抗菌肽CM4组分对K562癌细胞染色质DNA断裂作用的SCGE研究

单细胞凝胶电泳法（singe cell gel electrophoresis, SCGE)是一种快速,敏感的检测单个哺乳动物细胞DNA断裂的技术,也叫彗星实验（comet assay).此实验首次通过SCGE法观察抗菌肽CM4组分对人髓样白血病K562细胞和正常人白细胞核染色质DNA的影响,从而进一步研究抗菌肽抗癌作用的机制.荧光显微镜观察显示经抗菌肽CM4组分处理过的K562癌细胞核染色质DNA出现断裂,形成一个亮的荧光头部和彗星似的尾部,而经同样处理的正常人白细胞和未经抗菌肽处理的K562癌细胞核染色质DNA未出现断裂,核完整,呈圆形.经彗星尾长分析,前者DNA损伤率平均为73.62%,统计学处理P＜0.001,具高度显著性差异.这表明,抗菌肽CM4对K562癌细胞核染色质DNA有明显的断裂作用,而对正常人白细胞则没有断裂作用.     

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。     

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。     

家蚕抗菌肽对癌细胞的杀伤作用及其超微结构的观察

从家蚕蛹血淋巴中分离纯化的抗菌肽β组分对体外培养的癌细胞有选择性杀伤作用,而对正常人Ｂ淋巴细胞无不良影响。抗菌肽与巨噬细胞淋巴瘤细胞Ｕ９３７体外培养一段时间后,用扫描和透射电镜观察细胞膜和细胞器的变化,发现细胞膜和核被膜局部溶解,线粒体肿胀、发空,胞内物质大量外泄,从而导致细胞死亡。     

中国家蚕抗菌肽ABP-CM4在E.coli中的融合表达及活性分析

参照天然抗菌肽CM4(ABP-CM4)氨基酸序列和大肠杆菌偏爱密码子,采用rPCR法获得CM4基因后重组到表达载体pET32a上,在E.coli中融合表达。表达产物以可溶性存在,经Ni2 -NTA琼脂糖亲和层析获得融合蛋白,再经甲酸切割、亲和层析和阳离子交换层析,得到纯化的重组抗菌肽。琼脂糖扩散法和液相测定法证明了纯化的抗菌肽具有抗菌活性。     

抗菌肽CM Ⅳ突变体基因在E.coli中融合表达的研究

根据家蚕抗菌肽CMⅣ的氨基酸序列,利用E.coli偏爱的密码子设计并合成其突变体DNA,克隆到融合表达载体pEZZ318中,在E.coli中进行融合表达,经亲和层析得到26mg/L的融合表达产物.用化学方法裂解该融合表达产物,得到了具有抗菌活性的突变体抗菌肽CMⅣ.     

抗菌肽CM4对酿酒酵母原生质体再生抑制作用的研究*

抗菌肽CM4能抑制酿酒酵母原生质体的再生 ,随着加入抗菌肽浓度的增大 ,原生质体的再生率下降。用荧光素FITC标记的抗菌肽处理原生质体 ,激光共聚焦扫描电镜观察 ,抗菌肽分子覆盖于膜 ,破坏了原生质体的膜结构 ,改变了原生质体的膜渗透性。最终原生质体无法恢复细胞壁而死亡。     

抗菌肽CM4对酿酒酵母原生质体再生抑制作用的研究

抗菌肽CM4能抑制酿酒酵母原生质体的再生,随着加入抗菌肽浓度的增大,原生质体的再生率下降。用荧光素FTTC标记的抗菌肽处理原生质体,激光共聚焦扫描电镜观察,抗菌肽分子覆盖于膜,破坏了原生质体的膜结构,改变了原生质体的膜渗透性。最终原生质体无法恢复细胞壁而死亡。     

中国家蚕抗菌肽人工合成类CMⅣ基因在甜菜夜蛾虫体中的表达

将人工合成的中国家蚕抗菌肽类CMⅣ基因与抗菌肽信号肽基因连接 ,经EcoRⅠ、HindⅢ双酶切后 ,克隆于pFASTBacⅠ的EcoRⅠ、HindⅢ酶切位点之间 ,得到重组转座载体pFASTBac ABP ,经测序证明阳性克隆正确。将重组转座载体转化HD1 0Bac大肠杆菌 ,得到重组Bacmid ABP。将重组Bacmid转染sf2 1细胞及感染甜菜夜蛾 (Laphygmaexigua)幼虫 ,在培养细胞上清及虫体血淋巴中均测到抗菌活性。经Northernblotting证明感染甜菜夜蛾幼虫中有类CMⅣmRNA的存在。且表达产物在酸性电泳中电泳行为与天然抗菌肽CMⅣ组分相似。为进一步利用昆虫细胞及虫体生产抗菌肽药物打下了基础。     

抗菌肽CM4对串珠镰孢菌孢子的抑制杀伤效应

The antibacterial peptide CM4 inhibits growth of conidia of Fusaricum moniliforme. The observation by using the electron microscope showed that some cells were empty and interior structure twisted into helix. Conidia cells in control group were in good condition, normally grew up. The fungal conidia treated with the antibacterial peptide CM4, then confocal laser scanning microscope were performed. As a result, it was showed that FITC-labeled peptide assembled in cells, centre of which had more peptides than two ends. Then conidia split, became empty. Finally the cells plasma membrane disintegrated and died, lead to small fragments.     

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.     

Molecular design of antimicrobial peptides based on hemagglutinin fusion domain to combat antibiotic resistance in bacterial infection

Antimicrobial peptides are derived from the viral fusion domain of influenza virus hemagglutinin based on rational analysis of the intermolecular interaction between peptides and bacterial outer membrane. It is revealed that the isolated viral fusion domain is a negatively charged peptide HAfp^1‐23 that cannot effectively interact with the anionic membrane. Conversion of the native HAfp^1‐23 to a positively charged peptide HAfp^1‐23_KK by E11K/D19K mutation can promote the peptide‐membrane interaction substantially; this confers to the peptide a moderate antibacterial potency against antibiotic‐resistant bacterial strains. Cyclization of the linear peptide HAfp^1‐23_KK results in a cyclic peptide cHAfp^1‐23_KK, which can largely minimize entropy penalty upon the peptide‐membrane binding by pre‐stabilizing peptide hairpin configuration in solvent, where the linear peptide would incur in a considerable conformational change/folding from intrinsic disorder (in water) to the structured hairpin conformation (in lipid). As might be expected, the cyclization considerably improves peptide antibacterial activity with minimum inhibitory concentration of 67 and 34 μg/mL against multidrug‐resistant Pseudomonas aeruginosa and methicillin‐resistant Staphylococcus aureus, respectively.     

动物源杂合肽P18对白血病K562细胞的致死作用研究

通过其致死白血病K562细胞过程,对动物源杂合肽P18的分子结构与功能进行了研究.MTT实验表明P18显著致死K562细胞;Western blot实验和流式细胞分析提示P18导致 K562细胞坏死,而非依赖于caspases的传统凋亡;荧光探针标记后,观察到P18显著增加K562细胞的膜通透性;圆二(CD)图谱分析,在模拟细胞膜环境的溶液中,P18分子出现α-螺旋.综上所述,P18肽分子可能在细胞膜微环境的介导下产生α-螺旋,产生或暴露了肽分子的破膜结构,导致膜通透性显著增加,最终导致K562细胞的坏死.     

Cell-penetrating peptides: a comparative membrane toxicity study

Cell-penetrating peptides (CPPs) constitute a new class of delivery vectors with high pharmaceutical potential. However, the abilities of these peptides to translocate through cell membranes can be accompanied by toxic effects resulting from membrane perturbation at higher peptide concentrations. Therefore, we investigated membrane toxicity of five peptides with well-documented cell-penetrating properties, pAntp(43-58), pTAT(48-60), pVEC(615-632), model amphipathic peptide (MAP), and transportan 10, on two human cancer cell lines, K562 (erythroleukemia) and MDA-MB-231 (breast cancer), as well as on immortalized aortic endothelial cells. We studied the effects of these five peptides on the leakage of lactate dehydrogenase and on the fluorescence of plasma membrane potentiometric dye bis-oxonol. In all cell lines, pAntp(43-58), pTAT(48-60), and pVEC(615-632) induced either no leakage or low leakage of lactate dehydrogenase, accompanied by modest changes in bis-oxonol fluorescence. MAP and transportan 10 caused significant leakage; in K562 and MDA-MB-231 cells, 40% of total lactate dehydrogenase leaked out during 10 min exposure to 10 microM of transportan 10 and MAP, accompanied by a significant increase in bis-oxonol fluorescence. However, none of the CPPs tested had a hemolytic effect on bovine erythrocytes comparable to mastoparan 7. The toxicity profiles presented in the current study are of importance when selecting CPPs for different applications.     

家蝇幼虫抗菌肽MDL-2的电泳制备及其抗菌作用机理

通过体壁损伤和感染大肠杆菌同时诱导家蝇Musca domestica幼虫产生免疫血淋巴,经沸水浴热变性,透析浓缩处理,然后经Tricine-SDS-PAGE得到诱导前后家蝇幼虫血淋巴中蛋白差异表达条带,将该条带电泳回收,复性,抗菌活性检测等步骤,分离纯化得到抗菌肽MDL-2,其分子中富含Pro,Gly和碱性氨基酸,分子量为11 kD,对革兰氏阴性菌Escherichia coli和革兰氏阳性菌Staphylococcus aureus均有较强抗性,因此电泳制备抗菌肽的方法为此类生物微量活性物质的分离纯化提供一种行之有效的途径。通过MDL-2对大肠杆菌和金黄色葡萄球菌通透性和透射电镜超微结构的图谱分析,MDL-2首先与细菌的外膜结合,然后抗菌肽形成柔性的两亲空间构象与细胞内膜作用,扰乱了膜脂分子的排列,改变了细胞膜的通透性,影响细胞膜的结构和功能,细胞膜上形成了许多孔道,同时造成细胞内的原生质扩散,并从孔道向胞外渗漏,影响了细菌的代谢系统,最终引起细胞膜破碎,细胞完全解体,从而起到抑菌杀菌作用。     

Novel short antimicrobial peptide isolated from Xenopus laevis skin

A rich source of bioactive peptides, including a large number of antimicrobial peptides, has been found in amphibian skin. In this study, a novel short antimicrobial peptide was purified from Xenopus laevis skin and characterised through reversed‐phase high‐performance liquid chromatography, Edman degradation and matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry. The peptide was composed of six amino acids with a sequence of DEDLDE and thus named X. laevis antibacterial peptide‐P2 (XLAsp‐P2). Transmission electron microscopy revealed that this peptide showed potential antimicrobial abilities against bacteria by damaging the bacterial cell membrane. XLAsp‐P2 maybe inhibit bacterial growth by binding to the microbial genomic DNA. The peptide also exhibited a weak haemolytic activity against rabbit red blood cells. Therefore, XLAsp‐P2 is a novel short anionic antibacterial peptide with broad activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.