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21.
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
22.
Self-splicing group I and group II introns encode homologous (putative) DNA endonucleases of a new family. 总被引:17,自引:3,他引:14 下载免费PDF全文
A. E. Gorbalenya 《Protein science : a publication of the Protein Society》1994,3(7):1117-1120
A new family of protein domains consisting of 50-80 amino acid residues is described. It is composed of nearly 40 members, including domains encoded by plastid and phage group I introns; mitochondrial, plastid, and bacterial group II introns; eubacterial genomes and plasmids; and phages. The name "EX1HH-HX3H" was coined for both domain and family. It is based on 2 most prominent amino acid sequence motifs, each encompassing a pair of highly conserved histidine residues in a specific arrangement: EX1HH and HX3H. The "His" motifs often alternate with amino- and carboxy-terminal motifs of a new type of Zn-finger-like structure CX2,4CX29-54[CH]X2,3[CH]. The EX1HH-HX3H domain in eubacterial E2-type bacteriocins and in phage RB3 (wild variant of phage T4) product of the nrdB group I intron was reported to be essential for DNA endonuclease activity of these proteins. In other proteins, the EX1HH-HX3H domain is hypothesized to possess DNase activity as well. Presumably, this activity promotes movement (rearrangement) of group I and group II introns encoding the EX1HH-HX3H domain and other gene targets. In the case of Escherichia coli restrictase McrA and possibly several related proteins, it appears to mediate the restriction of alien DNA molecules. 相似文献
23.
Sixty-one isolates of Rhizobium meliloti from two field sites which had been previously classified into 15 phage types on the basis of sensitivity to 16 typing phages, were subjected to insertion sequence (IS) hybridization using DNA probes for ISR m 3 and ISR m 5. Isolates from all but one phage type contained ISR m 3 (apparent copy no. 1–11) and all isolates contained ISR m 5 (apparent copy no. 3–11). The isolates were placed into 24 IS classes based on differences in their respective ISR m 3 and ISR m 5 hybridization profiles. At either field site, isolates representing different phage types possessed IS hybridization profiles that differed from each other, while those comprising a specific type had identical or closely related profiles. Isolates from one phage type were unusual since they did not react with any of the typing phages and were shown by IS hybridization to constitute a heterogeneous group. Evidence for spatial effects were provided by isolates from two of six types present at both sites which fell into separate IS classes on the basis of their site of origin. These data have ecological implications and suggest that for a particular site, phage typing may be employed for the rapid assessment of the genetic diversity among field isolates. 相似文献
24.
25.
单链抗体(single chain antibody fragment,scFv)是由抗体重链可变区(variable region of heavy chain,VH)和轻链可变区(variable region of light chain,VL)通过柔性短肽连接组成的小分子,是具有完整抗原结合活性的最小功能片段,包含抗体识别及抗原结合部位。相比于其他抗体,scFv具有分子量小、穿透性强、免疫原性弱、易构建表达等优点。目前,scFv最常用的展示系统主要有噬菌体展示系统、核糖体展示系统、mRNA展示系统、酵母细胞表面展示系统和哺乳动物细胞展示系统等。近年来,随着scFv在医学、生物学、食品安全学等领域的发展,使得其在生物合成和应用研究方面备受关注。本文对近年来scFv展示系统的研究进展作一综述,以期为scFv的筛选及应用提供理论基础。 相似文献
26.
27.
人抗E.Coli J5噬菌体抗体制备的初步研究 总被引:1,自引:0,他引:1
以E.Coli J5株对合人Ig基因的噬菌体抗体库进行淘筛富集,免疫印迹筛选,以及ELISA检测,结果获得4株能与E.Coli J5株结合的阳性克隆,且阳性克隆结合抗原的活性可分别被E.Coli J5株、E.Coli Rc-LPS和抗E.Coli J5株核心糖脂域MAb抑制.PCR检测表明,4株阳性克隆均分别带有约660bp大小的重链和轻链基因片段.SDS-PAGE与蛋白质印迹的结果显示,经IPTG诱导的阳性克隆能表达分子量约为50000大小的蛋白,提示该4株阳性克隆能够表达具有一定抗原结合活性的人源Fab片段. 相似文献
28.
The cyanophage AN-15 was found to have a requirement for either 1 mM calcium or 1 mM magnesium ions to maintain viral stability,
whereas 1 mM calcium ions alone were essential for the infection process to proceed in Anabaena sp. strain PCC 7120. Following
prolonged incubation, phage-resistant cells were detected at a high frequency (approximately 10-5) in lysates, as either renewed growth in liquid cultures, or as colonies in confluently lysed lawns. Southern hybridisation
failed to detect AN-15 DNA in any of the resistant strains, implying that resistance is unlikely to be due to the presence
of temperate phages. A high rate of spontaneous mutation is therefore likely to be the cause of resistance. Two classes of
resistant cells were identified; those in which AN-15 failed to attach to host cells, and those in which attachment occurred,
but subsequent replication was defective. However, it was possible to overcome phage resistance by the isolation of spontaneous
mutants of AN-15, capable of infecting phage-resistant cells. These observations imply that if cyanophages are to be assessed
as a means of controlling cyanobacterial blooms in freshwater bodies, the ionic (notably calcium) concentration of the water
must be considered, together with the possible need to employ alternative cyanophage strains if resistance to the original
one arises.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
29.
Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): engineering of inhibitors with altered specificities. 总被引:1,自引:0,他引:1 下载免费PDF全文
A. J. Scheidig T. R. Hynes L. A. Pelletier J. A. Wells A. A. Kossiakoff 《Protein science : a publication of the Protein Society》1997,6(9):1806-1824
The crystal structures of the inhibitor domain of Alzheimer's amyloid beta-protein precursor (APPI) complexed to bovine chymotrypsin (C-APPI) and trypsin (T-APPI) and basic pancreatic trypsin inhibitor (BPTI) bound to chymotrypsin (C-BPTI) have been solved and analyzed at 2.1 A, 1.8 A, and 2.6 A resolution, respectively. APPI and BPTI belong to the Kunitz family of inhibitors, which is characterized by a distinctive tertiary fold with three conserved disulfide bonds. At the specificity-determining site of these inhibitors (P1), residue 15(I)4 is an arginine in APPI and a lysine in BPTI, residue types that are counter to the chymotryptic hydrophobic specificity. In the chymotrypsin complexes, the Arg and Lys P1 side chains of the inhibitors adopt conformations that bend away from the bottom of the binding pocket to interact productively with elements of the binding pocket other than those observed for specificity-matched P1 side chains. The stereochemistry of the nucleophilic hydroxyl of Ser 195 in chymotrypsin relative to the scissile P1 bond of the inhibitors is identical to that observed for these groups in the trypsin-APPI complex, where Arg 15(I) is an optimal side chain for tryptic specificity. To further evaluate the diversity of sequences that can be accommodated by one of these inhibitors, APPI, we used phage display to randomly mutate residues 11, 13, 15, 17, and 19, which are major binding determinants. Inhibitors variants were selected that bound to either trypsin or chymotrypsin. As expected, trypsin specificity was principally directed by having a basic side chain at P1 (position 15); however, the P1 residues that were selected for chymotrypsin binding were His and Asn, rather than the expected large hydrophobic types. This can be rationalized by modeling these hydrophilic side chains to have similar H-bonding interactions to those observed in the structures of the described complexes. The specificity, or lack thereof, for the other individual subsites is discussed in the context of the "allowed" residues determined from a phage display mutagenesis selection experiment. 相似文献
30.