日本血吸虫卵壳蛋白编码基因的扩增及克隆

目的:扩增和克隆日本血吸虫卵壳蛋白编码基因(SjESG),以研究其作为候选疫苗分子和药物靶点的可能性.方法:合成引物,以日本血吸虫雌、雄虫cDNA第一链为模板,RT-PCR扩增日本血吸虫卵壳蛋白编码基因,与带有硫氧还蛋白(Trx)基因的高效原核表达质粒pET32a(+)定向重组,构建原核表达重组质粒,并经限制性核酸内切酶酶切分析和PCR鉴定.结果:从雌虫cDNA中扩增出624bp SjESG基因,原核表达重组质粒pET32a(+)-SiESG经限制性核酸内切酶酶切和PCR均获得624bp的DNA片段.结论:成功构建原核表达重组质#ipET32a(+)-SjESG.     

分子标记及AFLP技术

分子标记类型可以基因表达的结果为基础,对基因的间接反映;分子标记则是DNA分子碱基序列变异的直接反映.早期利用限制性内切酶,酶切生物体DNA后来检测不同遗传位点等位变异(RFLP)和以一个碱基顺序随机排列的寡核苷酸序列为引物,利用对基因组DNA随机扩增来鉴别DNA多态性(RAPDs).真核生物基因组中普遍存在的重复序列产生了微卫星(microsatellites)标记技术.而RFLP与RAPDs有机结合形成了有着更为广阔的应用前景的AFLP技术.SNP标记利用大多数基因位点上都会有若干个等位型(alleles)为DNA芯片技术应用于遗传作图提供了基础.     

50例中国人IX因子基因的限制性片段长度多态性

用IX因子基因内探针F9(VⅢ)对TaqI,BamHI和EcoRI酶切的50例中国人基因组DNA进行杂交分析。结果表明,所有个体经TaqI酶切的杂交片段为4.5kb和1.8kb,BamHI和EcoRI酶切的杂交片段分别为23kb和5.0 kb。基因组DNA样本中未发现限制性片段长度多态性(RFLP),这与欧美国家的民族群体中存在着IX因子基因内TaqI和BamHI的RFLP的结论不同。造成不同种族间DNA水平差异的原因,很可能与长期在不同地理环境中的进化适应有关。     

茶毛虫核型多角体病毒DNA性质的研究

本文报道了对茶毛虫核型多角体病毒蒙山株系(EpNPV-M)核酸性质研究的结果。EpNPV-M含双链DNA分子,含量为6.85μgDNA/mgPIB,提纯的DNA具典型的紫外线吸收特性,琼脂糖凝胶电泳证明DNA分子是大小均一的。DNA的(G+C)％为36.6,限制性片段积加法测得该DNA分子量为75.61×10~6道尔顿,109.57Kb,电镜法测得DNA分子长度为35.8μm,相当于分子量为74.1×10~6道尔顿,107.4Kb.电镜观察证实该病毒含有一些超螺旋环状DNA分子。建立了三种限制性内切酶对该DNA的酶切图谱。三种酶的酶解片断数为:EcoRI,27个;BglⅡ,15个;BamHI,9个。     

基因工程中重组质粒的获得

用两种特异的限制性核酸内切酶同时酶切含目的基因的DNA片段与质粒,可以有效的降低或防止目的基因和质粒表达载体在酶切后的末端发生任意连接;双抗生素间接平板筛选,得到含有目的基因的质粒。     

Trichostatin A能导致小麦基因组DNA改变及甲基化修饰

为了研究组蛋白去乙酰化酶抑制剂的作用机制,本实验以组蛋白去乙酰化酶抑制剂Trichostatin A处理小麦根尖,通过随机扩增多态性DNA标记技术(random amplified polymorphic DNA,RAPD)和双限制性内切酶酶切-随机扩增法(Coupled Restriction Enzyme Digestion-Random Amplification,CRED-RA)进行探究的结果发现,与对照组相比,处理组10个随机引物的RAPD扩增条带出现83条多样性差异,多样性比例达到87.4%,同时处理组DNA甲基化比例增加.本研究证实Trichostatin A能使小麦基因组DNA发生改变,并出现了表观修饰,从而从分子和表观水平探讨了该抑制剂的作用机理.     

构建重组质粒的二步PCR方法

建立一种简单的重组质粒构建方法——二步PCR,不需要限制性内切酶对基因和载体进行酶切.第一步常规PCR中,用正向引物(IF)和反向杂合引物(IR)扩增黑曲霉木聚糖酶Xyn基因,IF与基因特异性匹配,IR引物3’端与基因序列匹配、5’端含有19 bp p ET20b一端正链序列;第二步反向PCR中,以扩增的Xyn基因作为正向引物,其3’端含有19 bp与环状p ET20b模板退火、并在高保真Q5 DNA聚合酶作用下延伸,与模板另一端互补的反向引物VR协同作用,重组质粒通过PCR扩增出来.反向PCR产物经T4 DNA连接酶环化,并经Dpn I限制性内切酶消解处理,可以直接转化BL21(DE3)感受态细胞.用这种方法将622、639、1125、1209 bp大小的基因重组p ET20b中,显示出有较广泛的普适性.     

PCR-RFLP技术应用于鉴定美洲斑潜蝇和番茄斑潜蝇的初步研究

本文试探运用 PCR- RFL P技术 ,从美洲斑潜蝇 (L iriomyza sativae)和番茄斑潜蝇 (L iriomyza bryoni-ae)中提取总的 DNA,根据实蝇 18S和 5 .8S保守序列设计一对引物扩增这两种斑潜蝇的 ITS1基因区域 ,用五种限制性内切酶对 PCR产物进行酶切 ,构建了酶切图谱。从酶切图谱中选择特异酶切位点作为区分两种斑潜蝇的标记。根据特异的酶切片段来区分这两种斑潜蝇     

基于线粒体DNA多态性鉴定烟台海鲜市场鱼种

由烟台海鲜市场随机采集具有相对重要商业价值的10种海鱼(小黄鱼、牙片、鲅鱼、面条鱼、鲐鱼、鲳鱼、鲈鱼、偏口、黑鱼、白姑鱼),利用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)技术对鱼种进行鉴定.首先提取10种海鱼的基因组DNA,利用自行设计的通用引物对其线粒体细胞色素C氧化酶亚基Ⅲ基因(MT-co3)进行PCR扩增,对扩增产物克隆、测序,用NCBI数据库检索,确定了10种海鱼的鱼种.依据测序结果进行限制性内切酶酶切分析,选用NlaⅢ和HinfⅠ2种内切酶进行酶切,获得了各种鱼类特异的酶切片段图谱.研究结果表明,PCR-RFLP能有效地区分以上10种海鱼,在鱼种鉴定上具有简便、准确的优势,可为鱼类食品检测提供科学依据.     

DNA分子标记的发展及主要技术

DNA分子标记技术是一种新的比较理想的遗传标记,本文综述了DNA分子标记技术的发展概况、种类及特点,以及广泛应用于农作物分子标记基因定位的分子标记技术：限制性片段长度多态性（RFLP）,随机扩增多态性DNA（RAPD）,简单序列长度多态性标记（SSR）和扩增的限制性内切酶片段长度多态性（AFLP）的原理、特点及他们的优缺点。     

New strategies of protein engineering——directed evolution of enzyme in vitro

New and highly effective strategies for directed enzyme evolution in vitro have been developed in the protein engineering field. They allow engineering all kinds of enzymes in vitro so that new ones with novel functions and features can be obtained by the methods of error-prone PCR, DNA shuffling (exon shuffling), hybrid enzyme, random-priming in vitro recombination (RPR), stagger extension process (StEP), random-directed mutagenesis in vitro, etc., even with little knowlodge of spatial structure and catalytic mechanism of enzymes in advance. The process that would take millions of years in nature can in principle be accomplished in the test within several years.     

用体外定向进化的方法对EPSPS合酶基因草苷膦抗性机制的研究

为了寻找EPSP合酶中某些与草苷膦抗性相关的位点，利用体外定向进化技术，对可变盐单胞菌HT7的EPSP合酶基因，荧光假单胞菌G2的EPSP合酶基因以及按植物偏爱密码子合成G2的EPSP合酶基因，进行DNA shuffling，通过EPSP合酶缺陷型菌株E.coil ER2799的功能互补筛选，获得了7个草苷膦抗性有较大变化的EPSP合酶突变体.     

利用EvolvR系统对酶基因实现多窗口定向进化的研究

定向进化包括随机基因文库生成、基因在合适宿主中的表达和筛选目标特性变体. EvolvR作为一种CRISPR介导的新型定向进化技术，可以使目标序列在一天内完成整个进化过程. 目前，EvolvR只报导用于抗性基因的突变，以及串联最多2个sgRNAs以扩大突变窗口长度. 本研究旨在探究EvolvR系统在酶基因序列定向进化中的适用性以及突变效率， 同时在此基础上扩大EvolvR的突变窗口长度. 该研究结果表明，将4个能高效表达的单个sgRNA 串联，成功检测到靶向同一基因的3个不同靶位点，证明了EvolvR具有在目标基因区域大范围制造突变的潜力，具有很高的应用价值. 本研究为利用EvolvR系统对目标酶基因实现定向进化的研究奠定了基础.     

人工酶与定向进化的前沿与挑战

 定向进化是在实验室环境中，在分子水平上模拟进化过程，得到具有期望特征的蛋白质的方法，目前已成为蛋白质设计改造的重要方法。定向进化不仅可以用于天然蛋白质的改造，也可以通过改造现有的酶，使其具有新的催化活性，从而构建人工酶。本文重点介绍工业生物催化、纳米酶设计和光催化3个方向的前沿成果，并讨论人工酶与定向进化领域存在的挑战和问题。     

Dynamics of DNA in vitro evolution

In vitro evolution has become a very important research area in recent years. From a practical point of view, it provides a powerful and reliable tool for engineering functional molecules (DNA, RNA or proteins) in reasonably short periods of time. From a theoretical point of view, since in vitro evolution is analogous to natural evolution in many respects, the study of the dynamic details of in vitro evolution may provide some instructive insights into the process of evolution. In this review, we summarize current theoretical and exper-imental studies, including several efforts made by our group, on the dynamics of DNA in vitro evolution.     

Dynamics of DNA in vitro evolution

In vitro evolution has been becoming a very important research area in recent years. From a practical point of view, it provides a powerful and reliable tool for engineering functional molecules (DNA, RNA or proteins) in reasonably short periods of time. From a theoretical point of view, since in vitro evolution is analogous to natural evolution in many respects, the study of the dynamic details of in vitro evolution may provide some instructive insights into the process of evolution. In this review, we summarize current theoretical and experimental studies, including several efforts made by our group, on the dynamics of DNA in vitro evolution.     

A novel method of DNA shuffling without PCR process

Most DNA shuffling methods currently used require PCR process. A novel method of DNA shuffling without PCR process is described, taking advantage of the feature of some restriction enzymes whose recognition sites differ from their cleavage sites, thus giving rise to different cohesive ends. These cohesive ends can be rejoined at their native sites from different parental sequences, generating new sequences with various combinations of mutations.     

Selection and evolution of enzymes from a partially randomized non-catalytic scaffold

Enzymes are exceptional catalysts that facilitate a wide variety of reactions under mild conditions, achieving high rate-enhancements with excellent chemo-, regio- and stereoselectivities. There is considerable interest in developing new enzymes for the synthesis of chemicals and pharmaceuticals and as tools for molecular biology. Methods have been developed for modifying and improving existing enzymes through screening, selection and directed evolution. However, the design and evolution of truly novel enzymes has relied on extensive knowledge of the mechanism of the reaction. Here we show that genuinely new enzymatic activities can be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity, with product formation as the sole selection criterion. We used messenger RNA display, in which proteins are covalently linked to their encoding mRNA, to select for functional proteins from an in vitro translated protein library of >10(12 )independent sequences without the constraints imposed by any in vivo step. This technique has been used to evolve new peptides and proteins that can bind a specific ligand, from both random-sequence libraries and libraries based on a known protein fold. We now describe the isolation of novel RNA ligases from a library that is based on a zinc finger scaffold, followed by in vitro directed evolution to further optimize these enzymes. The resulting ligases exhibit multiple turnover with rate enhancements of more than two-million-fold.     

Markov Model Applied to Gene Evolution

IntroductionWith the development of molecular biology,thetheory of evolution is being developed andchanged,especially at the molecular level.Theobjective of this research was to try to understandthe force behind sequence evolution and the rulesof evolution.The development of bioinformaticshas made itpossible to study the sequences directlyinstead of relying on complicated experiments.In the end of the1 96 0 s,Kimura and Ohta,and King and Jukes proposed the neutral theory ofmolecular evolutio…     

Directed evolution of new catalytic activity using the alpha/beta-barrel scaffold

In biological systems, enzymes catalyse the efficient synthesis of complex molecules under benign conditions, but widespread industrial use of these biocatalysts depends crucially on the development of new enzymes with useful catalytic functions. The evolution of enzymes in biological systems often involves the acquisition of new catalytic or binding properties by an existing protein scaffold. Here we mimic this strategy using the most common fold in enzymes, the alpha/beta-barrel, as the scaffold. By combining an existing binding site for structural elements of phosphoribosylanthranilate with a catalytic template required for isomerase activity, we are able to evolve phosphoribosylanthranilate isomerase activity from the scaffold of indole-3-glycerol-phosphate synthase. We find that targeting the catalytic template for in vitro mutagenesis and recombination, followed by in vivo selection, results in a new phosphoribosylanthranilate isomerase that has catalytic properties similar to those of the natural enzyme, with an even higher specificity constant. Our demonstration of divergent evolution and the widespread occurrence of the alpha/beta-barrel suggest that this scaffold may be a fold of choice for the directed evolution of new biocatalysts.