登革病毒变异性分析法简介

用变异性分析法对不同地域、不同年份的登革病毒分离株的遗传差异进行分析,有利于对该病毒的进一步深入了解。随着近年来分子生物技术的快速发展,新型的、更精确的分析方法不断涌现,如多聚酶链反应（ＰＣＲ）限制酶分析法和种系发生分析法等。本文对各种分析法作了简要介绍,并分别指出其优缺点。     

元素分析法鉴定肠道杆菌

用元素分析法鉴定细菌,曾报道了八种杆菌的鉴定。为了进一步探索本方法的可行性,和扩大元素分析法的应用范围,我们对某些生物样品,如放线菌、分枝杆菌、人体血液及植物根瘤菌等,进行了元素分析,特别是对肠道杆菌     

放射受体分析法

放射受体分析(Radioreceptor assay.RRA)是竞争性蛋白结合分析的一种.1970年Lefkowitz最先应用于促肾上腺皮质激素的测定,以后逐步推广到生长激素、促甲状腺激素、催乳素、促性腺激素、下丘脑释放激素、生长抑素、胰岛素等多种激素的测定,亦可进行去甲肾上腺素、多巴胺、5-羟色胺、乙酰胆碱等各种神经介质以及一些药物(如     

DNase的DNA-PAGE快速分析法

核酸酶,特别是DNase,往往和机体的生理状况及病变有密切的关系,如果能建立一种简便的核酸酶分析方法,将对临床诊断、探讨病变机制及筛选药物都有一定的价值。关于DNase的分析Boyd等先后建立了DNA-聚丙烯酰胺凝胶电泳(DNA-PAGE)法;Brown等又将它加以简化,并将它直接用于细胞抽提液中DNase的鉴定与比较。本文又作了改进,降低了分离胶中的DNA含量,并以溴化乙锭(Ethidum Bromid),代替焦宁Y(Pyronin Y)。结果表明,改进后保温与染色时间可以大大缩     

DNase的DNA-PAGE快速分析法

核酸酶,特别是DNase,往往和机体的生理状况及病变有密切的关系,如果能建立一种简便的核酸酶分析方法,将对临床诊断、探讨病变机制及筛选药物都有一定的价值。关于DNase的分析Boyd等先后建立了DNA-聚丙烯酰胺凝胶电泳(DNA-PAGE)法;Brown等又将它加以简化,并将它直接用于细胞抽提液中DNase的鉴定与比较。本文又作了改进,降低了分离胶中的DNA含量,并以溴化乙锭(Ethidium Bromid),代替焦宁Y(Pyronin Y)。结果表明,改进后保温与染色时间可以大大缩短,检测灵敏度提高。应用这种方法检定人和小鼠白细胞和肝细胞中的DNase,表明,不同类型的细胞中包含各自特征的DNase酶谱,而且它对反应条件有不同的要求,对抑制剂有不     

微量氨基酸分析法的改进

本文介绍一种用甲醇作为流动相的微量氨基酸分析方法,通过对17种氨基酸进行了实验观察、分析,结果表明,分离分析结果良好,氨基酸浓度在1.5～12.0pmol之间峰面积成良好的线性关系。     

神经生长因子的发光免疫分析法

介绍了一种灵敏的神经生长因子化学发光免疫测定方法。小鼠神经生长因子的抗体ＩｇＧ用亲和层析纯化并用吖啶酯标记。该法可用于大鼠组织中ＮＧＦ含量的测定。方法的灵敏度为１０ｐｇ／ｍＬＮＧＦ;批内批间变异系数分别为８．７％及１３．２％;回收率平均１０３％。ｍＥＧＦ抗体对人的ＮＧＦ也有极强的交叉反应,故本方法也能用于病人血清或脑脊液中ＮＧＦ含量的测定。     

神经生长因子的发光免疫分析法

介绍了一种灵敏的神经生长因子化学发光免疫测定方法。小鼠神经生长因子（ｍＮＧＦ）的抗体ＩｇＧ用亲和层析纯化并用吖啶酯标记。该法可用于大鼠组织中ＮＧＦ含量的测定。方法的灵敏度为１０ｐｇ／ｍＬＮＧＦ;批内批间变异系数分别为８．７％及１３．２％;回收率平均为１０３％。ｍＮＧＦ抗体对人的ＮＧＦ也有极强的交叉反应,故本方法也可能用于病人血清或脑脊液中ＮＧＦ含量的测定。     

髋骨性别分级判别分析法

现有骨骼性别判别分析方法无疑是判别率最好的方法之一 ,但是否能在原基础上再提高一步 ?为此 ,本文选取 1 6项髋骨指标 ,制作“分级判别分析表”对 82副髋骨 (男42副 ,女40副 )进行分级性别判别分析 ,并与传统单因素及多因素判别分析法进行比较 ,3种方法最高正确判别率分别为 97.0 %、85 .4 %和 92 .1 % ,结果显示 :分级判别分析法可提高正确判别分析率 ,为考古学和法医学工作者提供了一种骨骼性别判别分析的新方法。     

化学发光分析法变异因素的控制

化学发光分析法是一种高灵敏度的分析测试方法,但该法易受多种因素干扰,易使结果产生较大变异。针对这一问题,我们采取了一系列措施,在甾体激素的化学发光免疫测定中获得了满意的效果。     

An alternative nonradioactive method for labeling DNA using biotin

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.     

艾滋病毒核酸探针的制备

 采用PXY104(含化学合成的HIV-env26肽基因4重串联体结构)为材料。温和裂解法提取质粒DNA,制备电泳纯化,Hind Ⅲ和EcoR Ⅰ酶解,低融点琼脂糖凝胶电泳回收HIV-DNA片段作为核酸探针;用[α-~(32)P]dATP通过缺口移位法标记,比放射性为4.05—6.69×10~7cpm/μgDNA通过分子杂交能测出lPg的靶DNA,初步试验结果表明,在本文试验条件下,可用该探针检测与之互补的核酸分子。     

A specific DNA probe for the identification of Campylobacter jejuni

A 6.1 kb DNA probe for the human enteric pathogen Campylobacter jejuni has been isolated from a genomic library constructed in the plasmid vector pBR322 in Escherichia coli. The DNA sequence used as a probe was identified from recombinant plasmids following immunological screening of transformants using polyclonal antisera to whole cells and to membrane antigens of C. jejuni. Restriction endonuclease fragment mapping of C. jejuni DNA inserts from three of the recombinant plasmids showed an overlapping DNA fragment. One of these recombinant plasmids, when used as a DNA probe in Southern hybridization, specifically hybridized with chromosomal DNA from all of the C. jejuni strains tested. Hybridization was not detected at high stringency between the DNA probe and chromosomal DNA from any other Campylobacter species tested except weakly with the chromosomal DNA of strains of Campylobacter coli. Hybridization was also not detected with chromosomal DNA from a range of other enteric bacteria likely to be encountered in faecal material. The intensity of hybridization with C. coli could be increased by reducing the stringency of hybridization.     

Plasmid-encoded copper resistance in Lactococcus lactis

A 54-kb plasmid (pND306) from Lactococcus lactis subsp. lactis 1252D encoded resistance to both Cu and Sn . The copper resistance determinant was subcloned on a 12.8-kb PvuII DNA fragment and mapped using a number of restriction endonucleases. Six other copper resistant lactococcal strains were also identified and all contained multiple plasmids. Plasmids in five of these strains showed strong hybridization with a probe made using the 12.8-kb DNA fragment, however no chromosomal homologs were detected. The copper resistance determinant was further isolated as a 10.6-kb SphI fragment and used to construct pND968 that expresses resistance to both copper and nisin.     

Direct cloning of a long restriction fragment aided with a jumping clone.

Long-range physical mapping with rare-cutting restriction enzymes (rare cutters) is an important step for structural analysis of complex genomes. Combination of two types of DNA clones bearing the rare-cutter sites, linking clones and jumping clones (Fig. 1a), facilitates the physical mapping [Poustka et al., Nature 325 (1987) 353-355]. A step followed by the physical mapping is the cloning of the large (rare-cutter-generated) restriction fragment of interest. For facilitating this step, we devised a method to directly clone a long restriction fragment without constructing the whole genomic DNA library using the jumping clone as starting material. The short DNA segments of a jumping clone, which are derived from the 5' and 3' terminal regions of the large restriction fragment, are inserted into the yeast artificial chromosome plasmid (pYAC) vector, and then converted into single strands with T7 gene 6-encoded 5'----3' exonuclease. The total genomic DNA digested with the restriction enzyme is also treated with the exonuclease to convert the terminal regions of the restriction fragments into single strands. In the resulting products, only the fragment corresponding to the jumping clone can form hybrids with the just-mentioned, single-stranded DNAs, which are connected to the pYAC, and only this fragment is cloned in yeast. We describe the protocol of this method with Escherichia coli DNA as a model experiment. Judging from the cloning efficiency, this method could be applied to cloning single-copy regions of the human genome, provided a jumping clone is available. The instability of inserts in the pYAC vector is also discussed.     

肺炎支原体P1蛋白羧基端基因片段在大肠杆菌中克隆的研究

在大肠杆菌中克隆肺炎支原体P1蛋白羧基端基因片段,为P1蛋白基因片段的扩增、表达及探讨羧基端基因片段功能打基础.采用PCR扩增方法获取P1结构基因.扩增产物用SalI和EcoRI酶切消化,回收1kb大小的DNA片段并与pUC19DNA连接,转入大肠杆菌JM109菌株.用X-gal平板及质粒图谱分析方法筛选重组克隆株,再用限制性核酸内切酶酶切图谱分析鉴定.经PCR扩增MPDNA获得1条5.0kbDNA片段.重组质粒限制性内切酶指纹图谱显示出2条带,1条为pUC19载体DNA带,另1条是1kb的插入片段.实验获得肺炎支原体P1蛋白结构基因及含P1蛋白羧基端DNA片段的重组克隆株.     

3'-end labeling of DNA with [alpha-32P]cordycepin-5'-triphosphate

Cordycepin-5'-triphosphate (3'-deoxyadenosine-5'-triphosphate) can be incorporated into the 3'-ends of DNA fragments using terminal deoxynucleotidyl transferase from calf thymus (Bollum, 1974). Because cordycepin-5'-monophosphate lacks a 3'-OH group, only a single residue is incorporated. Furthermore, DNA molecules that contain cordycepin-5'-monophosphate at their 3'-ends become resistant to hydrolysis by exonucleases that require free 3'-OH ends. As an alternative to 5'-end labeling of complementary DNA strands, we have used [32P]cordycepin-5'-triphosphate labeling of 3'-ends to confirm the nucleotide sequence of a HhaI-endonuclease-generated pTU4-plasmid DNA fragment that contains several hot spots for insertions of the transposable genetic element Tn3. 3'-End labeling with [32P] cordycepin-5'-triphosphate has also proved useful in determining the sequence of the pTU4 DNA in the vicinity of a strategically located SstII endonuclease cleavage site in the replication region of the plasmid.     

Mapping muscle protein genes by in situ hybridization using biotin-labeled probes.

The genes coding for the myosin heavy chain isoforms (unc-54, myo-1, myo-2 and myo-3) and the actins (act-1,2,3 and act-4) have been mapped on the embryonic metaphase chromosomes of Caenorhabditis elegans by in situ hybridization. The genes were cloned in a cosmid vector and the entire cosmid was nick translated to incorporate biotin-labeled dUTP. This produced a probe DNA complementary to a 35-45 kb length of chromosomal DNA. The hybridization signal from the cosmid probe, detected by immunofluorescence, could be easily seen by eye. The clear signals and the specific hybridization of the cosmid probes provided a faster means of mapping these single copy genes than small probes cloned in plasmid or lambda vectors. The myosin heavy chain genes are not clustered. Only unc-54 and myo-1 mapped to the same chromosome; the unc-54 locus is at the extreme right end of linkage group I and myo-1 mapped 40-50% from the left end of linkage group I. Myo-2 mapped to the X, 52-75% from the left end. The myo-3 gene mapped to the middle of linkage group V near the cluster of three actin genes (act-1,2,3). The fourth actin gene, act-4 mapped to 20-35% from the left end of X.