / Zn-SOD and Mn-SOD. At present, copper-zinc superoxide dismutase is widely used in the field of medical and skin care products because of its high specific activity (>10000IU / mg). However, protein products currently require additional preservatives to ensure their stability during storage.

[0003] Antimicrobial peptides (AMPs) are a class of polypeptides with antimicrobial activity, usually composed of 20 to 60 amino acid residues. They are usually alkaline because they are rich in arginine. They have the characteristics of broad-spectrum antibacterial, high antibacterial efficiency, and high thermal stability. They are widely present in microorganisms such as animals, plants, and bacteria. Antimicrobial peptides selectively kill bacteria by binding to the cell membrane of bacteria to form pores that make them permeable. Therefore, they do not involve any specific target protein, are not prone to bacterial resistance, and are also effective against strains that have already developed resistance. They are an ideal substitute for antibiotics. However, antimicrobial peptides themselves have problems such as high production costs due to the difficulty in expression or synthesis, and they have multiple positive charges that make them difficult to use in combination. Summary of the invention

[0004] The purpose of the present invention is to provide an antimicrobial peptide fusion protein and a preparation method and application thereof to solve the problems existing in the above-mentioned prior art. The fusion protein has both antioxidant activity and antimicrobial activity.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides an antimicrobial peptide fusion protein. The antimicrobial peptide fusion protein consists of a SOD protein and two antimicrobial peptides CecropinB. Both ends of the SOD protein are connected with the antimicrobial peptide CecropinB through a flexible peptide segment.

[0007] Furthermore, the amino acid sequence of the antimicrobial peptide fusion protein is shown in SEQ ID NO.7.

[0008] The present invention also provides a gene sequence encoding the antimicrobial peptide fusion protein, and the gene sequence is shown as SEQ ID NO.4.

[0009] The present invention also provides a recombinant expression vector comprising the above gene sequence.

[0010] The present invention also provides a recombinant strain comprising the above gene sequence or recombinant expression vector.

[0011] The present invention also provides a method for preparing the above-mentioned antimicrobial peptide fusion protein, comprising the following steps:

[0012] (1) expressing the gene sequence shown in SEQ ID NO.4 in a host cell in the form of inclusion bodies to obtain inclusion body protein;

[0013] (2) The inclusion body protein is washed, dissolved, and renatured to obtain the antimicrobial peptide fusion protein.

[0014] Furthermore, in step (2), the specific operation of washing includes: washing the inclusion body protein with washing solution I, washing solution II and washing solution III in sequence; the washing solution I is: 50mM Tris-HCl, 10mM EDTA, 5% glycerol, 1% Triton X-100, 100mM β-mercaptoethanol, pH 8.0; the washing solution II is: the washing solution I + 0.3M Urea; the washing solution III is: the washing solution I + 0.6M Urea.

[0015] Furthermore, in step (2), the specific operation of dissolving includes: adding pure water to the washed inclusion body protein and mixing it evenly, and then adding urea solution to perform denaturation treatment.

[0016] The present invention also provides the use of the antimicrobial peptide fusion protein in the preparation of antimicrobial and antioxidant products.

[0017] Furthermore, the product is an antibacterial drug or a beauty skin care product.

[0018] The present invention discloses the following technical effects:

[0019] The present invention fuses the two ends of human zinc-copper superoxide dismutase with the antimicrobial peptide Cecropin B by designing a reasonable flexible peptide chain, thereby designing an antimicrobial peptide fusion protein Cb-SOD-Cb. The antimicrobial peptide fusion protein has both antioxidant and antimicrobial activity, and can be used in the preparation of antimicrobial drugs and cosmetic skin care products.

[0020] The present invention efficiently expresses the antimicrobial peptide fusion protein Cb-SOD-Cb in host cells, firstly adopts inclusion body expression to avoid the damage of the recombinant protein produced by soluble expression to the host, and adopts inclusion body washing, impurity removal and renaturation technology to prepare the recombinant protein with both antimicrobial activity and antioxidant activity. The method of the present invention can simply, quickly and efficiently prepare multifunctional fusion proteins, providing a new path for the preservation of enzymes and the application expansion of the non-preservative system. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 It is a schematic diagram of the structure of Cb-SOD-Cb fusion protein;

[0023] Figure 2This is the predicted structure of Cb-SOD-Cb fusion protein; green: SOD, blue: linker, red: CecropinB;

[0024] Figure 3 is a schematic diagram of the recombinant vector pET26-Cb-SOD-Cb;

[0025] Figure 4 The SDS-PAGE image of Cb-SOD-Cb protein and recombinant bacteria crushed precipitate; wherein M: standard molecular weight marker, 1: recombinant Cb-SOD-Cb protein after renaturation (0.5 mg / mL), 2: recombinant Cb-SOD-Cb protein after renaturation (0.5 mg / mL), 3: precipitate obtained by centrifugation after recombinant bacteria crushed (containing Cb-SOD-Cb inclusion bodies);

[0026] Figure 5 This is the antibacterial graph of Cb-SOD-Cb protein; A is Coccus aureus, B is Escherichia coli; in A and B, 0: PBS control, 1: kanamycin 0.5 mg / mL, 2: Cb-SOD-Cb protein 1 mg / mL, 3: Cb-SOD-Cb protein 0.5 mg / mL. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] The preparation of recombinant Cb-SOD-Cb antioxidant and antibacterial protein comprises the following specific steps:

[0034] (1) Construction of recombinant Cb-SOD-Cb antioxidant and antibacterial protein expression vector: The 3' end of the nucleotide sequence of CecropinB was linked to the DQYTD flexible peptide nucleotide sequence, which was then linked to the nucleotide sequence encoding human zinc-copper superoxide dismutase SOD protein, which was then linked to the DQYTD flexible peptide nucleotide sequence, and then to the nucleotide sequence of CecropinB, forming a CecropinB-DQYTD-SOD-DQYD-CecropinB cascade structure, referred to as Cb-SOD-Cb. The recombinant gene structure is shown in Figure 1 .

[0035] The spatial structure obtained by 3D structural simulation (https: / / zhanggroup.org / I-TASSER / ) of the recombinant protein Figure 2 ) showed that superoxide dismutase SOD and cecropin B at both ends were connected by flexible peptides to form a free and open state, which would not affect their respective functional areas. The recombinant protein neither destroyed the spatial structure of SOD nor could it attack the bacterial outer membrane to achieve the antibacterial purpose.

[0036] The Cb-SOD-Cb gene sequence was connected to the E. coli expression vector pET26b(+) through NdeⅠ and XhoI to obtain the recombinant vector pET26-Cb-SOD-Cb. The vector structure is shown in Figure 3 The synthetic gene was completed by Shanghai Jierui Bioengineering Co., Ltd.

[0037] The nucleotide sequence of CecropinB (SEQ ID NO.1):

[0038] AAGTGGAAAGTATTTAAAAAGATAGAAAAAATGGGTCGTAACATTCGTAACGGCATTGTAAAGGCGGGTCCGGCGATCGCCGTTCTGGGCGAAGCTAAAGCTCTG。

[0039] Nucleotide sequence encoding human zinc-copper superoxide dismutase SOD protein (SEQ ID NO.2):

[0040] GCAACCAAGGCGGTGTGCGTCTTGAAGGGTGACGGTCCGGTGCAGGGTATTATCAACTTCGAGCAGAAAGAGAGCAATGGCCCGGTGAAAGTTTGGGGTTCTATCAAAGGCTTGACCGAAGGTCTGCATGGTTTTCACGTGCACGAGTTCGGCGACAATACCGCGGGCTGCACCAGCGCAGGTCCGCATTTTAACCCGCTTTCGCGCAAACACGGCGGCCCAAAAGATGAGGAGCGCCACGTTGGTGACCTCGGCAACGTGACTGCGGATAAGGACGGCGTTGCCGACGTGTCCATTGAAGATAGCGTTATCAGCCTGTCCGGTGATCATTGCATCATTGGCCGTACGCTGGTTGTCCACGAGAAGGCGGACGATCTGGGAAAGGGCGGTAATGAAGAAAGCACCAAGACGGGTAACGCGGGTTCTCGTCTGGCTTGTGGTGTTATCGGCATCGCACAA。

[0041] Nucleotide sequence of the DQYTD flexible peptide segment (SEQ ID NO.3): GATCAATATACTGAC。

[0042] Cb-SOD-Cb gene sequence (SEQ ID NO.4):

[0043] ATGAAGTGGAAAGTATTTAAAAAGATAGAAAAAATGGGTCGTAACATTCGTAACGGCATTGTAAAGGCGGGTCCGGCGATCGCCGTTCTGGGCGAAGCTAAAGCTCTGGACCAATACACCGATGCAACCAAGGCGGTGTGCGTCTTGAAGGGTGACGGTCCGGTGCAGGGTATTATCAACTTCGAGCAGAAAGAGAGCAATGGCCCGGTGAAAGTTTGGGGTTCTATCAAAGGCTTGACCGAAGGTCTGCATGGTTTTCACGTGCACGAGTTCGGCGACAATACCGCGGGCTGCACCAGCGCAGGTCCGCATTTTAACCCGCTTTCGCGCAAACACGGCGGCCCAAAAGATGAGGAGCGCCACGTTGGTGACCTCGGCAACGTGACTGCGGATAAGGACGGCGTTGCCGACGTGTCCATTGAAGATAGCGTTATCAGCCTGTCCGGTGATCATTGCATCATTGGCCGTACGCTGGTTGTCCACGAGAAGGCGGACGATCTGGGAAAGGGCGGTAATGAAGAAAGCACCAAGACGGGTAACGCGGGTTCTCGTCTGGCTTGTGGTGTTATCGGCATCGCACAAGATCAATATACTGACAAGTGGAAAGTCTTTAAAAAAATCGAGAAAATGGGTCGCAACATCCGTAATGGTATTGTTAAGGCGGGCCCAGCGATCGCGGTGCTCGGCGAGGCGAAGGCGCTGTAA。

[0044] Amino acid sequence of Cecropin B (SEQ ID NO.5):

[0045] KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL。

[0046] Amino acid sequence of human zinc-copper superoxide dismutase SOD protein (SEQ ID NO.6):

[0047] ATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ.

[0048] Cb-SOD-Cb amino acid sequence (SEQ ID NO.7):

[0049] MKWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKALDQYTDATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKD EERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQDQYTDKWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL.

[0050] (2) Screening and expression of recombinant protein Cb-SOD-Cb expression strain BL21(DE3):

[0051] The recombinant vector pET26-Cb-SOD-Cb obtained in step (1) was transformed into the Escherichia coli strain BL21 (DE3) by heat shock method, and the positive transformants screened by LB solid medium plates containing 50 μg / mL kanamycin were cultured in LB medium until OD 600 =0.6-0.8, IPTG was used to induce expression. After 3-6 hours of induction, the bacteria were collected and the protein expression was detected by SDS-PAGE.

[0052] The specific operation is as follows: the inducible positive transformant selected from step (1), i.e., the pET26-Cb-SOD-Cb / BL21(DE3) strain, is streaked on an LB plate for 1 day, and a single colony is inoculated into a 5 ml centrifuge tube containing 1 ml LB medium containing 50 μg / mL kanamycin, and cultured at 37°C 200 rpm overnight. The next day, the inoculation amount is 1% and inoculated into a new 500 ml LB medium containing 50 μg / mL kanamycin, and cultured at 37°C 200 rpm until the OD reaches 0. 600=0.8, add isopropylthiogalactoside (IPTG) to a final concentration of 1 mmol, 37°C 200rpm for induction expression of recombinant protein, after 5 hours of induction, centrifuge at 4000rpm for 10min to collect the bacteria. Wherein, LB medium is made by adding water to 1000mL of yeast powder 5g, peptone 10g and sodium chloride 5g, and adjusting the pH value to 7.2-7.4.

[0053] (3) Preparation of recombinant Cb-SOD-Cb antioxidant and antibacterial protein

[0054] The E. coli strain with high expression of Cb-SOD-Cb antioxidant and antibacterial protein screened in step (2) was induced to express, and then centrifuged at 6000rpm / min for 10min, the bacteria were collected, placed under -20°C, and repeatedly frozen and thawed three times, lysozyme was added to make the final concentration of lysozyme 100μg / mL, incubated at 37°C for 20min, and ultrasonically disrupted, and the ultrasonic disruption conditions were: 300w, 4s, 6s, 20min. Bacterial disruption buffer: 20nM PBS (containing 150nM NaCl), pH=7.4. After the disruption, centrifuged at 20000rpm, 4°C for 20min, and the precipitate (mostly inclusion body protein) was retained.

[0055] Inclusion body washing: Use washing solution I (50mM Tris-HCl, 10mM EDTA, 5wt% glycerol, 1wt% Triton X-100, 100mM β-mercaptoethanol, pH 8.0), washing solution II (washing solution I + 0.3M urea), and washing solution III (washing solution I + 0.6M urea) to wash the inclusion body precipitate in sequence.

[0056] Inclusion body dissolution (denaturation): To dissolve the inclusion body precipitate, add pure water at a mass ratio of 2:1 and mix evenly with a small homogenizer. Add 8M urea solution at a mass ratio of 1:25 and stir on a magnetic stirrer for 4 hours to obtain a relatively clear solution. Centrifuge at 20,000 rpm and 4°C for 20 minutes. The clear supernatant is the denatured solution of Cb-SOD-Cb antioxidant and antibacterial protein.

[0057] Inclusion body renaturation: The denaturing solution of Cb-SOD-Cb antioxidant and antibacterial protein and buffer (50mM Tirs-HCl, 150mM sodium chloride, 1M urea, pH=8.4) were mixed in a volume ratio of 1:9, stirred evenly, and refrigerated in a 4°C refrigerator overnight. After centrifugation to remove a small amount of precipitate, a 10kDa ultrafiltration membrane was used for desalting and concentration to obtain the recombinant Cb-SOD-Cb antioxidant and antibacterial protein.

[0058] The recombinant protein was detected by SDS-PAGE. Figure 4After the recombinant bacteria were broken, the precipitate was electrophoresed to show an obvious band at 25 kDa (lane 3), indicating that the target protein was successfully expressed in inclusion bodies. After the precipitate was washed and the inclusion bodies were renatured, electrophoresis was performed at different concentrations, and an obvious band at 25 kDa was shown (lanes 1, 2), which was the recombinant Cb-SOD-Cb).

[0059] Example 2

[0060] The antioxidant activity of the recombinant Cb-SOD-Cb antioxidant and antibacterial protein was tested by measuring the self-oxidation rate of pyrogallol.

[0061] As shown in Table 1, keep the control tube reagent in a 25°C water bath for 20 minutes, quickly mix and shake according to the volume, and immediately pour into a colorimetric cup with an optical path of 1 cm. Measure the light absorption value every 60 seconds at a wavelength of 325 nm, and calculate the A per minute. 325 The increased value is the self-oxidation rate A0 of pyrogallol.

[0062] The recombinant Cb-SOD-Cb antioxidant and antibacterial protein obtained by renaturation was diluted to a concentration of 10 μmol / L to be the test sample. The sample tube reagent was kept in a 25°C water bath for 20 minutes, quickly mixed and shaken according to the volume, and immediately poured into a colorimetric cup with a light path of 1 cm. The light absorption value was measured every 60 seconds at a wavelength of 325 nm, and the A per minute was calculated. 325 The increase in value is the oxidation rate A of pyrogallol under inhibition conditions. m .

[0063] Table 1 Reagents used in the pyrogallol auto-oxidation method

[0064]

[0065] According to the definition of enzyme activity unit, enzyme activity is calculated according to the following formula:

[0066]

[0067] A0: pyrogallol self-oxidation rate; A m : The self-oxidation rate of pyrogallol after adding fusion protein; Total activity (U) = unit activity × V total protein stock solution.

[0068] The recombinant Cb-SOD-Cb antioxidant and antibacterial protein obtained by renaturation was diluted to a protein concentration of 10 μmol / L, and its activity was tested by pyrogallol self-oxidation. The recombinant Cb-SOD-Cb was 1587±30U / mL, and the enzyme activity of SOD at the same concentration was 1611±30U / mL, indicating that the antioxidant capacity of recombinant Cb-SOD-Cb is equivalent to that of SOD.

[0069] Inhibitory effect of recombinant Cb-SOD-Cb antioxidant and antibacterial protein on Staphylococcus aureus (G+) and Escherichia coli (G-). Before each test, the test bacteria were cultured in (LB) liquid nutrient medium at 37°C and 200 rpm for 12 h to obtain a preliminary bacterial suspension. Before the experiment, the bacterial suspension was re-inoculated into fresh medium and cultured until OD 600 The value is 0.4, where the bacterial concentration is 4×10 7 CFU / mL. The obtained bacterial solution was further diluted with sterile water by 10 5 Spread on a non-resistant LB solid plate, add a sterile filter paper, drop 5 μL of different concentrations of recombinant Cb-SOD-Cb antioxidant and antibacterial protein, and use kanamycin as a positive control. The results are shown in Figure 5 The results showed that the recombinant Cb-SOD-Cb antioxidant and antibacterial protein had a significant antibacterial effect on Staphylococcus aureus (G+) and Escherichia coli (G-).

[0070] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention. Sequence Listing <110> Hangzhou Shuibasha Biopharmaceutical Technology Co., Ltd.; Hangzhou Huawei Biotechnology Co., Ltd. <120> Antimicrobial peptide fusion protein and its preparation method and application <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 105 <212> DNA <213> Artificial Sequence <400> 1 aagtggaaag tatttaaaaa gatagaaaaa atgggtcgta acattcgtaa cggcattgta 60 aaggcgggtc cggcgatcgc cgttctgggc gaagctaaag ctctg 105 <210> 2 <211> 459 <212> DNA <213> Artificial Sequence <400> 2 gcaaccaagg cggtgtgcgt cttgaagggt gacggtccgg tgcagggtat tatcaacttc 60 gagcagaaag agagcaatgg cccggtgaaa gtttggggtt ctatcaaagg cttgaccgaa 120 ggtctgcatg gttttcacgt gcacgagttc ggcgacaata ccgcgggctg caccagcgca 180 ggtccgcatt ttaacccgct ttcgcgcaaa cacggcggcc caaaagatga ggagcgccac 240 gttggtgacc tcggcaacgt gactgcggat aaggacggcg ttgccgacgt gtccattgaa 300 gatagcgtta tcagcctgtc cggtgatcat tgcatcattg gccgtacgct ggttgtccac 360 gagaaggcgg acgatctggg aaagggcggt aatgaagaaa gcaccaagac gggtaacgcg 420 ggttctcgtc tggcttgtgg tgttatcggc atcgcacaa 459 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <400> 3 gatcaatata ctgac 15 <210> 4 <211> 705 <212> DNA <213> Artificial Sequence <400> 4 atgaagtgga aagtatttaa aaagatagaa aaaatgggtc gtaacattcg taacggcatt 60 gtaaaggcgg gtccggcgat cgccgttctg ggcgaagcta aagctctgga ccaatacacc 120 gatgcaacca aggcggtgtg cgtcttgaag ggtgacggtc cggtgcaggg tattatcaac 180 ttcgagcaga aagagagcaa tggcccggtg aaagttttggg gttctatcaa aggcttgacc 240 gaaggtctgc atggttttca cgtgcacgag ttcggcgaca ataccgcggg ctgcaccagc 300 gcaggtccgc atttaaccc gctttcgcgc aaacacggcg gcccaaaaga tgaggagcgc 360 cacgttggtg acctcggcaa cgtgactgcg gataaggacg gcgttgccga cgtgtccatt 420 gaagatagcg tttcagcct gtccggtgat cattgcatca ttggccgtac gctggttgtc 480 cacgagaagg cggacgatct gggaaagggc ggtaatgaag aaagcaccaa gacgggtaac 540 gcgggtctc gtctggcttg tggtgttac ggcatcgcac aagatcaata tactgacaag 600 tggaaagtct ttaaaaaaat cgagaaaatg ggtcgcaaca tccgtaatgg tattgttaag 660 gcgggcccag cgatcgcggt gctcggcgag gcgaaggcgc tgtaa 705 <210> 5 <211> 35 <212> PRT <213> Artificial Sequence <400> 5 Lys Trp Lys Val Phe Lys Lys Ile Glu Lys Met Gly Arg Asn Ile Arg 1 5 10 15 Asn Gly Ile Val Lys Ala Gly Pro Ala Ile Ala Val Leu Gly Glu Ala 20 25 30 Lys Ala Leu 35 <210> 6 <211> 153 <212> PRT <213> Artificial Sequence <400> 6 Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asp Gly Pro Val Gln Gly 1 5 10 15 Ile Ile Asn Phe Glu Gln Lys Glu Ser Asn Gly Pro Val Lys Val Trp 20 25 30 Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His 35 40 45 Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe 50 55 60 Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His 65 70 75 80 Val Gly Asp Leu Gly Asn Val Thr Ala Asp Lys Asp Gly Val Ala Asp 85 90 95 Val Ser Ile Glu Asp Ser Val Ile Ser Leu Ser Gly Asp His Cys Ile 100 105 110 Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys 115 120 125 Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu 130 135 140 Ala Cys Gly Val Ile Gly Ile Ala Gln 145 150 <210> 7 <211> 234 <212> PRT <213> Artificial Sequence <400> 7 Met Lys Trp Lys Val Phe Lys Lys Ile Glu Lys Met Gly Arg Asn Ile 1 5 10 15 Arg Asn Gly Ile Val Lys Ala Gly Pro Ala Ile Ala Val Leu Gly Glu 20 25 30 Ala Lys Ala Leu Asp Gln Tyr Thr Asp Ala Thr Lys Ala Val Cys Val 35 40 45 Leu Lys Gly Asp Gly Pro Val Gln Gly Ile Ile Asn Phe Glu Gln Lys 50 55 60 Glu Ser Asn Gly Pro Val Lys Val Trp Gly Ser Ile Lys Gly Leu Thr 65 70 75 80 Glu Gly Leu His Gly Phe His Val His Glu Phe Gly Asp Asn Thr Ala 85 90 95 Gly Cys Thr Ser Ala Gly Pro His Phe Asn Pro Leu Ser Arg Lys His 100 105 110 Gly Gly Pro Lys Asp Glu Glu Arg His Val Gly Asp Leu Gly Asn Val 115 120 125 Thr Ala Asp Lys Asp Gly Val Ala Asp Val Ser Ile Glu Asp Ser Val 130 135 140 Ile Ser Leu Ser Gly Asp His Cys Ile Ile Gly Arg Thr Leu Val Val 145 150 155 160 His Glu Lys Ala Asp Asp Leu Gly Lys Gly Gly Asn Glu Glu Ser Thr 165 170 175 Lys Thr Gly Asn Ala Gly Ser Arg Leu Ala Cys Gly Val Ile Gly Ile 180 185 190 Ala Gln Asp Gln Tyr Thr Asp Lys Trp Lys Val Phe Lys Lys Ile Glu 195 200 205 Lys Met Gly Arg Asn Ile Arg Asn Gly Ile Val Lys Ala Gly Pro Ala 210 215 220 Ile Ala Val Leu Gly Glu Ala Lys Ala Leu 225 230

Claims

1. An antimicrobial peptide fusion protein, characterized in that: The antimicrobial peptide fusion protein is composed of a SOD protein and two antimicrobial peptides CecropinB, and the two ends of the SOD protein are connected to the antimicrobial peptide CecropinB through a flexible peptide segment; The amino acid sequence of the antimicrobial peptide fusion protein is shown in SEQ ID NO.

7.

2. A gene encoding the antimicrobial peptide fusion protein according to claim 1, characterized in that: The sequence of the gene is shown in SEQ ID NO.

4.

3. A recombinant expression vector, characterized in that: Comprising the gene according to claim 2.

4. A recombinant strain, characterized in that: Comprising the gene according to claim 2 or the recombinant expression vector according to claim 3.

5. A method for preparing the antimicrobial peptide fusion protein according to claim 1, characterized in that: The following steps are involved: (1) expressing the gene shown in SEQ ID NO.4 in a host cell in the form of inclusion bodies to obtain inclusion body protein; (2) The inclusion body protein is washed, dissolved, and renatured to obtain the antimicrobial peptide fusion protein.

6. The preparation method according to claim 5, characterized in that: In step (2), the specific operation of washing includes: washing the inclusion body protein with washing solution I, washing solution II and washing solution III in sequence; the washing solution I is: 50 mM Tris-HCl, 10 mM EDTA, 5% glycerol, 1% Triton X-100, 100 mM β-mercaptoethanol, pH 8.0; the washing solution II is: the washing solution I + 0.3 M Urea; the washing solution III is: the washing solution I + 0.6 M Urea.

7. The preparation method according to claim 5, characterized in that: In step (2), the specific operation of dissolving includes: adding pure water to the washed inclusion body protein and mixing it evenly, and then adding urea solution to denature it.

8. Use of the antimicrobial peptide fusion protein according to claim 1 in the preparation of antimicrobial and antioxidant products, characterized in that: The product is an antibacterial drug or a beauty skin care product; The bacteria targeted by the antibacterial agent are Staphylococcus aureus and Escherichia coli.

Images