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.     

昆虫抗菌肽基因工程及其分子设计研究进展(英文)

昆虫抗菌肽是昆虫免疫后存在于血淋巴中的一类活性肽,具有广谱抗菌的生物活性,因而昆虫抗菌肽基因工程迅速成为生命科学领域的研究热点之一.介绍了昆虫抗菌肽的基因结构和表达,综述了昆虫抗菌肽的人工合成与改造以及转基因工程的研究成果,概述了昆虫抗菌肽的分子设计方法和现状,并展望了昆虫抗菌肽的应用前景.     

黄粉虫抗菌肽的提取及其生化特性的研究

【目的】测定黄粉虫Tenebrio molitor Linnaeus幼虫抗菌肽提取液的浓度、抑菌活性及其部分生化特性和凝血效应。【方法】本实验用浓度为1×108 CFU/m L大肠杆菌Escherichia coli诱导5龄黄粉虫幼虫,分别在诱导12、24、36、48、60和72 h后提取其中的抗菌肽,并用考马斯亮兰法测定抗菌肽粗提液蛋白的浓度,并用滤纸片法测定其抑菌活性,同时对其热稳定性、反复冻融稳定性、蛋白酶稳定性及不同p H对其活性的影响等生化特性及凝血效应进行了探究。【结果】经大肠杆菌诱导的黄粉虫抗菌肽粗提液的蛋白浓度均显著高于未诱导的黄粉虫组(P<0.01),且在诱导48 h时产生的抗菌肽提取液蛋白的浓度最高,产生的抑菌圈直径也显著高于未诱导的黄粉虫组(P<0.05),生化特性的测定结果显示,黄粉虫抗菌肽有较好的热稳定性、酶稳定性及酸碱稳定性,反复冻溶后对其抑菌活性影响不大,并且无凝血效应。【结论】大肠杆菌可以刺激黄粉虫的免疫系统,增加抗菌肽的表达量,使其产生浓度高、活性强的抗菌肽,且生化特性较稳定。本研究对黄粉虫抗菌肽作为绿色抗生素用于畜牧养殖业的进一步开发与利用提供了科学的理论依据。     

抗菌肽克隆基因的表达和转基因研究现状

抗菌活性肽具有抗细菌、真菌、病毒、原虫,还有杀精子活性和抑癌活性,具有潜在的应用价值。本文介绍国内外克隆抗菌肽基因的表达研究状况以及抗菌肽在基因植物、动物方面的应用成果存在的问题。     

小菜蛾Gloverin like抗菌肽对蝉拟青霉侵染的响应研究

抗菌肽是昆虫天然免疫的重要成分,在抵御病原物的侵染中发挥着重要的作用。昆虫Gloverin抗菌肽是一类仅在鳞翅目昆虫中存在的富含甘氨酸抗菌肽。本研究在转录组测序的基础上对小菜蛾抗菌肽gloverin like基因进行了克隆,其开放阅读框序列全长519 bp,编码172 aa,信号肽为1-17 aa。序列分析表明其序列中甘氨酸的比例为15%,与鳞翅目昆虫Gloverin抗菌肽具有较高的同源性。qRT-PCR结果表明小菜蛾抗菌肽gloverin like基因在脂肪体和血细胞组织中表达量较高。蝉拟青霉、金黄色葡萄球菌及大肠杆菌均可诱导小菜蛾抗菌肽gloverin like基因上调表达,蝉拟青霉诱导12 h后表达量达到最高。利用LC-MS方法在蝉拟青霉侵染的小菜蛾幼虫血淋巴中鉴定出小菜蛾Gloverin like、Gloverin抗菌肽以及Lysozyme II、Transferrin、Prophenoloxidase等抗菌效应蛋白。与对照相比,蝉拟青霉侵染后小菜蛾Toll通路4个免疫基因(βGBP、Toll、Cactus和Dorsal)表达量上升。本研究在抗菌肽方面为进一步研究小菜蛾抵御病原真菌入侵的分子机制提供基础信息,也为新的害虫防治方法提供了思路和靶标。     

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。     

抗菌肽克隆基因的表达和转基因研究现状

抗菌活性肽具有抗细菌、真菌、病毒、原虫,还有杀精子活性和抑癌活性,具有潜在的应用价值。本文介绍国内外克隆抗菌肽基因的表达研究状况以及抗菌肽在转基因植物、动物方面的应用成果及存在的问题。     

管氏肿腿蜂寄生和注射其毒液对黄粉虫过氧化氢酶基因表达的影响

为了探究管氏肿腿蜂Scleroderma guani寄生和注射其毒液对寄主黄粉虫Tenebrio molitor蛹中过氧化氢酶表达的影响,采用RT-PCR克隆黄粉虫过氧化氢酶基因,利用生物信息学软件分析基因序列结构特性,采用实时荧光定量PCR技术分析该基因的表达特征,使用试剂盒测定过氧化氢酶活性。克隆获得的黄粉虫过氧化氢酶基因编码阅读框序列长1 620 bp,编码539个氨基酸。该基因编码的氨基酸序列中含有催化氨基酸位点(His-71、Asn-183和Tyr-393)以及过氧化氢酶家族的3个保守基序：近端活性位点序列(FDRERIPERVVHAKGXG)、NADPH结合位点(XXHQXXXXFXD)和血红素配体结合位点(RXFXYXDXH)。被管氏肿腿蜂寄生和注射其毒液后,黄粉虫蛹中的过氧化氢酶基因的表达量显著上调,其血淋巴和脂肪体中的过氧化氢酶活性显著升高。研究结果表明,管氏肿腿蜂毒液能调控黄粉虫过氧化氢酶基因的表达。     

家蝇抗菌肽Domesticin在毕赤酵母中的表达及抑菌活性检测

利用毕赤酵母菌株表达家蝇抗菌肽domesticin基因并检测其抑菌活性。克隆家蝇domesticin基因与pPIC9k质粒相连,构建重组表达载体p PIC9K-domesticin,将其电击转化入毕赤酵母KM71中。甲醇诱导后利用Tricine-SDS-PAGE及Western Blot检测融合蛋白的表达,通过最小抑菌浓度测定表达产物的抑菌活性。结果显示家蝇抗菌肽Domesticin在毕赤酵母中成功表达,抑菌实验表明Domesticin对多种细菌具有抑制作用。Domesticin是一种对受试革兰氏阳性菌和革兰氏阴性菌均具有抑菌活性的广谱新型抗菌肽,有望成为新一代抗菌剂。     

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。     

鸡β-防御素7在大肠杆菌中的表达及活性分析

【目的】根据鸡β-防御素7(Gal-7)的成熟肽基因序列合成基因,构建表达Gal-7的大肠杆菌工程菌,研究重组鸡防御素Gal-7成熟肽的体外生物活性。【方法】将合成的gal-7基因克隆到大肠杆菌表达载体pGEX-6p-1中,得到重组质粒pGEX-6p-gal7,转化大肠杆菌BL21(DE3),经IPTG诱导表达得到含GST标签的融合蛋白GST-Gal7;之后用Prescission蛋白酶将GST标签切除,并对成熟肽进行质谱分析;再利用琼脂打孔扩散法检测Gal-7成熟肽的体外抑菌活性,用2倍稀释法测定对指示菌的最低抑菌浓度。【结果】成功构建Gal-7大肠杆菌异源表达工程菌,表达纯化的重组Gal-7成熟肽质谱鉴定分子量为5 516 Da,其对黄色微球菌(NCIB 8166)、金黄色葡萄球菌(ATCC 25923)、粪肠球菌(ATCC 29212)、大肠杆菌(CMCC 44102)均有抑菌活性,最低抑菌浓度分别为16.875、67.5、67.5、135 mg/L。【结论】获得表达鸡Gal-7成熟肽的大肠杆菌工程菌,并且切除GST标签的Gal-7成熟肽具有生物活性。     

果蝇dro基因的克隆与原核表达载体构建

目的:检测drosocin对农作物致病菌的抑菌作用,构建含drosocin基因dro的原核表达载体。方法:以黑腹果蝇(Drosophila melanogaster)DNA为模板,由特异引物通过PCR方法扩增dro基因的编码序列,将此片段连接在克隆载体pMD18-T上进行测序,再用酶切-连接的方法将目的片断亚克隆到携带有6×组氨酸二氢叶酸还原酶标签的原核表达载体pQE40上。结果:克隆得到大小为195bp的dro基因片段,并成功构建了原核表达载体pQE40/dro。结论:克隆到dro基因,构建了原核表达载体pQE40/dro,并获得了转化株M15[pREP4]/dro。     

Cloning,expression, and purification of a new antimicrobial peptide gene from Musca domestica larva

Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram − bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study.     

猪防御素PD基因表达载体的构建

目的：构建猪防御素PD基因表达载体。方法：化学合成经过适当改造的带有双酶切位点的PD基因,将该基因定向插入带有相同酶切位点的融合表达质粒PinPoin^TMXa-3的多克隆位点构建表达质粒。结果：构建的表达载体经双酶切电泳分析及插入基因片段序列分析,表明PD基因表达载体构建成功。结论：PD基因表达载体的构建,为进一步获得PD基因的表达产物,研究其抗菌活性、抗菌机理打下基础。     

Molecular cloning,expression in <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> of Attacin A gene from <Emphasis Type="Italic">Drosophila</Emphasis> and detection of biological activity

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.     

Stress responses as a tool To detect and characterize the mode of action of antibacterial agents.

Single-copy gene fusions between the lacZ reporter gene and Escherichia coli strains containing promoters induced by cold shock (cspA), cytoplasmic stress (ibp), or protein misfolding in the cell envelope (P3rpoH) were constructed and tested to determine their ability to detect antibacterial agents while simultaneously providing information on their cellular targets. Antibiotics that affect prokaryotic ribosomes selectively induced the cspA::lacZ or ibp::lacZ gene fusion, depending on their mode of action. The membrane-damaging peptide polymyxin B induced both the P3rpoH::lacZ and ibp::lacZ fusions, while the beta-lactam antibacterial agent carbenicillin activated only the P3rpoH promoter. Nalidixic acid, a compound that causes DNA damage, downregulated beta-galactosidase synthesis from P3rpoH but had little effect on expression of the reporter enzyme from either the cspA or ibp promoter. All model antibiotics could be identified over a wide range of sublethal concentrations with signal-to-noise ratios between 2 and 11. A blue halo assay was developed to rapidly characterize the modes of action of antibacterial agents by visual inspection, and this assay was used to detect chloramphenicol secreted into the growth medium of Streptomyces venezuelae cultures. This simple system holds promise for screening natural or combinatorial libraries of antimicrobial compounds.     

The molecular design of a recombinant antimicrobial peptide CP and its in vitro activity

Antibacterial peptides from various sources express different antibacterial activity. In order to obtain a high activity antibacterial peptide, the sequences of four antimicrobial peptides--Protegrin-1, 4 kDa Scorpion Defensin, Metalnikowin-2A and Sheep Myeloid Antibacterial Peptide SMAP-29--were exploited to generate a synthetic antimicrobial peptide cp gene, which was then cloned into the expression vector pPICZalpha-A. The constructed recombinant expression vector pPICZalpha-cp was transformed into Pichia pastoris X-33, in which the synthetic antimicrobial peptide (CP) could be expressed under the control of the inducible AOX1 promoter and secreted via the alpha mating factor leader of Saccharomyces cerevisiae. Results showed that recombinant plasmid is highly stable, and In vitro experiments showed that the recombinant antimicrobial peptide CP is heat and acid-stable, and it has high antibacterial activity against several Gram-positive and -negative bacteria. Only 1 microg of the recombinant antimicrobial peptide CP has an antibacterial activity equivalent to 64 U ampicillin. Thus, this recombinant antimicrobial peptide could serve as an attractive candidate for the development of therapeutic antimicrobial drugs.     

人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白的原核表达及纯化

目的:表达和纯化人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白。方法:利用PCR搭接方法及基因合成方法获得目的基因,插入带有6×His标签的原核高效可溶性表达载体pET32a中,构建重组表达质粒pET32a-T9-ac-9,将重组表达质粒转化大肠杆菌BL21(DE3),经IPTG诱导目的基因表达;对融合蛋白进行Ni2+金属螯合柱纯化。结果:构建的重组表达质粒经PCR、内切酶鉴定及基因序列测定证实;目的蛋白在大肠杆菌中获得表达,SDS-PAGE显示相对分子质量为22.917×103;对表达产物进行了亲和层析纯化,从上清中获得了纯度较高的人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白。结论:获得了可溶性的人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白,为其生物学功能研究奠定了基础。     

甾短杆菌胆固醇氧化酶基因在大肠杆菌中的表达

为了实现胆固醇氧化酶在大肠杆菌中的表达,将甾短杆菌Brevibacterium sp．DGCDC-82胆固醇氧化酶基因用PCR的方法去掉信号肽序列,连接到质粒pTrc99a,遗过筛选得到了表达胆固醇氧化酶的重组菌JMl09／pTrc99a—COD。经IPTG诱导后表达出相对分子质量约为5．5×10^4的蛋白质。分别考察了诱导温度、时间、IPTG浓度等因素对重组菌表达的胆固醇氧化酶的影响。在优化条件下,该胆固醇氧化酶的酶活可以达到700U／L。酶学特性分析表明其反应的最适pH为7．5,最适温度为40℃。