Induction, selection and antibacterial activity of the antibacterial peptides from lepidopteran insect cultured cell lines

We induced 3 cell lines that were in vitro cultured from Lepidoptera with heat inactivated Escherichia coli DH_5α to stimulate the antibacterial peptide followed by antibacterial activity assay, induction dynamic research and Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine SDS-PAGE) experiment. The antibacterial activity of the induced BTI-Tn-5B1 cell line was the highest, and the antibacterial activity increased gradually to the highest level in 16 hours after stimulation. A new antibacterial peptide with a molecular weight of about 8000 Da was preferentially induced in Trichoplusia ni BTI-Tn-5B1 cells in 16 hours after stimulation. Antibacterial activity assays indicated that it had inhibition against Staphylococcus aureus, Escherichia coli K₁₂D₃₁ and Salmonella derby. It has especially strong inhibition against Gram-negative bacteria such as Escherichia coli K₁₂D₃₁ and Salmonella derby. __________ Translated from Journal of Central China Normal University (Natural Sciences), 2006, 40(2): 240–243 [译自: 华中师范大学学报 (自然科学版]     

昆虫抗茵肽的生理活性及其转基因应用前景

昆虫抗菌肽是昆虫免疫后存在于血淋巴中的一类活性肽.根据分子的结构可分为5类天蚕素类、昆虫防御素、富含脯氨酸或精氨酸的抗菌肽、富含甘氨酸的抗菌肽、抗真菌肽.且具有广谱的抗菌、抗病毒、抑制肿瘤的生物活性.概述了昆虫抗菌肽的基因的克隆与表达及转基因研究方面的进展,并展望了抗菌肽在基因工程中的应用前景.     

昆虫抗菌肽的生理活性及其转基因应用前景

昆虫抗菌肽是昆虫免疫后存在于血淋巴中的一类活性肽。根据分子的结构可分为 5类 :天蚕素类、昆虫防御素、富含脯氨酸或精氨酸的抗菌肽、富含甘氨酸的抗菌肽、抗真菌肽。且具有广谱的抗菌、抗病毒、抑制肿瘤的生物活性。概述了昆虫抗菌肽的基因的克隆与表达及转基因研究方面的进展 ,并展望了抗菌肽在基因工程中的应用前景     

昆虫抗菌肽和抗真菌肽结构与功能的关系及分子设计

在对GenBank和EMBL数据库中登录的昆虫抗微生物肽 (antimicrobial peptide, AMP), 即昆虫抗菌肽 (antibacterial peptide) 和抗真菌肽 (antifungal peptide, AFP) 进行归类整理的基础上,对天蚕素族(cecropins )抗菌肽结构与功能的关系及人工改造的分子设计策略,特别是对目前新发现的一些昆虫抗真菌肽的已知结构与功能关系的研究进展、存在问题等进行了简要介绍和分析,为从事昆虫抗微生物肽的理论研究和发展新型抗生素药物提供了必要的信息。     

Expression of the antibacterial peptide, cecropin, in cultured mammalian cells

We have stably transfected a Chinese hamster lung cell line V79 with a recombinant gene construct where the Drosophila cecropin A2 cDNA is under the control of Rous sarcoma virus, long terminal repeat (RSV LTR). We have not only been able to demonstrate expression at the RNA level by Northern analysis but also have detected an unprocessed peptide using an antiserum raised against Hyalophora cecropin.     

从Granulysin抗菌机理出发合理设计抗菌肽

Granulysin是一种表达于人体毒性T淋巴细胞中的抗菌蛋白,其作用相当于广谱抗菌素。随着分子模拟技术的发展,人们已经初步掌握了其结构与功能的关系,有关这方面的研究也取得了较大的进展。根据Granulysin的覆膜作用机理重新设计出具有活性的抗菌小肽,并通过实验对该片段的活性进行检验。     

昆虫抗菌肽对病原微生物作用的研究进展

在诱导和非诱导情况下,昆虫能产生各种类型的具有体液免疫功能的小分子物质-抗菌肽,参与机体对入侵病原微生物的免疫应答反应,构成了机体独特的免疫系统和免疫机制。这类抗菌肽或抗菌蛋白也存在于其它动物。研究表明,抗菌肽对细菌、真菌、病毒和原虫都具有作用,甚至对癌细胞也具有杀伤作用。随着抗菌肽家族的不断扩大,其结构研究的深入,相继提出了一些崭新的杀菌方式和作用机制。本文从目前国内外这方面的研究入手,分析各抗菌肽的作用特点、杀菌作用模式,展望了基因工程及临床应用的前景。     

昆虫细胞系的培养和建立技术

迄今已经报道的昆虫细胞系有800株以上。昆虫细胞系在昆虫病理学、寄生虫学、内分泌学、遗传学和分子生物学等基础和应用研究中得到越来越广泛的应用。本文结合我们研究的结果和实践经验,概括了国内外昆虫细胞系建立技术的研究进展,包括昆虫细胞培养的发展、昆虫细胞系建立技术、不同昆虫组织来源细胞系的建立方法和过程,以及对昆虫细胞系特征的鉴定等方面。     

斜纹夜蛾Cecropin D成熟肽的原核表达及活性检测

采用RT-PCR方法从斜纹夜蛾Spodoptera litura脂肪体组织中扩增得到了Cecropin D成熟肽基因序列,分析发现Cecropin D成熟肽与斜纹夜蛾Cecropin B之间存在2个氨基酸残基的差异。将获得的基因序列连接入原核表达载体pGEX-4T-1,并在原核细胞中实现了该蛋白的融合表达。SDS-PAGE结果表明,诱导后的宿主菌比未诱导菌中多出了一条融合蛋白表达带,诱导后1 h就可以检测到该蛋白,从诱导后1 h到5 h该蛋白在表达量上没有明显的差异。生长曲线显示在IPTG诱导后宿主菌的生长受到明显的抑制,纯化后的蛋白对细菌具有一定的抑制作用。     

Characterization and culture of virus replicating continuous insect cell lines from the bollworm,Heliothis zea (boddie)

Summary A series of five discrete virus replicating insect cell lines were isolated from the ovarian and fat body tissues ofHeliothis zea pupae. Two of these cell lines (IPLB-HZ-1075 and-HZ-1079) were studied in depth as to their growth and virus replication responses to specific nutrients (acetyl-β-methylcholine, fresh glutamine) in a number of media. The same two cell lines were identified to species by serological (microimmunodiffusion) and isozyme (phosphoglucoisomerase and peptidase:glycyl-leucine) techniques. Distinguishing comparisons were made with other cell lines that have been confused with the present lines in the literature and with cell line and host pupal extracts from the same and other lepidopteran species studied concurrently in this laboratory. Sterility culture tests were negative for mycoplasmas. The present fiveH. zea lines were the first insect cell lines to replicate polyhedra from a unicapsid multiple embedded nuclear polyhedrosis virus (Baculovirus Group A), in this case the homologous virus obtained from larvae ofH. zea.     

Identification of the region that plays an important role in determining antibacterial activity of bovine seminalplasmin

Seminalplasmin (SPLN) is a 47-residue protein isolated from bovine seminal plasma having potent antimicrobial activity against a broad spectrum of microorganisms. SPLN, also known as caltrin, acts as a calcium transport regulator in bovine sperms. Analysis of the sequence of SPLN reveals a 27-residue stretch with the sequence SLSRYAKLANRLANPKLLETFLSKWIG more hydrophobic than the rest of the protein. It is demonstrated that a synthetic peptide corresponding to this 27-residue segment has antimicrobial activity comparable to that of SPLN. It does not exhibit hemolytic activity at concentrations where antibacterial activity is observed. Since P27 can be conveniently obtained in large amounts by chemical synthesis, it could serve not only as a starting compound to obtain peptides with improved antibacterial activity but also to understand the role of SPLN in reproductive physiology.     

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。     

Bt Cry1Ac毒素筛选的粉纹夜蛾离体抗性细胞与敏感细胞蛋白质组的差异分析

采用双向电泳技术和肽质量指纹图谱技术分析了Bt Cry1Ac毒素筛选的粉纹夜蛾Trichoplusia ni抗性BTI-Tn-5B1-4细胞与同源敏感细胞的蛋白质组的差。用Melanie ViewerⅡ软件在抗性细胞的双向电泳图谱上检测到的平均蛋白质点数为707±25个（n=3）,在敏感细胞的双向电泳图谱上检测到的平均蛋白质点数为637±19个（n=3）,其中分辨率高、重复性良好的显著差异点有10余个。对其中的一个敏感细胞特有的显著差异斑点进行了肽质量指纹图谱分析,经数据库 查询表明该蛋白质与胞质外周蛋白具有同源性。     

Induction of antibacterial activity in the blood of the migratory locust Locusta migratoria L.

Injections of low doses of living cells of Bacillus thuringiensis into larvae or adults of Locusta migratoria (Orthoptera) induce an in vivo protection against lethal doses of this pathogen (‘immunisation’). In the present paper we show that concomitant with the induction of the in vivo protection, an antibacterial activity appears in the plasma of the immunised insects. This activity is maintained for 10 days and disappears at the time the protective mechanism loses its efficiency. Antibacterial activity in the plasma of experimental insects is induced by injections of bacteria other than B. thuringiensis (Escherichia coli, Pseudomonas aeruginosa) and non-bacterial substances (ovalbumine, iron saccharate): it reduces growth of cultures of B. thuringiensis and also of E. coli or P. aeruginosa under in vitro conditions. Repeated immunising injections increase the level of plasma antibacterial activity. Preliminary biochemical studies indicate that the antibacterial activity is linked to the presence in the plasma of a low molecular weight (<5000 daltons), heat-stable, polar bacteriostatic compound which is not inactivated by use of conventional proteolytic or glycolytic enzymes.     

印楝素A与印楝素B对粉纹夜蛾BTI-Tn-5B1-4的细胞毒性

为了明确印楝素A和B活性差异的机理,本研究比较了印楝素A和印楝素B对粉纹夜蛾Trichoplusia ni离体培养胚胎细胞系BTI-Tn-5B1-4的毒性。结果表明：印楝素A与印楝素B对BTI-Tn-5B1-4细胞具有良好的增殖抑制活性,处理后3 d,其IC₅₀值分别为2.9 μg/mL和9.85 μg/mL,印楝素A的细胞毒力显著高于印楝素B。倒置显微镜观察发现,印楝素A和印楝素B处理可导致细胞变形,贴壁能力下降,并出现明显空泡,印楝素A的影响明显高于印楝素B。流式细胞仪检测结果表明,印楝素可导致BTI-Tn-5B1-4细胞体积显著膨大,印楝素A处理细胞体积增大程度显著高于印楝素B;印楝素可以明显影响BTI-Tn-5B1-4细胞膜电位,1.25 μg/mL印楝素A和印楝素B处理后3 d,细胞DiBAC4（3）荧光强度分别增加88.12%和55.37%,印楝素A的影响显著高于印楝素B。荧光显微镜观察发现,印楝素对BTI-Tn-5B1-4细胞核具有明显影响,印楝素B的影响明显高于印楝素A,印楝素B处理后,细胞核受损细胞数更多,受损程度更严重。结果显示印楝素A和印楝素B的细胞作用机理存在差异,本研究从细胞学水平解释了印楝素的生长发育抑制作用机理。     

Development and characterization of insect cell lines

Conclusions With the wide availability of insect cell culture media, it can generally be considered a routine process to develop new cell lines. Exceptions to this statement do exist, of course. Difficulties may arise when attempting to culture a specific cell type. For example, while there are a few cell lines from insect fat body and at least one from the midgut, it may not be possible to obtain cell lines from these tissues from all insect species due to terminal differentiation and other factors. Also, researchers have desired cell lines from certain species, such as the honey bee, for which no success has been obtained. As in the early days of tissue culture, it is difficult to discern why negative results occur. However, as more is learned about the physiology and nutrition of various insects and tissues, we may get clues which will help solve these questions.The remaining chapters in this book will provide the reader with exciting uses for insect cell culture. As I mentioned earlier, the baculovirus expression vector system has provided a stimulus to the field of insect cell culture not seen previously.Abbreviations ICD Isocitrate dehydrogenase - ME malic enzyme - PGI phosphoglucose isomerase - PGM phosphoglucose mutase     

Structure-activity relationship of an antibacterial peptide, maculatin 1.1, from the skin glands of the tree frog, Litoria genimaculata.

Maculatin 1.1 (Mac) is a cationic antibacterial peptide isolated from the dorsal glands of the tree frog, Litoria genimaculata, and has a sequence of GLFGVLAKVAAHVVPAIAEHF-NH2. A short peptide lacking the N-terminal two residues of Mac was reported to have no activity. To investigate the structure-activity relationship in detail, several analogs and related short peptides of Mac were synthesized. CD measurement showed that all the peptides took more or less an alpha-helical structure in the presence of anionic lipid vesicles. Analogs which are more basic than Mac had strong antibacterial and hemolytic activities, while short peptides lacking one or two terminal residues exhibited weak or no activity. Outer and inner membrane permeabilization activities of the peptides were also reduced with shortening of the peptide chain. These results indicate that the entire chain length of Mac is necessary for full activity, and the basicity of the peptides greatly affects the activity.     

黄粉甲幼虫抗菌物质的诱导及其抗菌活性

采用饥饿法、紫外线照射法和针刺法处理黄粉甲Tenebriomolitor 6龄幼虫后均能诱导其 产生抗菌物质,收集的血淋巴上清液对真菌有抑制作用,对细菌无抑制作用;经热处理后的血 淋巴上清液则对细菌有抑制作用,而对真菌无抑制作用。SDS-PAGE检测结果发现,与未诱导的 对照相比经诱导的黄粉甲幼虫血淋巴中,原有的一类大分子蛋白质如分子量分别为97kD、44 kD和37 kD左右的蛋白质缺失;而ESI-MS分析结果显示诱导后比诱导前黄粉甲幼虫血淋巴中有 小分子物质产生,推测可能是此类缺失蛋白质分解为小分子量的抗菌肽,从而表现出抗菌活性 。     

DNA content analysis of insect cell lines by flow cytometry

The DNA content of insect cell lines (6 lepidoptera, 1 coleoptera and 1 diptera) was determined by flow cytometry. The DNA profiles of the 8 cell lines tested were different. They were characterized by the presence of several peaks (2 to 7) corresponding to different ploidy levels, by differences in the fluorescence intensity of each peak and by the proportion of cells in each peak. Two cell lines (Cf124 and BmN) were constituted of 2 distinct populations of cells. The DNA profiles of the cell lines were stable among the passages and during the length of time culture. This technique was demonstrated to be useful for the detection of mixed cell lines and nucleopolyhedrovirus cell infection, using Autographa californica MNPV. The flow cytometry gives interesting results on the cell cycle and the ploidy level; it appears as a good tool for insect cell lines characterization. This revised version was published online in July 2006 with corrections to the Cover Date.