结核分枝杆菌同源重组基因敲除系统的构建和应用

【目的】结核分枝杆菌同源重组效率很低,突变株的构建需要半年之久。本研究的目的在于构建一种用于在结核分枝杆菌中进行基因快速敲除、且易于筛选的高效同源重组系统。【方法】野生型结核分枝杆菌转化含有SacB反向选择标记、且能诱导表达两种同源重组酶gp60和gp61的质粒pSL002。然后分别将靶基因的两个同源臂克隆入到含有hyg(潮霉素)抗性基因和gfp(绿色荧光蛋白)基因的重组质粒pSL001中,再将靶基因同源臂-loxP-hyg-gfp-loxP片段从pSL001切下,转化含有pSL002的野生型结核分枝杆菌,一步得到双交换突变株。再将含有SacB反向选择标记、且表达Cre重组酶的质粒pSL003转化入结核分枝杆菌双交换突变株中,切除两个loxP之间的hyg抗性基因和gfp基因,得到无痕缺失突变株。最后利用含有2%蔗糖的琼脂糖平板去除含有SacB反向选择标记的质粒pSL002和pSL003。【结果】在结核分枝杆菌中成功构建了高效同源重组系统,利用该系统构建了rv1364c、pstP跨膜区、pstP胞外区三个突变株,得到双交换突变株的效率为25%-62.5%,从双交换突变株得到无痕缺失突变株的效率为100%。通过gfp作为荧光标记基因,利用NightSea BlueStar蓝光手电筒和滤光眼镜,可以对平板上的基因缺失株直接进行快速判定。【结论】该同源重组系统利用gp60和gp61重组酶,在时间上将在结核分枝杆菌中无痕缺失突变株的构建从6个月缩短到3个月。这是目前为止在结核分枝杆菌中构建突变株最快且效率最高的方法,为加速分枝杆菌功能基因组的研究提供了新的遗传工具。     

重组分枝杆菌噬菌体检测分枝杆菌的发光研究

采用生物发光方法检测重组的分支杆菌噬菌体对不同细菌的发光反应,并比较了仅在特定的温度范围内才能繁殖的温敏噬菌体Phage 88和正常噬菌体Phage 40对分枝杆菌感染活力测定时发光强度的差异,以建立用不同类型重组噬菌体检测结核分枝杆菌耐药性的方法和条件.结果显示两种噬菌体对各种分枝杆菌作用后均有发光,对非分枝杆菌发光值很低,两者差异有显著性;不同的分枝杆菌发光值有差异：卡介苗的发光值最高,结核分枝杆菌的发光值最低;温敏噬菌体Phage 88的检测灵敏度大于正常噬菌体Phage 40,差异显著.因此可认为两株噬菌体均可特异地检测结核分枝杆菌,但Phage 88的效果优于Phage 40.     

分枝杆菌噬菌体的分子生物学研究进展及其应用

世界卫生组织估计,世界上有１／３多的人感染结核分枝杆菌ｕ砂ｃｏｂａｃｔｅｒｉｕｍｔａｂｅｒｃｕｌｏｓｉｓ,Ｔｈ）,并导致每年有３００万人死亡。最近几年,由于抗药性和多重抗药性结核杆菌（ＭＤＲ－－Ｔｈ）的出现以及人免疫缺损病毒（ＨＩＶ）侵染,Ｔｈ在世界范围内复苏,现行的医疗手段越来越无能为力,急需一种新的、有效的方法来预防、诊断和治疗结核［’］病原性分枝杆菌生长缓慢,ＭｔＵｂｅｒｃｕｌｏｓｉｓ繁殖一代大约需２４ｈ,在固体培养基上形成一个菌落至少需要３—６周时间。Ｍｌａｐｒａｅ不能体外培养,必须在鼠…     

重组工程及其应用

随着功能基因组研究的需要 ,新近建立起一项新型高效的基于体内同源重组的遗传工程技术———重组工程技术。重组工程可定义为 :基于噬菌体短同源序列重组功能的遗传工程 ,或者基于同源重组的遗传工程。λ噬菌体Red系统完全不同于传统的依赖RecA的大肠杆菌重组系统 ,特点是使用长度仅为 <5 0个碱基的同源臂高效率地催化体内同源重组反应。体内重组过程不再需要预先构建含有同源序列的质粒或噬菌体的中间产物 ,只需要简单在体外合成寡核苷酸同源序列 ,或者用PCR方法合成线性打靶序列。重组反应不依赖大肠杆菌RecA系统 ,不需要限制性内切核酸酶和连接酶 ,不需要复杂的体外重组操作 ,可在大肠杆菌体内对染色体DNA、对BAC和PAC质粒或普通质粒载体进行精确的修饰 ,包括真核或原核细胞基因组DNA的基因敲除、基因敲入、基因克隆和各种突变体的引入。由于该技术具有高效率、简单性和应用的广泛性等独特优点 ,将来完全有可能取代传统的遗传工程技术。主要介绍了λ噬菌体Red重组酶系统及重组工程在功能基因组研究方面的应用与进展     

噬菌体重组酶介导的DNA同源重组工程

来源于噬菌体的遗传操作工具在基因工程中具有非常重要的地位,例如位点特异性重组酶、柯斯质粒DNA文库及同源重组酶等。其中,来源于Lambda噬菌体的同源重组酶Redα/Redβ和来源于Rac原噬菌体的同源重组酶RecE/RecT能够高效地介导35–50bp短同源臂之间的重组。基于噬菌体同源重组酶Redα/Redβ和RecE/RecT开发的DNA同源重组工程(Recombineering)能够对靶标DNA分子进行快速、精准、高效的修饰,不受限制性内切酶识别位点和DNA分子大小限制,已发展成为一种新型的基因工程技术。本文主要综述了噬菌体同源重组酶及其作用机制、在大肠杆菌及其他细菌中的应用和开发,以及在微生物次级代谢产物的挖掘、动植物转基因、病毒基因组克隆和修饰等方面的应用。原位激活沉默基因簇需要宿主特异性的DNA同源重组工程进行启动子和调控元件的修饰;异源表达次级代谢产物的首要步骤一般是通过RecET直接克隆大的DNA片段;动植物转基因复杂载体的构建效率在有了Red同源重组系统以后有了革命性的发展;RecET直接克隆和Red同源重组介导的感染性克隆构建和修饰方法,不仅有利于病毒基因组功能研究...     

细菌重组系统及其应用研究进展

利用重组酶和辅助蛋白共同作用于DNA片段上,使不同基因重新组合以完成基因重组的现象在细菌中广泛存在,基因重组对于细菌的遗传多样性、进化等具有重要意义。目前,细菌基因重组主要分为同源重组、位点特异性重组和转座重组3种类型。本文主要对细菌重组系统重组酶的种类、作用机制及其在细菌遗传操作中的应用策略进行阐述。     

应用Red重组工程技术建立asd基因缺失的大肠杆菌DH108菌株

目的：建立一株新遗传表型的大肠杆菌DH10BAasd菌株。方法：应用pKD46介导的重组系统、kan／kil选择反选择系统、双链线性DNA重组技术和重叠引物介导的DNA重组技术,在菌株DH10B体内,对其染色体上的asd基因进行了基因敲除。结果：建立了一株二氨基庚二酸（DAP）营养缺陷型重组大肠杆菌DH10BAasd。结论：为进一步建立以大肠杆菌DH10B为载体的DNA疫苗或基因治疗载体奠定了基础。     

大肠杆菌重组工程

源于噬菌体的大肠杆菌同源重组系统不需要限制性内切酶和DNA连接酶就可以进行DNA克隆和亚克隆,还能快速地改造质粒、细菌人工染色体及细菌基因组染色体,是基因工程技术的一大突破,被称为重组基因工程或重组工程。该技术操作简单,效率较高,可望为功能基因组学研究提供一个有力的工具。     

分枝杆菌噬菌体生物学特性探讨

为了确定不同分枝杆菌噬菌体的宿主菌以及扩增方法和最佳保存方法,观察了七种分枝杆菌噬菌体对结核分枝杆菌、耻垢分枝杆菌的裂解情况,并分别于感染后 24、48小时采用离心 过滤、孵育 过滤方法收集噬菌体比较扩增效率,采用不同稳定剂对分枝杆菌噬菌体进行液体和冻干保存,在不同时间段采用琼脂双层法检测其效价。结果显示:①D29分枝杆菌噬菌体能同时较高效地裂解结核分枝杆菌和耻垢分枝杆菌;②感染 48小时后采用孵育 过滤方法收集的噬菌体效价高,方法简单;③液体 4℃保存的噬菌体稳定性好, 70℃液体保存和冻干后 4℃、室温、37℃保存依据不同稳定剂而相差较大。因此,在 48小时后采用孵育 过滤方法收集噬菌体具有高效率特性并且简单易行,噬菌体液体 4℃保存简单、有效,值得推荐。     

应用Red重组工程技术建立asd基因缺失的大肠杆菌DH10B菌株

目的:建立一株新遗传表型的大肠杆菌DH10BΔasd菌株。方法:应用pKD46介导的重组系统、kan/kil选择反选择系统、双链线性DNA重组技术和重叠引物介导的DNA重组技术,在菌株DH10B体内,对其染色体上的asd基因进行了基因敲除。结果:建立了一株二氨基庚二酸(DAP)营养缺陷型重组大肠杆菌DH10BΔasd。结论:为进一步建立以大肠杆菌DH10B为载体的DNA疫苗或基因治疗载体奠定了基础。     

分枝杆菌甾酮C1,2位脱氢酶基因敲除的研究

利用分枝杆菌对植物甾醇进行边链降解可产生4-AD（4-烯-雄甾-3,17-二酮）和ADD（1,4-二烯-雄甾-3,17-二酮）,ADD由4-AD在C1,2位脱氢酶(ksdD)作用下脱氢产生,这两种物质在化学结构上高度相似,难以分离。本文首先扩增出部分ksdD基因,大小为631bp,并以此为基础构建打靶载体pUC19-MK。将pUC19-MK电转分枝杆菌感受态,通过同源重组敲除分枝杆菌染色体上正常的ksdD基因,使C1,2位脱氢酶失活,以达到4-AD大量积累的目的。结果通过初筛筛选出5株转化子,进行甾体转化实验,发酵144h时,1号转化子的4-AD生成率达到17.52％,比出发菌株提高了192％,而此时ADD的生成率仅为6.12％,比出发菌株降低了89.9％。     

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.     

Production of unmarked mutations in mycobacteria using site-specific recombination

Gene disruption experiments play an important role in the functional characterization of genes in mycobacteria and rely mostly on the use of one or two antibiotic resistance markers. We have developed a system for mycobacteria which features both the advantages of the use of antibiotic resistance markers for gene disruption experiments and the ability to efficiently rescue the marker leaving an unmarked mutation on the chromosome. This new genetic tool relies on the transposon gammadelta site-specific recombination system. A res-OmegaKm-res cassette was used to generate an insertional mutation by allelic exchange both in Mycobacterium smegmatis and Mycobacterium bovis BCG. Upon expression in the mutated strains of tnpR, the transposon gammadelta resolvase gene, res-OmegaKm-res, was excised efficiently leaving behind a single res sequence at the mutated locus. A plasmid was engineered allowing expression of tnpR from an easily curable mycobacterial vector. This system will be useful for simple construction of unmarked mutations or repeated use of the same antibiotic marker to generate multiple mutants.     

结核分枝杆菌EF4敲除菌株的构建

EF4是一个由 lepA 基因编码的与蛋白质翻译密切相关的延伸因子,在细菌中高度保守,但其确切功能和分子机制尚不清楚,在结核分枝杆菌中的功能至今未见报道。为探索EF4在结核分枝杆菌中的功能,需构建一株结核分枝杆菌 lepA 基因敲除株。本研究以结核分枝杆菌H37Ra全基因组DNA为模板,设计并通过聚合酶链反应(polymerase chain reaction,PCR)扩增 lepA 基因左、右臂,连接到p0004S质粒,构建同源重组质粒p0004S-Δ lepA 。然后,通过噬菌体体外包装,将p0004S-Δ lepA 质粒连接到phAE159质粒,构建phAE159-Δ lepA 噬菌体包装质粒。在耻垢分枝杆菌mc 2155中大量扩增噬菌体并受结核分枝杆菌侵染进行同源重组,筛选阳性克隆,从基因组和蛋白质表达水平检测该突变株中 lepA 基因及EF4蛋白表达。PCR结果显示,敲除株基因组中 lepA 基因已被潮霉素抗性基因成功替换,蛋白免疫印迹结果显示该敲除株中无EF4表达,表明其为成功构建的Ra Δ lepA 。生长曲线分析显示,正常培养条件下,结核分枝杆菌野生株与敲除株生长趋势一致。敲除株与野生株在菌落形态上有一定差异,相比于野生株,Ra Δ lepA 菌落颜色发黄,凸起偏厚,生长过程中生物膜皱褶较少。耐胁迫能力分析显示,与野生株相比,Ra Δ lepA 耐热、抗去垢剂、抗氧化能力无显著差异,但耐酸性环境能力明显增强。本研究利用噬菌体介导的重组法成功构建了结核分枝杆菌 lepA 基因敲除株,为后续研究结核分枝杆菌EF4的功能提供了重要基础。     

基因克隆及组装技术的研究进展 *

随着测序技术的发展,已知的DNA序列数量呈指数性增加,为了能更快的探索其未知的生物功能,一些简化组装流程的DNA克隆及组装新技术争相发展起来。其中大部分需要在菌体外构建重组体,但重组酶纯化过程复杂,运送和保存方法要求严格,致使成本较高。最近研究者开发了一些在菌体内进行DNA组装的简易、低成本的新方法。主要对各类基因克隆及组装方法的研究现状、原理和优缺点等进行综述,并结合实际的工作内容展望了未来的发展趋势,希望能为进一步研究开发新技术提供参考。     

应用大规模测序技术和生物信息学研究造血干/祖细胞的基因表达及新基因 的识别和克隆

从新生儿脐血和成人骨髓中分选出造血干/祖细胞(HSC/HPC),构建成cDNA文库,对其进行大规模表达序列标签(EST)测序,通过生物信息学等手段分析基因表达谱,并进行新基因的全长cDNA克隆。在所测的10512条可分析E ST序列中,有9866条来自脐血CD34+|细胞,其中4697条(47.6%)为已知基因,2603条(26.4%)为已知EST,1415条(14.3%)代表未知EST。在已知基因中,8.2%基因与造血相关,22.7%涉及细胞代谢、结构和迁移,13.0%与细胞分裂和防御相关,26.2%与RNA、蛋白质的合成相关,10.6%和细胞信号传递有关。对一些已知和未知的EST,综合测序、生物信息学等方法,进行全长克隆,已获得23个新基因的全长cDNA。 Abstract:Hematopoietic stem/progenitor cells were isolated from umbilical cord blood and adult bone marrow,and subject to cDNA library construction.The gene expression pattern in CD34+ cells and the identification and cloning of novel genes were performed by sequencing ESTs and analyzing them with the tools of bioinformatics.Among the obtained 10 512 ESTs which could be further analyzed,9,866 were from umbilical cord blood where 4 697(67.6%)were known genes,2 603(26.4%)were known ESTs and 1415(14.3%)represented novel ESTs.Within the identified genes,8.2% was involved in hematopoiesis,22.7% was associated with cell metabolism,structure and mobility,13.0% was linked to cell division and defence,26.2% was related to RNA protein synthesis and 10.6% was related with cell signal transduction.In parallel,we developed an efficient working system combining sequencing,bioinformatics,etc.and obtained 23 full-length cDNAs from both known and novel ESTs identified in this work.     

‘Yeast Mail’: A Novel Saccharomyces Application (NSA) to Encrypt Messages

The universal genetic code is used by all life forms to encode biological information. It can also be used to encrypt semantic messages and convey them within organisms without anyone but the sender and recipient knowing, i.e., as a means of steganography. Several theoretical, but comparatively few experimental, approaches have been dedicated to this subject, so far. Here, we describe an experimental system to stably integrate encrypted messages within the yeast genome using a polymerase chain reaction (PCR)‐based, one‐step homologous recombination system. Thus, DNA sequences encoding alphabetical and/or numerical information will be inherited by yeast propagation and can be sent in the form of dried yeast. Moreover, due to the availability of triple shuttle vectors, Saccharomyces cerevisiae can also be used as an intermediate construction device for transfer of information to either Drosophila or mammalian cells as steganographic containers. Besides its classical use in alcoholic fermentation and its modern use for heterologous gene expression, we here show that baker's yeast can thus be employed in a novel Saccharomyces application (NSA) as a simple steganographic container to hide and convey messages.     

A Novel Possible Mechanism for the Genesis of Genomic Duplications and Its Experimental Test

Duplication of genomic regions is an important biological process associated with the appearance of gene families, the origin of alternative splicing, and the etiopathogenesis of genetic diseases. Different mechanisms for the genesis of duplications have been suggested, based mainly on structural analyses. However, experimental confirmation of those mechanisms is scarce, mostly because of a lack of information about the circumstances that triggered the rearrangements. Here, I characterize a duplication of about 300 kbp (kilobase pairs) that occurred in the course of a gene targeting experiment. Considering the structure of the locus and the triggering event, I suggest a likely mechanism for the genesis of this duplication which involves anomalous processing of contiguous Okazaki fragments during lagging strand replication. Most importantly, I provide experimental evidence to substantiate that the proposed mechanism can indeed lead to duplication of genomic segments. The model presented represents a novel mechanistic pathway that can explain a variety of rearrangements, including genomic tandem duplications and deletions.[Reviewing Editor: Dr. Jonathon A. Eisen]