DNA改组的最新动态及应用前景

DNA改组(DNA shuffling)是目前最方便、有效的一种分子水平的体外定向进化技术,该技术同倾向错误PCR (Error-prone PCR) 相结合,通过对单基因或相关基因家族的靶序列进行多轮随机诱变、重组和高通量的筛选,可以有效富集正突变,去除负突变,提高突变文库的丰度,创造新基因和获得期望功能的蛋白质。DNA改组技术已在新药物等领域取得了广泛的应用,极大地推动了现代生物科学和生物技术的发展。该技术同计算机强大的数据分析系统相结合,将会为后基因组学的发展提供强有力的技术平台。     

微生物酶DNA shuffling的改进及应用

介绍了DNA shuffling技术的基本原理以及技术改进,对此技术在提高微生物酶的活性、稳定性、抗性以及改变底物专一性等方面的应用进行了综述,并展望了应用前景。     

改进重叠延伸PCR技术构建定点双突变

目的:目的DNA片段中快速构建位点不同的定点双突变体。方法: 借鉴DNA shuffling技术中DNA小片段延伸扩增获得全长DNA片段的工作原理,与常规基因定点突变技术相结合,改进重叠延伸PCR技术构建定点双突变。结果:对嗜酸热脂肪杆菌(Alicyclobacillus acidocaldarius)Tc-12-31的甘露聚糖酶基因AamanA中两个可能的活性位点E151和E231进行双点突变,先后经过无引物和有引物两步PCR,扩增获得全长DNA,测序结果表明得到预期的定点双突变体;酶活性检测和薄层层析结果表明双点突变体丧失了酶的活性。结论: 改良的重叠PCR技术,能经济、简便、高效地获得双点定点突变体,在酶的催化机理的阐述、蛋白质结构改造等分子生物学领域中具有较高的应用价值。     

用交错延伸剪接PCR技术扩增油菜花粉育性基因MS2Bnap全长cDNA序列

交错延伸剪接PCR是一种用于分子进化研究的DNA改组技术.本实验利用其原理,将已获得的油菜花粉育性基因MS2Bnap有部分序列重叠的三段PCR产物作为模板进行扩增,获得了花粉育性基因MS2Bnap的全长cDNA序列。 Abstract:SOE by PCR is one of DNA shuffling technologies for molecular evolution engineering.Using the theory of SOE by PCR,the full-length cDNA sequence of pollen fertility gene MS2Bnap of Brasscia napus was amplified with three obtained fragments as templates which have part overlapping of MS2Bnap sequence.     

利用DNA Shuffling技术构建重组PRRSV ORF5基因

通过DNA shuffling技术对PRRSV ORF5基因进行改组,研究其对异源毒株交叉中和能力。将获得的嵌合基因△2ORF5与载体pET-32a连接,转化到大肠杆菌Escherichia coli BL21中,经诱导表达获得约42 kDa重组蛋白。将纯化的重组△2ORF5蛋白免疫BALB/c小鼠,制备多克隆抗体,Western blotting试验表明其与4株亲本毒株重组ORF5蛋白具有良好的反应性。病毒感染抑制试验结果表明,多克隆抗体对4株亲本毒株具有较高的抑制率(53%)。研究结果为研制新型PRRSV疫苗奠定了基础。     

分段DNA shuffling: 一种大分子海藻糖合酶有效的定向进化方法

[目的]红色亚栖热菌(Meiothermus ruber)海藻糖合酶(Trehalose synthase,M-TreS)将麦芽糖转化生成海藻糖只需一步反应,且具有很好的热稳定性及pH耐受性,是潜在的工业生产海藻糖的酶源.为了提高该酶的性能,有必要对其进行定向进化.[方法]M-TreS基因(M-treS)大小为2 889bp.该蛋白质分子本身具有很大的进化空间,但是却不宜进行全长基因Shuffling.分段DNA shuffling是为大分子蛋白质(基因≥2 000 bp)的进化而设计的一种方法.该方法分为三步:(1)用两对引物分别扩增目的基因的上游片段和下游片段;(2)上下游片段各自进行Shuffling; (3)利用重叠延伸PCR连接上下游突变群,建立完整基因的突变文库.[结果]结合易错PCR,通过该方法经一轮进化获得一株酶活力是野生型1.6倍、催化效率是野生型2倍的突变株.序列分析表明,该突变株共有6个位点发生了氨基酸的替代,其中一个来自易错突变,2个来自同源重组,3个为随机突变.[结论]分段DNA shuffling是进化大分子蛋白质的有效方法.     

利用DNA改组技术改造aacC1基因启动子活性的研究

从表达质粒pYPX251(GenBank 登陆号：AY178046)中获得aacC1基因启动子,采用DNA改组（DNA shuffling）技术在体外获得突变体。以lacZ作为报告基因,筛选获得活性明显改变的启动子。经过验证,对其中活性变化明显的7个启动子用邻硝基苯基β半乳糖苷(ONPG)作为底物进行表达活性测定。结果表明,获得的强启动子比原来的提高了3～8倍,而弱启动子则活性下降明显,其中3个几乎无活性。进一步对这7个启动子进行了序列分析。     

从噬菌体抗体库中淘选血纤维蛋白特异的单链抗体

为构建小鼠噬菌体抗体库 ,以获得对人血纤维蛋白特异的抗体 ,由小鼠脾脏提取 m RNA,经反转录 PCR扩增出抗体重链、轻链可变区基因片段 ,将二者和一段编码十五肽 (Gly4 Ser) 3的 DNA接头借助重组 PCR组装成为单链抗体 (single- chain antibody,Sc Ab)基因 .将单链抗体基因插入噬菌体展示载体 p CANTAB- 5E,通过电击法转化大肠杆菌 TG1细胞 ,用辅助噬菌体 M1 3K0 7超感染 ,构建了库容量在 1 0 8以上的噬菌体单链抗体库 .利用亲和选择方法 (淘选 ) ,从噬菌体抗体库中选得血纤维蛋白特异的单链抗体 .模拟抗体成熟过程 ,用 DNA改组 (DNA shuffling)技术使抗体基因重新组合 ,构建新的改组抗体库 ,并从中选择到提高了亲和力的噬菌体单链抗体 .抗体基因在大肠杆菌中表达 ,表达蛋白经 Sephadex G- 75柱层析分离 ,得到初步纯化的单链抗体蛋白 .     

基因组改组技术及其在微生物育种中的应用研究进展

Genome shuffling(基因组改组)技术是借助递归式原生质体融合策略对微生物基因组进行遗传改良的一种新兴微生物育种方法。自2002年首次被用来培育tylosin高产菌株以来,目前已为育种工作者广泛采用。对Genome shuffling技术的原理及最近的应用研究进展进行了综述,探讨了其局限性,并展望了其发展的趋势。     

全局转录调控及其在代谢工程中的应用

全局转录调控是一种全新的改进细胞表型的定向进化方法,通过error-prone PCR、DNA shuffling等技术对细胞中的σ因子和其他转录元件进行多轮突变修饰,改变RNA聚合酶的转录效率和对启动子的亲和能力,使细胞的转录在整体水平上发生改变,导致许多由多种基因控制的细胞表型得以改进。全局转录调控可以对代谢途径快速优化,在代谢工程中已被成功地应用于各种代谢产物的生物合成中。随着全局转录调控理论的不断完善,其应用前景也将越来越广阔。     

Modeling DNA shuffling.

In vitro evolution is a new, important laboratory method to evolve molecules with desired properties. It has been used in a variety of biological studies and drug development. In this paper, we study one important mutagenesis method used in in vitro evolution experiments called DNA shuffling. We construct a mathematical model for DNA shuffling and study the properties of molecules after DNA shuffling experiments based on this model. The model for DNA shuffling consists of two parts. First we apply the Lander-Waterman model for physical mapping by fingerprinting random clones to model the distribution of regions that can be reassembled through DNA shuffling. Then we present a model for recombination between different DNA species with different mutations. We compare our theoretical results with experimental data. Finally we propose novel applications of the theoretical results to the optimal design of DNA shuffling experiments and to physical mapping using DNA shuffling.     

DNA改组(DNA shuffling)及其研究进展

自1994年首次提出至今,DNA改组已经成为定向进化最为有效的方法之一.无论DNA、蛋白质还是生物体的进化,DNA改组都有十分突出的表现.通过对十余年来DNA改组的发展作以简要综述,为日后相关研究的开展提供理论基础.     

Versatile capacity of shuffled cytochrome P450s for dye production

DNA family shuffling is a relatively new method of directed evolution used to create novel enzymes in order to improve their existing properties or to develop new features. This method of evolution in vitro has one basic requirement: a high similarity of initial parental sequences. Cytochrome P450 enzymes are relatively well conserved in their amino acid sequences. Members of the same family can have more than 40% of sequence identity at the protein level and are therefore good candidates for DNA family shuffling. These xenobiotic-metabolising enzymes have an ability to metabolise a wide range of chemicals and produce a variety of products including blue pigments such as indigo. By applying the specifically designed DNA family shuffling approach, catalytic properties of cytochrome P450 enzymes were further extended in the chimeric progeny to include a new range of blue colour formations. This mini-review evokes the possibility of exploiting directed evolution of cytochrome P450s and the novel enzymes created by DNA family shuffling for the production of new dyes.     

Degenerate oligonucleotide gene shuffling (DOGS) and random drift mutagenesis (RNDM): two complementary techniques for enzyme evolution

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Many gene shuffling techniques result predominantly in the regeneration of unshuffled (parental) molecules. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled, and reduces the regeneration of unshuffled parental genes. This shuffling procedure avoids the use of endonucleases for gene fragmentation prior to shuffling and allows the inclusion of random mutagenesis of selected portions of the chimeric genes as part of the procedure. We illustrate the use of the shuffling technique with a family of beta-xylanase genes that possess widely different G+C contents. In addition, we introduce a new method (RNDM) for rapid screening of mutants from libraries where no adaptive selection has been imposed on the cells. They are identified only by their retention of enzymatic activity. The combination of RNDM followed by DOGS allows a comprehensive exploration of a protein's functional sequence space.     

Correcting errors in synthetic DNA through consensus shuffling

Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1–3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from ~60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of ~1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences.     

Genome shuffling of Lactobacillus plantarum C88 improves adhesion

Genome shuffling is an important method for rapid improvement in microbial strains for desired phenotypes. In this study, ultraviolet irradiation and nitrosoguanidine were used as mutagens to enhance the adhesion of the wild-type Lactobacillus plantarum C88. Four strains with better property were screened after mutagenesis to develop a library of parent strains for three rounds of genome shuffling. Fusants F3-1, F3-2, F3-3, and F3-4 were screened as the improved strains. The in vivo and in vitro tests results indicated that the population after three rounds of genome shuffling exhibited improved adhesive property. Random Amplified Polymorphic DNA results showed significant differences between the parent strain and recombinant strains at DNA level. These results suggest that the adhesive property of L. plantarum C88 can be significantly improved by genome shuffling. Improvement in the adhesive property of bacterial cells by genome shuffling enhances the colonization of probiotic strains which further benefits to exist probiotic function.     

Analysis of shuffled gene libraries

In vitro recombination of homologous genes (family shuffling) has been proposed as an effective search strategy for laboratory evolution of genes and proteins. Few data are available, however, on the composition of shuffled gene libraries, from which one could assess the efficiency of recombination and optimize protocols. Here, probe hybridization is used in a macroarray format to analyze chimeric DNA libraries created by DNA shuffling. Characterization of hundreds of shuffled genes encoding dioxygenases has elucidated important biases in the shuffling reaction. As expected, crossovers are favored in regions of high sequence identity. A sequence-based model of homologous recombination that captures this observed bias was formulated using the experimental results. The chimeric genes were found to show biases in the incorporation of sequences from certain parents, even before selection. Statistically different patterns of parental incorporation in genes expressing functional proteins can help to identify key sequence-function relationships.     

Applications of DNA shuffling to pharmaceuticals and vaccines

DNA shuffling is a practical process for directed molecular evolution which uses recombination to dramatically accelerate the rate at which one can evolve genes. Single and multigene traits that require many mutations for improved phenotypes can be evolved rapidly. DNA shuffling technology has been significantly enhanced in the past year, extending its range of applications to small molecule pharmaceuticals, pharmaceutical proteins, gene therapy vehicles and transgenes, vaccines and evolved viruses for vaccines, and laboratory animal models.