CN107459569B

CN107459569B - Cecropin B antibacterial peptide mutant

Info

Publication number: CN107459569B
Application number: CN201710742441.2A
Authority: CN
Inventors: 王嘉榕; 孙红宾; 赵宏鑫
Original assignee: Hefei Institutes of Physical Science of CAS
Current assignee: Hefei Institutes of Physical Science of CAS
Priority date: 2017-08-25
Filing date: 2017-08-25
Publication date: 2020-09-29
Anticipated expiration: 2037-08-25
Also published as: CN107459569A

Abstract

The invention discloses a cecropin B antibacterial peptide mutant and a preparation method and application thereof. The antibacterial peptide has an inhibiting effect on escherichia coli, pseudomonas aeruginosa, bacillus subtilis and the like, can inhibit the proliferation of lung cancer cells, and can be used for preparing antibacterial and lung cancer medicaments. The antibacterial peptide can also effectively inhibit the increase of the level of the body proinflammatory cytokines caused by lipopolysaccharide, and can be used for developing anti-inflammatory drugs.

Description

Cecropin B antibacterial peptide mutant

Technical Field

The invention relates to an antibacterial peptide, in particular to a mutant containing 32 amino acids and derived from a Chinese oak silkworm moth antibacterial peptide cecropin B. The invention also relates to a genetic engineering preparation method of the antibacterial peptide and application of the antibacterial peptide in preparation of antibacterial drugs, anticancer drugs and anti-inflammatory drugs, belonging to the technical field of biochemistry.

Background

In recent years, the drug resistance problem caused by abuse of antibiotics is becoming serious, and the problem poses a great threat to human diseases, and as an increasingly prominent international problem, the current and future of people are influenced. In order to cope with drug-resistant bacterial infection, people on the one hand structurally modify traditional antibiotics to reduce the drug resistance of bacteria, and on the other hand, continuously research and develop novel antibacterial drugs. The antimicrobial peptide (ABP) appeared in recent years is a novel antimicrobial drug with great development potential. The antibacterial peptide is an amphipathic micromolecular polypeptide coded by genes, generally consists of 12-100 amino acid residues, and is an ancient and important component in an organism innate immune system. The electropositivity and amphiphilicity of the antimicrobial peptide can mediate the interaction with electronegative substances on the surface of bacterial cell membranes, and generate transmembrane pores or destroy lipid systems of the bacterial cell membranes, thereby causing cell damage or death. In addition, the antibacterial peptide can penetrate bacterial cell membranes to inhibit the synthesis of certain proteins and nucleic acids, so that cells can die.

For gram-negative bacteria, lipopolysaccharides are a major component of their cell wall, providing structural integrity to the bacteria and protecting the bacterial membrane from attack by certain chemicals. Lipopolysaccharide, called endotoxin, is the major pathogenic component of gram-negative bacteria and can cause a strong immune response, resulting in morphological, metabolic and gene expression changes in almost all eukaryotic cells, stimulating uncontrolled expression of host cytokines, and severe infections (Schulte et al, 2013, mediatos inflam.2013: 165974). It has been reported that various antibacterial peptides can neutralize the cytotoxicity of lipopolysaccharide (Ding et al, 2003, biochemistry.42(42):12251-12559), inhibit activation of mononuclear macrophages, reduce the production of proinflammatory cytokines, and act as monocyte and T lymphocyte chemokines to rapidly gather at inflammatory reaction sites, thereby eliminating inflammation as soon as possible, and the remarkable characteristic of the antibacterial peptides makes the antibacterial peptides a new star of a new generation of anti-infective agents.

Disclosure of Invention

The invention provides an antibacterial peptide, which is derived from an antibacterial peptide cecropin B secreted by a Chinese oak silkworm moth named Antheraea pernyi, wherein the cecropin B contains 35 amino acids. The inventors removed AGP of 3 amino acids in the hinge region to obtain the antimicrobial peptide (KL32) of the present invention, whose amino acid sequence is shown below:

KWKIFKKIEKVGRNIRNGIIKAVAVLGEAKAL(SEQ ID NO.1)。

the invention also provides a nucleic acid which encodes the cecropin B antibacterial peptide mutant.

The invention also provides a vector comprising a nucleic acid of the invention. In a preferred embodiment, the vector is an expression vector, more preferably a prokaryotic expression vector.

The invention also provides a host cell comprising a vector of the invention or a nucleic acid of the invention. In a preferred embodiment, the host cell is E.coli.

The invention also provides a method for preparing KL32, which comprises the steps of operably connecting the coding gene of KL32 with the expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into a host cell; culturing the recombinant host cell, inducing the recombinant protein to express, collecting and purifying the expressed target protein.

In a preferred embodiment, the method comprises sequentially joining together a Sumo tag gene (6 histidine tags at the N-terminus of the Sumo tag gene) and a nucleic acid sequence encoding said KL 32. Operably linking the gene with an expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into a host expression cell, culturing the recombinant host cell, inducing recombinant protein expression, collecting and purifying the expressed protein, cutting a Sumo label by using Sumo enzyme, and performing reverse high performance liquid chromatography to purify KL 32.

In a preferred embodiment, for the mutants of the invention, the conditions for inducing expression are: culturing the cells to OD at 37 ℃₆₀₀When the concentration reached 0.6, IPTG was added to a final concentration of 0.5mM, and expression was induced at 37 ℃ for 4 hours.

In one embodiment, the purification method of the present invention comprises subjecting the collected bacterial cells to a lysis buffer (50mM NaH)₂PO₄300mM NaCl, pH 8.0), carrying out cell ultrasonication, centrifuging, collecting supernatant, mixing the supernatant with Ni purification filler, incubating for 2h at 4 ℃, transferring the mixture to a chromatographic column, carrying out protein purification, and carrying out enzyme digestion on purified Sumo-KL32 by using Sumo enzyme, wherein the enzyme digestion conditions comprise: the temperature was 4 ℃ and the buffer 50mM Tris, 100mM NaCl, pH 8.0, cut for 6h to give KL32 using reverse phase high performance liquid chromatography.

In another aspect, the invention provides the use of the antimicrobial peptide KL32 in the preparation of a medicament for the treatment of bacterial diseases, lung cancer and inflammatory conditions of the body caused by lipopolysaccharide.

More specifically, the present invention provides the following:

1. an cecropin B antibacterial peptide mutant derived from Oak Bombycis Mori has amino acid sequence shown in SEQ ID NO. 1.

2. A nucleic acid encoding the mutant antimicrobial peptide of 1.

3. A vector comprising the nucleic acid of 2.

4. A host cell comprising the nucleic acid of 1 or the vector of 3.

5.1 the use of the mutant antibacterial peptide, the nucleic acid of 2, the vector of 3 or the host cell of 4 in the preparation of a medicament for preventing or treating bacterial infection diseases.

6.5, wherein the bacteria comprise Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis.

7.1 the use of the mutant antimicrobial peptide, the nucleic acid of 2, the vector of 3 or the host cell of 4 in the preparation of a medicament for preventing or treating lung cancer.

8.1 the use of the mutant antimicrobial peptide, the nucleic acid of 2, the vector of 3 or the host cell of 4 in the preparation of a medicament for preventing or treating an inflammatory response mediated by upregulation of pro-inflammatory factors or nitric oxide by lipopolysaccharide.

9.1 the antibacterial peptide mutant, 2 the nucleic acid, 3 the carrier or 4 the host cell for preventing or treating bacterial infection diseases.

10.1 the antibacterial peptide mutant, the nucleic acid of 2, the vector of 3 or the host cell of 4 for preventing or treating lung cancer.

11.1 the mutant antibacterial peptide, 2 the nucleic acid, 3 the vector or 4 the host cell for preventing or treating inflammatory response mediated by upregulation of pro-inflammatory factors or nitric oxide caused by lipopolysaccharide.

12. A method for preventing or treating a bacterial infection disease, the method comprising administering a therapeutically effective amount of the mutant antibacterial peptide of 1 to a subject in need thereof.

13. A method for preventing or treating lung cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the mutant antimicrobial peptide of 1.

14. A method for preventing or treating an inflammatory response mediated by upregulation of pro-inflammatory factors or nitric oxide by lipopolysaccharide, the method comprising administering to a subject in need thereof a therapeutically effective amount of the mutant antimicrobial peptide of claim 1.

Drawings

FIG. 1 is a schematic diagram of a Sumo-KL32 expression vector.

FIG. 2 is an SDS-PAGE pattern of KL32 protein purified by reverse phase high performance liquid chromatography.

FIG. 3 shows the minimum inhibitory concentration of KL32 protein on Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis.

FIG. 4. effect of salt ion concentration on the minimum inhibitory concentration of KL32 protein in inhibiting E.coli.

FIG. 5 shows the toxicity test results of KL32 protein on human lung cancer cells.

FIG. 6. effect of KL32 protein on nitric oxide production by lipopolysaccharide-induced macrophages.

FIG. 7 is a graph showing the effect of KL32 protein on LPS-induced macrophage expression of inflammatory factors.

Detailed Description

The advantages and features of the present invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings. These examples are only illustrative and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Example 1 construction of recombinant cecropin B antimicrobial peptide mutant KL32 expression vector

Firstly, optimizing the nucleotide sequence of the cecropin B antibacterial peptide mutant gene according to an escherichia coli preferred codon, and then obtaining a target gene by a primer splicing method, wherein the nucleotide sequence optimization and the primer are both completed by Nanjing Kingsler Biotechnology GmbH (the cecropin B which is antibacterial peptide from Chinese oak silkworm moth is taken as a template to carry out nucleotide sequence optimization, 3 amino acids AGP of the hinge region are removed, and the amino acid sequence of the cecropin B is KWKIFKKIEKVGRNIRNGIIKAGPAVAVLGEAKAL (SEQ ID NO. 2)). The KL32 primer sequence was as follows:

upstream primer (KL 32-F):

5′-GCATGCGAATTCAAGTGGAAGATTTTTAAGAAGATTG-3′(SEQ ID NO.3)

intermediate primer (KL 32-M):

5′-TTAAGAAGATTGAAAAGGTCGGTCGTAACATTCGTAACGGCATTATCAAG-3′(SEQ ID NO.4)

downstream primer (KL 32-R):

5′-CGTAGCCTCGAGTCACAGGGCTTTCGCTTCGCCCAGCACTGCCACAGCCTTGATAATGCCGT-3′(SEQ ID NO.5)

the underlined parts are restriction endonuclease sites Nde I (upstream primer) and Xho I (downstream primer) respectively, the primers are directly added into a PCR system, the gene fragment of KL32 (the gene sequence of KL32 is AAGTGGAAGATTTTTAAGAAGATTGAAAAGGTCGGTCGTAACATTCGTAACGGCATTATCAAGGCTGTGGCAGTGCTGGGCGAAGCGAAAGCCCTG (SEQ ID NO.6)) is obtained by PCR through mutual splicing of the primers, and the PCR annealing temperature is 63 ℃.

The KL32 gene fragment was then subcloned into an expression vector containing a Sumo tag. Plasmid pET22b containing a Sumo tag is modified in the laboratory, a His-tag sequence is introduced into the N-terminal of Sumo, and no restriction enzyme sites exist between the Sumo tag gene and a foreign protein gene, so that the Sumo tag gene and the KL32 gene need to be fused together by a PCR method. Taking pET22b plasmid containing Sumo label and exogenous protein in the laboratory and the PCR product as templates, designing primers, and obtaining KL32 gene containing Sumo fusion label through PCR, wherein the downstream primer is unchanged and is KL32R, and the upstream primer and the intermediate primer have the following sequences:

upstream primer (Sumo-up):

5′-GGAATTCCATATGCATCATCATCATCAT-3′(SEQ ID NO.7)

middle primer (Sumo-KL 32):

5′-CAGAGAACAGATTGGTGGTAAGTGGAAGATTTTTAAGAAGATTG-3′(SEQ ID NO.8)

the underlined part is a restriction endonuclease site Nde i (upstream primer), and the slashed part is a nucleotide sequence of the 3' end of the Sumo tag gene. The PCR annealing temperature is 63 ℃, and the PCR product is recovered by the Tiangen agarose gel recovery kit. Performing double enzyme digestion on PCR (polymerase chain reaction) recovery products of Nde I and Xho I and a pET22b vector for 6h at 37 ℃, recovering digested plasmids by a Tiangen agarose gel recovery kit, and recovering digested gene fragments by a Tiangen centrifugal column type common DNA product recovery kit; then, the double enzyme-digested gene fragment and the linearized vector are connected according to the molar ratio of the vector to the insert fragment of 1:5 to obtain a pET22bSumo-KL32 target gene expression vector (the construction of a cloning vector is shown in figure 1), the TAKARA connection kit is connected for 2h in a water bath at 16 ℃, a reaction result system directly converts competent escherichia coli Top10 cells, a colony PCR detects positive recombinants, and the positive recombinants are sent to the Shanghai engineering for sequencing. Sequencing of the correct transformants transformed E.coli expression strain BL21(DE3)/pLysS competent cells.

Example 2 expression and purification of recombinant cecropin B antimicrobial peptide mutant KL32

The overnight culture of the correct transformant was sequenced, transferred to a freshly prepared LB medium containing ampicillin (100. mu.g/mL) and chloramphenicol (34. mu.g/mL) at an inoculum size of 1%, cultured at 37 ℃ with shaking at 250rpm for about 3 hours, and the OD thereof was determined₆₀₀When the concentration reached 0.6, IPTG was added to a final concentration of 0.5mM, the mixture was cultured at 37 ℃ and 250rpm for 4 hours with shaking, and the cells were collected and resuspended in a lysate (50mM NaH)₂PO₄300mM NaCl, pH 8.0), ultrasonication of disrupted cells (power 30%, over 3s, stop 4s, 3min repeated four times), centrifugation at 12000rpm at 4 ℃ for 30min, taking the supernatant, binding to Ni (binding buffer pre-equilibrated, binding buffer identical to lysis buffer) resin, binding at 4 ℃ for 2h, collecting the permeate, washing the resin with 10 column volumes of binding buffer containing 30mM imidazole to remove contaminating proteins, eluting the target protein with 4 column volumes of binding buffer containing 300mM imidazole, collecting the eluate, obtaining Sumo-KL32 fusion protein. Imidazole was removed by dialysis at 4 ℃ in 50mM Tris, 100mM NaCl, pH 8.0. Measuring A280 value, obtaining protein concentration according to A280/protein extinction coefficient,sumo protease (namely W) is added according to the mass ratio of 1:50_SumoProtease: w_Sumo-KL321:50), and cutting at 4 ℃ for 6 h. And (3) separating KL32 by reverse high performance liquid chromatography, wherein HPLC mobile phases are as follows: buffer A, 100% H₂O, 0.1% TFA; buffer B, 90% acetonitrile, 0.1% TFA; the purification result is detected by 12% SDS-PAGE electrophoresis, the gel electrophoresis result is shown in figure 2, and the KL32 protein with higher purity is obtained through purification.

Example 3. broth dilution method to determine the Minimum Inhibitory Concentration (MIC) of antimicrobial peptides and the effect of salt ions on the MIC:

single colonies of test bacteria were picked from MHA plates and inoculated into a triangular flask containing 10ml of fresh sterile MHB medium, incubated overnight at 37 ℃ with shaking at 180rpm, antimicrobial peptides were prepared as 2000. mu.M stock solution in sterile water, serially diluted with 0.01% acetic acid (0.2% BSA) to a concentration 10 times the concentration at the time of the test (1000. mu.M to 1.85. mu.M), and overnight cultures of test bacteria were diluted to 2 × 10⁵CFU/ml～7×10⁵CFU/ml, for determining the effect of salt ions on the action of KL32 on the MIC of E.coli, salt solutions (NaCl, MgCl) 50 times the final concentration of salt ions₂、CaCl₂) Adding into diluted bacteria solution to Na⁺The final concentration is 100mM, Ca²⁺Final concentration 2mM, Mg²⁺The final concentration was 1 mM. Respectively adding the diluted test bacterium liquid into a 96-hole cell culture plate with No.1 to No. 11 holes, wherein each hole is 100 mu l, adding 100 mu l of fresh MHB culture medium into No. 12 holes to serve as reference control, and making 3 parallel strains; correspondingly adding 11 mul of test antibacterial peptide solution into the No.1 to No. 10 holes; culturing at 37 deg.C and 180rpm at constant temperature overnight, and measuring 490nm absorbance with enzyme labeling instrument; the concentration of the antibacterial peptide in the holes with the absorbance being more than 50% lower than that of the No. 11 control hole is the MIC of the antibacterial peptide to the test bacteria, the MIC result of KL32 is shown in figure 3, the influence of the salt ion concentration on the KL32MIC is shown in figure 4, and the result shows that KL32 has strong inhibition effect on escherichia coli, pseudomonas aeruginosa and bacillus subtilis; while KL32 inhibited E.coli, 2mM Ca²⁺Has no effect on its MIC, and is at 100mM Na⁺And 1mM Mg²⁺In the presence of the salt ions, the KL32 has stronger bacteriostatic effect than the salt ions in the absence of the salt ions.

Example 4 toxicity of antimicrobial peptides to human Lung cancer cells

The human lung cancer cell line pc9 was seeded in 96-well plates at 1.16 × 10 cells per well⁴The culture medium is RPMI 1640 (containing 10% FBS, 100U/mL penicillin and 100 mu g/mL streptomycin), and the cells are cultured in an incubator at 37 ℃ for 24 hours; adding antibacterial peptide solution prepared by PBS until the final concentration is 2 μ M, 10 μ M and 50 μ M respectively, taking the hole only containing cells and not containing antibacterial peptide as a control hole, and continuously culturing for 24h in an incubator at 37 ℃; each well was added with 10. mu.l of CCK-8 reagent in the cell counting kit, and OD was measured after 2.5h of incubation at 37 ℃₄₅₀And (3) calculating the light absorption value, calculating the influence of the antibacterial peptide on the cell growth rate, wherein the experimental result is shown in figure 5, and compared with the wild antibacterial peptide cecropin B, the mutant KL32 has an inhibiting effect on the lung cancer cells.

Example 5 inhibition of lipopolysaccharide induced nitric oxide production by mouse macrophage RAW264.7 by antimicrobial peptides

The content of nitrite in the culture medium can be detected to indirectly reflect the nitric oxide generated when lipopolysaccharide induces macrophage to generate immune response. Mouse macrophage RAW264.7 at 1.5 x 10⁵The cells are inoculated in a 96-well plate, the culture medium is DMEM (containing 10% FBS, 100U/mL penicillin and 100 mu g/mL streptomycin), and the cells are cultured in an incubator at 37 ℃ until the cells adhere to the wall; adding antibacterial peptide solution prepared by PBS to the final concentration of 2 muM, 10 muM and 50 muM respectively, culturing for 30min in an incubator at 37 ℃, then adding lipopolysaccharide to the final concentration of 200ng/ml, taking the hole only containing the cell as a negative control hole, taking the hole containing the cell and the lipopolysaccharide as a positive control hole, and continuously culturing for 24h in the incubator at 37 ℃; and (3) detecting the nitrite content in the sample by using a nitric oxide detection kit in Biyun. The inhibition result of KL32 on the generation of nitric oxide by lipopolysaccharide-induced mouse macrophages is shown in figure 6, and compared with the wild type, the inhibition effect of the mutant KL32 is better and is more than 50% higher than that of the wild type.

Example 6 inhibitory Effect of antibacterial peptides on lipopolysaccharide-induced macrophage production of inflammatory factors at the transcriptional level

At 5 × 10 cells per well⁵Mouse macrophage RAW264.7 was inoculated in 6-well plates in DMEM (containing 10% FBS, 100U/mL cyan)Streptomycin and 100. mu.g/mL streptomycin), incubated overnight in an incubator at 37 ℃; adding antibacterial peptide to the final concentration of 10 mu M, culturing in an incubator at 37 ℃ for 1h, adding lipopolysaccharide to the final concentration of 200ng/ml, taking the hole only containing the cell as a negative control hole and the hole containing the cell and the lipopolysaccharide as a positive control hole, and continuously culturing in the incubator at 37 ℃ for 3 h; respectively extracting total RNA in cells by using a Tiangen RNA extraction kit, and quantifying the extracted total RNA; with RNAse-free H₂O, diluting the RNA sample to the same concentration, and carrying out reverse transcription on the RNA diluted to the same concentration by using a Thermo reverse transcription kit to obtain cDNA; with Nuclease-free H₂O all cDNAs were diluted 5-fold, 2. mu.l was used as a PCR template, and the PCR reaction system and conditions were as follows:

the reaction conditions are as follows: 5min at 94 ℃; 94 ℃ for 1min, 55 ℃ for 1min30s, 72 ℃ for 1min, 35 cycles; 10min at 72 ℃; keeping at 4 ℃.

The PCR product is detected by 1% agarose gel electrophoresis, the result is shown in figure 7, and compared with the wild type antibacterial peptide, the mutant KL32 of the invention has inhibition effect on the expression of 6 inflammatory factors, and the inhibition effect is better.

Sequence listing

<110> institute of science of fertilizer combination and substance science of Chinese academy of sciences

<120> cecropin B antibacterial peptide mutant

<130>IB178491

<160>8

<170>SIPOSequenceListing 1.0

<210>1

<211>32

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Lys Trp Lys Ile Phe Lys Lys Ile Glu Lys Val Gly Arg Asn Ile Arg

1 5 10 15

Asn Gly Ile Ile Lys Ala Val Ala Val Leu Gly Glu Ala Lys Ala Leu

20 25 30

<210>2

<211>35

<212>PRT

<213> Oak Bombycis mori (Antheraea pernyi)

<400>2

Lys Trp Lys Ile Phe Lys Lys Ile Glu Lys Val Gly Arg Asn Ile Arg

1 5 10 15

Asn Gly Ile Ile Lys Ala Gly Pro Ala Val Ala Val Leu Gly Glu Ala

20 25 30

Lys Ala Leu

35

<210>3

<211>37

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

gcatgcgaat tcaagtggaa gatttttaag aagattg 37

<210>4

<211>50

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ttaagaagat tgaaaaggtc ggtcgtaaca ttcgtaacgg cattatcaag 50

<210>5

<211>62

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

cgtagcctcg agtcacaggg ctttcgcttc gcccagcact gccacagcct tgataatgcc 60

gt 62

<210>6

<211>96

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

aagtggaaga tttttaagaa gattgaaaag gtcggtcgta acattcgtaa cggcattatc 60

aaggctgtgg cagtgctggg cgaagcgaaa gccctg 96

<210>7

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

ggaattccat atgcatcatc atcatcat 28

<210>8

<211>44

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

cagagaacag attggtggta agtggaagat ttttaagaag attg 44

Claims

2. A nucleic acid encoding the mutant antimicrobial peptide of claim 1.

3. A vector comprising the nucleic acid of claim 2.

4. A host cell comprising the nucleic acid of claim 2 or the vector of claim 3.

5. Use of the mutant antimicrobial peptide of claim 1, the nucleic acid of claim 2, the vector of claim 3, or the host cell of claim 4 in the preparation of a medicament for preventing or treating an e.

6. Use of the mutant antimicrobial peptide of claim 1, the nucleic acid of claim 2, the vector of claim 3, or the host cell of claim 4 in the preparation of a medicament for treating lung cancer.

7. Use of the mutant antimicrobial peptide of claim 1, the nucleic acid of claim 2, the vector of claim 3, or the host cell of claim 4 in the preparation of a medicament for inhibiting upregulation of pro-inflammatory factors or nitric oxide by lipopolysaccharide.