抗菌肽CM4对串珠镰孢菌孢子的抑制杀伤效应

家蚕抗菌肽CM4可抑制串珠镰孢菌孢子的生长发育.扫描电镜超微结构观察显示孢子空泡化,内部结构萎缩成团,对照组的孢子结构则完好无损,发育正常.家蚕抗菌肽与真菌作用,用激光共聚焦扫描图象分析,发现荧光素FITC标记抗菌肽先是聚集包围细胞,呈中间浓度大,两头浓度小状态分布,进而两头断裂,或形成空泡,最后连空泡的膜也破裂,细胞死亡,形成一堆碎片.     

抗菌肽CM4抗K562癌细胞的超微结构研究

报道了家蚕抗菌肽CM4抗K562癌细胞的体外实验研究.结果表明：纯化后的家蚕抗菌肽CM4对培养的K562(人髓样白血病细胞)有很强的杀伤作用.用扫描和透射电镜观察超微结构以及用激光共聚集显微断层图像分析,表明微量纯化的抗菌肽CM4能使K562癌细胞产生一系列的病理变化,可造成细胞高度肿胀,膜与胞质分离,细胞器和膜结构排列紊乱,细胞表面微绒毛消失,出现不规则的孔洞,细胞骨架严重破坏,膜局部结构破裂,缺损,胞浆内容物大量外泄,最终细胞解体,崩解成碎片.     

家蚕抗菌肽CM4对大肠杆菌超微结构影响的研究*(简报)

The effect of antibacterial peptide CM4 of Bombyx mori against E. coll K12 was investigated using scanning electron microscopy(SEM) and transmission electron microscopy (TEM). The ultrastructural changes of E. coli K12 were observed by the challenge of the purified antibacterial peptide CM4. The results showed that the antibacterial peptide caused a series of pathological changes on E. coli. SEM and TEM revealed aggregates of bacteria and SEM revealed wrin-kled bacterial surfaces in the early stage. Thereafter, plasmolysis was observed with irregular holes appearing in the two ends of bacteria and the cytoplasmic contents of the cells leaking out. Finally, bacteria became empty vesicles and disintegrated into small fragments subsequently. Comparatively, the bacterial membrane was normal and the bacterial structure remained intact in the control group.     

家蚕抗菌肽CM4对大肠杆菌超微结构影响的研究（简报）

The effect of antibacterial peptide CM4 of Bombyx mori against E. coli K12 was investigated using scanning electron microscopy(SEM) and transmission electron microscopy (TEM). The ultrastructural changes of E. coli K12 were observed by the challenge of the purified antibacterial peptide CM4. The results showed that the antibacterial peptide caused a series of pathological changes on E. coli. SEM and TEM revealed aggregates of bacteria and SEM revealed wrinkled bacterial surfaces in the early stage. Thereafter, plasmolysis was observed with irregular holes appearing in the two ends of bacteria and the cytoplasmic contents of the cells leaking out. Finally, bacteria became empty vesicles and disintegrated into small fragments subsequently. Comparatively, the bacterial membrane was normal and the bacterial structure remained intact in the control group.     

Expression and purification of antimicrobial peptide CM4 by Npro fusion technology in E. coli

Antimicrobial peptide CM4 is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells. Different strategies have been developed to produce small antibacterial peptides using recombinant techniques. To date, no efforts to obtain large quantities of active recombinant CM4 have been reported. In order to establish a bacterium-based CM4 production system, CM4 was cloned into pET28a and expressed with N^pro mutant (EDDIE) fusion. CM4 expressed as EDDIE are deposited as inclusion bodies. On in vitro refolding by switching from chemotropic to kosmotropic conditions, the fusion partner is released from the C-terminal end of the autoprotease by self-cleavage, leaving CM4 protein with an authentic N terminus. Purified CM4 was separated on Ni²⁺-chelating chromatography column and cation-exchange chromatography column. Mass spectroscopic analysis indicated the protein to be 4132.56 Dalton, which equalled the theoretically expected mass. N-terminal sequencing of CM4 showed the sequence corresponded to the native protein. The recombinant CM4 exhibited the same antimicrobial and anti-tumor activity as reported previously. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant CM4 with native sequences.     

抗菌肽CM4与人可溶性B淋巴细胞因子hsBAFF在大肠杆菌中的融合表达

为进一步探讨抗菌肽CM4的原核表达及其生物学功能,本实验研究了抗菌肽CM4与人可溶性B淋巴细胞刺激因子hsBAFF的融合表达及抗菌肽CM4的生物学活性。运用PCR把B淋巴细胞因子hsBAFF和家蚕抗菌肽CM4进行基因融合,构建了融合表达载体pET28a (+)/CM4-hsBAFF,并在大肠杆菌中获得高可溶性表达的融合靶蛋白,且存在于超声破碎后的上清,经分子筛Sephadex G-75纯化后的重组融合蛋白用SDS-PAGE和Western blot分析鉴定.SDS-PAGE分析表明：可以通过分子筛一步纯化得到融合蛋白,该重组融合蛋白的分子量约22.0 KDa。Western blot结果显示该重组蛋白能与鼠抗人hsBAFF的抗体发生特异性反应.运用基因工程的方法获得CM4-hsBAFF重组融合蛋白,并具有很好的抑菌生物学活性。     

Biodiversity in aerobic granule membrane bioreactor at high organic loading rates

Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of cationic antibacterial peptide ABP-CM4. The fusion protein expressed in a soluble form was purified to a purity of 90% by Ni-IDA chromatography and 112 mg protein of interest was obtained per liter of fermentation culture. After the SUMO–CM4 fusion protein was cleaved by the SUMO protease at 30 °C for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally, about 24 mg recombinant CM4 was obtained from 1 l fermentation culture with no less than 96% purity and the recombinant CM4 had similar antimicrobial properties to the synthetic CM4. Thus, the SUMO-mediated peptide expression and purification system potentially could be employed for the production of recombinant cytotoxic peptides.     

抗菌肽CM4与绿色荧光蛋白融合基因的真核载体构建及表达

目的：构建绿色荧光蛋白（GFP）与抗菌肽CM4融合基因的真核表达载体。方法：采用递归PCR（rPCR）将GFP基因与CM4基因通过一段核苷酸片段连接成GFP-CM4融合基因,构建真核表达载体pcDNA3-GFP-CM4,用脂质体介导转染人K562白血病细胞,用MTT检测其活性。结果：融合基因可在K562细胞中表达,抗菌肽CM4的表达可抑制K562细胞的生长。结论：为全面了解抗菌肽的生物学活性及其在基因治疗中的可能作用提供了重要的理论依据。     

Investigation of the antibacterial activity of a short cationic peptide against multidrug-resistant Klebsiella pneumoniae and Salmonella typhimurium strains and its cytotoxicity on eukaryotic cells

With the growing microbial resistance to conventional antimicrobial agents, the development of novel and alternative therapeutic strategies are vital. During recent years novel peptide antibiotics with broad spectrum activity against many Gram-positive and Gram-negative bacteria have been developed. In this study, antibacterial activity of CM11 peptide (WKLFKKILKVL-NH2), a short cecropin–melittin hybrid peptide, is evaluated against antibiotic-resistant strains of Klebsiella pneumoniae and Salmonella typhimurium as two important pathogenic bacteria. To appraise the antibacterial activity, minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and bactericidal killing assay were utilized with different concentrations (2–128 mg/L) of peptide. To evaluate cytotoxic effect of peptide, viability of RAJI, Hela, SP2/0, CHO, LNCAP cell lines and primary murine macrophage cells were also investigated with MTT assay in different concentrations (3–24 and 0.5–16 mg/L, respectively). MICs of K. pneumoniae and S. typhimurium isolates were in range of 8–16 and 4–16 mg/L, respectively. In bactericidal killing assay no colonies were observed at 2X MIC for K. pneumoniae and S. typhimurium isolates after 80–90 min, respectively. Despite the fact that CM11 reveals no significant cytotoxicity on RAJI, Hela, SP2/0, and CHO cell lines beneath 6 mg/L at first 24 and 48 h, the viability of LNCAP cells are about 50 % at 3 mg/L, which indicates strong cytotoxicity of the peptide. In addition, macrophage toxicity by MTT assay showed that LD₅₀ of CM11 peptide is 12 μM (16 mg/L) after 48 h while in this concentration after 24 h macrophage viability was about 70 %.     

Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners

It has been reported that it is difficult to express cationic antibacterial peptides in engineered bacteria because such peptides are highly toxic to the host bacteria cells and sensitive to intracellular proteases. Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi and tumor cells, which may possibly be used as an antimicrobial agent. Here we tried to express ABP-CM4 in Escherichia coli cells using either the GST fusion system or the intein-mediated fusion expression system. In order to investigate the possible use of these two fusion partners in cationic small peptide expression and purification, a mutant ABP-CMt, which is a highly positively charged peptide with +9 charges at neutral pH, was designed. In the present study, we have shown that both ABP-CM4 and ABP-CMt peptides can be expressed and purified by the intein-mediated expression system but not by the GST fusion expression system. Thus the intein-mediated peptide expression and purification system potentially could be employed for the production of recombinant protease-sensitive and cytotoxic peptides.