黑水虻抗菌肽的研究进展

黑水虻是近几年昆虫资源研究领域的热点,目前其应用研究主要集中在饲料添加剂和有机垃圾处理等方面.然而,近年来研究发现,黑水虻幼虫自身免疫系统产生的抗菌肽对革兰氏阴性菌、革兰氏阳性菌以及真菌都具有广谱抗菌活性,有着广泛的应用前景.为了促进黑水虻抗菌肽的开发和应用,本文主要综述了黑水虻抗菌肽的结构特征、抑菌活性、作用机制、生...     

Cu2+胁迫对黑水虻幼虫抗菌肽组分及抑菌活性的影响

研究不同浓度Cu~(2+)胁迫对黑水虻5龄幼虫抗菌肽分离纯化组分及抑菌活性影响,为黑水虻无害化处理粪便技术的有效实施提供有力的理论依据,为其副产品在饲料、食品及医药研发中的应用提供有价值的实验数据。本文在人工饲料中添加不同浓度Cu~(2+)(0、150、1 200 mg/kg)以饲喂黑水虻幼虫,采用金黄色葡萄球菌针刺法诱导5龄幼虫,断头收集血淋巴,高速冷冻离心结合超滤离心制备抗菌肽粗提物;利用RP-HPLC对三组抗菌肽粗提物进行分离纯化,收集各纯化峰对应组分,采用纸片琼脂扩散法测定各纯化峰对应组分的抑菌效果,以阐述Cu~(2+)胁迫对黑水虻抗菌肽的影响。结果表明,经Cu~(2+)胁迫,抗菌肽粗提物分离纯化后各组分的出峰时间及峰面积所占比例均不同,不同处理浓度下各分离组分第2峰面积均大于第1峰面积,其中150-2峰面积最大,为73.31%;分离纯化后所得6个组分对金黄色葡萄球菌、大肠杆菌及白色念珠菌均有抑菌活性,对金黄色葡萄球菌、白色念珠菌的抑菌活性显著高于对大肠杆菌抑菌活性,但其对金黄色葡萄球菌及白色念珠菌的抑菌活性未见显著差异;与其他5个组分相比,组分150-2对金黄色葡萄球菌、白色念珠菌及大肠杆菌抑菌活性最强,抑菌直径分别为27.85±0.74 mm、28.34±0.76 mm、21.97±0.54 mm。由此可见,不同浓度的Cu~(2+)胁迫对黑水虻幼虫抗菌肽组分及抑菌活性均产生显著影响,其中组分150-2抑菌活性最强,具有很好的开发潜能。     

黄粉虫抗菌肽在大肠杆菌中表达条件优化及活性分析

为了提高黄粉虫抗菌肽基因tmAMP1m在大肠杆菌中的表达量,研究了培养温度、诱导时间及IPTG浓度等不同条件对HIS-TmAMP1m融合蛋白表达量和活性的影响。通过Tricine-SDS-PAGE分析确定最佳表达条件,同时,通过琼脂孔穴扩散法检测其抑菌活性。结果表明,含有重组质粒的大肠杆菌在37℃,使用终浓度为0.1 mmol/L IPTG培养4 h时,融合蛋白表达量较高,可占细菌总蛋白40%以上,抗菌活性最好。用Ni2+亲和层析纯化获得较纯的融合蛋白,Western blotting分析表明其能与His单克隆抗体起特异性反应。诱导表达的融合蛋白对宿主菌生长产生一定程度抑制。融合蛋白经100℃煮沸10 h,在20℃反复冻融10次,与强酸强碱缓冲液、不同的有机溶剂和蛋白酶混合后都具有极强的稳定性,仍然表现出良好的抗菌活性。此外,最小抑菌浓度(MIC)测定结果表明,融合蛋白对5种菌具有良好的抗菌活性。研究结果为昆虫抗菌肽推广应用和进一步研究奠定了基础。     

抗菌肽活性及天然抗菌肽的改造

抗菌肽具有抗菌谱广、热稳定性强、分子量小及免疫原性小等特点,其杀菌机制独特,病原菌不易产生耐药性,有望开发成新一代肽类抗生素。本文主要综述了影响抗菌肽生物活性的生化性质,即螺旋度、疏水性、两亲性、正电荷数等,并从结构的角度论述了其对抗菌肽抑菌活性的影响。部分抗菌肽具有空间结构不稳定、溶血活性等缺点,限制了其临床应用。因此,对天然抗菌肽的改造也成为目前抗菌肽的研究热点,本文还综述了天然抗菌肽的改造方法。     

黑水虻抗菌肽HI-3对RAW264.7细胞免疫调控作用研究

为了明确黑水虻Hermetia illucensL.抗菌肽HI-3对RAW264.7细胞的免疫调控作用,拓展黑水虻抗菌肽在免疫领域的应用潜能,本文研究了抗菌肽HI-3对RAW264.7细胞形态、增殖、吞噬功能、分泌NO和细胞因子以及抗氧化活性的影响。结果表明,20、40、80、160、320μg/mL的抗菌肽HI-3对RAW264.7细胞形态及增殖指数均无显著影响,但均能显著增强RAW264.7细胞的吞噬功能,且随着HI-3浓度增加吞噬能力逐渐增强,当HI-3浓度达到320μg/mL时RAW264.7细胞的吞噬能力最强(P<0.01);而在160μg/mL以下处理浓度时,对NO含量无显著性影响(P>0.05);抗菌肽HI-3可以上调RAW264.7细胞分泌IL-6、TNF-α和IL-1β,下调抑炎因子IL-10的分泌量,且具有一定的浓度依赖性,浓度达到320μg/mL时效果最为显著(P<0.01);另外,抗菌肽HI-3亦能够显著提高RAW264.7细胞超氧化物歧化酶(SOD)活力和总抗氧化能力(T-AOC)含量,尤其是320μg/mL时,效果最为明显(P<0.0...     

正交优化诱导蚯蚓抗菌肽的研究

采用电激诱导蚯蚓产生抗菌肽,考察了影响诱导率的因素。在单因素实验的基础上,通过正交试验优化了诱导工艺。结果表明:最佳诱导工艺条件为:电压5V、pH6.0、诱导时间为2min,诱导率达95.2%。     

抗菌肽RIKL的分子设计及生物学活性分析

天然抗菌肽具有较强的杀菌能力,但高生物相容性抗菌肽的构建一直阻碍着该领域的发展.为了提高抗菌肽的选择特异性,通过分子动力学分析探讨了抗菌肽的结构特性,并检测其生物学活性.首先以(RXKY)2(YRY)2 (X代表Ile,Y代表Leu)为模板设计新型抗菌肽分子RIKL.通过圆二色谱(circular dichroism,...     

家蝇幼虫抗菌肽的超声诱导及分离纯化和活性研究

目的:分离纯化家蝇(Musca domestica)幼虫免疫血淋巴中的抗菌肽,研究抗菌肽的性质和抑菌特性.为进一步研究家蝇幼虫抗菌肽的产生及应用提供实验基础.方法:通过超声处理诱导家蝇幼虫产生免疫血淋巴,经沸水浴热变性,结合高速离心,两步CM-Sepharose离子交换层析,冷冻干燥浓缩等步骤,分离得到一种具抗菌活性的蛋白质.用Tricine-SDS-PAGE鉴定抗菌肽的纯度和性质,抑菌试验分析抗菌肽的抑菌特性.结果:300W,50Hz超声处理60s能够诱导家蝇幼虫产生抗菌肽,经分离纯化后Tricine-SDS-PAGE凝胶电泳结果显示为一条带,相对分子量大约为5.8kD.纯化蛋白对阴沟肠杆菌,绿脓杆菌等G-杆菌的抑菌活性较强,而对金黄色葡萄球菌效果不显著.结论:诱导和纯化了一种家蝇幼虫抗菌肽,为此类活性物质的分离纯化提供了有效的方式.     

人源抗菌肽LL-37的原核表达及活性鉴定

目的:实现大肠杆菌高效可溶表达人源抗菌肽LL-37。方法:LL-37基因克隆至原核载体pET32a,于大肠杆菌BL21(DE3)中诱导表达。运用相关生物信息学软件分析重组蛋白Trx-LL-37的理化性质、亲/疏水性、蛋白质二级结构及其可溶表达概率。实验还考察了不同诱导温度对重组蛋白可溶表达比例的影响。结果:生物信息学分析显示,Trx-LL-37分子量21.5kD,理论等电点6.3,物理性质稳定,二级结构简单,具有可溶表达倾向。重组蛋白最佳诱导温度为17℃,与37℃相比,可溶表达比例由37.2%提高至50.2%,并且总表达量也提高了5%左右。抑菌结果显示纯化产物对多种常见细菌的生长具有抑制作用。结论:可采用融合方式通过原核系统高效可溶表达LL-37,为LL-37的功能研究打下基础。     

新疆家蚕抗菌肽在毕赤酵母中表达及活性研究

目的:在毕赤酵母中表达新疆家蚕抗菌肽基因(Cecropin-XJ)并检测其活性.方法:根据作者实验室已克隆获得的新疆家蚕抗菌肽(Cecropin-XJ)基因设计引物,通过PCR方法扩增Cecropin-XJ,将PCR产物和表达载体pPIC9K用EcoR Ⅰ及Not Ⅰ双酶切,构建重组表达质粒pPIC9K-(Cecropin-XJ),酶切及测序正确后,电转化到毕赤酵母GS115,对分泌表达的重组蛋白进行活性检测.结果:PCR扩增获得192 bp Cecropin-XJ,成功构建pPIC9K-Cecropin-XJ,优化诱导条件证明在pH 6的BMMY培养液中,0.5%甲醇诱导约48h后,获得的表达产物活性较强,对多种革兰氏阴性菌和阳性菌具有抗菌活性,在100℃条件下,其活性可维持100min以上.结论:新疆家蚕抗菌肽在毕赤酵母中分泌表达,为大规模发酵生产奠定了基础.     

Black soldier fly (Diptera: Stratiomyidae) larval heat generation and management

Mass production of black soldier fly, Hermetia illucens (L.) (Diptera: Stratiomyidae), larvae results in massive heat generation, which impacts facility management, waste conversion, and larval production. We tested daily substrate temperatures with different population densities (i.e., 0, 500, 1000, 5000, and 10 000 larvae/pan), different population sizes (i.e., 166, 1000, and 10 000 larvae at a fixed feed ratio) and air temperatures (i.e., 20 and 30 °C) on various production parameters. Impacts of shifting larvae from 30 to 20 °C on either day 9 or 11 were also determined. Larval activity increased substrate temperatures significantly (i.e., at least 10 °C above air temperatures). Low air temperature favored growth with the higher population sizes while high temperature favored growth with low population sizes. The greatest average individual larval weights (e.g., 0.126 and 0.124 g) and feed conversion ratios (e.g., 1.92 and 2.08 g/g) were recorded for either 10 000 larvae reared at 20 °C or 100 larvae reared at 30 °C. Shifting temperatures from high (30 °C) to low (20 °C) in between (∼10-d-old larvae) impacted larval production weights (16% increases) and feed conversion ratios (increased 14%). Facilities should consider the impact of larval density, population size, and air temperature during black soldier fly mass production as these factors impact overall larval production.     

Antimicrobial activity of Wedelia trilobata crude extracts.

A biological screening of activity against Gram-positive and Gram-negative bacteria, yeasts, and fungi of crude extracts from Wedelia trilobata is reported. The n-hexane extract showed antibacterial activity against Bacillus subtilis, Mycobacterium smegmatis, Staphylococcus aureus, and Staphylococcus epidermidis (Gram-positive bacteria); along with Proteus vulgaris, Pseudomonas aeruginosa, Salmonella group C, Salmonella paratyphi, and Shigella sonnei (Gram-negative bacteria). The ethyl acetate extract was active only against Salmonella group C; and the aqueous extract was inactive against the tested bacteria. None of the tested extracts showed biological activity against the yeasts (Candida albicans, Candida tropicalis, Rhodotorula rubra) or the fungi (Aspergillus flavus, Aspergillus niger, Mucor sp., Trichophyton rubrum).     

DNA ligase activity in crude extracts of fibroblasts and lymphocytes

DNA ligase activity was determined in crude cell extracts using a new assay which measures the retention of double stranded circular phage λ DNA on nitrocellulose filters, and allows accurate determinations of the enzyme activity with cell concentration corresponding to 0.1 μg of proteins. Using this assay, we show that the DNA ligase activity varies greatly among mammalian cell lines. The higher activity is found in actively growing fibroblasts where it is stimulated by dimethyl sulfate pretreatment of the cells, whereas the low activity measured in resting lymphocytes is not modified by dimethyl sulfate. The DNA ligase activity correlates with the cells sensitivity towards ionizing radiations.     

Anthelmintic and antimicrobial activity of Indigofera oblongifolia leaf extracts

Due to the endless emergence of drug resistant pathogens, there is a constant need for new therapeutic agents for clinical use. The identification of active components in natural products and determining the efficacy of these active components has become the current focus of pharmacological research. The present study aimed to evaluate the anthelmintic and antimicrobial activities of Indigofera oblongifolia leaf extract (ILE) against the earthworm Allolobophora caliginosa, the gram-positive bacteria (Bacillus cereus, Streptococcus pneumoniae, and Staphylococcus aureus), the gram-negative bacteria (Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli) and the yeast Candida albicans. Methanolic extract of I. oblongifolia leaf was obtained and the total phenolics and flavonoids in ILE were determined. The anthelmintic study was carried out to determine the time to paralysis and time to death of worms using three doses (100, 200, and 300 mg/mL) of ILE. Also, Kirby-Bauer disk diffusion susceptibility method was used to determine the antimicrobial activity of ILE. The results showed that ILE induces paralysis and death of A. caliginosa at all concentration tested faster than the reference drug, Albendazole. Additionally, ILE exhibited prominent antimicrobial activity against all gram-positive bacteria tested but almost no significant activity against the gram-negative bacteria, except K. pneumoniae. ILE showed close similarity to the spectrum of chloramphenicol and cefoxitin activities. Furthermore, C. albicans was highly susceptible to the leaf extracts. Our results showed that ILE is an effective anthelmintic and antimicrobial agent.     

黑水虻肠道细菌抗菌筛选及其活性物质分子鉴定

【目的】从昆虫黑水虻分离的肠道细菌进行抗植物病原菌的拮抗菌筛选,对获得有拮抗活性的肠道细菌进行活性物质的分子鉴定。【方法】用稀释涂布法从水虻肠道中分离菌株,采用平板对峙法进行抗菌筛选,对有抗菌活性的菌株通过生理生化实验、16S rRNA鉴定和进化树分析确定其种属。参考已知脂肽合成关键基因设计引物,以拮抗菌总DNA为模板进行PCR扩增,对目的片段进行测序。【结果】通过抗菌筛选获得一株对水稻黄单胞菌以及小麦纹枯病病原菌等有很强抑制效果的水虻肠道细菌BSF-CL,经鉴定为枯草芽胞杆菌。脂肽合成关键基因PCR结果显示BSF-CL菌株具有脂肽Iturin和Surfactin合成的关键基因。推测BSF-CL很可能合成脂肽Iturin和Surfactin。【结论】从水虻肠道中分离出对水稻黄单胞菌有很强抑菌活性的菌株,分离菌被鉴定为一种枯草芽胞杆菌,通过活性物质的分子克隆鉴定初步推测其活性物质可能为脂肽Iturin和Surfactin。     

Antimicrobial activity of crude extracts from mangrove fungal endophytes

The aim of this work was to select endophytic fungi from mangrove plants that produced antimicrobial substances. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) or minimal fungicidal concentrations (MFC) of crude extracts from 150 isolates were determined against potential human pathogens by a colorimetric microdilution method. Ninety-two isolates (61.3%) produced inhibitory compounds. Most of the extracts (28–32%) inhibited Staphylococcus aureus (MIC/MBC 4–200/64–200 μg ml⁻¹). Only two extracts inhibited Pseudomonas aeruginosa (MIC/MBC 200/>200 μg ml⁻¹). 25.5 and 11.7% inhibited Microsporum gypseum and Cryptococcus neoformans (MIC/MFC 4–200/8–200 μg ml⁻¹ and 8–200/8–200 μg ml⁻¹, respectively), while 7.5% were active against Candida albicans (MIC/MFC 32–200/32–200 μg ml⁻¹). None of the extracts inhibited Escherichia coli. The most active fungal extracts were from six genera, Acremonium, Diaporthe, Hypoxylon, Pestalotiopsis, Phomopsis, and Xylaria as identified using morphological and molecular methods. Phomopsis sp. MA194 (GU592007, GU592018) isolated from Rhizophora apiculata showed the broadest antimicrobial spectrum with low MIC values of 8–32 μg ml⁻¹against Gram-positive bacteria, yeasts and M. gypseum. It was concluded that endophytic fungi from mangrove plants are diverse, many produce compounds with antimicrobial activity and could be suitable sources of new antimicrobial natural products.     

Interactions of lactoferricin-derived peptides with LPS and antimicrobial activity

Synthetic peptides derived from human and bovine lactoferricin, as well as tritrpticin sequences, were assayed for antimicrobial activity against wild-type Escherichia coli and LPS mutant strains. Antimicrobial activity was only obtained with peptides derived from the bovine lactoferricin sequence and peptides corresponding to chimeras of human and bovine sequences. None of the peptides corresponding to different regions of native human lactoferricin showed any antimicrobial activity. The results underline the importance of the content of tryptophan and arginine residues, and the relative location of these residues for antimicrobial activity. Results obtained for the same assays performed with LPS mutants suggest that lipid A is not the main binding site for lactoferricin which interacts first with the negative charges present in the inner core. Computer modelling of the most active peptides led to a model in which positively charged residues of the cationic peptide interact with negative charges carried by the LPS to disorganise the structure of the outer membrane and facilitate the approach of tryptophan residues to the lipid A in order to promote hydrophobic interactions.     

Measurement of lipoxygenase activity in crude and partially purified potato extracts

Two assay systems, one a spectrophotometric assay at 234 nm, the other based on the oxygen electrode, were compared as methods for the routine analysis of lipoxygonase activity in crude and partially purified potato extracts. The spectrophotometric assay was unsuitable for the analysis of crude extracts and only gave meaningful results under very restricted reaction conditions. The oxygen electrode provided a reliable method for routine analysis of lipoxygenase activity.     

Pilot-scale extraction and antimicrobial activity of crude extract from ovine neutrophils

Antimicrobial peptides have potential to be a high-value product purified from waste ovine blood. Previous work characterised antimicrobial peptides isolated from ovine neutrophils and determined the mechanisms of action. Here it is shown that the crude antimicrobial extract can be produced on a pilot-scale while retaining the antimicrobial activity. This crude extract could be used as a biopreservative for chilled lamb meat products or in a topical cream for treating cuts and grazes.