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51.
Evaluation of mass spectrometric techniques for charaterization of engineered proteins 总被引:7,自引:0,他引:7
Roepstorff Peter Schram Karl H. Andersen Jens S. Rafn Kate Baldursson Trausti Krøll Jenny Poulsen Kjeld Knudsen Jens Kristiansen Karsten 《Molecular biotechnology》1995,3(1):1-7
A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described.
The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used
as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation.
The various aspects of the procedure and its application is described in detail. 相似文献
52.
Ralph Rapley 《Molecular biotechnology》1995,3(2):139-154
The exquisite specificity of monoclonal antibodies (MAb) has long provided the potential for creating new reagents for the
in vivo delivery of therapeutic drugs or toxins to defined cellular target sites or improved methods of diagnosis. However,
many difficulties associated with their production, affinity, specificity, and use in vivo have largely confined their application
to research or in vitro diagnostics. This situation is beginning to change with the recent developments in the applied molecular
techniques that allow the engineering of the genes that encode antibodies rather than the manipulation of the intact antibodies
themselves. Techniques, such as the polymerase chain reaction, have provided essential methods with which to generate and
modify the genetic constituents of antibodies, allow their conjugation to toxins or drugs, provide ways of humanizing murine
antibodies, and allow discrete modular antigen binding components to be produced. More recent developments of in vitro expression
systems and powerful phage surface display technologies will without doubt play a major role in future antibody engineering
and in the successful development of new diagnostic and therapeutic antibody-based reagents. 相似文献
53.
Abstract The firefly luciferase gene, luc , was demonstrated to hold promise as a specific marker for monitoring of genetically modified bacteria in the environment. PCR amplification and bioluminescence procedures were modified and compared for environmental monitoring of luc -tagged bacteria, using Escherichia coli as a model. The methods were used to track luc -tagged bacterial cells added to intact sediment core microcosms. Detection limits for the luc -tagged cells were the following, expressed as cells per 0.5 g of sediment: 102 , by PCR amplification; 103 , by whole cell luminescence; and 103 −104 , by measurement of luminescence in cell extracts. 相似文献
54.
Hermann Schmidt Thomas Dresselhaus Friedrich Buck Ernst Heinz 《Plant molecular biology》1994,26(2):631-642
A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5 end of the cDNA was amplified by a 5-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen. 相似文献
55.
56.
DNA sequencing with arbitrary primer pairs 总被引:8,自引:0,他引:8
57.
旋毛虫肌幼虫ES抗原的基因克隆及高效表达 总被引:7,自引:0,他引:7
作者对编码旋毛虫肌幼虫ES抗原的部分结构基因进行了克隆、鉴定和表达。用RNA PCR技术直接从旋毛虫肌幼虫总RNA中反转录并扩增出0.7kh的靶DNA,酶切分析后将其克隆到融合表达载体pEx3lC中。SDS—PAGE电泳表明,含重组子的大肠杆菌能够表达出一分子量为37kDa的融合蛋白(P37),后者占菌体总蛋白的22%以上,并以包含体形式存在于菌体中。经对纯化后表达蛋白的ELlSA检测,证明它能被猪旋毛虫病阳性血清和抗旋毛虫单克隆抗体识别。研究结果揭示,重组蛋白P37对于研制旋毛虫病诊断抗原和免疫抗原具有潜在的应用价值。 相似文献
58.
59.
Yoshihiro Imamura Nobuyuki Hamada Takayuki Nagai Masahisa Shingu 《Microbiology and immunology》1994,38(8):673-676
Stool specimens from infants who visited a clinic because of acute gastroenteritis were tested for the presence of human rotavirus. Among the samples obtained were specimens taken from seven patients who had visited the clinic at two different times. In six of these seven children, human rotavirus (HRV) was detected in only one of the specimens taken (i.e. during only one of the two visits). One patient was shown to have excreted HRV twice; in both cases the HRV was serotyped to be type 1. The present results indicate that the symptomatic reinfection of HRV was not a widely occurring phenomenon in the group of infants tested. 相似文献
60.
杂交水稻及其“三系”线粒体DNA的AP—PCR指纹图谱 总被引:23,自引:1,他引:22
为了研究水稻(Oryza sativa L.)细胞质雄性不育(CMS)与线粒体基因组的关系,应用AP-PCR 分析,用7 个任意单引物对6 种水稻品系线粒体DNA 进行了扩增。水稻线粒体DNA 的AP-PCR 产物可分为三种类型:(1)所有供试品系均能扩增的片段,它们代表了线粒体DNA 在进化上的保守性序列。有4 个引物检测到这类片段。(2)2 个以上水稻品系共同出现而在全部供试材料间存在差异的扩增片段,这类片段是检测水稻线粒体DNA多态性的主要来源。(3)一种细胞质类型所特有的扩增片段,从引物R2 和V5 的扩增产物中发现了这类片段,它们可能与CMS有关联。另外,WA型不育系珍汕97A 与其杂种之间在6 个引物的扩增图谱上均存在不同程度的差异,说明两者的线粒体DNA序列结构可能存在某种差别 相似文献