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61.
62.
Kinetic screening of antibody-Im7 conjugates by capture on a colicin E7 DNase domain using optical biosensors 总被引:1,自引:0,他引:1
Antibody generation by phage display and related in vitro display technologies routinely yields large panels of clones detected in primary end-point screenings such as enzyme-linked immunosorbent assay (ELISA). However, for the development of clinical lead candidates, rapid determination of secondary characteristics such as kinetics and thermodynamics is of nearly equal importance. Surface plasmon resonance-based biosensors are ideal tools for carrying out such high-throughput secondary screenings, allowing preliminary but confident ranking and identification of lead clones. A key feature of these assays is the stable and reversible capture of antibody fragments from crude samples leading to high-resolution kinetic analysis of library outputs. Here we exploit the high-affinity interaction between the naturally occurring nuclease domain of E. coli colicin E7 (DNaseE7) and its cognate partner, the immunity protein 7 (Im7), to develop a ligand capture system suitable for accurate kinetic ranking of library clones. We demonstrate generic applicability for a range of antibody formats: scFv antibodies, diabodies, antigen binding fragments (Fabs), and shark VNAR single domain antibodies. The system is adaptable and reproducible, with comparable results achieved for both the Biacore T100 and ProteOn XPR36 array biosensors. 相似文献
63.
Complex multimeric recombinant proteins such as therapeutic antibodies require a eukaryotic expression system. Transgenic
plants may serve as promising alternatives to the currently favored mammalian cell lines or hybridomas. In contrast to prokaryotic
systems, posttranslational modifications of plant and human proteins resemble each other largely, among those, protein N-glycosylation
of the complex type. However, a few plant-specific sugar residues may cause immune reactions in humans, representing an obstacle
for the broad use of plant-based systems as biopharmaceutical production hosts. The moss Physcomitrella patens represents a flexible tissue-culture system for the contained production and secretion of recombinant biopharmaceuticals
in photobioreactors. The recent synthesis of therapeutic proteins as a scFv antibody fragment or the large and heavily modified
complement regulator factor H demonstrate the versatility of this expression system. A uniquely efficient gene targeting mechanism
can be employed to precisely engineer the glycosylation machinery for recombinant products. In this way, P. patens lines with non-immunogenic optimized glycan structures were created. Therapeutic antibodies produced in these strains exhibited
antibody-dependent cellular cytotoxicity superior to the same molecules synthesized in mammalian cell lines. 相似文献
64.
65.
Nitrogen yield advantage from grass–legume mixtures is robust over a wide range of legume proportions and environmental conditions 下载免费PDF全文
Matthias Suter John Connolly John A. Finn Ralf Loges Laura Kirwan Maria‐Teresa Sebastià Andreas Lüscher 《Global Change Biology》2015,21(6):2424-2438
Current challenges to global food security require sustainable intensification of agriculture through initiatives that include more efficient use of nitrogen (N), increased protein self‐sufficiency through homegrown crops, and reduced N losses to the environment. Such challenges were addressed in a continental‐scale field experiment conducted over 3 years, in which the amount of total nitrogen yield (Ntot) and the gain of N yield in mixtures as compared to grass monocultures (Ngainmix) was quantified from four‐species grass–legume stands with greatly varying legume proportions. Stands consisted of monocultures and mixtures of two N2‐fixing legumes and two nonfixing grasses. The amount of Ntot of mixtures was significantly greater (P ≤ 0.05) than that of grass monocultures at the majority of evaluated sites in all 3 years. Ntot and thus Ngainmix increased with increasing legume proportion up to one‐third of legumes. With higher legume percentages, Ntot and Ngainmix did not continue to increase. Thus, across sites and years, mixtures with one‐third proportion of legumes attained ~95% of the maximum Ntot acquired by any stand and had 57% higher Ntot than grass monocultures. Realized legume proportion in stands and the relative N gain in mixture (Ngainmix/Ntot in mixture) were most severely impaired by minimum site temperature (R = 0.70, P = 0.003 for legume proportion; R = 0.64, P = 0.010 for Ngainmix/Ntot in mixture). Nevertheless, the relative N gain in mixture was not correlated to site productivity (P = 0.500), suggesting that, within climatic restrictions, balanced grass–legume mixtures can benefit from comparable relative gains in N yield across largely differing productivity levels. We conclude that the use of grass–legume mixtures can substantially contribute to resource‐efficient agricultural grassland systems over a wide range of productivity levels, implying important savings in N fertilizers and thus greenhouse gas emissions and a considerable potential for climate change mitigation. 相似文献
66.
67.
Filip Vandenbussche Ana C Fierro Gertrud Wiedemann Ralf Reski Dominique Van Der Straeten 《BMC plant biology》2007,7(1):65
Background:
Gibberellins (GA) are plant hormones that can regulate germination, elongation growth, and sex determination. They ubiquitously occur in seed plants. The discovery of gibberellin receptors, together with advances in understanding the function of key components of GA signalling in Arabidopsis and rice, reveal a fairly short GA signal transduction route. The pathway essentially consists of GID1 gibberellin receptors that interact with F-box proteins, which in turn regulate degradation of downstream DELLA proteins, suppressors of GA-controlled responses. 相似文献68.
Christiane Wloczyk Achim Kröger Thomas Göbel Gabriele Holdt Ralf Steudel 《Archives of microbiology》1989,152(6):600-605
Wolinella succinogenes grown on formate and elemental sulphur was found to use the polysulphide derivatives 2,2-tetrathiobispropionate (R2S4) or pentathionate (S5O
6
=
) as acceptors for formate oxidation. The specific activities of formate oxidation with these acceptors were similar to those with elemental sulphur. The main reaction products of R2S4 reduction were 2,2-dithiobispropionate (R2S2) and sulphide. Pentathionate was converted to thiosulphate and some elemental sulphur. The electrochemical proton potential
across the cytoplasmic membrane of the bacterium was measured in the steady state of electron transport from formate to R2S4. The electrical proportion () of the
determined through the distribution of labeled tetraphenylphosphonium cation was obtained as 0.17 Volt. The was zero, when a protonophore was present. The pH-difference across the membrane was negligible. Thus the
generated by sulphur respiration is close to that measured earlier with fumarate as the terminal acceptor of electron transport.Abbreviations DMO
5,5-dimethyloxazolidine-2,4-dione
- R2Sn (n=2–5)
2,2-polythiobispropionate
- TTFB
4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazol
- TPP
tetraphenylphosphonium cation 相似文献
69.
Quantitative PCR Method for Sensitive Detection of Ruminant Fecal Pollution in Freshwater and Evaluation of This Method in Alpine Karstic Regions 下载免费PDF全文
Georg H. Reischer David C. Kasper Ralf Steinborn Robert L. Mach Andreas H. Farnleitner 《Applied microbiology》2006,72(8):5610-5614
A quantitative TaqMan minor-groove binder real-time PCR assay was developed for the sensitive detection of a ruminant-specific genetic marker in fecal members of the phylum Bacteroidetes. The qualitative and quantitative detection limits determined were 6 and 20 marker copies per PCR, respectively. Tested ruminant feces contained an average of 4.1 × 109 marker equivalents per g, allowing the detection of 1.7 ng of feces per filter in fecal suspensions. The marker was detected in water samples from a karstic catchment area at levels matching a gradient from negligible to considerable ruminant fecal influence (from not detectable to 105 marker equivalents per liter). 相似文献
70.
Song Y Friebe P Tzima E Jünemann C Bartenschlager R Niepmann M 《Journal of virology》2006,80(23):11579-11588
The positive-strand RNA genome of the hepatitis C virus (HCV) is flanked by 5'- and 3'-untranslated regions (UTRs). Translation of the viral RNA is directed by the internal ribosome entry site (IRES) in the 5'-UTR, and subsequent viral RNA replication requires sequences in the 3'-UTR and in the 5'-UTR. Addressing previous conflicting reports on a possible function of the 3'-UTR for RNA translation in this study, we found that reporter construct design is an important parameter in experiments testing 3'-UTR function. A translation enhancer function of the HCV 3'-UTR was detected only after transfection of monocistronic reporter RNAs or complete RNA genomes having a 3'-UTR with a precise 3' terminus. The 3'-UTR strongly stimulates HCV IRES-dependent translation in human hepatoma cell lines but only weakly in nonliver cell lines. The variable region, the poly(U . C) tract, and the most 3' terminal stem-loop 1 of the highly conserved 3' X region contribute significantly to translation enhancement, whereas stem-loops 2 and 3 of the 3' X region are involved only to a minor extent. Thus, the signals for translation enhancement and for the initiation of RNA minus-strand synthesis in the HCV 3'-UTR partially overlap, supporting the idea that these sequences along with viral and possibly also cellular factors may be involved in an RNA 3'-5' end interaction and a switch between translation and RNA replication. 相似文献