基因组改组选育蔗糖利用型L-乳酸高产菌株

对干酪乳杆菌进行紫外诱变和NTG诱变,得到蔗糖耐受性高的突变菌株,以此作为基因组改组的出发菌株,制得原生质体,将原生质体分别于紫外线和热灭活,致死率分别为89%和91.6%,在再生平板中培育,将存活的原生质体进行融合,获得的融合子通过蔗糖YE平板筛选,获得F1代,然后以F1代为出发菌,经过上述步骤得到了能够高效利用蔗糖发酵的F2代菌株.与野生型菌株比较发现,在15.0%蔗糖浓度条件下菌体旺盛生长,OD600达到3.11,较原始菌提高了0.70,发酵产酸量提高了61.0%,而且蔗糖酶活性比野生型有很大的提高,从0.54 U/mg cells提高到1.93 U/mg cells,提高了近4倍.     

基因组重排与传统育种相结合选育大观霉素高产菌株

以壮观链霉菌(Streptomyces spectabilis)为研究对象,采用基因组重排技术与传统诱变育种相结合的方法选育大观霉素的高产菌株.通过原生质体紫外诱变获得壮观链霉菌突变体群体,高产突变菌株间进行两轮的基因组重排,筛选的高产菌株用NTG诱变得新霉素和链霉素的抗性突变菌株,抗性突变菌株间进行两轮基因组重排,从...     

Anticipatory evolution and DNA shuffling

DNA shuffling has proven to be a powerful technique for the directed evolution of proteins. A mix of theoretical and applied research has now provided insights into how recombination can be guided to more efficiently generate proteins and even organisms with altered functions.     

DNA vaccine strategies: candidates for immune modulation and immunization regimens

DNA vaccine strategies can differ greatly, with significant effects on the outcome of immunization. In this article, we discuss plasmid design strategies and vaccine regimens. Effectiveness against a pathogen can be affected by the choice of antigen and inclusion of multiple antigens. Gene expression and the resulting immune response can be improved by gene modification and choice of promoters. In designing vaccine regimens, one must consider further dose, timing of doses, adjuvants, and routes of vaccination. Many vaccines are enhanced by combining DNA with other vaccines in "prime-boost" regimens, in which the second vaccine is often a recombinant viral vector or purified protein subunit. Prime-boost vaccines including DNA can elicit immune responses that differ in magnitude, quality, and balance of cellular and humoral responses from those elicited by single components and thus provide further enhancement for DNA immunizations.     

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.     

DNA改组的发展及应用

DNA改组自1994年首次提出至今,逐渐成为基因和蛋白质体外定向进化的高效方法,总结了DNA改组的技术路线、特点及发展,并对其应用作简要综述.     

DNA重排及体外分子进化

ＤＮＡ重排是目前为止最简便、最有效的体外定向进化技术,可以对单一基因、质粒、代谢途径、部分甚至整个基因组进行改造。本综述了ＤＮＡ重排的基本原理、特点、与其它体外进化技术的不同,着重介绍了其在体外分子进化上的广泛应用,并对应用前景进行了展望。     

Optimization of DNA shuffling for high fidelity recombination.

A convenient 'DNA shuffling' protocol for random recombination of homologous genes in vitro with a very low rate of associated point mutagenesis (0.05%) is described. In addition, the mutagenesis rate can be controlled over a wide range by the inclusion of Mn2+or Mg2+during DNase I digestion, by choice of DNA polymerase used during gene reassembly as well as how the genes are prepared for shuffling (PCR amplification versus restriction enzyme digestion of plasmid DNA). These protocols should be useful for in vitro protein evolution, for DNA based computing and for structure-function studies of evolutionarily related genes.     

Predicting out-of-sequence reassembly in DNA shuffling

We present an analysis for calculating the frequency of out-of-sequence reassembly in DNA shuffling experiments. Out-of-sequence annealing events are undesirable since they typically encode non-functional proteins with missing or repetitive regions. The approach builds on the e Shuffle framework for the prediction of crossover formation using equilibrium thermodynamics and complete sequence information to model the reassembly process. An in silico case study of a set of subtilases reveals that, as expected, the presence of significant sequence identity between distant portions of the parental sequences gives rise to out-of-sequence annealing events that upon reassembly generate sequences with missing or repetitive DNA segments. The frequency of these events increases as the fragment length decreases. Interestingly, out-of-sequence annealing events are at a minimum near the annealing temperature of 55 degrees C used in the original DNA shuffling protocol. Neither parental sequence identity nor number of shuffled parents significantly alter the extent of out-of-sequence reassembly     

DNA shuffling method for generating highly recombined genes and evolved enzymes

We introduce a method of in vitro recombination or "DNA shuffling" to generate libraries of evolved enzymes. The approach relies on the ordering, trimming, and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold. We generated chimeric libraries averaging 14.0 crossovers per gene, a several-fold higher level of recombination than observed for other methods. We also observed an unprecedented four crossovers per gene in regions of 10 or fewer bases of sequence identity. These properties allow generation of chimeras unavailable by other methods. We detected no unshuffled parental clones or duplicated "sibling" chimeras, and relatively few inactive clones. We demonstrated the method by molecular breeding of a monooxygenase for increased rate and extent of biodesulfurization on complex substrates, as well as for 20-fold faster conversion of a nonnatural substrate. This method represents a conceptually distinct and improved alternative to sexual PCR for gene family shuffling.     

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.     

Experimental vaccine strategies for cancer immunotherapy

Recently, cancer immunotherapy has emerged as a therapeutic option for the management of cancer patients. This is based on the fact that our immune system, once activated, is capable of developing specific immunity against neoplastic but not normal cells. Increasing evidence suggests that cell-mediated immunity, particularly T-cell-mediated immunity, is important for the control of tumor cells. Several experimental vaccine strategies have been developed to enhance cell-mediated immunity against tumors. Some of these tumor vaccines have generated promising results in murine tumor systems. In addition, several phase I/II clinical trials using these vaccine strategies have shown extremely encouraging results in patients. In this review, we will discuss many of these promising cancer vaccine strategies. We will pay particular attention to the strategies employing dendritic cells, the central player for tumor vaccine development.     

Breeding of retroviruses by DNA shuffling for improved stability and processing yields

Manufacturing of retroviral vectors for gene therapy is complicated by the sensitivity of these viruses to stress forces during purification and concentration. To isolate viruses that are resistant to these manufacturing processes, we performed breeding of six ecotropic murine leukemia virus (MLV) strains by DNA shuffling. The envelope regions were shuffled to generate a recombinant library of 5 x 106 replication-competent retroviruses. This library was subjected to the concentration process three consecutive times, with amplification of the surviving viruses after each cycle. Several viral clones with greatly improved stabilities were isolated, with the best clone exhibiting no loss in titer under conditions that reduced the titers of the parental viruses by 30- to 100-fold. The envelopes of these resistant viruses differed in DNA and protein sequence, and all were complex chimeras derived from multiple parents. These studies demonstrate the utility of DNA shuffling in breeding viral strains with improved characteristics for gene therapy.     

DNA shuffling技术及其在基因工程疫苗中的应用

DNA shuffling技术是一项全新的体外人工进化模式,它通过基因在分子水平上的重组,再定向筛选具有预期性状的突变体,获得同时具有多个亲本基因的特征的突变基因。该文介绍DNA shuffling技术的基本原理,并列举了由该技术发展而来的新技术及其在基因工程疫苗领域的应用,展望了DNAshuffling技术的发展方向。     

Full length antigen priming enhances the CTL epitope-based DNA vaccine efficacy

Although CD8⁺ cytotoxic T lymphocyte (CTL) epitope-based DNA vaccination is valuable experience on vaccine research but many attempts are still continued to achieve acceptable protective response. To study the role of full length antigen in CTL epitope immunization, we evaluated cellular immunity of diverse patterns of complete Herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) and the immunodominant CTL epitope (498–505) DNA injection in C57BL/6 mice. Optimal immune response was observed in the group immunized with the full length of gB in the first injection and CTL epitope in the second and third vaccination as assessed by lymphocyte proliferation assay (MTT), cytokine assay (ELISA) and CTL assay. B cell and spatially CD4⁺ T cell epitopes in full length protein might be important for appropriate priming of CTL immune response. These findings may have important implication for the improvement of CTL epitope based DNA vaccine against HSV and other pathogens.     

An effective family shuffling method using single-stranded DNA

Family shuffling, which is one of the most powerful techniques for in vitro protein evolution, always involves the problem of reassembling the gene fragments into parental gene sequences, because such a process prevents the formation of chimeric sequences. In order to improve the efficiency of hybrid formation in family shuffling, single-stranded DNAs (ssDNAs) were used as templates. The ssDNAs of two catechol 2,3-dioxygenase genes, nahH and xylE, were prepared, the xylE strand being complementary to the nahH strand. When these ssDNAs were digested by DNase I and reassembled, chimeric genes were obtained at a rate of 14%, which was much higher than the rate of less than 1% obtained by shuffling with double-stranded DNAs. Chimeric catechol 2,3-dioxygenases that were more thermally stable than the parental enzymes, XylE and NahH, were obtained by this ssDNA-based DNA shuffling.