高细胞密度发酵技术的研究进展

随着基因重组技术和发酵技术的发展,为实现菌体的低成本规模化生产,高细胞密度发酵技术已成为发酵领域的研究热点。该研究阐述了影响高细胞密度发酵的主要因素和建立高细胞密度发酵技术的优化策略,并对高细胞密度发酵技术的应用前景进行了展望。     

膜荚黄芪苯丙氨酸解氨酶在大肠杆菌中的表达和纯化

为了解膜荚黄芪苯丙氨酸解氨酶(AmPAL)基因的功能及生物活性,用基因重组技术构建了pQE30-AmPAL原核表达载体,在大肠杆菌M15中表达和纯化融合蛋白,并对其进行PAL酶活性测定.结果表明,成功地构建了pQE30-AmPAL原核表达载体,并在大肠杆菌大量表达,表达的重组AmPAL经纯化后进行SDS-PAGE电泳显示1条蛋白条带.酶活性测定表明,携带6个组氨酸的重组蛋白并不影响蛋白质的功能,可保持PAL原有的生物活性.     

向量化编程技术及其在大型电网潮流计算中的应用

提出一种基于向量计算的编程技术,应用该技术开发大规模程序,工作周期短,程序代码精炼,数学概念清晰,程序执行效率高.应用这种向量化编程技术,分别采用MATLAB和C语言编写了可计算大型电网的潮流计算程序.以3个不同规模的电网为测试算例,对程序的执行效率分别进行了分析比较.     

Administration divided over OECD biotech plan

For the past two years, members of the Organization for Economic Cooperation and Development (OECD)--which includes the United States, most of its European allies, and Japan--have been discussing how to set up international guidelines to regulate the emerging biotechnology industry. A May 1985 first draft of the guidelines spawned many objections, and now a revised draft, called "Safety and Regulations in Biotechnology," has encountered mixed responses from U.S. federal agencies. The Environmental Protection Agency generally supports the document, but the State Department, Food and Drug Administration, and Department of Agriculture have significant objections to its tone and substance, particularly in regard to the specificity of safety controls over the commercial development of recombinant DNA organisms used in large-scale production and for agricultural purposes.     

基于RPA技术对转CP4-EPSPS基因产品的快速检测

核酸检测在农产品安全检测中的应用十分广泛，随着转基因作物的商业化种植，迫切需要一种快速、特异、灵敏的转基因作物检测方法。利用重组酶聚合酶扩增（recombinase polymerase amplification，RPA）技术对含有转基因产品及其制品中CP4-EPSPS基因进行检测，共设计5对引物来筛取最佳引物，并对反应体系和反应温度进行优化。结果表明，该方法采用20 μL体系在37 ℃恒温反应15 min即可对CP4-EPSPS基因进行快速检测。该方法检测转基因的灵敏度阈值为45拷贝，灵敏度高、特异性强，为大规模筛查CP4-EPSPS基因提供了一种新的途径。     

小尾寒羊骚痒病单抗制备及免疫组化检测方法的初步建立

【目的】通过表达羊PrPc核心片段、单抗制备，最后建立免疫组织化学检测羊痒病方法。【方法】用PCR方法从小尾寒羊基因组DNA中扩增编码PrPc核心片段DNA， 并克隆到硫氧还原蛋白融合表达载体pET-32a（+）上，转化至E.coli BL21，IPTG诱导融合表达。以纯化的重组小尾寒羊PrPc核心片段免疫PrPc基因敲除小鼠，利用淋巴细胞杂交瘤技术，制备羊核心片段单克隆抗体。【结果】通过上述方法，得到相对分子质量约为35 kD的重组小尾寒羊PrPc核心片段，筛选出六株能稳定分泌针对小尾寒羊PrPc核心片段特异单抗的杂交瘤细胞株。免疫组化试验表明,其中4株可特异性识别羊痒病脑组织切片中耐PK酶消化的PrPsc，经5次重复试验表明：所制备单抗与对照单抗F89结果完全一致。【结论】利用原核表达，单抗制备等技术制备出PrP特异性单克隆抗体，用笔者自己研制的单抗作为核心试剂初步建立了羊痒病免疫组化检测方法。     

牛分枝杆菌RecA基因的克隆与序列分析

以牛分枝杆菌(Mycobacterium bovis)Valleelll株染色体DNA为模板,以RecA基因特异性引物进行PCR扩增,获得约240 bp的DNA片段.将PCR产物克隆至pMDTM18-T Vector中,通过α-互补法筛选、质粒PCR鉴定及序列分析鉴定,成功构建出了重组质粒pMDTM18-T-RecA,为进一步研究RecA基因及其在牛结核病诊断中的应用奠定了基础.     

工商资本下乡种粮的增收机制——基于案例的实证分析

目前学术界对于工商资本务农的效益存在许多争议,尤其是在种粮领域。以安徽、山东和河北的新型农业经营主体为研究对象,通过实地调研,从工商资本投资农业生产的技术、资本、管理、加工、销售以及效益等方面探讨了其下乡种粮的增收机制。结果表明:1)先进的技术与精细的规模化管理模式是保证种粮增收的重要前提。与一般的粮食规模经营主体相比,工商资本通过先进的粮食生产技术和精细的规模化管理获得了规模经济,保障了粮食的产量和品质,从而增加了收入。2)农业产业链的延伸以及品牌化营销是保证种粮增收的关键。工商资本主导的粮食规模经营主体通过开展粮食加工和创建品牌增加了农产品附加值,从而使其在种粮领域取得较高的收益。基于此,提出要通过考察涉农工商资本的农业背景、监督工商资本的生产行为和土地流转过程、完善农村金融和农产品市场,以及引进农业技术和管理人才等措施促进资本务农实现增收。     

奶牛β-防御素5基因的克隆与原核表达

根据已发表的β-防御素5基因序列设计引物,采用RT-PCR技术扩增奶牛白细胞β-防御素5基因片段,构建原核表达载体并进行诱导表达.结果表明:将PCR产物插入pGEM-T easy载体后,经琼脂糖凝胶电泳、PCR、酶切及DNA测序证明构建的重组克隆载体完全正确,以该重组质粒为模板扩增目的基因的成熟肽片段,产物用EcoR I+NotⅠ双酶切并与原核表达载体PET28a连接,获得了预期的重组表达质粒.将重组表达质粒转化到BL21(DE3)宿主菌中,用异丙基--βD-硫代半乳糖苷诱导表达,经尿素-SDS-PAGE检测表明表达产物为6.4 kda的融合蛋白.     

Chelex-100法快速提取白粉菌基因组DNA及ITS2序列的克隆

[目的]建立快速提取及克隆白粉病菌基因组DNA的方法。[方法]以豌豆白粉菌分生孢子为材料,用Chlex-100法快速提取白粉菌基因组DNA,并以此为模板对内转录间隔区Ⅱ（ITS2）序列进行了巢式PCR扩增、克隆、测序及分析。[结果]Chlex-100法可高效的提取白粉病菌基因组DNA,适用于PCR扩增及相关分析。[结论]为快速有效地对白粉病资源进行大规模分子分类鉴定奠定了基础。     

Gene transfer in cereals

Until recently, gene transfer in plants was achieved only by sexual hybridization. Now, in addition, plant genetic manipulation, with the use of both recombinant DNA and protoplast fusion technology, is being applied to an increasing range of plants. The soil bacterium Agrobacterium tumefaciens, with its associated plasmid, is used as a vector for introducing DNA into the genomes of dicotyledonous plants, but it has not proved suitable for cereals. Instead, the direct uptake of plasmid DNA into cereal protoplasts is being used for the transformation of cells in rice, wheat, and maize. Transformation efficiencies, in some cases, are becoming comparable to those obtained in dicotyledons with Agrobacterium. In rice it is now possible to regenerate efficiently whole plants from protoplasts, and this capability may soon be extended to the other cereals. By means of direct interaction of cereal protoplasts with plasmids, coupled with improved procedures for the regeneration of plants from their protoplasts, gene transfer in the cereals is becoming established at the frontiers of recombinant DNA technology.     

Disease diagnosis by recombinant DNA methods

Recombinant DNA procedures have now been applied to the problem of the identification of molecular defects in man that account for heritable diseases, somatic mutations associated with neoplasia, and acquired infectious disease. Thus recombinant DNA technology has rapidly expanded our ability to diagnose disease. Substantial advances in the simplification of procedures for diagnostic purposes have been made, and the informed physician has gained in diagnostic accuracy as a consequence of these developments. The wide application of recombinant DNA diagnostics will depend on simplicity, speed of results, and cost containment.     

A single recombinant plasmid expressing two major outer surface proteins of the Lyme disease spirochete

A gene bank of DNA from the Lyme disease spirochete was constructed in the plasmid pBR322. Plasmid pTRH32, a recombinant that in Escherichia coli expresses the two major outer surface proteins of the Lyme disease spirochete, was identified. One of the recombinant products, designated OspA, represents a surface protein that appears to be common to all Lyme disease spirochetes, whereas the other recombinant product, designated OspB, represents a more variable surface protein. This recombinant plasmid provides a foundation for future studies on the epidemiology and pathogenesis of Lyme disease as well as on the genetic organization of the etiologic agent.     

Congress ponders rDNA and environmental risks

The federal government's role in regulating recombinant DNA technology was the topic of a 22 June 1983 hearing held by Representatives Albert Gore and Doug Walgren. Scientists testified that potential hazards from the release of rDNA organisms into the environment are low, and that voluntary guidelines, like those of the National Institutes of Health's Recombinant DNA Advisory Committee, are sufficient to control the new technology. Some government officials concur with this assessment. Should increased federal oversight be decided upon, it has yet to be established whether the Environmental Protection Agency or the Agriculture Department has regulatory authority.     

副结核分枝杆菌hsp65基因的克隆及序列分析

以副结核分枝杆菌C-2株基因组DNA为模板,以hsp65基因特异性引物进行PCR扩增,获得了1 626 bp的DNA片段.通过T-A克隆技术,将PCR产物克隆至pGEM-T载体.经鉴定,成功地构建出了重组质粒pGEM-T-hsp65.序列分析结果表明:该序列与GenBank中副结核分枝杆菌K～10株的同源性为99.2%.     

Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins

Clones of complementary DNA encoding the human lymphokine known as granulocyte-macrophage colony-stimulating factor (GM-CSF) were isolated by means of a mammalian cell (monkey COS cell) expression screening system. One of these clones was used to produce recombinant GM-CSF in mammalian cells. The recombinant hematopoietin was similar to the natural product that was purified to apparent homogeneity from medium conditioned by a human T-cell line. The human T-cell GM-CSF was found to be 60 percent homologous with the GM-CSF recently cloned from murine lung messenger RNA.     

碧冬茄花特异表达基因启动子PchsA的克隆与序列分析

根据Ingrid M．报道的碧冬茄花特异表达基因CHSA启动子的序列设计并合成一对特异引物，以碧冬茄总DNA为模板，通过PCR扩增出约370bp大小的DNA片段，回收后克隆到pUCm—T质粒载体上，经转化、筛选确定重组子，酶切鉴定后送上海生工生物工程公司测序，得到片段长度为370bp．采用DSgene分析软件进行序列分析发现其基本具有启动子的所有保守序列，TATAbox，CCAATbox，Anther box，G—box，TACPyAT box，box1，box2，Capsite等，且经InternetBLAST程序和DSgene分析软件进行同源性对比和序列分析，显示序列与已报道序列同源性为96％，登陆GenBank，ID号为AY360358。     

耐盐基因Hal1的克隆及表达载体的构建

以酵母菌株BWG1-7A基因组DNA为模板,利用PCR方法,扩增出耐盐基因Hal1,测序表明该基因全长为885个核苷酸,与已发表的序列NC_001148比较,同源性99．3％。将该基因插入表达载体pYES2的BamHI和EcoRI酶切位点之间。构建表达载体pYES2-Hal1,序列测定完全正确,为植物表达载体的构建打下基础。     

单核细胞增生性李斯特氏菌溶血素基因克隆及序列分析

构建单核细胞增生性李斯特氏菌 (L isteria Monocytogenes,L MO)溶血素基因重组质粒。文章采用 PCR方法扩增出 L MO 0 5 86株溶血素 (Hemolysin,hly)基因 ,将其克隆到 p MD18- T中 ,转化 E.coil TGI。经酶切及 PCR鉴定 ,而后进行测序。 hly基因体外扩增产物大小约为 16 4 6 bp。重组质粒经酶切及 PCR鉴定表明为正确重组子。核苷酸序列鉴定表明 ,其核苷酸序列与国外报道的 L MO F6 789株、L MO F2 36 5株同源性分别为 99.70 %和 99.39%。推导出的氨基酸序列与其相应菌株比较 ,同源性分别为 99.82 %和 98.90 %。在国内首次克隆到 L MO hly全基因 ,为研究hly的功能和探讨 hly蛋白作为特异性诊断靶抗原的研究奠定了基础。     

基于OSG数字林分景观可视化技术研究

大规模林分场景建模和实时绘制对于模拟林分生长具有重要意义。本文定义了一个基于树龄分枝的树木表达模型,该模型可构建形态结构不规则、灵活的树木模型;在林分场景绘制中,提出了一种基于OSG大规模林分场景的实时绘制技术,利用存储在XML文件中真实的林分调查数据可以实时绘制三维林分场景。林分树木场景数据量大,为了提高场景渲染的效率,利用OSG场景图来组织和优化场景数据;在林分单元格的绘制中引入3D树木的LOD网格简化和PageLOD数据库分页机制实现大规模数据调度。实例证明,所绘制的大规模林分场景能满足用户的实时交互需求。