噬菌体重组酶系统在合成生物学中的应用*

合成生物学技术采用工程化设计理念,对生物体进行有目标的设计、改造乃至重新合成,对重塑非自然功能的“人造生命”具有重要意义。噬菌体重组系统具有高效、精确和广谱适用性等特点,在基因工程、代谢工程以及生物治疗等合成生物学领域得到了广泛的应用。从基因电路、体内遗传改造和体外重组等方面全面阐述了噬菌体重组系统在合成生物学研究的现状及热点,对当前该系统的局限性进行了探讨,并就未来的研究和发展趋势进行了展望。     

噬菌体gp17基因2种重组产物的抗菌效应比较

通过重组技术获得大肠埃希菌噬菌体内溶素纯化蛋白和表面展示噬菌体,并观察产物的生物效应。将肠侵袭性大肠埃希菌EIEC 8401噬菌体LSB-1内溶素基因gp17构建到质粒pET300中,并在大肠埃希菌BL21中诱导表达,通过Ni柱纯化系统纯化产物;利用噬菌体展示技术构建T7-LSB-gp17重组噬菌体,通过双层琼脂法纯化噬菌体,并观察2种产物的抗菌效应。2 139 bp的gp17基因通过重组技术表达出78.3 ku的可溶性蛋白,纯化后浓度为2.38 mg/mL,其对EIEC8401有良好的抑菌活性,但对其他试验菌无抗性;通过噬菌体展示技术构建的重组噬菌体T7-LSB-gp17通过SDS-PAGE电泳显示在78 ku处有表达增强,对EIEC8401无感染、裂解作用,但对EIEC8401及其他试验菌有明显溶菌作用,宿主谱增加。通过重组技术获得的噬菌体LSB-1内溶素基因gp17的产物对LSB-1噬菌体原宿主具有明显的抑制效应。其中gp17表达的纯化蛋白具有明显的宿主专一性,重组噬菌体悬液有较宽种类的抗菌作用。这可能是因为gp17蛋白与噬菌体表面复杂空间结构的相互作用产生的生物效应。     

鸡白痢沙门氏菌及其噬菌体vB-SpuS-Spp4的分离鉴定

分离鸡白痢沙门氏菌及其噬菌体,对分离到的噬菌体进行生物学特性分析。采用沙门氏菌选择性培养基进行鸡白痢沙门氏菌的分离,并进行特异性PCR鉴定。以鸡白痢沙门氏菌分离株为宿主菌,采用双层平板法进行噬菌体的分离,通过负染法电镜观察噬菌体的形态和大小,并对噬菌体进行裂解谱、最佳MOI、一步生长曲线、对温度、pH、紫外线以及氯仿的稳定性等生物学特性的研究。分离到噬菌体vB-SpuS-Spp4为长尾噬菌体,对29株鸡白痢沙门氏菌的裂解率为100%;最佳感染复数为0.001时噬菌体的效价可达1.47×10~9 PFU/mL。该噬菌体潜伏期为15min,暴发期为45min,暴发量为127;vB-SpuS-Spp4温度耐受性较强,在pH2～13环境中仍有活性。噬菌体vB-SpuS-Spp4对紫外照射不敏感,对氯仿不敏感。噬菌体vB-SpuS-Spp4作为一株烈性噬菌体,对鸡白痢沙门氏菌具有广泛的裂解作用,为临床鸡白痢的噬菌体控制奠定了基础。     

一株新分离的肠杆菌噬菌体的快速遗传鉴定及其宿主识别基因的快速克隆

以大肠杆菌8099为宿主自医院污水中分离出一株肠杆菌噬菌体IME08,其遗传物质经RNA酶、DNA酶处理证实其为DNA,用限制性内切酶处理该DNA证实其为双链DNA。利用随机引物PCR技术扩增并克隆该噬菌体基因组的随机片段,经测序后同源比对,判断该噬菌体是一株新的T4-like噬菌体。根据4株T4-like噬菌体 (T4,JS98,T2及K3) 宿主识别基因 (g37) 5'端的高度保守序列,采用随机PCR与巢式PCR结合的“基因组跳跃”策略快速克隆出了该噬菌体的宿主识别基因g37和g38。     

自然环境中T4型噬菌体g23基因多样性研究进展

摘要：过去的20多年,伴随着分子生物学技术在环境微生物研究中的应用,环境中细菌和真菌群落基因多样性及与其生存环境间的关系逐渐被揭示,但对于地球上广泛存在且数量巨大的生命体-噬菌体基因多样性研究还很少。本文以编码T4型噬菌体主要壳蛋白基因g23为目标,综述了近年来T4型噬菌体在海洋、湖泊和稻田中基因多样性的研究进展。研究结果表明T4型噬菌体g23基因分布与其生存环境关系很大,许多g23基因按获取环境不同划分为几个新类群。同时文中也指出了针对环境中T4型噬菌体g23基因研究应该注意的几点问题及未来的研究发展趋势。     

利用λRed重组系统敲除鼠伤寒沙门氏菌sopB基因

利用λRed重组系统敲除鼠伤寒沙门氏菌LT2的(Salmonella enterica serovar typhimurium LT2,S.typhimurium LT2)sopB基因。以pKD4质粒为模板,扩增得到中间带有卡那霉素抗性基因且两端各带有59 bp分别与sopB基因上下游序列同源的同源打靶片段,将其转化至表达Red重组酶的S.typhimurium LT2感受态细胞中;在抗生素压力和λRed重组系统帮助下,同源片段和菌体sopB基因发生同源重组,通过卡那霉素筛选得到带有抗性标记的阳性重组菌;转入重组酶表达质粒pCP20以除去抗性标记,得到保留单一FRT位点的突变菌株;利用PCR技术鉴定重组菌,并通过检测沙门氏菌效应蛋白SopB的分泌以及沙门氏菌感染HeLa细胞后pAKT的激活反应来鉴定sopB基因是否被敲除。构建的ΔsopB突变菌株失去了分泌SopB蛋白的能力,且不能够像野生型菌株那样在感染HeLa细胞的过程中激活pAkt。本研究获得了S.typhimurium LT2的sopB基因缺失突变株,为沙门氏菌感染宿主过程中SopB的功能研究提供工具,同时也为进一步探索其他类型细菌的基因敲除提供了线索。     

Mu噬菌体中的一个新的基因拼接(模型)

一般认为基因表达是通过蛋白的作用,如阻遏物和效应物来控制的。近来报道了一个不同的控制模型。借此,一个失活的基因通过某种重组过程重新置于一个新的位点,并由此产生转录。酵母的交配型,沙门氏菌的相变和Mu噬菌体的宿主范围都是重组控制的例子。     

人Ⅰ型免疫缺陷病毒gag-env嵌合基因在重组痘苗中的表达

Gag和Env蛋白是人Ⅰ型免疫缺陷病毒(Humanimmunodeficiencyvirustype1,HIV1)的结构蛋白,是HIV1诱导机体产生体液免疫和细胞免疫的主要抗原。本实验通过多次亚克隆,将env基因以正确的三联密码读框插入gag基因的下游,制备了HIV1gagenv嵌合基因,并将嵌合基因分别置于痘苗病毒p75启动子和牛痘病毒A型包涵体(ATI)启动子的下游,经过同源重组和红细胞吸附试验筛选,获得了2株重组痘苗病毒。免疫荧光试验和酶免疫试验证明,两株重组痘苗病毒均能正确地表达HIV1gagenv嵌合基因。动物实验表明,gagenv嵌合基因重组痘苗病毒可诱导小鼠产生抗HIV特异性抗体。这些结果为艾滋病颗粒化疫苗的研制提供了借鉴。     

Red同源重组在大肠杆菌基因组修饰中的应用

Red同源重组技术发展迅速,已广泛应用于大肠杆菌基因组修饰,在点突变、基因敲除、序列整合等方面发挥着重要作用。简要综述了Red同源重组的重组机制和操作策略等研究进展,并介绍了Red同源重组在大肠杆菌基因组减小及多基因代谢途径优化方面的应用情况。     

基因组装技术在合成生物学中的应用

基因组装技术是合成生物学领域近年来发展起来的新型技术。它基于大规模基因组数据分析,发现新型的或隐藏的生物活性物质合成基因簇。利用基因组装技术,可提高或激活沉默的生物合成基因簇在微生物中的表达,从而合成潜在的、有价值的生物活性物质。本文旨在阐明最新的体内和体外基因组装技术的设计原理、关键策略及其应用。基因组装技术是合成生物学、代谢工程和功能基因组学研究的重要工具,对生物活性物质的高效生产及合成具有重要意义。     

Site-specific recombination of T2 phage using IP008 long tail fiber genes provides a targeted method for expanding host range while retaining lytic activity

The application of bacteriophages (phages) in therapy urgently requires the production of wide-host-range recombinant phages that possess strong lytic activity. The wide-host-range IP008 phage was classified by transmission electron microscopy analysis as an A2 morphotype member of the Myoviridae family of the order Caudovirales . IP008 showed a high homology (99.4% similarity in the amino acid alignment of the major capsid protein Gp 23) with KEP10, another wide-host-range phage. The long tail fiber genes (genes 37 and 38 ) from the genome of T2 were replaced with those of the IP008 phage by homologous recombination. The host range of the recombinant phages was identical to that of IP008. Furthermore, the recombinant phage bacterial lytic activity was restored. Future analyses of host-range mutants of the closely related phages T2 and IP008 could lead to a more precise localization of the genetic factors responsible for receptor specificity.     

Community composition, host range and genetic structure of the fungal entomopathogen Beauveria in adjoining agricultural and seminatural habitats

Although intensively investigated for biological control of insect pests, little is known about the ecology of the fungal entomopathogenic genus Beauveria in natural or agricultural habitats. In this study, we used molecular phylogenetic and genotypic information to infer species diversity, reproductive potential and genetic structure of Beauveria occurring within a single arable field and bordering hedgerow in Denmark. Isolates were sampled from cultivated field and hedgerow soils, from insects harbouring latent fungal infections, and from the phylloplanes of three plant species common in the hedgerow flora. A nuclear phylogeny of this local Beauveria assemblage resolved seven phylogenetic species, including (i) five phylogenetic species within Beauveria bassiana sensu stricto ; (ii) Clade C, a taxonomically uncharacterized species that is morphologically indistinguishable but phylogenetically distant from B. bassiana s.s. ; and (iii) Beauveria brongniartii. All seven species were present throughout the hedgerow habitat, including as infections in insects. Significantly, only B . bassiana s.s. phylogenetic species Eu_1 was isolated from tilled soils. Mating type polymerase chain reaction assays demonstrated that all five B. bassiana s.s. phylogenetic species possess bipolar outcrossing mating systems. Of these, only the Eu_1 population contained two mating types; however, a 31:2 skew in MAT1:MAT2 mating types suggests a low frequency of sexual reproduction in this population. The four remaining B. bassiana s.s. phylogenetic species were fixed for single mating types and these populations are evidently clonal. Multilocus microsatellite genotyping revealed polymorphism in all five phylogenetic species of B. bassiana s.s. ; however, all show evidence of clonal genetic structure.     

食源性致病性大肠杆菌O157:H7和O55:H7特异性噬菌体的分离与鉴定

【背景】肠出血性大肠杆菌(enterohemorrhagic Escherichia coli,EHEC) O157:H7和肠致病性大肠杆菌(enteropathogenic E. coli,EPEC) O55:H7是2株常见食源性致病菌,能导致腹泻及肠道外疾病,其特异性噬菌体具有制备新型抗菌制剂的应用前景。【目的】分离能特异裂解O157:H7和O55:H7的噬菌体,并分析其生物学特性和基因组特征,探索致病性大肠杆菌防控的抗生素替代方法。【方法】利用双层平板法从环境水样中分离噬菌体,对其形态、感染复数、宿主范围、一步曲线等生物学特性进行鉴定,使用Illumina MiSeq平台对其全基因组进行测序,利用RAST、Prokka、BLASTp等软件进行生物信息学分析。【结果】分别以E.coli O157:H7和O55:H7为宿主分离出2株特异性烈性噬菌体:vB＿EcoM＿P251和vB＿EcoM＿P255,均属于肌尾病毒科(Myoviridae)。最佳感染复数均为1,在培养15min内能以91.9%和90.8%的速率吸附到宿主细胞上,而且在37-60℃、pH4.0-11.0条件下保持高且稳...     

The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny

The CRISPR/Cas nuclease is becoming a major tool for targeted mutagenesis in eukaryotes by inducing double‐strand breaks (DSBs) at pre‐selected genomic sites that are repaired by non‐homologous end joining (NHEJ) in an error‐prone way. In plants, it could be demonstrated that the Cas9 nuclease is able to induce heritable mutations in Arabidopsis thaliana and rice. Gene targeting (GT) by homologous recombination (HR) can also be induced by DSBs. Using a natural nuclease and marker genes, we previously developed an in planta GT strategy in which both a targeting vector and targeting locus are activated simultaneously via DSB induction during plant development. Here, we demonstrate that this strategy can be used for natural genes by CRISPR/Cas‐mediated DSB induction. We were able to integrate a resistance cassette into the ADH1 locus of A. thaliana via HR. Heritable events were identified using a PCR‐based genotyping approach, characterised by Southern blotting and confirmed on the sequence level. A major concern is the specificity of the CRISPR/Cas nucleases. Off‐target effects might be avoided using two adjacent sgRNA target sequences to guide the Cas9 nickase to each of the two DNA strands, resulting in the formation of a DSB. By amplicon deep sequencing, we demonstrate that this Cas9 paired nickase strategy has a mutagenic potential comparable with that of the nuclease, while the resulting mutations are mostly deletions. We also demonstrate the stable inheritance of such mutations in A. thaliana.     

Macroptilium atropurpureum (siratro) host specificity genes are linked to a nodD-like gene in the broad host range Rhizobium strain NGR234

Summary A 6.7 kb HindIII fragment from the Sym-plasmid of strain NGR234 was found to code a nodD-like gene flanked by two loci which were required for siratro host range. Transfer of the 6.7 kb fragment from NGR234 to R. trifolii strain ANU843 conferred extended host range ability to this strain on siratro plants but not to other plants normally nodulated by strain NGR234. Tn5 mutagenesis of the 6.7 kb fragment showed that insertions located into loci flanking the nodD-like gene abolished the extended host range phenotype. A hybridization probe spanning one of the host specificity loci was shown to hybridize to three specific bands in the NGR234 genome. Complementation and DNA hybridization data showed that the nodD-like gene of strain NGR234 was functionally similar to that in R. trifolii. The introduction to R. trifolii of the 6.7 kb HindIII fragment containing Tn5 insertions located in the nodD-like gene did not abolish the ability to extend the host range of R. trifolii to siratro plants. However, transfer of the 6.7 kb HindIII to R. trifolii derivatives containing Tn5 insertions into either nodA, B or C or other R. trifolii nod genes failed to confer siratro nodulation to these recipients. Reconstruction experiments showed that the 6.7 kb fragment from strain NGR234 and the 14 kb nodulation region of R. trifolii could induce the nodulation of siratro plants when introduced together into Sym-plasmid-cured Rhizobium strains.     

新疆阿克苏地区一株羊源沙门菌及其小菌落变异株的生物学特性

【背景】小菌落变异株(smallcolonyvariant,SCV)是一种具有独特的表型及致病特征且生长缓慢的细菌亚群,而国内鲜有关于食源性沙门菌SCV的研究报道。【目的】为食源性沙门菌的防治及动物性食品安全提供实验数据。【方法】使用氨基糖苷类抗生素对羊源胆汁中分离的沙门菌进行实验室诱导得到SCV,然后分别对野生株和诱导株的菌落形态、生长、生化特性、营养缺陷型检测、运动性、耐药性检测及耐药基因、毒力基因、生物被膜形成能力进行比较和分析。【结果】经卡那霉素诱导获得一株血红素依赖型沙门菌SCV,与野生株相比,诱导株生长缓慢,低于野生株84%,不利用柠檬酸盐,溶血能力增强40%,对磺胺类和氨基糖苷类药物的耐受性增强,生物被膜形成能力减弱45%,运动能力减弱78%。【结论】沙门菌SCV的生长和生理生化特性与野生株相比有显著差异,使得沙门菌SCV的分离鉴定尤为困难;并且SCV的致病性与耐药性等方面的变化可能给沙门菌病的防治带来更大挑战,其机制还有待深入研究。     

Post-release survey to assess impact and potential host range expansion by Amitus hesperidum and Encarsia perplexa, two parasitoids introduced for the biological control of the citrus blackfly, Aleurocanthus woglumi in Dominica

Four years after the release of two exotic parasitoids, Amitus hesperidum Silvestri (Hymenoptera: Platygasteridae) and Encarsia perplexa Huang and Polaszek (Hymenoptera: Aphelinidae) for the classical biological control of the citrus blackfly (CBF), Aleurocanthus woglumi Ashby (Hemiptera: Aleyrodidae) in Dominica, a survey was conducted to assess establishment as well as potential nontarget effects especially on Aleyrodidae and other related taxa. CBF populations were low to non-existent in 50 of 51 field sites examined. At the site where CBF was encountered, both E. perplexa and A. hesperidum were present and CBF populations were declining. The two parasitoids were not among the several species collected on nontarget Aleryodidae and Hemiptera. It is concluded that E. perplexa and A. hesperidum have kept CBF populations under effective biological control in Dominica and there is no evidence of any nontarget effects on other Aleyrodidae or their natural enemies. Handling Editor: Dirk Babendreier.