WO2019210845A1 - 生物活性重组人hnp-1蛋白及应用 - Google Patents

生物活性重组人hnp-1蛋白及应用 Download PDF

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WO2019210845A1
WO2019210845A1 PCT/CN2019/085130 CN2019085130W WO2019210845A1 WO 2019210845 A1 WO2019210845 A1 WO 2019210845A1 CN 2019085130 W CN2019085130 W CN 2019085130W WO 2019210845 A1 WO2019210845 A1 WO 2019210845A1
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hnp
protein
mature
recombinant human
recombinant
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French (fr)
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徐平
谢琦
武军驻
王银
魏威
肖伟弟
王一豪
常蕾
高慧英
刘艳红
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北京谱峰源生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the present invention relates to genetic engineering, and in particular to the expression of a biologically active recombinant human HNP-1 protein (human alpha-defensin-1) using a prokaryotic expression system.
  • the invention also relates to the use of the recombinant human HNP-1 protein.
  • Antimicrobial peptides are important sources of new antibiotics (Mishra B, Reiling S, Zarena D, & Wang G, Host defense antimicrobial peptides as antibiotics: design and application strategies. Current Opinion in Chemical Biology, 2017, 38 :87-96).
  • There are two types of antimicrobial peptides such as cathelicidins and defensins in animals (Zanetti M, Cathelicidins, multifunctional peptides of the innate immunity. Journal of Leukocyte Biology, 2004, 75(1): 39-48; Ganz T, Defensins: antimicrobial peptides of innate immunity. Nature Reviews. Immunology, 2003, 3(9): 710-720; Selsted ME & Ouellette AJ, Mammalian defensins in the antimicrobial immune response. Nature Immunology, 2005, 6(6): 551-557).
  • Defensins are a class of small molecule short peptides that are widely found in animals, plants and insects. It is resistant to microbial invasions such as bacteria, fungi or viruses and is part of the body's immune system.
  • Swedish scientist Hulmark et al. induced the first bactericidal peptide Cecropin from the sputum of Hyatophora Cecropins in 1975, opening up this new field (Hulmark D.Insect immunity: purification and properties of three inducible bactericidal proteins from Hemolymph of immunized pupae of Hyalophora cecropia. European Journal of Biochemistry, 1980, 106: 7-16).
  • defensins usually consist of 29 to 54 amino acids, including 6 to 8 conserved cysteines, which form an antiparallel ⁇ -sheet or alpha-helical structure through a cysteine intramolecular disulfide bond.
  • defensins can be divided into animal defensins, plant defensins and insect defensins; according to molecular structure characteristics, animal defensins can be divided into ⁇ -defensins, ⁇ -defensins and ⁇ -defensins. .
  • Alpha-defensins were first isolated from rabbit lung macrophages. These defensins are mainly found in mammalian tissues and cells, consisting of 29 to 35 amino acids, rich in arginine and very conserved 6 cysteines with Cys-l-Cys-6 and Cys-2. -Cys-4, Cys-3-Cys-5 mode to form 3 pairs of intramolecular disulfide bonds, wherein Cys-l-Cys-6 is a disulfide bond connecting the N-terminal and C-terminal cysteine to form a circular macrocycle .
  • rabbit neutrophils contain six kinds of ⁇ -defensins, namely RabNP-1, -2, -3a, -3b, -4, -5. The total amount of these defensins accounts for 15% of the total cellular protein. ⁇ 20%.
  • alpha-defensin-1 (HNP-1) is the major defensin molecule of human neutrophils. These defensins are mainly found in the lysosomal granules of human neutrophils. They are the most abundant and active bactericidal molecules found to date, against Gram-positive and Gram-negative bacteria, as well as adenoviruses and influenza viruses. HIV, HIV, etc.
  • defensins There are two main hypotheses for the antibacterial mechanism of defensins, one is the independent membrane mechanism, and the other is the mechanism by which defensin binds to intracellular complexes.
  • the independent membrane mechanism hypothesis suggests that positively charged defensins and negatively charged bacterial cell membranes attract each other and bind to each other, thereby destroying the integrity of the phospholipid bilayer and causing fissures in the target cell membrane.
  • This antibacterial process is divided into three phases. First, the defensin binds to the target cell membrane by electrostatic attraction. Since the defensin has a positive charge, it can be combined with a negatively charged bacterial membrane lipid layer by electrostatic action. The second step forms a channel.
  • the positively charged defensin molecule or its multimer interacts with the negatively charged phospholipid head and water molecules on the bacterial plasma membrane, significantly increasing the permeability of the biofilm. Defensins act on the membrane to form a stable plurality of channels. Finally, the contents leaked and the cells died. After the channel is formed, while the defensin enters the cell, other extracellular molecules such as peptides, proteins or inorganic ions are also accompanied, and the important ions such as salt ions and macromolecules of the target cells are exuded, so that the permeability of the cell membrane and The cell's energy state is destroyed, resulting in depolarization of the cell membrane, inhibition of respiration and a decrease in cellular ATP content.
  • defensins also have immunomodulatory effects in acquired immune challenge by inducing chemotaxis of T lymphocytes and immature dendritic cells (Yang D, Chen Q, Chertov O, & Oppenheim JJ, Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells, Journal of Leukocyte Biology, 2000, 68(1): 9-14; Hancock RE, Haney EF, & Gill EE, The immunology of host kissing peptides: beyond antimicrobial activity, Nature Reviews. Immunology, 2016, 16(5): 321-334).
  • HNP-1 has an attractive use prospect. Direct extraction can obtain defensins, but due to the complicated process route, the cost is extremely high and has no application value (Hancock RW, Robert L. Cationic peptides: a new source of antibiotics [J]. Tibtech, 1998, 16: 82-88 ).
  • chemical synthesis of HNP-1 because it contains three pairs of disulfide bonds in the molecule, it is difficult to ensure that the peptides are properly paired when folded, thereby affecting their biological activity. Therefore, the production of defensins by genetic engineering methods has become a research hotspot in the field.
  • HNP-1 In human cells, HNP-1 is first translated into 94 amino acid residues of preproHNP-1 (including a hydrophobic signal sequence consisting of 19 amino acid residues, a negatively charged anterior sequence consisting of 45 amino acid residues and C The terminal contains a positively charged mature peptide consisting of 30 amino acids).
  • PreproHNP-1 enters the endoplasmic reticulum to obtain protoprotective protein (proHNP-1) containing 56 amino acid residues, which is then cleaved one or more times in lysosomes to form a cationic mature peptide containing 30 amino acid residues.
  • HNP-1 (Valore, EV & Ganz, T. Posttranslational processing of defensins in immature human myeloid cells.
  • HNP-1 Due to the toxicity of defensins, most of the current research focuses on the modification of expression vectors, and attempts to adopt the strategy of fusion expression. Since the positive electrophoresis carried by mature HNP-1 is the chemical basis for killing microorganisms, the negatively charged polypeptide is expressed by fusion, and its positive polarity is neutralized to reduce the bactericidal activity, thereby efficiently expressing HNP-1. Piers attempts to express human defensin HNP-1 in bacteria via the fusion protein pathway, but the resulting HNP-1 is not biologically active (Piers K L, Brown M H, Robert EHRecombinant DNA procedures for producing small antimicrobial etching peptides in bacteria , Gene, 1993, 134: 7-13).
  • Xu Zhinan and other domestic laboratories express the ⁇ -human defensin-glutathione transferase (GST) fusion protein in E. coli through the fusion protein pathway, but the recombinant strain not only expresses GST- ⁇ -human defensin very much. Low, the fusion protein itself is still unknown or not (Xu Zhinan, Peng Li, Xiang Ming et al, recombinant expression and optimization of ⁇ -human defensin-2 in E. coli, Fine and Specialty Chemicals, 2002, (Supplement): 39 -42; Li Xia, Shen Yi, Wang Yiqin et al. Expression and purification of rabbit defensin in E.
  • GST ⁇ -human defensin-glutathione transferase
  • Inclusion body form can produce HNP-1, but it is difficult to form HNP-1 correctly in purification (Pazgier, M. & Lubkowski, J. Expression and purification of recombinant human alpha-defensins in Escherichia coli.
  • HNP-1 precursor protein in endothelial cells, it is suggested that during the maturation process of HNP-1, the anterior region sequence not only neutralizes the positive charge of mature HNP-1, but also the cells appearing during the blocking process. Toxic, and important for the correct folding of HNP-1 (Wu, Z. et al. Impact of pro segments on the folding and function of human neutrophil alpha-defensins. Journal of molecular biology, 2007, 368: 537-549) . Although the prokaryotic expression of HNP-1 has been a hot and difficult problem, we have learned from the above studies that E. coli may also have a HNP-1 activation mechanism similar to human body. The precursor protein has a certain bactericidal ability. The illusion, but through the activation of mature HNP-1.
  • Another object of the present invention is to provide the use of the above recombinant human HNP-1 protein.
  • the recombinant human HNP-1 protein of the present invention expresses a human defensin HNP-1 precursor protein by using an E. coli expression system, and is isopropyl- ⁇ -D-1-thiopyr
  • the galactogalglycoside induces the production of a mature human HNP-1 protein having antibacterial activity.
  • An E. coli expression system for expressing a mature human HNP-1 protein of the present invention comprises an E. coli expression vector and a HNP-1 precursor protein encoding gene operably linked to the expression vector.
  • the human defensin HNP-1 precursor protein gene sequence is derived from the human genome sequence (NM_004084.3) and includes a signal peptide, a propeptide and mature HNP-1. Further, a 6 ⁇ His tag was introduced at its N-terminus to obtain an open reading frame (ORF) encoding 100 amino acids, and the coding sequence and corresponding amino acid sequence are shown in FIG.
  • the E. coli expression vector for the E. coli expression system may be various E. coli expression vectors commonly used, and these expression vectors include, but are not limited to, pET series vectors, pGEX series vectors, pTWIN1 vectors, pET-28a (+) vectors, and the like.
  • a preferred E. coli expression vector can be a pET-28a (+) vector.
  • the construction process of the E. coli expression system of the present invention comprises inserting the HNP-1 precursor protein coding gene sequence into the EcoR I of the pET-28a(+) vector and A HNP-1 precursor protein recombinant expression vector was obtained between the Xho I restriction sites, and the expression vector was introduced into E. coli BL21 (DE3) competent cells by chemical transformation to obtain a HNP-1 precursor protein gene. Recombinant strain.
  • a method for preparing a biologically active mature human HNP-1 protein using the above E. coli expression system comprises the steps of:
  • step 3 adding isopropyl- ⁇ -D-1-thiogalactopyranoside to the culture solution of step 2) to induce culture, and collecting the cells;
  • the mature human HNP-1 protein is isolated and purified from the cells.
  • IPTG isopropyl- ⁇ -D-1-thiogalactopyranoside
  • the induction culture time is preferably 0.5- 3 hours, preferably, when the bacterial solution OD 600 is grown to 0.4-0.6 (in LB liquid medium as a blank control), the final concentration is 1 mM isopropyl- ⁇ -D-1-thiopyran.
  • Galactoside (IPTG) induction culture for 3 h. Induction culture parameters: culture temperature was 37 ° C, and shaker rotation speed was 220 rpm.
  • the present invention uses a filtration technique to retain large molecular proteins, thereby separating and purifying, and obtaining mature Small molecular weight HNP-1 protein.
  • the use of the recombinant human HNP-1 protein for the preparation of an antibacterial agent is provided.
  • the recombinant protein of the present invention can be prepared as an antibacterial agent by adding a pharmaceutically acceptable adjuvant. More preferably, the recombinant human HNP-1 protein of the present invention is prepared into a liposome-coated pharmaceutical form to enhance the bactericidal effect.
  • the recombinant human HNP-1 protein of the present invention has an antibacterial effect against the drug-resistant bacteria, and the preferred drug-resistant bacteria type is resistant to Staphylococcus aureus, resistant to Klebsiella pneumoniae or resistant The drug Acinetobacter baumannii, more preferably methicillin-resistant Staphylococcus aureus.
  • HNP-1 is a cationic mature peptide obtained by cleavage of preproHNP-1 in the endoplasmic reticulum and lysosomes.
  • prokaryotic system due to the lack of mechanism for cleavage of preproHNP-1 to produce mature HNP-1, it is usually designed to fuse the negatively charged polypeptide with the positively charged HNP-1, and then the fusion protein is cleaved to obtain HNP- 1.
  • the prior art does not have a prokaryotic system capable of expressing biologically active mature HNP-1.
  • the present invention constructs an E. coli expression system expressing a biologically active mature human HNP-1 protein, and establishes a method for preparing human HNP-1 protein, and successfully expressed and prepared an active HNP-1 protein.
  • Figure 1 Sequence encoding human defensin HNP-1 precursor protein gene and corresponding amino acid sequence
  • the gray shaded part is the excised part of the protein during maturation, ie mature HNP-1; the double solid line part is the introduced 6 ⁇ His tag; the dotted line part is the signal peptide; the solid line part is the precursor part of the protein, ie pro HNP -1.
  • Figure 2 Induced expression of recombinant E. coli BL21-preproHNP-1 strain.
  • FIG. 3 Tricine gel electrophoresis analysis of recombinant preproHNP-1 and mature HNP-1;
  • Lane M is a protein molecular weight standard with a molecular weight of 3.4-100 kD
  • lane-IPTG and +IPTG are the cell lysate supernatants after 3 hours of induction and induction, respectively.
  • Lane M is the molecular weight standard, and lanes 30, 90, and 180 are induction products for 30 minutes, 90 minutes, and 180 minutes after IPTG induction, respectively, and the amount of mature HNP-1 is shown at a position of molecular weight of 3.4 kDa.
  • Figure 5 Targeted quantitative proteome analysis of the amount of mature HNP-1 induced by IPTG, (*, p ⁇ 0.05);
  • the abscissa is the induction time (minutes) and the ordinate is the relative amount.
  • Figure 7 Changes in the number of bacterial colonies after IPTG induced expression of PreproHNP-1.
  • the time of IPTG induction is shown in the figure.
  • the number of viable cells is determined by the number of colonies grown for 16 hours on the LB solid medium, expressed as mean ⁇ variance (**, p ⁇ 0.01). .
  • Figure 8 Analysis of purified recombinant prepro HNP-1His affinity chromatography by Tricine gel electrophoresis
  • Lane M is a molecular weight standard of molecular weight of 3.4-100 kD
  • lanes TCL, SN, IS, wash1, wash2, ET1, and ET2 are total cell lysate, cell lysate supernatant, insoluble protein, washing 1, washing 2 , elute sample 1 and elute sample 2.
  • Figure 9 A diagram of a recombinant mature HNP-1 purification device
  • the protein supernatant was added to the inner tube, 5,000 g, and centrifuged at room temperature for 10 minutes, and the filtrate was collected, and the above procedure was repeated 4 times.
  • FIG. 10 Tricine gel electrophoresis analysis of purified mature recombinant HNP-1
  • Lane M is the molecular weight standard of protein with a molecular weight of 3.4-100 kDa.
  • Lane TCL is the cell lysate supernatant after 3 hours of induction by IPTG, and the purified protein of the lane is the filtrate obtained by ultracentrifugation.
  • FIG. 11 Tricine gel electrophoresis analysis of recombinant mature HNP-1
  • Lane M is the molecular weight standard of protein with a molecular weight of 3.4-100 kDa
  • lane-IPTG and +IPTG are the supernatants of the cell lysis after 3 hours of induction and induction, respectively.
  • Figure 12 Mass spectrometric identification of recombinant mature HNP-1
  • E. coli XPX-1 induced the expression of PreproHNP-1 with 1 mM IPTG, and the growth curve of mature HNP-1 was positive.
  • E. coli XPX-1 had no IPTG-induced growth curve as a negative control; purified PreproHNP-1 and mature HNP The concentration of -1 was 40 ⁇ g/mL.
  • Figure 15 Quantitative proteome study of antibacterial activity of endogenous mature active HNP-1.
  • Figure 16 Quantitative proteomic comparison to identify the process profile of upregulated proteins in Gene Ontology analysis.
  • Figure 17 Quantitative proteomic comparison to identify upregulated proteins for endonuclease enrichment.
  • Figure 18 Quantitative proteomic comparison to identify the process profile of down-regulated proteins in Gene Ontology analysis.
  • Figure 19 Quantitative proteomic comparison to identify down-regulated proteins in NAD(P)H-reactive enzyme enrichment.
  • Figure 21 Annexin V staining analysis, values are expressed as mean ⁇ SD, **, p ⁇ 0.01. XPX-1 expressing perproHNP-1 and XPX-2 only from the control plasmid were induced with 1 mM IPTG for 3 hours, respectively. 10 ⁇ g/mL gentamicin was used as a positive control for inducing apoptosis.
  • Figure 22 Tris-Tricine gel and silver staining analysis of HNP-1 purified under non-denaturing conditions.
  • FIG. 23 Fractional analysis of HNP-1 interacting proteins, including biological replicates. HNP-1 itself and highly enriched RecA are labeled with arrows, respectively.
  • FIG. 24 Histidine-tagged fusion of HNP-1 and RecA interactions.
  • HNP-1 was purified by nickel-NTA packing by a 6-histidine tag, and separated by SDS-PAGE gel electrophoresis and then hybridized with RecA antibody (Abeam, Cambridge, United Kingdom).
  • Figure 25 Direct interaction of preproHNP-1 and RecA with mature HNP-1 but not full-length 6 histidine tagged histidine tag fusion.
  • HNP-1 expressing cells were harvested at the indicated time points, and RecA was affinity-purified with an anti-RecA antibody and subjected to Western blot analysis with an anti-HNP-1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA).
  • FIG. 26 HNP-1 inhibits ATPase activity of single-chain activated RecA.
  • the ATPase activity was detected by measuring the amount of inorganic phosphate released by the malachite green phosphate reaction. The experiment was repeated 3 times (*, p ⁇ 0.05), the abscissa was the HNP-1 concentration, and the ordinate was the free phosphoric acid concentration ( ⁇ M).
  • FIG. 27 HNP-1 inhibits single-stranded DNA binding of RecA. RecA-bound single-stranded DNA and free single-stranded DNA were analyzed by agarose gel electrophoresis migration assay.
  • Figure 28 Particle size distribution of liposome coated HNP-1. Particle size analysis was done using a Zetasizer Nano ZS (Malvern, UK).
  • Figure 29 HNP-1 kills methicillin-resistant Staphylococcus aureus by apoptosis.
  • FIG 30 Liposomal coated HNP-1 killing pneumonia mouse model methicillin-resistant Staphylococcus aureus (MRSA).
  • MRSA was isolated from the patient's lung lavage fluid and divided into 7 groups: (a) healthy control group (Health); (b) MRSA-induced pneumonia model mice (MRSA); (c) 12 ⁇ g/mL penicillin treatment MRSA-induced pneumonia model mice, as a control for penicillin-resistant strains of MRSA (PCN); (d) pneumonia model mice 0.1 mL 20 ⁇ g/mL HNP-1 treatment group (HNP-1); (e) small pneumonia model Mouse blank liposome treatment control group (Lipo); (f) pneumonia model mice 0.1 mL 5 ⁇ g/mL liposome coated HNP-1 treatment group (5Lipo-H); (g) pneumonia model mice 0.1 mL 10 ⁇ g The /mL liposome was coated with the HNP-1 treatment group (10 Lipo-H); the experiment was repeated 3 times (**, p ⁇ 0.01).
  • Figure 31 Liposomal coating of HNP-1 kills resistant Klebsiella pneumoniae.
  • the drug-resistant Klebsiella pneumoniae strain number is ATCC BAA2146 and is provided by the National Center for Disease Control and Prevention. The experiment was divided into 6 groups, which were (a) 12 ⁇ g/mL penicillin-killing Klebsiella pneumoniae group, as a control (PEN) for penicillin-resistant strains of Klebsiella pneumoniae; (b) Klebsiella pneumoniae 16 ⁇ g/mL HNP-1 bactericidal experimental group (HNP-1 (16 ⁇ g/mL)); (c) drug-resistant Klebsiella pneumoniae 32 ⁇ g/mL HNP-1 bactericidal experimental group (HNP -1 (32 ⁇ g/mL)); (d) Klebsiella pneumoniae 16 ⁇ g/mL liposome coated HNP-1 bactericidal experimental group (Lipo-H (16 ⁇ g/mL)); (e) resistant Klebsiella pneumoniae
  • Figure 32 Liposomal coating of HNP-1 kills resistant Acinetobacter baumannii.
  • the drug-resistant Acinetobacter baumannii strain No. NO801 is provided by the National Center for Disease Control and Prevention. The experiment was divided into 6 groups, namely (a) 12 ⁇ g/mL penicillin killing drug-resistant Acinetobacter baumannii group, as a control (PEN) for penicillin-resistant strains resistant to Acinetobacter baumannii; (b) resistance Acinetobacter baumannii 16 ⁇ g/mL HNP-1 bactericidal experimental group (HNP-1 (16 ⁇ g/mL)); (c) drug-resistant Acinetobacter baumannii 32 ⁇ g/mL HNP-1 bactericidal experimental group (HNP-1 (32 ⁇ g) /mL)); (d) drug-resistant Acinetobacter baumannii 16 ⁇ g / mL liposome coated HNP-1 bactericidal experimental group (Lipo-H (16 ⁇ g / mL)
  • the human defensin HNP-1 precursor protein gene sequence of the present invention is derived from the human genome sequence (NM_004084.3) and includes a signal peptide, a propeptide and mature HNP-1. We introduced a 6 ⁇ His tag at its N-terminus to obtain an open reading frame (ORF) encoding 100 amino acids.
  • ORF open reading frame
  • the coding sequence and corresponding protein sequence are shown in Figure 1, wherein the coding base sequence is shown in SEQ NO.
  • the protein sequence is shown in SEQ NO. 2.
  • the sequence SEQ NO. 1 was inserted between the EcoR I and Xho I restriction sites of the pET-28a (+) vector (Novagen, Cat. No. 69864) to obtain a HNP-1 precursor protein recombinant expression vector, which was named pET-preproHNP- 1.
  • the pET-preproHNP-1 was introduced into E. coli BL21 (DE3) competent cells by chemical transformation to obtain a recombinant strain containing the HNP-1 precursor protein gene, and the strain was named recombinant Escherichia coli BL21(DE3)-preproHNP. -1 (hereinafter referred to as BL21-preproHNP-1 strain).
  • Example 2 Culture and collection of BL21-preproHNP-1 strain
  • the recombinant strain BL21-preproHNP-1 strain was activated on a solid LB plate containing kanamycin to obtain a monoclonal clone of the BL21-preproHNP-1 strain.
  • the monoclonal of BL21-preproHNP-1 strain was inoculated into 5 mL of LB liquid medium (kanamycin concentration of 50 ⁇ g/mL), and cultured overnight at 37 ° C with shaking to obtain the first seed of BL21-preproHNP-1 strain. .
  • the primary seed of the BL21-preproHNP-1 strain was diluted 1:100 and inoculated into fresh LB liquid medium containing 50 ⁇ g/mL kanamycin.
  • Culture parameters after inoculation the culture temperature was 37 ° C, and the shaker rotation speed was 220 rpm.
  • IPTG isopropyl- ⁇ -D-1-thiogalactopyranoside
  • the bacterial suspension was lysed by a sonicator (JY92-IIN, Ningbo Xinzhi).
  • the ultrasound condition was set to a power of 25% and the ultrasound was stopped for 3 seconds for 3 seconds for a total of 5 minutes.
  • the crushed cells were centrifuged at 10,000 g for 20 min at 4 ° C, and the supernatant was collected, and 50 ⁇ g of protein was loaded and separated by 12% Tricine gel.
  • ⁇ 5% of the gel 2 mL of distilled water, 3 ⁇ gel buffer (36.3g Tris base, 0.3g SDS, distilled water to 100mL, HCl adjusted pH 8.45) 1.33mL, 30% acrylamide 0.67mL, 10% ammonium persulfate 40uL, tetramethylethylenediamine (TEMED) 4uL; And 12% separation gel: distilled water 6.4 mL, 3 ⁇ gel buffer 5 mL, glycerol 1.5 g, 49.5% acrylamide 3.6 mL, 10% ammonium persulfate 150 uL, TEMED 10 uL ⁇ .
  • the initial voltage was 40V for electrophoresis. After the sample entered the separation gel, the voltage was changed to 200V and electrophoresis was carried out for 2 hours.
  • Tricine gel was rinsed with distilled water, fixed solution (50% methanol, 10% acetic acid, distilled water) for 10 minutes, and Coomassie Brilliant Blue G250 stained for 1 h. After decolorization, the image was scanned with a scanner. Tricine gelatin results showed that two distinct bands were generated at 14.8 kDa and 3.4 kDa after induction with IPTG compared to the group without IPTG induction (Fig. 3), with prepro HNP-1 and mature HNP-1, respectively. The theoretical molecular weights are consistent, and the two bands are presumed to be prepro HNP-1 and mature HNP-1.
  • preproHNP-1 induced by IPTG was detected by Tris-Tricine gel and Coomassie blue staining. It was found that the amount of mature HNP-1 increased gradually at the molecular weight of 3.4 kDa with the prolongation of IPTG induction (Fig. 4), indicating maturity. HNP-1 is efficiently induced to be expressed by IPTG.
  • the content of mature HNP-1 was quantified by the targeted quantitative proteomic technique, and it was found that the IPTG-induced expression of mature HNP-1 for 3 hours was 2-fold higher than that of the induction 0.5 hour (Fig. 5).
  • the column was pre-equilibrated with buffer A (20 mM Tris, 150 mM NaCl, pH 7.5) and the sample was loaded. The loading flow rate was 5 mL/min. After the loading was completed, the heteroprotein was eluted with 10 column volumes of buffer A and 10 column volumes of 5% buffer solution B (20 mM Tris, 100 mM NaCl, 0.5 M imidazole, pH 7.5), followed by 15 column volumes of 5 -80% buffer solution B linear gradient elution. All purification work was carried out at 20-25 °C. The purified preproHNP-1 solution was stored at 4 ° C ( Figure 8).
  • High performance liquid chromatography is a common technique for protein purification. Based on the characteristics of mature HNP-1 containing more basic amino acids, we tried the tandem purification analysis technique of ion exchange chromatography and molecular sieve, but did not get the mature HNP-1 band visible by SDS-PAGE gel electrophoresis, and tried other Separation method.
  • mature HNP-1 Since mature HNP-1 has no purification label and it also needs to maintain its antibacterial activity during purification, it can only be purified under some mild natural conditions. Based on the analysis of Tricine gel, it was found that the molecular weight of mature HNP-1 (3.4 kDa) was much smaller than that of E. coli itself. Proteomic analysis revealed that the total cell protein of the BL21-prepro-HNP1 strain induced by IPTG was less than that of mature HNP-1. Therefore, we attempted to isolate and purify the large molecular weight HNP-1 by filtration technology to retain large molecular weight proteins.
  • the cells were induced by IPTG at 50 OD 600 , resuspended in PBS buffer, and pipetted with a pipette.
  • the bacterial suspension was lysed by the sonicator and the disruption conditions described in Example 3. After the cells were broken, 10,000 g, centrifuged at 4 ° C for 20 min, and the supernatant was collected.
  • the Amicon Ultra-0.5 (3kDa, MWCO, Merckmillipore, item number: UFC500396) ultrafiltration centrifuge tube was used for separation and purification.
  • the Amicon Ultra-0.5 ultrafiltration inner tube was inserted into a microcentrifuge tube, 400 ⁇ L of double distilled water, 5,000 g was added to the inner tube, and centrifuged at room temperature for 10 minutes, and the ultrafiltration tube was pre-washed and repeated three times. Then, 400 uL of PBS, 5,000 g, was added to the inner tube, and the mixture was centrifuged at room temperature for 10 minutes to carry out filter activation, and the reaction was repeated three times. Add no more than 500 ⁇ L of protein supernatant to the Amicon Ultra inner tube, 5,000 g, and centrifuge at room temperature for 10 minutes.
  • the total cellular protein induced by IPTG was subjected to 12% separation of Tricine gel to obtain mature HNP-1 with a molecular weight of 3.4 kDa (Fig. 11).
  • the target strip (marked in black frame in Fig. 11) was cut and cut into 1 mm 3 colloidal particles.
  • the extract was repeated 3 times, and after centrifugation, 30 ⁇ L of acetonitrile was added, and the operation of centrifugation was repeated, and after centrifugation, the supernatant was taken out and continued to be added to the corresponding tube.
  • the acetonitrile extraction operation was repeated until the micelles became hard granules.
  • the extracted peptide solution was centrifuged at 13,300 rpm for 3 minutes and transferred to a new Eppendorf tube. The vacuum dryer is dried, and after the liquid is completely evaporated to dryness, the product is a dry powder of the peptide mixture.
  • the digested peptides were dissolved using mass spectrometry loading buffer of 1% acetonitrile, 1% formic acid, 98% water, and separated by Waters Corporation's ultra-high pressure liquid chromatography (NanoAcquity Ultra Permance LC, Waters). A column of 75 ⁇ m, 15 cm long C 18 reverse phase chromatography. The autosampler automatically injects a sample volume of 3 ⁇ L and a loading flow rate of 300 nL/min. The liquid phase separation was carried out for 60 minutes with a liquid phase gradient from 98% phase A (2% ACN + 0.1% FA) to 35% phase B (100% ACN + 0.1% FA).
  • the eluted components are passed directly into the mass spectrometer through a nanospray ion source.
  • Mass spectrometry was performed using Thermo Scientific's LTQ Orbitrap Velos high resolution mass spectrometer.
  • the mass spectrometry data was acquired by the first-order mass spectrometry data in the Data Dependent MS/MS Scan (DDA) mode.
  • the mass spectrometric scanning ion mass-to-charge ratio ranged from 300 to 1600 (m/z), and the primary parent ion scan was in Orbitrap.
  • the full scan resolution is set to 30,000
  • the Automatic Gain Control (AGC) sets 1 ⁇ 10 6 ions
  • MIT maximum ion injection time
  • the second spectrum is used.
  • CID fragmentation mode normalized collision energy is 35%.
  • the first 20 ions with the highest abundance and charge >1 were selected for secondary (MS/MS) fragmentation, the AGC was set to 1 ⁇ 10 4 and the MIT was set to 25 ms.
  • the resulting mass spectral data was identified using the software MaxQuant (1.5.6.0).
  • the complete proteome sequence of the target E. coli strain K12 obtained from UniProt (version 2016.01) plus 6x his-tag preproHNP-1 (Uniprot ID: 59665) and common contamination.
  • the protein identification parameters were set as follows: Carbamidomethylation was set as a fixed modification on cysteine, and methionine oxidation was set as a variable modification. Whole trypsin digestion allows up to two missed cuts.
  • the parent ion error is set at 20 ppm, the secondary fragment ion error is 0.5 Da, the peptide length is 7 amino acids, and the false discovery rate (FDR) of the peptide and protein is controlled below 1%.
  • Mature HNP-1 can be cleaved by trypsin to produce two theoretical peptides, IPACIAGER (SEQ NO. 3) and YGTCIYQGR (SEQ NO. 4).
  • IPACIAGER SEQ NO. 3
  • YGTCIYQGR SEQ NO. 4
  • the results of mass spectrometry showed that the peptide IPACIAGER was eluted within the retention time of 15.43 minutes, and the parent ion of +2 valence was obtained.
  • the signal intensity was 769,620.
  • CID fragmentation 7 pairs of continuous b, y ions, MS were obtained. 2 matched well (Fig.
  • the dried micelles were directly added to 40 ⁇ L of extract (5% FA + 50% ACN, ie formic acid and acetonitrile) for protein extraction, and the extraction procedure was the same as above. After the extraction step is completed, 30 ⁇ L of acetonitrile is added, and the centrifugation is repeated, and the supernatant is taken out and added to the corresponding Eppendorf tube; the acetonitrile extraction operation is repeated until the micelles become hard granules.
  • the extracted solution was centrifuged at 13,300 rpm for 1 minute, transferred to a new Eppendorf tube, and dried by a vacuum dryer to obtain a separated and purified protein.
  • the protein obtained above was dissolved by the same mass spectrometry loading buffer (1% acetonitrile, 1% formic acid, 98% water) as above. Detected using the same LC-MS/MS platform. A 60 minute liquid phase gradient elution was used with a gradient from 4% phase A (0.1% acetic acid, 2% acetonitrile) to 35% phase B (0.1% acetic acid, 100% acetonitrile) at a flow rate of 0.3 [mu]L/min.
  • the primary parent ion scan is done in Orbitrap, the full scan resolution is set to 30,000, the ion automatic gain control sets 1 ⁇ 10 6 ions, and the maximum ion injection time (MIT) is set to 150 ms.
  • the three ions with the highest ionic strength were isolated in a window of m/z of 2, fragmented at 32-34% collision energy, AGC set to 1x10 4 and MIT set to 25 ms.
  • the precursor ion peak is manually assigned based on the state of charge, the derived mass, and the liquid phase retention time.
  • HNP-1 mature peptide sequence (Cat. No. d2043) was purchased from Sigma and subjected to Topdown analysis in the same manner.
  • a standard of HNP-1 mature peptide sequence (Cat. No. d2043) was purchased from Sigma and subjected to Topdown analysis in the same manner.
  • an elution peak was identified at 35.40 minutes, and the retention time was substantially the same as that of our purified HNP-1.
  • the experimental molecular weight of this peak is 3445.554Da, which is only 0.004D difference from the theoretical molecular weight. It is also consistent with the molecular weight of the purified HNP-1 we have identified. It is further proved that BL21-prepro HNP-1 does induce the expression of mature HNP by IPTG. -1.
  • Example 8 Determination of antibacterial activity of recombinant mature HNP-1
  • the antibacterial activity was compared using purified mature HNP-1 and PreproHNP-1, and the growth curve was determined (Fig. 14). It was found that even in the case of purifying preproHNP-1 at a higher concentration (40 ⁇ g/mL), the growth of Escherichia coli XPX-1 was identical to that without IPTG induction, and was not affected, indicating that preproHNP-1 had no bacteriostatic activity. Using 40 ⁇ g/mL of purified HNP-1, the growth curve did not increase or even decreased slightly after reaching the plateau, indicating that HNP-1 purified under non-denaturing conditions had certain antibacterial activity.
  • the quantitative proteomics technique was used to compare the changes of cell proteome of E. coli XPX-1 with and without IPTG induction, aiming to elucidate the bactericidal mechanism of endogenous active HNP-1 (Fig. 15).
  • the equal amount of total cellular protein induced by 1 mM IPTG and without IPTG induction was subjected to mass spectrometry and trypsin digestion to obtain a peptide fragment, and subjected to non-standard quantitative proteome analysis.
  • Genomic DNA fragmentation is a major feature of apoptosis.
  • TUNEL technology terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labelling, BD Bioscience kit, Oakland, CA, USA
  • Bayles KW Bacterial programmed cell death: making sense of a paradox, Nature reviews, Microbiology , 2014, 12(1): 63-69)
  • apoptotic cells the cytoplasmic phosphatidylserine in the cell membrane is everted, which can be determined by specific binding to a negatively charged phospholipid, such as phosphatidylserine Annexin V stain (Bayne KW, Bacterial programmed). Cell death:making sense of a paradox, Nature reviews, Microbiology, 2014, 12(1): 63-69). We found that 1 mM IPTG induced positive expression of Annexin V in cells expressing biologically active mature HNP-1, whereas even IPTG induced no control cells in the control cells without HNP-1 gene coding sequence, indicating that mature HNP-1 elicited significant cells. Apoptosis ( Figure 21).
  • Example 10 Interaction proteomics reveals a novel antibacterial mechanism by which mature HNP-1 induces bacterial cell apoptosis by inhibiting RecA binding to single-stranded DNA.
  • HNP-1 interacting protein was affinity-purified under non-denaturing conditions, and HNP-1 itself and its interacting proteins were identified by mass spectrometry. Equal amounts of total cellular proteins from IPTG-induced expression of preproHNP-1 and no HNP-1 control bacteria were affinity-enriched with histidine tag, respectively, and Tris-Tricine gel and silver staining analysis indicated that preproHNP-1 and histidine-tagged proteins Significantly enriched ( Figure 22).
  • RecA is a known SOS-reactive protein after DNA damage in bacteria (Erental A, Kalderon Z, Saada A, Smith Y, & Engelberg-Kulka H, Apoptosis-like death, an extreme SOS response in Escherichia coli.mBio, 2014, 5 ( 4): e01426-01414.), but it is not clear how preproHNP-1 or mature HNP-1 itself regulates apoptosis through RecA.
  • Time series affinity purification of the total protein of HNP-1 expressing cells induced by IPTG at different time points revealed that preproHNP-1 first accumulated in the cells and then produced mature HNP-1. This is in line with our expectations.
  • RecA is a DNA-dependent ATPase that hydrolyzes ATP when it binds to single-stranded DNA, and its efficiency in hydrolyzing ATP is related to the amount of single-stranded DNA it binds.
  • the ability of mature HNP-1 to inhibit the ATPase activity of RecA in the presence of single-stranded DNA was tested (Yakimov A, et al., Blocking the RecA activity and SOS-response in bacteria with a short alpha-helical peptide, Nucleic Acids Research, 2017, 45(16): 9788-9796).
  • HNP-1 does achieve its function by inhibiting the binding of RecA to single-stranded DNA ( Figure 27).
  • HNP-1 Since the bactericidal activity of endogenous mature HNP-1 was significantly higher than that of mature HNP-1 added to the medium, we tried to coat HNP-1 with liposomes.
  • Soy lecithin was purchased from Shanghai Taiwei Pharmaceutical Co., Ltd., and cholesterol was purchased from Shanghai Sinopharm Holding Reagent Company.
  • the appropriate PBS is added and the gel is suspended while stirring to form a liposome.
  • the resulting liposomes were passed through a 0.45 ⁇ m sterile membrane (Pall, New York, NY, USA) three times to obtain a liposome finished product, which was detected by a laser particle detector and had an average particle diameter of 116.3 nm (Fig. 28).
  • Example 12 HNP-1 kills resistant Staphylococcus aureus by causing bacterial cell apoptosis
  • Example 13 Liposomal coating of HNP-1 kills mouse pneumonia model resistant to Staphylococcus aureus
  • HNP-1 Bacillus subtilis bactericidal activity against Klebsiella pneumoniae was tested. Significant bactericidal activity was exhibited when the concentration of HNP-1 reached 32 ⁇ g/mL (Fig. 31). Liposomal-coated HNP-1 exhibited bactericidal activity when the concentration was 16 ⁇ g/mL; when the concentration of HNP-1 coated with liposome increased, the bactericidal activity increased, showing significant drug concentration-bactericidal activity. The dose-effect relationship.
  • Example 15 HNP-1 kills resistant Acinetobacter baumannii

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Abstract

一种重组人HNP-1蛋白，通过构建并使用大肠杆菌表达体系制备具有生物活性的成熟人HNP-1蛋白，所述重组人HNP-1蛋白具有抗菌活性，即利用原核体系表达出具有活性的HNP-1蛋白。

Description

生物活性重组人HNP-1蛋白及应用 技术领域
本发明涉及基因工程，具体涉及利用原核表达体系表达有生物活性的重组人HNP-1蛋白(人α-防御素-1)。
本发明还涉及所述重组人HNP-1蛋白的应用。
背景技术
抗生素的广泛滥用使得细菌针对抗生素的抗性成为细菌感染的新的重大威胁(Sommer MOA,Munck C,Toft-Kehler RV,& Andersson DI,Prediction of antibiotic resistance:time for a new preclinical paradigm？Nature Reviews.Microbiology,2017,15(11):689-696)。人类面临正确使用已有的抗生素或者发展新的抗生素的重大选择(Mishra B,Reiling S,Zarena D,& Wang G,Host defense antimicrobial peptides as antibiotics:design and application strategies.Current Opinion in Chemical Biology,2017,38:87-96)。抗微生物肽(Antimicrobial peptides,AMPs)即是新型抗生素的重要来源(Mishra B,Reiling S,Zarena D,&Wang G,Host defense antimicrobial peptides as antibiotics:design and application strategies.Current Opinion in Chemical Biology,2017,38:87-96)。在动物体内存在抗菌肽(cathelicidins)和防御素(defensins)等两类抗微生物肽(Zanetti M,Cathelicidins,multifunctional peptides of the innate immunity.Journal of Leukocyte Biology,2004,75(1):39-48；Ganz T,Defensins:antimicrobial peptides of innate immunity.Nature Reviews.Immunology,2003,3(9):710-720；Selsted ME &Ouellette AJ,Mammalian defensins in the antimicrobial immune response.Nature Immunology,2005,6(6):551-557)。
防御素是广泛存在于动物、植物及昆虫体内的一类小分子短肽。它能抵御细菌、真菌或病毒等微生物入侵，是生物体免疫系统的组成部分。瑞典科学家Hulmark等1975年从惜古比天蚕(Hyatophora Cecropins)蛹中诱导分离出了第一个杀菌肽Cecropin，开辟了这个新的领域(Hulmark D.Insect immunity:purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of Hyalophora cecropia.European Journal of Biochemistry,1980,106:7～16)。1980年，美国Robert Lehrer实验室从兔肺巨噬细胞中首先分离得到了一种类似的阳离子性极强的小分子抗菌肽，定名为防御素(Lehrer,R.I.& Lu,W.alpha-Defensins in human innate immunity.Immunological reviews,2012,245:84～112)。
防御素通常由29～54个氨基酸组成，包括6～8个保守半胱氨酸，可通过半 胱氨酸分子内二硫键使肽环形成反向平行的β片状或α螺旋结构。根据来源的不同，防御素可分为动物防御素、植物防御素和昆虫防御素；根据分子结构特征，动物防御素又可分为α-防御素、β-防御素和θ-防御素3种。
α-防御素最早从兔肺巨噬细胞中分离得到。这类防御素主要存在于哺乳动物有关组织和细胞中，由29～35个氨基酸组成,富含精氨酸和非常保守的6个半胱氨酸以Cys-l-Cys-6、Cys-2-Cys-4、Cys-3-Cys-5方式形成3对分子内二硫键,其中Cys-l-Cys-6对二硫键连接N端和C端的半胱氨酸形成圆形分子大环。已知兔的中性粒细胞中含有6种α-防御素,即RabNP-1、-2、-3a、-3b、-4、-5,这些防御素总量占细胞蛋白质总量的15％～20％。与兔类似，α-防御素-1(α-defensin-1，HNP-1)是人中性粒细胞的主要防御素分子。这类防御素主要存在于人中性粒细胞的溶酶体颗粒内，是迄今发现含量最多、活性最强的杀菌分子，对革兰氏阳性菌和革兰氏阴性菌以及腺病毒、流感病毒、艾滋病毒等均有很强的杀伤活性(Selsted,M.E.& Ouellette,A.J.Mammalian defensins in the antimicrobial immune response.Nature immunology,2005,6:551-557,doi:10.1038/ni1206；Lehrer,R.I.&Lu,W.alpha-Defensins in human innate immunity.Immunological reviews,2012,245:84-112,doi:10.1111/j.1600-065X.2011.01082.x)。如此广谱的抗性可能是生物长期进化过程中与疾病斗争的结果。其抗微生物的作用机理可以分为抗菌作用、抗病毒的作用、细胞毒作用、免疫调节功能四个方面。
防御素抗菌机理主要有两种假说,一种是独立膜机制，另一种是防御素结合细胞内复合物的机制。独立膜机制假说认为带正电荷的防御素与带负电荷的细菌细胞膜相互吸引，并相互结合，从而破坏了磷脂双分子层的完整性，引起靶细胞膜出现裂隙。这个抗菌过程分为三个阶段。首先通过静电吸引，防御素与靶细胞膜结合。由于防御素带正电荷，可通过静电作用与带负电荷的细菌膜脂层结合。第二步形成通道。带正电荷的防御素分子或其多聚体则与细菌质膜上带负电荷的磷脂头部和水分子相互作用，显著地增加生物膜的通透性。防御素作用于膜上，可形成稳定的多个通道。最后内容物外泄，细胞死亡。通道形成后，防御素进入细胞内的同时，其他胞外分子，如肽、蛋白质或无机离子也伴随进入，而靶细胞的盐离子和大分子等重要物质渗出，使得细胞膜的通透性及细胞能量状态受到破坏，导致细胞膜去极化，呼吸作用受到抑制以及细胞ATP含量下降,离子和大分子物质通过细菌的细胞膜,最终导致靶细胞死亡(Jurgen Harder&Jens-M Schroder,Antimicrobial Peptides：Role in Human Health and Disease,Springer,2016；Hilchie,A.L.,Wuerth,K.& Hancock,R.E.Immune modulation by multifaceted cationic host defense(antimicrobial)peptides.Nature chemical biology,2013,9:761-768)。学界普遍认为防御素通过这个机制具备广谱抗微生物活性，包括抵抗细菌、真菌和一些具有包膜的病毒(Lehrer RI,et al. Interaction of human defensins with Escherichia coli.Mechanism of bactericidal activity,The Journal of Clinical Investigation,1989,84(2):553-561；Yeaman MR,Bayer AS,Koo SP,Foss W,& Sullam PM,Platelet microbicidal proteins and neutrophil defensin disrupt the Staphylococcus aureus cytoplasmic membrane by distinct mechanisms of action,The Journal of Clinical Investigation,1998,101(1):178-187)。此外，一些防御素通过诱导T淋巴细胞和未成熟的树突状细胞的趋化性，在获得性免疫激发中还具有免疫调节剂的作用(Yang D,Chen Q,Chertov O,& Oppenheim JJ,Human neutrophil defensins selectively chemoattract naive T and immature dendritic cells,Journal of Leukocyte Biology,2000,68(1):9-14；Hancock RE,Haney EF,& Gill EE,The immunology of host defence peptides:beyond antimicrobial activity,Nature Reviews.Immunology,2016,16(5):321-334)。
防御素广泛的抗菌谱、极强的抗菌活性和独特的杀菌机制，使其有可能成为新一代抗菌药物，有助于解决细菌的耐药性和抗生素的毒副作用等问题。2008年全球首个防御素多肽Brilacidin(PMX-30063)在加拿大获得批准并进行一期临床试验。PMX-30063通过破坏细菌细胞膜而起作用。这类抗菌肽尽管研究较多，但至今尚无商业化的成功先例。
尽管HNP-1具有诱人的使用前景。直接提取法可获得防御素,但由于工艺路线复杂,成本极高,不具有应用价值(Hancock RW,Robert L.Cationic peptides:a new source of antibiotics[J].Tibtech,1998,16:82—88)。而化学合成HNP-1则由于其分子内含有3对二硫键，难以保证多肽折叠时它们的正确配对，从而影响其生物活性。因此,通过基因工程方法生产防御素成为本领域的研究热点。
在人细胞内，HNP-1首先翻译成94个氨基酸残基的preproHNP-1(包括19个氨基酸残基组成的疏水性信号序列，45个氨基酸残基组成的带负电荷的前区序列和C末端含30个氨基酸组成的带正电荷的成熟肽)。preproHNP-1进入内质网后得到含56个氨基酸残基的原防御素(proHNP-1)，再在溶酶体中经一次或多次裂解，加工成为含30个氨基酸残基的阳离子成熟肽HNP-1(Valore,E.V.&Ganz,T.Posttranslational processing of defensins in immature human myeloid cells.Blood,1992,79,1538-1544；Wu,Z.et al.From pro defensins to defensins:synthesis and characterization of human neutrophil pro alpha-defensin-1and its mature domain.The journal of peptide research,2003,62:53-62)。
由于防御素本身具有毒性，目前的研究多集中在对表达载体的改造上，试图采用融合表达的策略。因为成熟的HNP-1所携带的正电性是杀灭微生物的化学基础，因此通过融合表达带负电荷的多肽，中和其正电性以降低杀菌活性，从而高效表达HNP-1。Piers试图通过融合蛋白途径在细菌中表达人的防御素 HNP-1,但得到的HNP-1没有生物学活性(Piers K L,Brown M H,Robert E.W.Recombinant DNA procedures for producing small antimicrobial cationic peptides in bacteria,Gene,1993,134:7—13)。徐志南等国内多个实验室通过融合蛋白途径在大肠杆菌中表达β-人防御素-谷胱甘肽转移酶(GST)融合蛋白,但该重组菌株不仅GST-β-人防御素的表达量很低，融合蛋白本身尚不知是否有活性(徐志南,彭力,方向明等，β-人防御素-2在大肠杆菌中的重组表达及优化，精细与专用化学品,2002,(增刊):39-42；李霞,沈昕,王义琴等，利用自剪切融合蛋白在大肠杆菌中表达并纯化兔防御素，高技术通讯,2005,15(4):67-70)。张满朝等利用pGT-2、pΩ等表达载体调查了prepro-HNP-1的cDNA和部分AFP的融合蛋白以及部分pro序列和完整的HNP-1的表达情况，Tricine-SDS-PAGE显示所构建的部分大肠杆菌可能可以诱导表达prepro-HNP-1。尽管作者描述依稀看到3.4kD大小的条带，但没能显示可见的结果。作者也没能观察到成熟HNP-1应该具有的杀菌特性，表明没有产生活性HNP-1(张满朝,郑国,许政凯等，在大肠杆菌中表达人防御素-1，生物化学杂志,1997,13(3):264-269)。胡云龙等采用核酸定向点突变技术将Cecropin B突变体编码基因与pGEX-24T表达载体中的谷胱甘肽转移酶(GST)基因融合,在大肠杆菌中可进行表达，但没有得到Cecropin B活性肽本身(胡云龙,胡泰山.Cecropin B抗菌肽基因的定向诱变与表达，药物生物技术,1999,6(4):193～197)。
一般认为，造成融合表达效率不高的原因是前体蛋白也具有一定的杀菌效果(Schneider,J.J.,Unholzer,A.,Schaller,M.,Schafer-Korting,M.&Korting,H.C.Human defensins.Journal of molecular medicine,2005,83:587-595,doi:10.1007/s00109-005-0657-1；Jenssen,H.,Hamill,P.& Hancock,R.E.Peptide antimicrobial agents.Clinical microbiology reviews,2006,19:491-511,doi:10.1128/CMR.00056-05)。包涵体形式可生成HNP-1，但在纯化中很难使HNP-1形成正确的折叠(Pazgier,M.& Lubkowski,J.Expression and purification of recombinant human alpha-defensins in Escherichia coli.Protein expression and purification,2006,49:1-8,doi:10.1016/j.pep.2006.05.004；Tongaonkar,P.,Golji,A.E.,Tran,P.,Ouellette,A.J.& Selsted,M.E.High fidelity processing and activation of the human alpha-defensin HNP1 precursor by neutrophil elastase and proteinase 3.PloS one,2012,7,e32469,doi:10.1371/journal.pone.0032469；Wilson,C.L.et al.Differential Processing of{alpha}-and{beta}-Defensin Precursors by Matrix Metalloproteinase-7(MMP-7).The Journal of biological chemistry,2009,284:8301-8311)。Chen等用电激法将兔防御素基因成功地导入小球藻细胞中,是至今为止唯一有效的人工重组兔防御素基因工程细胞株。体外离体抑菌实验证明兔防御素基因已稳定整合到小球藻基因组中,并进行了正确转录和表达(Chen Y, Wang Y Q,Sun Y R,et al.Highly efficient expression of rabbit neutrophil peptide-1gene in Chlorella ellipsoidea cells,Curr.Genet,2001,39:365—370)。但小球藻发酵后生产的兔防御素纯化难，目前停留在饲料添加剂的使用上。综上，国内外尚无改造大肠杆菌宿主直接高效表达活性HNP-1的研究报道。
在研究内皮细胞HNP-1前体蛋白激活机制时，有研究认为在HNP-1的成熟加工过程中，前区序列不仅中和成熟HNP-1所带的正电荷，封闭加工过程中出现的细胞毒性，而且对于HNP-1的正确折叠具有重要作用(Wu,Z.et al.Impact of pro segments on the folding and function of human neutrophil alpha-defensins.Journal of molecular biology,2007,368:537-549)。尽管HNP-1的原核高效表达一直是研究热点和难点问题，但我们从上述研究中获得启示，即大肠杆菌可能也存在与人体相似的HNP-1激活机制，前体蛋白具有一定的杀菌能力是假象，而是通过激活的成熟HNP-1起作用。
发明内容
本发明的目的在于提供一种具有抗菌活性的重组人HNP-1蛋白。
本发明的另一目的在于提供上述重组人HNP-1蛋白的应用。
根据本发明的一个方面，本发明所述重组人HNP-1蛋白是通过用大肠杆菌表达体系表达人防御素HNP-1前体蛋白，并以异丙基-β-D-1-硫代吡喃半乳糖苷诱导产生的成熟人HNP-1蛋白，所述人HNP-1蛋白具有抗菌活性。
本发明表达成熟人HNP-1蛋白的大肠杆菌表达体系包括大肠杆菌表达载体和可操作地连接于所述表达载体的HNP-1前体蛋白编码基因。所述编码人防御素HNP-1前体蛋白基因序列来源于人基因组序列(NM_004084.3)，包括信号肽、前肽和成熟HNP-1三部分。进一步在其N末端引入6×His标签，得到编码100个氨基酸的开放阅读框(ORF)，其编码序列和对应的氨基酸序列如图1所示。
用于大肠杆菌表达体系的大肠杆菌表达载体可以是常用的各种大肠杆菌表达载体，这些表达载体包括但不限于：pET系列载体、pGEX系列载体、pTWIN1载体、pET-28a(+)载体等，优选的大肠杆菌表达载体可以为pET-28a(+)载体。
根据本发明的一种较佳的具体实施方式，本发明所述的大肠杆菌表达体系的构建过程包括，将HNP-1的前体蛋白编码基因序列插入pET-28a(+)载体的EcoR I和Xho I酶切位点间，得到HNP-1前体蛋白重组表达载体，通过化学转化法，将所述表达载体导入大肠杆菌BL21(DE3)感受态细胞中，得到含有HNP-1前体蛋白基因的重组菌株。
利用上述大肠杆菌表达体系制备具有生物活性的成熟人HNP-1蛋白的方法包括下述步骤：
1)将所述大肠杆菌表达体系导入大肠杆菌宿主，获得含有HNP-1前体蛋白 基因的重组菌株；
2)活化并培养所述重组菌株；
3)向步骤2)的培养液中加入异丙基-β-D-1-硫代吡喃半乳糖苷诱导培养后，收集菌体；
4)从所述菌体中分离并纯化成熟人HNP-1蛋白。
在本发明所述方法中，异丙基-β-D-1-硫代吡喃半乳糖苷(IPTG)的加入可诱导大肠杆菌产生成熟的HNP-1蛋白，诱导培养的时间优选为0.5-3小时，优选条件为，当接种后菌液OD 600生长到0.4-0.6(以LB液体培养基为空白对照)时，加入终浓度为1mM异丙基-β-D-1-硫代吡喃半乳糖苷(IPTG)，诱导培养3h。诱导培养参数：培养温度为37℃，摇床转动速度为220rpm。
鉴于成熟HNP-1无纯化标签，且需要在纯化过程中维持其抗菌活性，只能在一些温和的天然条件下进行纯化，本发明使用过滤技术，截留大分子蛋白质，从而分离纯化，获得成熟的小分子量的HNP-1蛋白。
根据本发明的另一方面，提供所述重组人HNP-1蛋白在制备抗菌药物中的应用。可以将本发明的重组蛋白添加药学上可接受的辅料制备成抗菌药物。更优选的是，将本发明的重组人HNP-1蛋白制备成脂质体包被的药物形式，可提高杀菌效果。
根据本发明的又一方面，本发明的重组人HNP-1蛋白更对耐药菌具有抗菌作用，优选的耐药菌类型为耐药金黄色葡萄球菌，耐药肺炎克雷伯氏菌或耐药鲍曼不动杆菌,更优选的是耐甲氧西林金黄色葡萄球菌。
有益效果：HNP-1是一种由preproHNP-1在内质网和溶酶体中经过裂解后才能得到的阳离子成熟肽。而在原核体系表达中，由于认为缺乏使得preproHNP-1裂解产生成熟HNP-1的机制，通常设计带负电荷的多肽与带正电荷的HNP-1融合表达，再进而剪切融合蛋白得到HNP-1。但目前现有技术没有原核体系能表达出具有生物活性的成熟HNP-1。
本发明构建了表达有生物学活性的成熟人HNP-1蛋白的大肠杆菌表达体系，并建立了制备人HNP-1蛋白的方法，成功地表达并制备出了有活性的HNP-1蛋白。
附图说明
图1：编码人防御素HNP-1前体蛋白基因序列及对应的氨基酸序列；
灰色阴影部分为该蛋白成熟过程中的切除部分，即成熟HNP-1；双实线部分为引入的6×His标签；虚线部分为信号肽；实线部分为该蛋白的前体部分即pro HNP-1。
图2：重组大肠杆菌BL21-preproHNP-1菌株的诱导表达。
图3：重组preproHNP-1和成熟HNP-1的Tricine凝胶电泳分析图；
泳道M为分子量为3.4-100kD的蛋白质分子量标准，泳道-IPTG、+IPTG分别为未诱导和诱导3小时后菌体裂解上清液。
图4：成熟HNP-1的诱导表达；
泳道M为分子量标准，泳道30,90,180分别为IPTG诱导30分钟，90分钟和180分钟后的诱导产物，在3.4kDa分子量大小的位置显示成熟HNP-1的量。
图5：靶向定量蛋白质组分析IPTG诱导表达的成熟HNP-1的量，(*,p<0.05)；
横坐标为诱导时间(分钟)，纵坐标为相对量。
图6：PreproHNP-1随IPTG诱导时间延长而变化；诱导时间表示于图中。
图7：IPTG诱导表达PreproHNP-1后细菌菌落数的变化。
IPTG诱导的时间如图中所示，活菌数目由培养液在LB固体培养基上的生长16小时的菌落数决定，以平均值±方差(mean±SD)表示(**,p<0.01)。
图8：纯化的重组prepro HNP-1His亲和层析Tricine凝胶电泳分析图；
泳道M为分子量为3.4-100kD的蛋白质分子量标准，泳道TCL、SN、IS、wash1、wash2、ET1、ET2分别为总细胞裂解物、菌体裂解上清液、不可溶蛋白、洗涤1、洗涤2、洗脱样品1和洗脱样品2。
图9：重组成熟HNP-1纯化装置图；
向内管中加入蛋白上清液，5,000g，室温离心10分钟，收集滤液，重复上述步骤4次。
图10：纯化的成熟重组HNP-1的Tricine凝胶电泳分析图；
泳道M为分子量为3.4-100kDa的蛋白质分子量标准，泳道TCL为经IPTG诱导3小时后菌体裂解上清液，泳道purified protein为经过超速离心技术所得滤液。
图11：重组成熟HNP-1的Tricine凝胶电泳分析图；
泳道M为分子量为3.4-100kDa的蛋白质分子量标准，泳道-IPTG、+IPTG分别为未诱导和诱导3小时后菌体裂解上清液。
图12：重组成熟HNP-1的质谱鉴定谱图；
(A)肽段IPACIAGER的MS 2质谱图；(B)肽段YGTCIYQGR的MS 2质谱图。
图13：重组HNP-1及其标准品的质谱Topdown检测；
(A)重组HNP-1；(B)HNP-1的Sigma标准品。
图14：纯化HNP-1的抗菌活性；
大肠杆菌XPX-1以1mM IPTG诱导表达PreproHNP-1，并产生成熟HNP-1的生长曲线为阳性对照，大肠杆菌XPX-1没有IPTG诱导的生长曲线为阴性对照；纯化的PreproHNP-1和成熟HNP-1的浓度为40μg/mL。
图15：内源成熟活性HNP-1的抗菌活性定量蛋白质组研究。
图16：定量蛋白质组学比较鉴定上调蛋白在Gene Ontology分析中的过程分布。
图17：定量蛋白质组学比较鉴定上调蛋白在内切核酸酶富集。
图18：定量蛋白质组学比较鉴定下调蛋白在Gene Ontology分析中的过程分布。
图19：定量蛋白质组学比较鉴定下调蛋白在NAD(P)H反应酶富集。
图20：TUNEL分析IPTG诱导XPX-1表达成熟HNP-1 3小时细胞，数值以mean±SD表示,**,p<0.01。
图21：膜联蛋白V染色分析，数值以mean±SD表示,**,p<0.01。表达perproHNP-1的XPX-1和仅由对照质粒的XPX-2经1mM IPTG分别诱导3小时。10μg/mL庆大霉素作为引发细胞凋亡的阳性对照。
图22：Tris-Tricine gel和银染分析非变性条件下纯化的HNP-1。
Wash1,20mM imidazole buffer.Wash 2,40mM imidazole buffer.Elution,250mM imidazole buffer。
图23：HNP-1相互作用蛋白的分部分析，实验包括生物学重复。HNP-1本身和高富集的RecA分别用箭头标记。
图24：组氨酸标签融合的HNP-1和RecA互作。HNP-1通过6组氨酸标签用镍NTA填料纯化，SDS-PAGE凝胶电泳分离后用RecA抗体(Abcam,Cambridge,United Kingdom)进行杂交。
图25：成熟HNP-1而非全长的6组氨酸标签的组氨酸标签融合的preproHNP-1和RecA直接互作。
在图示的时间点收集表达HNP-1的细胞，RecA用抗RecA抗体进行亲和纯化，并用抗HNP-1抗体(Santa Cruz Biotechnology,Dallas,TX,USA)进行蛋白印迹分析。
图26：HNP-1抑制单链激活RecA的ATP酶活性。ATP酶活性利用孔雀石绿磷酸反应测定所释放的无机磷酸盐的量来检测。实验重复3次(*,p<0.05),横坐标为HNP-1浓度，纵坐标为游离磷酸浓度(μM)。
图27：HNP-1抑制RecA的单链DNA结合。RecA结合单链DNA和自由单链DNA用琼脂糖凝胶电泳迁移实验分析。
图28：脂质体包被HNP-1的粒径分布。粒径分析利用Zetasizer Nano ZS(Malvern,UK)完成。
图29：HNP-1通过细胞凋亡杀灭耐甲氧西林金黄色葡萄球菌。
A.不同浓度成熟HNP-1对耐甲氧西林金黄色葡萄球菌的杀菌效果(*,p<0.05；**,p<0.01)；B.HNP-1处理3小时后耐甲氧西林金黄色葡萄球菌TUNEL染色阳 性菌百分数分布(mean±SD,**,p<0.01)；C.HNP-1处理3小时后耐甲氧西林金黄色葡萄球菌annexin V标记阳性菌百分数分布(mean±SD,**,p<0.01)。
图30：脂质体包被HNP-1杀灭肺炎小鼠模型耐甲氧西林金黄色葡萄球菌(MRSA)。MRSA分离自患者肺灌洗液，小鼠分成7组，分别是(a)健康对照小鼠组(Health)；(b)MRSA诱导肺炎模型小鼠(MRSA)；(c)12μg/mL青霉素治疗MRSA诱导肺炎模型小鼠组，作为MRSA的青霉素抗性菌株的对照(PCN)；(d)肺炎模型小鼠0.1mL 20μg/mL HNP-1治疗组(HNP-1)；(e)肺炎模型小鼠空白脂质体处理对照组(Lipo)；(f)肺炎模型小鼠0.1mL 5μg/mL脂质体包被HNP-1治疗组(5Lipo-H)；(g)肺炎模型小鼠0.1mL 10μg/mL脂质体包被HNP-1治疗组(10Lipo-H)；实验重复3次(**,p<0.01)。
图31：脂质体包被HNP-1杀灭耐药肺炎克雷伯氏菌。耐药肺炎克雷伯氏菌菌株号为ATCC BAA2146，由国家疾病预防控制中心提供。实验分为6组，分别是(a)12μg/mL青霉素杀灭耐药肺炎克雷伯氏菌组，作为耐药肺炎克雷伯氏菌的青霉素抗性菌株的对照(PEN)；(b)耐药肺炎克雷伯氏菌16μg/mL HNP-1杀菌实验组(HNP-1(16μg/mL))；(c)耐药肺炎克雷伯氏菌32μg/mL HNP-1杀菌实验组(HNP-1(32μg/mL))；(d)耐药肺炎克雷伯氏菌16μg/mL脂质体包被HNP-1杀菌实验组(Lipo-H(16μg/mL))；(e)耐药肺炎克雷伯氏菌32μg/mL脂质体包被HNP-1杀菌实验组(Lipo-H(32μg/mL))；(f)耐药肺炎克雷伯氏菌64μg/mL脂质体包被HNP-1杀菌实验组(Lipo-H(64μg/mL))。实验重复3次(**,p<0.01)。
图32：脂质体包被HNP-1杀灭耐药鲍曼不动杆菌。耐药鲍曼不动杆菌菌株号为NO801，由国家疾病预防控制中心提供。实验分为6组，分别是(a)12μg/mL青霉素杀灭耐药鲍曼不动杆菌组，作为耐药鲍曼不动杆菌的青霉素抗性菌株的对照(PEN)；(b)耐药鲍曼不动杆菌16μg/mL HNP-1杀菌实验组(HNP-1(16μg/mL))；(c)耐药鲍曼不动杆菌32μg/mL HNP-1杀菌实验组(HNP-1(32μg/mL))；(d)耐药鲍曼不动杆菌16μg/mL脂质体包被HNP-1杀菌实验组(Lipo-H(16μg/mL))；(e)耐药鲍曼不动杆菌32μg/mL脂质体包被HNP-1杀菌实验组(Lipo-H(32μg/mL))；(f)耐药鲍曼不动杆菌64μg/mL脂质体包被HNP-1杀菌实验组(Lipo-H(64μg/mL))。实验重复3次(**,p<0.01)。
具体实施方式
下面结合具体实施方式对本发明做进一步说明，但不限制本发明权利要求范围。本发明所用试剂均为市售。
实施例1：preproHNP-1大肠杆菌表达载体的构建
本发明所述的编码人防御素HNP-1前体蛋白基因序列来源于人基因组序列 (NM_004084.3)，包括信号肽、前肽和成熟HNP-1三部分。我们在其N末端引入6×His标签，得到编码100个氨基酸的开放阅读框(ORF)，其编码序列和对应的蛋白质序列如图1所示，其中编码碱基序列如SEQ NO.1所示，蛋白质序列如SEQ NO.2所示。
将该序列SEQ NO.1插入pET-28a(+)载体(Novagen，货号69864)的EcoR I和Xho I酶切位点间，得到HNP-1前体蛋白重组表达载体，命名为pET-preproHNP-1。通过化学转化法，将pET-preproHNP-1导入大肠杆菌BL21(DE3)感受态细胞中，得到含有HNP-1前体蛋白基因的重组菌株，将该菌株命名为重组大肠杆菌BL21(DE3)-preproHNP-1(下文中简称BL21-preproHNP-1菌株)。
实施例2：BL21-preproHNP-1菌株的培养和收集
将重组菌株BL21-preproHNP-1菌株在含有卡那霉素的固体LB平板上活化，获得BL21-preproHNP-1菌株的单克隆。将BL21-preproHNP-1菌株的单克隆接种于5mL LB液体培养基中(卡那霉素的浓度为50μg/mL)，37℃摇床震荡过夜培养，得到BL21-preproHNP-1菌株的一级种子。将BL21-preproHNP-1菌株的一级种子以1：100的比例稀释，接种到含有50μg/mL卡那霉素的新鲜LB液体培养基。接种后的培养参数：培养温度为37℃，摇床转动速度为220rpm。当接种后菌液OD 600生长到0.4-0.6(以LB液体培养基为空白对照)时，加入优化的终浓度为1mM异丙基-β-D-1-硫代吡喃半乳糖苷(IPTG)(见表1)，诱导培养3h。诱导培养参数：培养温度为37℃，摇床转动速度为220rpm。采用水平转子对IPTG诱导3h后的BL21-preproHNP-1菌株培养液在3,000g条件下离心10min，收集菌体，得到BL21-preproHNP-1菌体。
表1.诱导prepro-HNP1的异丙基-β-D-1-硫代吡喃半乳糖苷(IPTG)浓度优化
Figure PCTCN2019085130-appb-000001
由图2可知，加入IPTG诱导后，大肠杆菌的生物量的增加幅度减少，表明营养流向胞内产物的表达，或是出现菌体毒性作用，可能出现具有活性的HNP-1蛋白。
实施例3：重组表达成熟HNP-1蛋白质的检测
取50OD 600上述菌体重悬于PBS缓冲液，用移液枪吹吸混匀。菌体混悬液以超声破碎仪(JY92-IIN，宁波新芝)裂解。超声条件设定为功率为25％，超声3秒停3秒，共5分钟。破碎后的菌体经10,000g，4℃离心20min，收集上清液，取50微克蛋白上样，以12％的Tricine胶分离{其中有5％浓缩胶：蒸馏水2mL，3×凝胶缓冲液(36.3g Tris base，0.3g SDS，蒸馏水定容至100mL，HCl调节pH为8.45)1.33mL，30％丙烯酰胺0.67mL，10％过硫酸铵40uL，四甲基乙二胺(TEMED)4uL；和12％分离胶：蒸馏水6.4mL，3×凝胶缓冲液5mL，甘油1.5g，49.5％丙烯酰胺3.6mL，10％过硫酸铵150uL，TEMED10uL}。起始电压40V进行电泳，待样品进入分离胶后，电压改为200V，电泳2h。
Tricine胶经蒸馏水漂洗，固定液(50％甲醇，10％乙酸，蒸馏水)固定10分钟，考马斯亮蓝G250染色1h，脱色后用扫描仪扫描保存图像。Tricine胶胶图结果显示，与未加IPTG诱导组相比，IPTG诱导后，在14.8kDa和3.4kDa处产生两条明显的条带(图3)，分别与prepro HNP-1和mature HNP-1的理论分子量相符，推测这两条带分别为prepro HNP-1和mature HNP-1。
实施例4：IPTG诱导时间的研究
利用Tris-Tricine gel和考马斯亮蓝染色检测IPTG诱导表达preproHNP-1产物，发现随着IPTG诱导时间的延长，在3.4kDa分子量大小的位置成熟HNP-1的量逐渐增加(图4)，表明成熟HNP-1可被IPTG有效诱导表达。
进而利用靶向定量蛋白质组技术定量了成熟HNP-1的含量，发现IPTG诱导表达3小时的成熟HNP-1比诱导0.5小时的增加了2倍(图5)。
与此相反，发现IPTG诱导1.5小时后preproHNP-1的量逐渐减少(图6)，表明成熟的HNP-1来自IPTG诱导表达的preproHNP-1。
进而研究了存活的宿主大肠杆菌数目随IPTG诱导时间变化而变化的情况，发现随着IPTG诱导时间延长，培养液中活菌数目显著减少(图7)。这种成熟HNP-1与培养液中活菌数目负相关表明成熟HNP-1具有抗菌活性。
实施例5：重组表达preproHNP-1蛋白质的纯化
取200g上述菌体按照1:10(w/v)的比例重悬于20mM Tris pH 7.5，150mM NaCl缓冲液，用高速组织剪切机混合均匀，高压匀浆破碎(APV-1000匀浆破碎仪，SPX FLOW,Inc)。高压匀浆压力设定800bar，连续破碎3次后10,000g，4℃离心30min，收集上清液在AKTA Purifier(GE healthcare)上进行金属鳌和亲和层析(HisTrap FF 5mL预装柱，GE healthcare，货号17-5255-01)。
层析柱用缓冲液A(20mM Tris,150mM NaCl,pH 7.5)预先平衡后进行样品上样。上样流速5mL/min。上样结束后，用10个柱体积缓冲液A和10个柱体积5％缓冲溶液B(20mM Tris,100mM NaCl,0.5M imidazole,pH 7.5)洗脱杂蛋白，然后用15个柱体积的5-80％缓冲溶液B线性梯度洗脱。所有纯化工作均在20-25℃进行。将纯化的preproHNP-1溶液置于4℃保存(图8)。
实施例6：重组成熟HNP-1蛋白质纯化
高效液相色谱是蛋白质纯化的常用技术。我们根据成熟HNP-1含有较多碱性氨基酸的特性，尝试了离子交换色谱和分子筛的串联纯化分析技术，但没有得到SDS-PAGE凝胶电泳可见的成熟HNP-1条带，转而尝试其它分离方法。
由于成熟HNP-1无纯化标签，而且还需要在纯化过程中维持其抗菌活性，只能在一些温和的天然条件下进行纯化。基于Tricine胶分析，发现成熟HNP-1(3.4kDa)的分子量远小于大肠杆菌本身表达的蛋白质分子量。蛋白质组学分析发现，从IPTG诱导的BL21-prepro-HNP1菌株总细胞蛋白中和成熟HNP-1分子量接近的蛋白少。因此，我们试图通过过滤技术，截留大分子量蛋白质，从而分离纯化获得成熟的小分子量HNP-1。
取50 OD 600经IPTG诱导后的菌体，重悬于PBS缓冲液中，用移液枪吹吸混匀。菌体混悬液以实施例3所述的超声破碎仪和破碎条件裂解。菌体破碎后10,000g，4℃离心20min，收集上清液。选用Amicon Ultra-0.5(3kDa，MWCO，Merckmillipore，货号：UFC500396)超滤离心管进行分离纯化。
将Amicon Ultra-0.5超滤内管插入到微量离心管中，向内管中加入400μL双蒸水，5,000g，室温离心10分钟，进行超滤管预清洗，重复3次。再向内管中加入400uL PBS，5,000g，室温离心10分钟，进行滤膜活化，重复3次。向Amicon Ultra内管中加入不超过500μL的蛋白上清液，5,000g，室温离心10分钟。收集过滤液至一新的Eppendorf管内，重复上述步骤4次，合并过滤液(图9)。取50μL滤液蛋白进行上述相同的12％的Tricine胶分析，考马斯亮蓝G250染色，脱色。结果显示与IPTG诱导后所得的全蛋白相比，通过此种超滤离心的方法可纯化得到足量纯化的成熟HNP-1(图10)。
实施例7：重组成熟HNP-1的鉴定
7.1 HNP-1的Bottom-up质谱分析
IPTG诱导的总细胞蛋白进行12％的Tricine胶分离，得到条带分子量大小为3.4kDa的成熟HNP-1(图11)。切取目的条带(图11黑框标注)，切成1mm 3的胶粒。加入胶粒脱色液(30％乙腈，7％碳酸氢铵，蒸馏水)，涡旋混合仪上缓慢涡旋，当胶粒中考马斯亮蓝脱尽，胶粒变为无色时，弃去脱色液。加入乙腈， 涡旋混合仪上缓慢涡旋，弃去上清后再次加入乙腈，直到胶粒中液体脱尽，胶粒完全变硬。胶粒真空干燥40分钟后，加入终浓度为10ng/μL胰蛋白酶消化液使胶粒重新溶胀，至胶粒略被酶解液覆盖。37℃放置12-14小时。
蛋白样品消化完毕后，13,300rpm离心1分钟，将上清转移至新的Eppendorf管，做好标记。加入40μL抽提液(5％甲酸，50％乙腈，蒸馏水)，轻弹管底部进行混匀，终止消化。13,300rpm离心1分钟，静置4分钟，重复4次；13,300rpm离心1分钟，取出上清，加入到相对应的Eppendorf管。重复3次抽提液，离心静置操作后，加入30μL乙腈，重复离心静置的操作，离心后取出上清，继续加入到相对应的管中。重复乙腈抽提的操作，直至胶粒变为硬的颗粒状。将抽提得到的肽段溶液13,300rpm离心3分钟，转移至新的Eppendorf管。真空干燥仪进行干燥，液体完全蒸干后，产物即为肽段混合物干粉。
消化后的肽段使用1％乙腈，1％甲酸，98％水的质谱上样缓冲液进行溶解，Waters公司的超高压液相色谱(nanoAcquity Ultra Permance LC,Waters)进行分离，其中分离柱采用内径75μm，长15cm的C 18反相色谱分析柱。自动进样器自动进样，上样体积为3μL，上样流速为300nL/min。在线液相分离60分钟，液相梯度从98％A相(2％ACN+0.1％FA)到35％B相(100％ACN+0.1％FA)。
洗脱下来的组分经过纳喷离子源直接进入质谱检测。质谱使用Thermo公司的LTQ Orbitrap Velos高分辨质谱。质谱数据采用一级质谱数据的二级质谱扫描模式(Data Dependent MS/MS Scan，DDA)模式采集，质谱扫描离子质荷比范围为300-1600(m/z)，一级母离子扫描在Orbitrap内完成，全扫描分辨率设置为30,000，离子自动增益控制(Automatic Gain Control，AGC)设置1×10 6个离子，最大离子注入时间(Max injection time，MIT)设置为150ms；二级谱图采用CID碎裂模式，归一化碰撞能量为35％。选取丰度最高且电荷>1的前20个离子进行二级(MS/MS)碎裂，AGC设置为1×10 4，MIT设置为25ms。动态排
产生的质谱数据使用软件MaxQuant(1.5.6.0)进行鉴定。从UniProt(2016.01版本)获得的目标大肠杆菌菌株K12完整蛋白质组序列加上6x his标签的preproHNP-1(Uniprot ID：59665)和常见污染。蛋白鉴定参数设置如下：半胱氨酸上设置Carbamidomethylation为固定修饰，蛋氨酸氧化设置为可变修饰。全胰蛋白酶酶切，最多允许两个漏切。母离子误差搜库时设定20ppm，二级碎片离子误差为0.5Da，肽段最小长度为7个氨基酸，肽段和蛋白的鉴定假阳性率(false discovery rate，FDR)控制在1％以下。
成熟HNP-1经胰蛋白酶完全酶切后可产生IPACIAGER(SEQ NO.3)和YGTCIYQGR(SEQ NO.4)两个理论肽段。质谱鉴定结果显示，肽段IPACIAGER在15.43分钟的保留时间内被洗脱出来，得到+2价的母离子，信号强度为769,620，经CID碎裂后，得到7对连续的b、y离子，MS 2匹配较好(图12A)， 共得到1张谱图；肽段YGTCIYQGR在16.15分钟的保留时间内被洗脱出来，得到+2价的母离子，信号强度为438,830，得到7对连续的b、y离子，MS 2匹配较好(图12B)，共得到1张谱图。质谱结果表明，在3.4kDa条带位置能够鉴定到成熟HNP-1理论酶切肽段。
7.2 HNP-1的Topdown质谱分析
为进一步确认目的蛋白的确切分子量大小，对成熟HNP-1进行了Topdown分析。IPTG诱导的总细胞蛋白在12％Tricine胶上电泳分离，利用考马斯亮蓝G250染色，将分子量大小为3.4kDa位置处的条带切成1mm 3的胶粒，脱至无色。加入液氮，研磨成粉末状胶粒，离心浓缩干燥。
干燥后的胶粒直接加入40μL抽提液(5％FA+50％ACN，即甲酸和乙腈)进行蛋白抽提，抽提步骤同上文。抽提步骤完成后，加入30μL乙腈，重复离心静置操作，取出上清，加入到相应的Eppendorf管中；重复乙腈抽提操作，直至胶粒变成硬的颗粒状。将抽提得到的溶液13,300rpm离心1分钟，转移至新的Eppendorf管中，真空干燥仪进行干燥得到分离纯化的蛋白质。
将上述所得蛋白质通过上文相同的质谱上样缓冲液(1％乙腈，1％甲酸，98％水)溶解。利用相同的LC-MS/MS平台检测。使用60分钟的液相梯度洗脱，梯度从4％A相(0.1％乙酸,2％乙腈)至35％B相(0.1％乙酸,100％乙腈)，流速为0.3μL/min。一级母离子扫描在Orbitrap内完成，全扫描分辨率设置为30,000，离子自动增益控制设置1×10 6个离子，最大离子注入时间(Max injection time，MIT)设置为150ms。离子强度最高的3个离子在m/z为2的窗口内被隔离，在32-34％的碰撞能量下被碎裂，AGC设置为1x10 4，MIT设置为25ms。根据电荷状态，衍生质量和液相保留时间手动分配前体离子峰。
质谱结果显示，在36.22分钟的保留时间鉴定到一个洗脱峰，分子量为3445.556Da，与HNP-1 3445.558Da的理论分子量仅相差0.002Da，误差仅0.58ppm(图13A)，表明我们鉴定得到了成熟的HNP-1。
为进一步证实得到成熟HNP-1，从Sigma购买了HNP-1成熟肽序列的标准品(货号：d2043)并以同样的方式进行Topdown分析。结果如图13B所示，在35.40分钟鉴定到了一个洗脱峰，其保留时间与我们纯化得到的HNP-1基本一致。该峰的实验分子量为3445.554Da，和其理论分子量仅相差0.004Da，也和我们鉴定到的纯化HNP-1的分子量基本一致，进一步证明BL21-prepro HNP-1确实经IPTG诱导表达了成熟的HNP-1。
实施例8：重组成熟HNP-1抗菌活性测定
当BL21-preproHNP-1菌株培养至OD 600为0.4-0.6后，将上述纯化所得的Prepro HNP-1和成熟HNP-1分别加入细菌培养基中，无IPTG诱导，测定细菌 的生长曲线。结果表明无IPTG诱导的菌生长正常；施加纯化所得的prepro HNP-1对应的生长曲线与没有IPTG诱导的正常生长培养基(-IPTG)几乎相同；菌体加入纯化的成熟HNP-1后菌体生长受到明显抑制(图14)。这些结果显示纯化的成熟HNP-1具有抗菌活性，而preproHNP-1没有抗菌活性。
利用纯化的成熟HNP-1和PreproHNP-1比较了抗菌活性，测定了生长曲线(图14)。发现即使在较高浓度(40μg/mL)纯化preproHNP-1的情况下，大肠杆菌XPX-1的生长也和没有IPTG诱导时一致，没有受到任何影响，表明preproHNP-1没有抑菌活性。使用40μg/mL的纯化HNP-1，生长曲线在达到平台期以后不再增长，甚至略有下降，表明在非变性条件下纯化的HNP-1具有一定的抗菌活性。但其抑制效果不如使用IPTG诱导表达preproHNP-1并产生细胞内成熟HNP-1的抑菌效果明显。这种内源性成熟HNP-1和外加非变性条件下纯化的成熟HNP-1在抑菌效果上显著的差异表明HNP-1可能不是经由原先认为的穿膜破坏细胞(Lehrer RI,et al.,Interaction of human defensins with Escherichia coli.Mechanism of bactericidal activity,The Journal of Clinical Investigation,1989,84(2):553-561；Yeaman MR,Bayer AS,Koo SP,Foss W,& Sullam PM,Platelet microbicidal proteins and neutrophil defensin disrupt the Staphylococcus aureus cytoplasmic membrane by distinct mechanisms of action,The Journal of Clinical Investigation,1998,101(1):178-187)，导致内容物渗漏这一机制杀菌的，而可能具有不同的抑菌机制。
我们也比较了Sigma等多家公司来源的商品化化学合成HNP-1，发现尽管氨基酸序列完全正确，但只有Sigma来源的HNP-1具有相当的生物学活性。
实施例9：定量蛋白质组学揭示成熟HNP-1引发细菌细胞凋亡的抗菌新机制
采用定量蛋白质组学技术比较大肠杆菌XPX-1在有和无IPTG诱导条件下细胞蛋白质组的变化，旨在阐述内源性活性HNP-1的杀菌机制(图15)。来自1mM IPTG诱导和没有IPTG诱导的等量总细胞蛋白经质检、胰蛋白酶消化后得到肽段，并经非标定量蛋白质组分析。
共鉴定了1559个蛋白，其中1431个蛋白在两个样品中均有定量信息。以p-value<0.05为卡值，共鉴定到152个差异蛋白，其中66个在IPTG诱导后上调，86个下调。Gene Ontology分析显示上调蛋白主要和水解酶和核酸结合蛋白关联(图16)，特别是内切核酸酶相关(图17)；下调蛋白主要和氧化还原酶活性(图18)，特别是NAD(P)H反应的酶相关(图19)。
先前的研究表明羟基自由基可损伤DNA和蛋白质，对细胞具有较强的毒性，并由此导致细胞凋亡(Kohanski MA,Dwyer DJ,Hayete B,Lawrence CA,& Collins JJ,A common mechanism of cellular death induced by bactericidal antibiotics.Cell,2007,130(5):797-810；Dwyer DJ,Camacho DM,Kohanski MA,Callura JM,&Collins JJ,Antibiotic-induced bacterial cell death exhibits physiological and biochemical hallmarks of apoptosis,Molecular Cell,2012,46(5):561-572)。因此猜测成熟的具有生物活性的HNP-1可能引发细菌的细胞程序性死亡或凋亡。
基因组DNA片段化是细胞凋亡的一个主要特征。利用TUNEL技术(terminal deoxynucleotidyl transferase(TdT)-mediated dUTP-biotin nick end labelling,BD Bioscience试剂盒,Oakland,CA,USA)测试(Bayles KW,Bacterial programmed cell death:making sense of a paradox,Nature reviews,Microbiology,2014,12(1):63-69)，发现成熟的HNP-1可有效诱导DNA片段化(图20)。
在凋亡细胞，在细胞膜的本来面向细胞质的磷脂酰丝氨酸被外翻，这可用特异性结合带负电磷脂，比如磷脂酰丝氨酸膜联蛋白V染色(Annexin V stain)进行测定(Bayles KW,Bacterial programmed cell death:making sense of a paradox,Nature reviews,Microbiology,2014,12(1):63-69)。我们发现1mMIPTG诱导表达有生物活性成熟HNP-1的细胞膜联蛋白V染色阳性，而即使IPTG诱导，没有HNP-1基因编码序列的对照细胞没有凋亡发生，表明成熟HNP-1引发了显著的细胞凋亡(图21)。
上述系列结果表明成熟的HNP-1可以引发大肠杆菌宿主的细胞凋亡，从而杀菌。
实施例10：相互作用蛋白质组学揭示成熟HNP-1通过抑制RecA结合单链DNA引发细菌细胞凋亡的抗菌新机制
为揭示成熟HNP-1引发细菌凋亡的机制，在非变性条件下亲和纯化HNP-1相互作用蛋白，质谱鉴定HNP-1本身及其相互作用蛋白。等量的来自IPTG诱导表达preproHNP-1和无HNP-1对照细菌的总细胞蛋白分别利用组氨酸标签亲和富集，Tris-Tricine gel和银染分析表明preproHNP-1和组氨酸标签蛋白被显著富集(图22)。
为降低偶然鉴定的误差，我们同时做了生物学重复实验。利用液相色谱和质谱对这些富集样品进行蛋白质鉴定，共鉴定180余个可能的相互作用蛋白(图23)，包括最为丰富的阳性对照preproHNP-1。数据分析发现RecA也是其中最为富集的蛋白。进一步用蛋白质印迹实验证实(图24)。这些结果表明HNP-1和RecA具有相互作用。
RecA是细菌中已知的DNA损伤后SOS反应蛋白(Erental A,Kalderon Z,Saada A,Smith Y,&Engelberg-Kulka H,Apoptosis-like death,an extreme SOS response in Escherichia coli.mBio,2014,5(4):e01426-01414.)，但preproHNP-1或者成熟的HNP-1本身如何通过RecA调节细胞凋亡并不清楚。通过对不同时间 点IPTG诱导表达HNP-1的细胞总蛋白利用RecA抗体进行时间系列亲和纯化(图25)，发现刚开始时preproHNP-1先在细胞中积累，并进而产生成熟的HNP-1，这与我们的预期相符。随着RecA抗体免疫亲和沉淀的preproHNP-1的逐渐减少，在投料的RecA基本不变的情况下，亲和富集的成熟HNP-1逐渐增多。这些结果证实RecA和成熟HNP-1相互作用，而非preproHNP-1。
RecA是DNA依赖的ATP酶，当其结合单链DNA时可水解ATP，其水解ATP的效率和其结合单链DNA的量相关。为此测试了在单链DNA存在时成熟HNP-1抑制RecA的ATP酶活性的能力(Yakimov A,et al.,Blocking the RecA activity and SOS-response in bacteria with a short alpha-helical peptide,Nucleic Acids Research,2017,45(16):9788-9796)。结果显示在没有任何抑制剂时，ATP水解活性较高，当加入成熟HNP-1时ATP水解活性降低，且随着加入HNP-1量的增加，ATP水解活性呈现较好的负相关(图26)。
更为特异的是HNP-1确实通过抑制RecA和单链DNA的结合来实现其功能(图27)。
上述系列结果表明成熟的HNP-1通过抑制RecA结合单链DNA，从而引发细菌细胞凋亡。RecA在DNA损伤修复中的功能在细菌中广泛存在。抗微生物肽或者防御素抑制RecA的机制也未见报道，不仅解释了这类碱性氨基酸较多的抗微生物肽的新机制，修正了其穿膜导致细胞渗漏从而杀菌的假说，而且为抗微生物肽的新用途开发创造了条件。
实施例11：HNP-1脂质体包被
由于内源性成熟HNP-1的杀菌活性显著高于在培养基中加入的成熟HNP-1的，我们尝试脂质体包被HNP-1。大豆卵磷脂购自上海太伟药业公司，胆固醇购自上海国药控股试剂公司，N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethy-lammonium(DOTAP)购自Sigma试剂公司(St.Louis,MO,USA)。使用逆相蒸发法制备脂质体包被HNP-1(Kim BK,et al.,DOTAP/DOPE ratio and cell type determine transfection efficiency with DOTAP-liposomes,Biochimica et Biophysica Acta,2015,1848(10Pt A):1996-2001)。大豆卵磷脂、胆固醇和阳离子型DOTAP-脂按照10:1:1的比例混合，并在超声条件下溶于绿化二甲基中。成熟的HNP-1溶于pH6.8的PBS后加入到上述的脂类溶液中。温和超声形成水乳剂。真空条件下蒸发有机溶剂，形成凝胶。加入适当的PBS，边搅拌，悬浮凝胶，形成脂质体。所得脂质体过0.45μm无菌膜(Pall,New York,NY,USA)三次，得到脂质体成品，经激光颗粒检测仪检测，平均粒径为116.3nm(图28)。
实施例12：HNP-1通过引起细菌细胞凋亡杀死耐药金黄色葡萄球菌
由于成熟HNP-1通过抑制RecA引发凋亡杀灭细菌的机制保守，我们测试了其对其它耐药菌的杀菌活性。测试了HNP-1对耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)的杀菌活性。当HNP-1的浓度到10μg/mL时呈现出显著的杀菌活性(图29A)。成熟HNP-1处理的MRSA具有显著的细胞凋亡特征(图29B和C)，证明成熟HNP-1确实也可以通过细菌细胞凋亡杀灭耐甲氧西林金黄色葡萄球菌。
实施例13：脂质体包被HNP-1杀灭小鼠肺炎模型耐药金黄色葡萄球菌
按照文献构建了MRSA诱导的小鼠肺炎模型(Li M,Zhang T,Zhu L,Wang R,& Jin Y,Liposomal andrographolide dry powder inhalers for treatment of bacterial pneumonia via anti-inflammatory pathway.Int J Pharm,2017,528(1-2):163-171)。检测了脂质体包被HNP-1肺炎小鼠体内耐药金黄色葡萄球菌的清除情况。脂质体包被HNP-1处理肺炎模型小鼠体内耐药金黄色葡萄球菌克隆形成单位显著低于非处理的肺炎模型小鼠(图30)。更为重要的是克隆形成单位降低呈现脂质体包被HNP-1的剂量依赖关系，表明脂质体包被HNP-1的杀菌作用的特异性。我们也看到HNP-1本身以及青霉素都无法杀灭MRSA诱导肺炎模型小鼠的MRSA。这些结果证明脂质体包被HNP-1可杀灭小鼠体内的MRSA。
进一步利用免疫荧光共定位技术分析肺炎模型小鼠脂质体包被HNP-1的杀菌机制，发现细胞凋亡标志，膜联蛋白V染色仅和HNP-1脂质体组的HNP-1共定位，而在HNP-1直接处理组不明显。这些结果显示脂质体包被HNP-1通过引发细胞凋亡杀灭耐甲氧西林金黄色葡萄球菌。
实施例14：HNP-1杀灭耐药肺炎克雷伯氏菌
我们测试了其对其它临床难治耐药菌的杀菌活性。测试了HNP-1对耐药肺炎克雷伯氏菌(Klebsiella pneumoniae)的杀菌活性。当HNP-1的浓度到32μg/mL时呈现出显著的杀菌活性(图31)。利用脂质体包被的HNP-1，当浓度在16μg/mL时就呈现出杀菌活性；当脂质体包被的HNP-1浓度的增加，杀菌活性增强，呈现显著的药物浓度-杀菌活性的量效关系。
实施例15：HNP-1杀灭耐药鲍曼不动杆菌
我们还测试了其对临床难治耐药鲍曼不动杆菌(Acinetobacter baumannii)的杀菌活性。当HNP-1的浓度到32μg/mL时呈现出显著的杀菌活性(图32)。利用脂质体包被的HNP-1，当浓度在16μg/mL和32μg/mL时就呈现出显著的杀菌活性，但两者间差异不显著；当脂质体包被的HNP-1浓度进一步增加到64μg/mL时，杀菌活性进一步增强。

Claims (16)

  1. 一种重组人HNP-1蛋白，其特征在于所述重组人HNP-1蛋白是通过用大肠杆菌表达体系表达人防御素HNP-1前体蛋白，并以异丙基-β-D-1-硫代吡喃半乳糖苷诱导产生的成熟人HNP-1蛋白，所述重组人HNP-1蛋白具有抗菌活性。
  2. 权利要求1所述的重组人-HNP蛋白，其特征在于所述大肠杆菌表达体系包括大肠杆菌表达载体和可操作地连接于所述表达载体的HNP-1前体蛋白编码基因，所述HNP-1前体蛋白编码基因包括信号肽、前肽和成熟HNP-1。
  3. 如权利要求2所述的重组人HNP-1蛋白，其特征在于所述HNP-1前体蛋白编码基因的N端进一步引入His标签。
  4. 如权利要求1～3任一项所述的重组人HNP-1蛋白，其特征在于所述大肠杆菌表达载体选自pET系列载体、pGEX系列载体、pRSET系列载体、pMAL系列载体、pTWIN1载体、pET-28a(+)载体。
  5. 如权利要求4所述的重组人HNP-1蛋白，其特征在于所述大肠杆菌表达载体为pET-28a(+)。
  6. 如权利要求1-5任一项所述的重组人HNP-1蛋白，其特征在于所述蛋白通过下述方法制备：
    1)将所述大肠杆菌表达体系导入大肠杆菌宿主，获得重组菌株；
    2)活化并培养所述重组菌株；
    3)向步骤2)的培养液中加入异丙基-β-D-1-硫代吡喃半乳糖苷诱导培养后，收集菌体；
    4)从所述菌体中分离并纯化成熟人HNP-1蛋白。
  7. 如权利要求6所述的重组人HNP-1蛋白，其特征在于步骤3)所述诱导培养时间为0.5～3小时。
  8. 如权利要求7所述的重组人HNP-1蛋白，其特征在于步骤3)所述的诱导培养包括当步骤2)培养重组菌株的菌液OD 600生长到0.4-0.6时，加入终浓度为1mM异丙基-β-D-1-硫代吡喃半乳糖苷，培养温度为37℃，摇床转动速度为220rpm，诱导培养3h。
  9. 如权利要求7所述的重组人HNP-1蛋白，其特征在于步骤4)所述的纯化包括将分离的成熟HNP-1蛋白经过滤截留大分子蛋白进行纯化。
  10. 权利要求1所述的重组人HNP-1蛋白在制备抗菌药物中的应用。
  11. 如权利要求10所述的应用，其中所述抗菌药物为抗耐药菌药物。
  12. 如权利要求11所述的应用，其中所述的耐药菌为耐药金黄色葡萄球菌。
  13. 如权利要求11所述的应用，其中所述的耐药菌为耐药肺炎克雷伯氏菌。
  14. 如权利要求11所述的应用，其中所述的耐药菌为耐药鲍曼不动杆菌。
  15. 如权利要求10所述的应用，其中所述重组人HNP-1蛋白经脂质体包被。
  16. 如权利要求10所述的应用，其中所述抗菌药物通过抑制RecA与单链DNA的结合引发细菌细胞凋亡。
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