Antimicrobial activity and structure of a consensus human β-defensin and its comparison to a novel putative hBD10

The spread of multidrug resistant bacteria owing to the intensive use of antibiotics is challenging current antibiotic therapies, and making the discovery and evaluation of new antimicrobial agents a high priority. The evaluation of novel peptide sequences of predicted antimicrobial peptides from different sources is valuable approach to identify alternative antibiotic leads. Two strategies were pursued in this study to evaluate novel antimicrobial peptides from the human β-defensin family (hBD). In the first, a 32-residue peptide was designed based on the alignment of all available hBD primary structures, while in the second a putative 35-residue peptide, hBD10, was mined from the gene DEFB110. Both hBDconsensus and hBD10 were chemically synthesized, folded and purified. They showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis, but were not hemolytic on human red blood cells. The NMR-based solution structure of hBDconsensus revealed that it adopts a classical β-defensin fold and disulfide connectivities. Even though the mass spectrum of hBD10 confirmed the formation of three disulfide bonds, it showed limited dispersion in ¹H NMR spectra and structural studies were not pursued. The evaluation of different β-defensin structures may identify new antimicrobial agents effective against multidrug-resistant bacterial strains.     

Preferential codons enhancing the expression level of human beta-defensin-2 in recombinant Escherichia coli

Human beta-defensin-2 (hBD2) is a small antimicrobial peptide with potential as a therapeutic agent. The effect of codon usage on the expression of hBD2 in Escherichia coli was studied. Two coding sequences encoding the same hBD2 precursor were both expressed as fusion protein with thioredoxin in E. coli BL21 (DE3). One is the wild-type human cDNA and the other is a gene synthesized by a PCR-based method in which rare codons were altered to those frequently used in E. coli. The expression level of recombinant hBD2 was over 50% of the total cellular protein when the synthetic gene with preferential codons was employed which was a 9-fold enhancement over the wild-type cDNA. The result shows the codon bias of the host was a major barrier in high-level expression of recombinant hBD2 and suggests a similar approach may be used in the expression of other defensins in E. coli.     

High-level expression of human beta-defensin-2 gene with rare codons in E. coli cell-free system

Human beta-defensin-2 (hBD2) is A small cationic peptide with A broad range of antimicrobial activity. An E. coli cell-free system was employed to express the hBD2 fusion protein by using the hBD2 gene with 14 rare codons. The results showed that the expression level of trxA-hBD2 fusion protein was 0.35 mg/ml, which is the same as that obtained with A synthetic codon-optimized gene. By using another fusion partner (GFP), similar high-level expression was also achieved in this cell-free system. This meant that human beta-defensin-2 gene could be directly used to express hBD2 fusion protein efficiently in an E. coli cell-free system without the optimization of codons. The expression level of hBD2 fused with thioredoxin could be further improved up to 2.0 mg/ml by adopting A continuous exchange cell-free system. A simple one-stage affinity purification procedure was also developed to recover this fusion protein efficiently.     

棘胸蛙抗菌肽Spinosan-C的串联表达与活性检测

为克服抗菌肽易被蛋白酶降解及对宿主大肠杆菌的杀伤作用,并进一步提高大肠杆菌系统的表达能力,以棘胸蛙Paa spinosa抗菌肽Spinosan-C为研究对象,按照大肠杆菌密码子利用频率进行密码子优化,设计合成8拷贝的串联8×Spinosan-C基因,将合成的串联基因克隆到大肠杆菌表达载体p ET-28a,利用大肠杆菌感受态细胞Rosetta进行原核表达,获得高效表达的串联8×Spinosan-C重组蛋白,用甲酸专一性切割得到抗菌肽Spinosan-C单体。体外抑菌试验表明,切割后的抗菌肽Spinosan-C单体对测试菌生长具有抑制作用,为蛙类抗菌肽的规模化制备提供了参考。     

High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression

Human beta-defensin-4 (hBD4) is a cationic 50-amino acid antimicrobial peptide with three conserved cysteine disulfide bonds. It exhibits a broad antimicrobial spectrum. This study describes the synthesis of hBD4 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released hBD4. A PCR-based gene SOEing (splicing by overlap extension) synthesis method was used in the synthesis of the hBD4 gene with optimized codons. By constructing the expression plasmid (pET32-smhBD4), high concentration of soluble hBD4 fusion protein (1.9 g/l) can be obtained in E. coli. Further optimization studies showed that the expression system was very efficient to produce soluble target protein, and the solubility of the target protein could attain more than 99% even when the culture temperature was as high as 37°C. The highest productivity (2.68 g/l) of the hBD4 fusion protein was achieved by cultivating the E. coli (pET32-smhBD4) in MBL medium at 34°C, inducing the culture at the mid-exponential phase with 0.4-mM isopropyl β-d-galactopyranoside (IPTG), and collecting the broth after 6-h expression. The soluble target protein accounted for 64.6% of the total soluble proteins, and the mature hBD4 expression level was stoichiometrically estimated to be 0.689 g/l. This fusion protein was then purified and cleaved to get the mature hBD4 peptide that showed antimicrobial activity against E. coli and Pseudomonas aeruginosa.     

稳定表达禽β防御素6的DF-1细胞系的建立及其抑菌活性

【背景】禽β防御素6是禽体内分泌的一类抗菌肽,在抵抗病原入侵和免疫调节中发挥着重要作用,但其常规表达方式效率较低,难以在产业化生产中加以应用。【目的】建立稳定表达AvBD6的细胞系,并检测其表达产物对耐药大肠杆菌的抗菌活性,为其他防御素表达提供参考。【方法】利用显微镜观察构建真核重组表达载体pLOV-eGFP-AvBD6转染至293T细胞后的转染效率;收集293T细胞上清液并感染DF-1细胞,通过嘌呤霉素加压筛选稳定表达株;利用RT-PCR和Western Blot分别检测目的基因在转录水平和蛋白水平的表达情况;利用扫描电镜观察细胞培养上清液对耐药大肠杆菌的抗菌效果及其对菌体的损伤。【结果】成功构建重组表达载体pLOV-eGFP-AvBD6,筛选出稳定表达AvBD6的DF-1细胞系,而且目的基因在转录水平和蛋白水平均有表达;细胞培养上清显著降低大肠杆菌和副伤寒沙门菌存活率,对金黄色葡萄球菌的抗菌活性较低。【结论】建立了稳定表达AvBD6的DF-1细胞系,其表达产物对耐药大肠杆菌具有良好的抗菌效果,对推动防御素的应用提供技术支持。     

Gingipains produced by Porphyromonas gingivalis ATCC49417 degrade human-β-defensin 3 and affect peptide's antibacterial activity in vitro

Porphyromonas gingivalis, one of the major pathogen associated with periodontitis, is a highly proteolytic bacterial species. Production of proteases is a common microbial virulence factor that enables the destruction of host tissues and evasion from host defense mechanisms. Antimicrobial peptides are important effector molecules of the innate immune system with a broad range of antimicrobial and immunoregulatory activities. We and others have previously demonstrated that P. gingivalis is relatively resistant to the bactericidal activity of the human β-defensin 3 (hBD3). In this study, ability of proteases released by the pathogenic strain of P. gingivalis ATCC 49417 to degrade hBD3 and to affect the antibacterial properties of the peptide was assessed. P. gingivalis culture supernatants (CS) were found to degrade hBD3 in a concentration- and time-dependent manner. Such degradation was mainly due to the activity of Arg and Lys-gingipains, as pretreatment of CS with inhibitors selective for this class of proteases abolished CS ability to degrade hBD3. Importantly, preincubation of hBD3 with CS reduced peptide's antibacterial activity against a susceptible strain of Staphylococcus aureus, while the presence of gingipain inhibitors in the bactericidal assay increased P. gingivalis susceptibility to hBD3. Altogether these results suggest that gingipains may have a role in the resistance of P. gingivalis ATCC 49417 to hBD3.     

Structural Basis for the Interaction of Human β-Defensin 6 and Its Putative Chemokine Receptor CCR2 and Breast Cancer Microvesicles

Human β-defensins (hBDs) are believed to function as alarm molecules that stimulate the adaptive immune system when a threat is present. In addition to its antimicrobial activity, defensins present other activities such as chemoattraction of a range of different cell types to the sites of inflammation. We have solved the structure of the hBD6 by NMR spectroscopy that contains a conserved β-defensin domain followed by an extended C-terminus. We use NMR to monitor the interaction of hBD6 with microvesicles shed by breast cancer cell lines and with peptides derived from the extracellular domain of CC chemokine receptor 2 (Nt-CCR2) possessing or not possessing sulfation on Tyr26 and Tyr28. The NMR-derived model of the hBD6/CCR2 complex reveals a contiguous binding surface on hBD6, which comprises amino acid residues of the α-helix and β2–β3 loop. The microvesicle binding surface partially overlaps with the chemokine receptor interface. NMR spin relaxation suggests that free hBD6 and the hBD6/CCR2 complex exhibit microsecond-to-millisecond conformational dynamics encompassing the CCR2 binding site, which might facilitate selection of the molecular configuration optimal for binding. These data offer new insights into the structure–function relation of the hBD6–CCR2 interaction, which is a promising target for the design of novel anticancer agents.     

Antimicrobial activity of two peptides casecidin 15 and 17, found naturally in bovine colostrum

Aims:  To isolate and characterize peptides from bovine colostrum with antimicrobial activity.
Methods and Results:  Three peptides were purified from fresh colostrum by a range of chromatographic methods using antimicrobial activity against Escherichia coli DH5α to screen for the most active fractions. Two peptides, with antimicrobial activity, casecidin 17 and casecidin 15, were identical to sequences in the C-terminal of bovine β-casein (YQEPVLGPVRGPFPIIV and YQEPVLGPVRGPFPI) and had corresponding molecular masses of 1881·00 and 1669·06 Da, respectively. The third peptide was the known peptide isracidin which has a mass of 2763·80 Da and sequence of RPKHPIKHQGLPQEVLNENLLRF. Casecidin 17 and casecidin 15 had identical minimal inhibition concentrations (MICs) against E. coli DPC6053 of 0·4 mg ml⁻¹. Structural modelling suggested amphiphilic structures having identical inhibitory and structural properties. The MIC value of isracidin against E. coli DPC6053 was 0·2 mg ml⁻¹.
Conclusions:  This study shows the presence of three antimicrobial peptides in colostrum which may contribute to a bioprotective role to limit pathogen contamination. Furthermore, the discovery of casecidin 17 and 15 may provide the basis for novel antimicrobial peptide design.
Significance and Impact of the Study:  This is the first study to characterize peptides with antimicrobial activity present in fresh bovine colostrum.     

人b防御素3和植物des-pGLu1-brazzein融合蛋白表达菌的诱导条件优化及其活性分析

对人b防御素3和植物甜蛋白des-pGlu1-Brazzein嵌合基因的工程菌株BL-pET-hBD3-Bra的IPTG诱导表达条件进行了研究, 同时对所表达的目的蛋白进行了纯化和活性分析。IPTG浓度、诱导时间和诱导温度对菌株生长和目的蛋白表达的试验结果显示: 所取的IPTG浓度(0.2~1.0 mmol/L)对菌株生长和目的蛋白的表达无显著影响(P>0.05); 菌株的生物量随着诱导时间的延长而增加, 6 h优于4 h(P<0.01), 但是蛋白的表达量无明显增加(P>0.05); 其中温度是重要的影响因素, 在30°C诱导时, 目的蛋白的表达量占总蛋白的35%左右。进一步的研究表明, 菌株在30°C~32°C生长, 在 30°C诱导最优。对目的蛋白的活性分析表明, 所得到的hBD3-Bra融合蛋白有甜味, 其甜度大约是蔗糖的200倍, 但是其杀菌活性很弱, 经凝血酶切割后, des-pGlu1-Brazzein的甜度大大提高, 大约为蔗糖甜度的600倍, 重组hBD3对大肠杆菌和金黄色葡萄球菌有明显的抑菌活性。     

Efficient production of a soluble fusion protein containing human beta-defensin-2 in E. coli cell-free system

Human beta-defensin-2 (hBD2), a small cationic peptide, exhibits a broad range of antimicrobial activity and does not cause microbial resistance. In order to produce hBD2 efficiently, an Escherichia coli cell-free biosynthesis system has been developed as an alternative method. A specific plasmid pIVEX2.4c-trxA-shBD2 was constructed for the cell-free expression of fusion protein (hBD2 linked with His-Tag and Trx-Tag). This allowed enhancement of protein stability and facilitated downstream purification process. Significant amount of target fusion protein was synthesized in the batch-mode bioreactor by optimizing the reaction conditions. About five-fold improvement of productivity (ca. 2.0 mg/ml soluble fusion protein) could be achieved by using a continuous exchange cell-free (CECF) system compared to batch system. One-step affinity chromatographic process was developed to recover high purity fusion protein (95.2%) with overall recovery ratio of about 50%. The fusion protein was cleaved by cyanogens bromide (CNBr), and the mature hBD2 had demonstrated strong inhibition on the growth of E. coli D31 at low concentration.     

Inhibition of frog antimicrobial peptides by extracellular products of the bacterial pathogen Aeromonas hydrophila

Aims:  To determine whether the extracellular products (ECPs) from Aeromonas hydrophila , a frog bacterial pathogen that is resistant to skin antimicrobial peptides of three different frog species Xenopus laevis , Litoria aurea and Litoria raniformis , can modulate the activity of these peptides.
Methods and Results:  ECPs were collected from cultures of Klebsiella pneumoniae , a pathogen susceptible to skin antimicrobial peptides of all three tested frog species, and from cultures of Aer .  hydrophila . They were tested for protease activity and for inhibition of the antimicrobial activity of natural peptide mixtures and single peptides of all three frog species against Escherichia coli ATCC 25922. ECPs from cultures of Aer .  hydrophila grown for 16, 24 and 36 h showed protease activity and inhibited the antibacterial activity of all peptides against E .  coli ATCC 25922. In contrast, the ECPs from cultures of Kl .  pneumoniae neither had protease activity nor inhibited the activity of any peptides.
Conclusion:  The proteolytic ECPs of Aer .  hydrophila have the ability to inhibit the skin antimicrobial peptides of frogs.
Significance and Impact of the Study:  The results of this study provide new information on the association of ECPs with the resistance of Aer .  hydrophila to frog antimicrobial peptides.     

抗菌肽人beta防御素3 对铜绿假单胞菌PAO-1 株的抑制作用

目的:分析抗菌肽人β防御素3(humanβdefensin 3,hBD3)对铜绿假单胞菌PAO-1株的抑制作用。方法:合成抗菌肽hBD3,分别通过最低抑菌浓度(minimal inhibitory concentration,MIC)检测、直接杀菌试验、重要功能基因检测分析其对PAO-1的直接抑制作用;并将其与阿奇霉素、四环素、利福平、氯霉素、链霉素、环丙沙星联合施用,观察对抗生素MIC的影响。结果:HBD3对PAO-1的MIC为32μg/mL;在浓度达到8μg/mL时即有明显杀菌作用。HBD3上调PAO-1株的ahpF基因表达,下调aprA和rhlR基因表达。在联用5μg/mL的hBD3后,四环素、利福平、氯霉素、链霉素、环丙沙星的MIC值均有降低。结论:抗菌肽hBD3对铜绿假单胞菌PAO-1株有显著的抑制作用。     

Toll-like receptor 2-mediated expression of beta-defensin-2 in human corneal epithelial cells

We previously showed that human corneal epithelial cells (HCECs) express Toll-like receptors (TLRs), which recognize gram-positive bacteria and respond to Staphylococcus aureus infection by the expression and secretion of proinflammatory cytokines and beta-defensin-2 (hBD2). In this study, we further elucidated the underlying mechanisms regulating hBD-2 expression and its role in innate defense in HCECs in response to S. aureus challenge. Exposure of HUCL cells, a telomerase-immortalized HCEC line, to S. aureus, its exoproducts (1:10 dilution), or synthetic lipopeptide Pam3Cys (10 microg/ml) resulted in the up-regulation of hBD-2, but not hBD1 and hBD3. Similar to HUCL cells, primary HCECs responded to S. aureus-exoproducts and Pam3Cys challenge by expressing hBD2 mRNA and secreting hBD2 into the culture media. Furthermore, these stimuli induced the expression of TLR2 at both mRNA and protein levels. Consistently with its role as a major pattern-recognizing receptor, TLR2 was located at the cell surface by cell surface biotinylation. The treatment of HUCL cells with TLR2 neutralizing antibody resulted in a significant decrease in Pam3Cys-induced hBD2 production as well as IL-6, IL-8, and TNF-alpha secretion. The Pam3Cys-induced hBD2 expression was completely blocked by NF-kappaB inhibitors and partially inhibited by p38 MAP kinase and the JNK inhibitors. Conditioned media derived from HCECs challenged with S. aureus-exoproducts or Pam3Cys exhibited antibacterial activity against S. aureus, Pseudomonas aeruginosa and Escherichia coli. These findings suggest that S. aureus induces hBD2 production through TLR2-mediated pathways in HCECs and that pathogen-challenged, TLR-activated HCECs possess antimicrobial activity. Thus, the epithelium might play a role in innate defense against bacterial infection by directly killing bacteria in the cornea.     

抗菌肽GK1在大肠杆菌中的融合表达

为高效表达抗菌肽GK1并避免GK1的高抗菌活性对大肠杆菌宿主菌的致命影响, 将经改造后的人胰岛素原(mhPI)与GK1的融合基因(mhPI-GK1)克隆到表达载体pET28a中, 构建出表达质粒pET28a-mhPI-GK1, 转化至大肠杆菌BL21(DE3)中进行表达。融合蛋白在大肠杆菌中以包涵体形式表达, 表达量占菌体总蛋白的20%。经CNBr裂解、阳离子交换层析和RP-HPLC纯化后, 每升发酵液可获得5.7 mg纯度大于97%的重组GK1。质谱检测显示重组GK1的分子量为2794.0 D, 抑菌活性实验表明纯化后的重组GK1和化学合成GK1具有相同的抗菌活性。为利用基因工程方法大规模生产GK1奠定了基础。     

A Novel Lipopeptide from Skin Commensal Activates TLR2/CD36-p38 MAPK Signaling to Increase Antibacterial Defense against Bacterial Infection

Staphylococcus epidermidis (S.epidermidis) plays important protective roles by directly producing or by stimulating hosts to produce antimicrobial peptides (AMPs) against pathogenic infections. Although several AMPs from S.epidermidis have been identified, molecules that stimulate hosts to produce AMPs remain largly unknown. Here we demonstrate that a new lipopeptide (named LP01) purified from S.epidermidis culture media has a unique structure with heneicosanoic acid (21 carbons) binding to lysine¹¹ of a peptide chain. In vitro LP01 increased the expression of β-defensin 2(hBD2) and hBD3 in neonatal human epidermal keratinocytes(NHEK), leading to increased capacity of cell lysates to inhibit the growth of S.aureus. In vivo LP01 induced the expression of mouse β-defensin 4(mBD4) to decrease the survival of local S.aureus in skin and systemic S.aureus survival in liver. The induction of beta-defensins by LP01 was dependent on TLR2 as Tlr2-deficient mice had decreased mBD4. Furthermore, knockdown of CD36 decreased the expression of hBD2 and hBD3, and p38 MAPK inhibitor significantly inhibited the expression of hBDs induced by LP01.Taken together, these findings demonstrate that lipopeptide LP01 from normal commensal S.epidermidis increases antimicrobial peptide hBD2 and hBD3 expression via the activation of TLR2/CD36-p38 MAPK, thus enhancing antimicrobial defense against pathogenic infections.     

Lipid-specific membrane activity of human beta-defensin-3

Defensins represent a major component of innate host defense against bacteria, fungi, and enveloped viruses. One potent defensin found, e.g., in epithelia, is the polycationic human beta-defensin-3 (hBD3). We investigated the role of the lipid matrix composition, and in particular the presence of negatively charged lipopolysaccharides (LPS) from sensitive (Escherichia coli, Salmonella enterica serovar Minnesota) or resistant (Proteus mirabilis) Gram-negative bacteria or of the zwitterionic phospholipids of human cells, in determining the action of polycationic hBD3 on the different membranes, and related to their biological activity. The main focus was directed on data derived from electrical measurements on a reconstitution system of the OM as a planar asymmetric bilayer composed on one side of LPS and on the other of a phospholipid mixture. Our results demonstrate that the antimicrobial activity and the absence of cytotoxicity can be explained by the lipid-specificity of the peptide. A clear correlation between these aspects of the biological activity of hBD3 and its interaction with lipid matrices could be found. In particular, hBD3 could only induce lesions in those membranes resembling the lipid composition of the OM of sensitive bacterial strains. The permeation through the membrane is a decisive first step for the biological activity of many antimicrobial peptides. Therefore, we propose that the lipid-specificity of hBD3 as well as some other membrane-active antimicrobial peptides is important for their activity against bacteria or mammalian cells.     

Identification and Biological Characterization of Mouse beta-defensin 14, the orthologue of human beta-defensin 3

beta-Defensins are small antimicrobial polypeptides that are mainly expressed by epithelial cells and play an important role in the antimicrobial innate immune response. In addition to the direct microbicidal effects of these polypeptides, it became evident that certain members of the beta-defensin super family have the capacity to promote local innate inflammatory and systemic adaptive immune responses by interacting with the CC-chemokine receptor CCR6. We have identified mouse beta-defensin 14 (mBD14, Defb14) as an orthologue of human beta-defensin 3 (hBD3 or DEFB103). Based on primary structural analysis, mBD14 demonstrates greater (68%) homology to its human orthologue, containing three conserved cystein linkages, characteristic for the beta-defensin super family. mBD14 is expressed in a wide variety of tissues including spleen, colon, and tissues of the upper and lower respiratory tract. Interestingly, we also detected mBD14 expression in immature CD11c+ bone marrow-derived dendritic cells. The expression of mBD14 can be induced by Toll-like receptor agonists such as lipopolysaccharide and poly(I:C) and by pro-inflammatory stimuli e.g. tumor necrosis factor and interferon-gamma. Furthermore, expression of mBD14 seems to be regulated by activation of the intracellular pattern recognition receptor NOD2/CARD15 as revealed by reporter gene analysis. We prepared a recombinant mBD14-Ig fusion protein that retained potent antimicrobial activity against several Escherichia coli strains but not against various Gram-positive Staphylococcus aureus strains. hBD3 and also the newly identified mBD14 were chemotactic for cells expressing the mouse CC-chemokine receptor CCR6. In addition, both hBD3 and mBD14 were chemotactic for freshly isolated mouse resident peritoneal cells. Thus, mBD14, based on structural and functional similarities, appears to be an orthologue of hBD3.     

内生枯草芽孢杆菌SWB8 菌株β-1，3-1，4-葡聚糖酶的抗菌作用及细胞毒性

【目的】本文研究从药用植物黄姜中分离的内生枯草芽孢杆菌菌株SWB8分泌的β-1,3-1,4-葡聚糖酶的抗菌活性和细胞毒性。【方法】利用液体发酵、凝胶渗透色谱(GPC)、十二烷基-聚丙烯酰胺凝胶电泳(SDS-PAGE)和液相层析串联质谱(LC-MS/MS)等方法纯化和鉴定枯草芽孢杆菌株SWB8合成的β-1,3-1,4-葡聚糖酶;利用纸片扩散法,检测葡聚糖酶抑制临床致病性细菌和真菌生长的活性;应用MTT法和流式细胞术(FCM)评估此葡聚糖酶对人肺腺癌细胞(A549)和骨髓间质干细胞(MSCs)的细胞毒性。【结果】细菌性β-1,3-1,4-葡聚糖酶显示了广谱的抗菌活性;抗肿瘤活性主要以细胞凋亡的方式选择性的抑制人肺腺癌细胞系A549细胞的增殖,而对人骨髓间质干细胞系MSC细胞无明显影响。【结论】首次报道β-1,3-1,4-葡聚糖酶的抗菌和抗肿瘤细胞的活性。内生枯草芽孢杆菌SWB8菌株有可能成为抗菌和高效低毒的抗肿瘤药物的潜在来源。     

利用ELP自断裂标签在大肠杆菌中生产抗菌肽Oxysterlin 1

抗菌肽是目前最有希望的抗生素替代品,但是使用重组技术生产抗菌肽的策略大多步骤烦琐且价格昂贵,不利于抗菌肽的规模化生产。Oxysterlin 1是一种新型的天蚕素抗菌肽,主要对革兰氏阴性菌有抗菌活性,具有较低的细胞毒性。文中利用一种简单经济的方法在大肠杆菌中实现Oxysterlin 1的表达和纯化。将Oxysterlin 1基因克隆到含有弹性蛋白样多肽Elastin-like polypeptide (ELP) 和蛋白质内含肽 (Intein) 的载体中,构建重组表达质粒pET-ELP-I-Oxysterlin 1。重组蛋白在大肠杆菌中主要以可溶性形式表达,进而通过简单的盐析和pH改变便可对目标小肽进行纯化。最终得到的Oxysterlin 1的产量约为1.2 mg/L,抑菌试验显示出预期活性,为抗菌肽的规模化生产及深入研究其抑菌机理奠定基础。