全文获取类型
收费全文 | 1046篇 |
免费 | 63篇 |
国内免费 | 162篇 |
出版年
2024年 | 2篇 |
2023年 | 19篇 |
2022年 | 32篇 |
2021年 | 49篇 |
2020年 | 20篇 |
2019年 | 45篇 |
2018年 | 19篇 |
2017年 | 26篇 |
2016年 | 30篇 |
2015年 | 36篇 |
2014年 | 47篇 |
2013年 | 94篇 |
2012年 | 32篇 |
2011年 | 35篇 |
2010年 | 37篇 |
2009年 | 38篇 |
2008年 | 44篇 |
2007年 | 54篇 |
2006年 | 72篇 |
2005年 | 57篇 |
2004年 | 53篇 |
2003年 | 62篇 |
2002年 | 56篇 |
2001年 | 32篇 |
2000年 | 22篇 |
1999年 | 24篇 |
1998年 | 13篇 |
1997年 | 14篇 |
1996年 | 18篇 |
1995年 | 11篇 |
1994年 | 8篇 |
1993年 | 7篇 |
1992年 | 16篇 |
1991年 | 5篇 |
1990年 | 6篇 |
1989年 | 9篇 |
1988年 | 4篇 |
1987年 | 8篇 |
1986年 | 6篇 |
1985年 | 25篇 |
1984年 | 28篇 |
1983年 | 7篇 |
1982年 | 13篇 |
1981年 | 7篇 |
1980年 | 4篇 |
1979年 | 8篇 |
1978年 | 10篇 |
1976年 | 4篇 |
1974年 | 1篇 |
1973年 | 1篇 |
排序方式: 共有1271条查询结果,搜索用时 421 毫秒
991.
Turan S Galla M Ernst E Qiao J Voelkel C Schiedlmeier B Zehe C Bode J 《Journal of molecular biology》2011,407(2):193-17240
Traditional DNA transduction routes used for the modification of cellular genomes are subject to unpredictable alterations, as the cell-intrinsic repair machinery may affect both the integrity of the transgene and the recipient locus. These problems are overcome by recombinase-mediated cassette exchange (RMCE) approaches enabling predictable expression patterns by the nondisruptive insertion of a gene cassette at a pre-characterized genomic locus. The destination is marked by a “tag” consisting of two heterospecific recombination target sites (RTs) at the flanks of a selection marker. Provided on a circular donor vector, an analogous cassette encoding the gene of interest can cleanly replace the resident cassette under the influence of a site-specific recombinase. RMCE was first based on the yeast integrase Flp but had to give way to the originally more active phage-derived Cre enzyme. To be effective, both Tyr-recombinases have to be applied at a considerable concentration, which, in the case of Cre, triggers endonucleolytic activities and therefore cellular toxicity. This review addresses the particularities of both recombination routes depending on the structure of the synaptic complex and on improved integrase and RT variants. While the performance of Flp-RMCE can now firmly rely on optimized Flp variants and multiple sets of functional target sites (FRTs), the Cre system suffers from the promiscuity of its RT mutants, which is explained in molecular terms. At present, RMCE enters applications in the stem cell field. Remarkable efforts are noted in the framework of various mouse mutagenesis programs, which, in their first phase, have targeted virtually all genes and now start to shift their emphasis from gene trapping to gene modification. 相似文献
992.
Dyson MR Zheng Y Zhang C Colwill K Pershad K Kay BK Pawson T McCafferty J 《Analytical biochemistry》2011,(1):25-35
Mapping protein interactions by immunoprecipitation is limited by the availability of antibodies recognizing available native epitopes within protein complexes with sufficient affinity. Here we demonstrate a scalable approach for generation of such antibodies using phage display and affinity maturation. We combined antibody variable heavy (VH) genes from target-specific clones (recognizing Src homology 2 (SH2) domains of LYN, VAV1, NCK1, ZAP70, PTPN11, CRK, LCK, and SHC1) with a repertoire of 108 to 109 new variable light (VL) genes. Improved binders were isolated by stringent selections from these new “chain-shuffled” libraries. We also developed a predictive 96-well immunocapture screen and found that only 12% of antibodies had sufficient affinity/epitope availability to capture endogenous target from lysates. Using antibodies of different affinities to the same epitope, we show that affinity improvement was a key determinant for success and identified a clear affinity threshold value (60 nM for SHC1) that must be breached for success in immunoprecipitation. By combining affinity capture using matured antibodies to SHC1 with mass spectrometry, we identified seven known binding partners and two known SHC1 phosphorylation sites in epidermal growth factor (EGF)-stimulated human breast cancer epithelial cells. These results demonstrate that antibodies capable of immunoprecipitation can be generated by chain shuffling, providing a scalable approach to mapping protein–protein interaction networks. 相似文献
993.
Stern A Sorek R 《BioEssays : news and reviews in molecular, cellular and developmental biology》2011,33(1):43-51
Bacteria, the most abundant organisms on the planet, are outnumbered by a factor of 10 to 1 by phages that infect them. Faced with the rapid evolution and turnover of phage particles, bacteria have evolved various mechanisms to evade phage infection and killing, leading to an evolutionary arms race. The extensive co-evolution of both phage and host has resulted in considerable diversity on the part of both bacterial and phage defensive and offensive strategies. Here, we discuss the unique and common features of phage resistance mechanisms and their role in global biodiversity. The commonalities between defense mechanisms suggest avenues for the discovery of novel forms of these mechanisms based on their evolutionary traits. 相似文献
994.
Akshat Mullerpatan Erin Kane Ronit Ghosh André Nascimento Henrik Andersen Steven Cramer Pankaj Karande 《Biotechnology and bioengineering》2020,117(12):3775-3784
Affinity precipitation using stimulus-responsive biopolymers such as elastin-like polypeptides (ELPs) have been successfully employed for the purification of monoclonal antibodies. In the current work, we extend these studies to the development of an ELP-peptide fusion for the affinity precipitation of the therapeutically relevant small non-mAb biologic, AdP. A 12-mer affinity peptide ligand (P10) was identified by a primary phage biopanning followed by a secondary in-solution fluorescence polarization screen. Peptide P10 and AdP interacted with a KD of 19.5 µM. A fusion of P10 with ELP was then shown to be successful in selectively capturing the biologic from a crude mixture. While pH shifts alone were not sufficient for product elution, the use of pH in concert with fluid-phase modifiers such as NaCl, arginine, or ethylene glycol was effective. In particular, the use of pH 8.5 and an arginine concentration of 500 mM enabled >80% product recovery. The overall process performance evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase ultra-performance liquid chromatography analyses indicated successful single-step purification of the biologic from an Escherichia coli lysate resulting in ∼90% purity and >80% recovery. These results demonstrate that phage display can be readily employed to identify a peptide ligand capable of successfully carrying out the purification of a non-antibody biological product using ELP-based affinity precipitation. 相似文献
995.
《MABS-AUSTIN》2013,5(2):422-436
While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGenTM) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling® platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs. 相似文献
996.
Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase
《MABS-AUSTIN》2013,5(5):1327-1339
A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes. 相似文献
997.
Hiroaki Tani Jane K. Osbourn Edward H. Walker Robert A. Rush Ian A. Ferguson 《MABS-AUSTIN》2013,5(3):471-478
The neurotrophin receptor p75NTR is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75NTR antibody or phage scFv library pre-panned against p75NTR are internalized by neurons expressing p75NTR; (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75NTR antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75NTR expression is upregulated in motor neurons in response to injury and in disease, the p75NTR antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier. 相似文献
998.
Ailan Guo Lianet Lopez Alevtina Pavlenco Adenrele Akintobi Yingnan Zhang Bridget Currell Somasekar Seshagiri Tong Hao Xinping Yang Yun A Shen Jingjing Li Aaron T Cheng Dryden Bouamalay Adrien Lugari David E Hill Mark L Grimes David G Drubin Barth D Grant Marc Vidal Charles Boone Sachdev S Sidhu Gary D Bader 《Molecular systems biology》2013,9(1)
Src homology 3 (SH3) domains bind peptides to mediate protein–protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two‐hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain‐mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form. 相似文献
999.
Honglei Zhao Xin Feng Wenyu Han Yuwen Diao Dong Han Xiaofeng Tian Yu Gao Shanshan Liu Seng Zhu Cuimei Yao Jingmin Gu Changjiang Sun Liancheng Lei 《Journal of peptide science》2013,19(10):639-650
A random phage 12‐mer peptide library and a whole‐cell subtractive biopanning protocol against HepG2 cells were used to select a novel peptide‐specific binding to hepatocellular carcinoma cells. As a result, peptide SLSLITMLKISR (AM‐2) was screened as a novel homing peptide to hepatocellular carcinoma cells, tested by immunofluorescence and immunochemistry assays. Subsequently, peptide AM‐2 was linked to melittin by A(EAAAK)2A, and the antitumor effect of this ligation product was detected by MTT assay, fluorescence‐activated cell sorting, and scanning electron microscopy methods. Results of cell growth inhibition tests confirmed that the affinity of melittin was increased after being incorporated into AM‐2, and AM‐2‐melittin specifically targeted and killed HepG2 cells in vitro. Thus, AM‐2 is a valuable ligand for tumor targeting, which leads to increased binding and killing effect of hepatocellular carcinoma cells in vitro when ligated to melittin, and AM‐2‐melittin has a clinical potential application as target agents for the treatment of human hepatocellular carcinoma. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
1000.
Ariane Toussaint Marie-José Gama Jamal Laachouch Geneviève Maenhaut-Michel Amina Mhammedi-Alaoui 《Genetica》1994,93(1-3):27-39
Bacteriophage Mu is a transposon and a temperate phage which has become a paradigm for the study of the molecular mechanism of transposition. As a prophage, Mu has also been used to study some aspects of the influence of the host cell growth phase on the regulation of transposition. Through the years several host proteins have been identified which play a key role in the replication of the Mu genome by successive rounds of replicative transposition as well as in the maintenance of the repressed prophage state. In this review we have attempted to summarize all these findings with the purpose of emphasizing the benefit the virus and the host cell can gain from those phage-host interactions. 相似文献