全文获取类型
收费全文 | 2848篇 |
免费 | 202篇 |
国内免费 | 444篇 |
出版年
2024年 | 4篇 |
2023年 | 23篇 |
2022年 | 34篇 |
2021年 | 54篇 |
2020年 | 52篇 |
2019年 | 60篇 |
2018年 | 65篇 |
2017年 | 65篇 |
2016年 | 78篇 |
2015年 | 71篇 |
2014年 | 135篇 |
2013年 | 206篇 |
2012年 | 110篇 |
2011年 | 126篇 |
2010年 | 125篇 |
2009年 | 158篇 |
2008年 | 181篇 |
2007年 | 162篇 |
2006年 | 175篇 |
2005年 | 171篇 |
2004年 | 127篇 |
2003年 | 146篇 |
2002年 | 145篇 |
2001年 | 155篇 |
2000年 | 105篇 |
1999年 | 110篇 |
1998年 | 73篇 |
1997年 | 61篇 |
1996年 | 59篇 |
1995年 | 46篇 |
1994年 | 56篇 |
1993年 | 44篇 |
1992年 | 53篇 |
1991年 | 26篇 |
1990年 | 18篇 |
1989年 | 13篇 |
1988年 | 21篇 |
1987年 | 9篇 |
1986年 | 9篇 |
1985年 | 34篇 |
1984年 | 29篇 |
1983年 | 14篇 |
1982年 | 21篇 |
1981年 | 16篇 |
1980年 | 15篇 |
1979年 | 21篇 |
1978年 | 12篇 |
1976年 | 1篇 |
排序方式: 共有3494条查询结果,搜索用时 16 毫秒
31.
E. A. Walker J. P. Ride S. Kurup V. E. Franklin-Tong M. J. Lawrence F. C. H. Franklin 《Plant molecular biology》1996,30(5):983-994
The S
3 allele of the S gene has been cloned from Papaver rhoeas cv. Shirley. The sequence predicts a hydrophilic protein of 14.0 kDa, showing 55.8% identity with the previously cloned S
1 allele, preceded by an 18 amino acid signal sequence. Expression of the S
3 coding region in Escherichia coli produced a form of the protein, denoted S3e, which specifically inhibited S3 pollen in an in vitro bioassay. The recombinant protein was ca. 0.8 kDa larger than the native stigmatic form, indicating post-translational modifications in planta, as was previously suggested for the S1 protein. In contrast to other S proteins identified to date, S3 protein does not appear to be glycosylated. Of particular significance is the finding that despite exhibiting a high degree of sequence polymorphism, secondary structure predictions indicate that the S1 and S3 proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the P. rhoeas S alleles share some limited homology with the SLG and SRK genes from Brassica oleracea. Previously, cross-classification of different populations of P. rhoeas had revealed a number of functionally identical alleles. Probing of western blots of stigma proteins from plants derived from a wild Spanish population which contained an allele functionally identical to the Shirley S
3 allele with antiserum raised to S3e, revealed a protein (S
3
s) which was indistinguishable in pI and M
r from that in the Shirley population. A cDNA encoding S
3
s was isolated, nucleotide sequencing revealing a coding region with 99.4% homology with the Shirley-derived clone at the DNA level, and 100% homology at the amino acid level. 相似文献
32.
Large scale production of recombinant mouse and rat growth hormone by fed-batch GS-NSO cell cultures
Zhou W Bibila T Glazomitsky K Montalyo J Chan C Distefano D Munshi S Robinson D Buckland B Aunins J 《Cytotechnology》1996,22(1-3):239-250
Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×109 cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions. 相似文献
33.
The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented. 相似文献
34.
The antitumor effect of the combined administration with recombinant human interleukin-2 (rIL-2) and sizofiran (SPG), a single
glucan of Shizophyllum commune Fries, was studied in vivo in C57BL/6 mice intraperitoneally inoculated with EL-4 lymphoma.
The effect was evaluated by a) comparing the survival time of the mice, b) analysis of the intraperitoneal cell population
in Giemsa-stained specimens, c) surface marker analysis of peritoneal exudative cells with flow cytometry, d) cytotoxic assay
of cells against EL-4 and Yac-1 lymphoma, and e) elimination of some cell populations by monoclonal antibodies, to identify
the antitumor-effector cells showing cytotoxic activity. The survival of mice given both rIL-2 and SPG was significantly longer
than the control mice or those given SPG alone or rIL-2 alone. It was demonstrated that the administration of SPG and/or rIL-2
to the EL-4 lymphoma-bearing mice activated immune-response cells in the peritoneal cavity such as T lymphocytes, NK cells,
or macrophages, which might be effective in reducing lymphoma cells. The combination of rIL-2 and SPG administration appears
to activate the antitumor- immune response at the tumor site more effectively than when either agent was administered alone. 相似文献
35.
Summary The glycosylation and subsequent processing of native and recombinant glycoproteins expressed in established insect cell lines
and insect larvae were compared. TheSpodoptera frugiperda (Sf21) andTrichoplusia ni (TN-368 and BTI-Tn-5B1-4) cell lines possessed several intrinsic glycoproteins that are modified with both N- and O-linked
oligosaccharides. The N-linked oligosaccharides were identified as both the simple (high mannose) and complex (containing
sialic acid) types. Similarly, theT. ni larvae also possessed intrinsic glycoproteins that were modified with O-linked and simple and complex N-linked oligosaccharides.
Additionally, human placental, secreted alkaline phosphatase (SEAP) produced during replication of a recombinant baculovirus
inT. ni larvae was modified with complex oligosaccharide having sialic acid linked α(2–6) to galactose. 相似文献
36.
Casein kinase II is composed of two catalytic (a) and two regulatory () subunits, the amino acid sequences of the and subunits are highly conserved between species. To examine whether heterologous casein kinase II could be formed, recombinant and subunits from human andDrosophila were reconstituted from inclusion bodies. Casein kinase II containing either human andDrosophila orDrosophila and human subunits exhibited enzymatic properties similar to those of the homologous holoenzymes with regard to specific activity, salt optima, and autophosphorylation. However, renaturation and reconstitution of casein kinase II was dependent on the type of subunits and the redox conditions, with theDrosophila subunits requiring more reduced conditions. Chimeric subunits prepared from human andDrosophila cDNA revealed that the N-terminal region was responsible for the requirement for the reduced redox state during renaturation. TheN-terminal region also affected solubility and electrophoretic mobility of the subunit. 相似文献
37.
Konieczny and Ausubel have described a technique whereby Arabidopsis thaliana loci can be rapidly mapped to one of the ten chromosome arms using a small number of F2 progeny from crosses between the ecotypes Landsberg erecta and Columbia. The technique involves the use of 18 co-dominant, cleaved amplified polymorphic sequence (CAPS) markers which are evenly distributed throughout the Arabidopsis genome. We have mapped these 18 markers using recombinant inbred (RI) lines generated in our laboratory. These data enable a better integration of loci mapped relative to the CAPS markers into the restriction fragment length polymorphism (RFLP) map generated using Arabidopsis RI lines. 相似文献
38.
Abstract.
- 1 The effects of resource limitation and the lethal and sublethal effects of a granulosis virus on a lepidopteran host, the Indian meal moth, Plodia interpunctella, were examined.
- 2 The food quality was manipulated by the addition of an inert bulking agent (methyl cellulose) which caused the size, development rate and fecundity of the moths to be reduced.
- 3 The resource quality had no effect on the mortality due to the virus. In contrast, sublethal effects of the virus on pupal weight were more apparent under conditions of resource limitation.
- 4 Considerable variation between the sublethal effects after challenge with different doses of the virus was found. The balance between deleterious sublethal effects of the virus and the selection of more robust individuals by the bioassays is proposed as a mechanism to explain this variation.
- 5 Implications for the dynamics of insect hosts and their pathogens are discussed.
39.
The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization 总被引:2,自引:0,他引:2
Si Lok Joseph L. Kuijper Laura J. Jelinek Janet M. Kramer Theodore E. Whitmore Cindy A. Sprecher Shannon Mathewes Francis J. Grant Shaula H. Biggs Gary B. Rosenberg Paul O. Sheppard Patrick J. O''Hara Donald C. Foster Wayne Kindsvogel 《Gene》1994,140(2):203-209
Characterization of the human glucagon-receptor-encoding gene (GGR) should provide a greater understanding of blood glucose regulation and may reveal a genetic basis for the pathogenesis of diabetes. A cDNA encoding a complete functional human glucagon receptor (GGR) was isolated from a liver cDNA library by a combination of polymerase chain reaction and colony hybridization. The cDNA encodes a receptor protein with 80% identity to rat GGR that binds [125I] glucagon and transduces a signal leading to increases in the concentration of intracellular cyclic adenosine 3′,5′-monophosphate. Southern blot analysis of human DNA reveals a hybridization pattern consistent with a single GGR locus. In situ hybridization to metaphase chromosome preparations maps the GGR locus to chromosome 17q25. Analysis of the genomic sequence shows that the coding region spans over 5.5 kb and is interrupted by 12 introns. 相似文献
40.
Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells 总被引:6,自引:0,他引:6
Alain Garnier Johanne Côté Isabelle Nadeau Amine Kamen Bernard Massie 《Cytotechnology》1994,15(1-3):145-155
Human 293S cells, a cell line adapted to suspension culture, were grown to 5×106 cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×105 cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×106 cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM
calcium-free DMEM
- CS
bovine calf serum
- hpi
hours post-infection
- J+
enriched Joklik medium
- MLP
major late promoter
- MOI
multiplicity of infection (# of infectious viral particle/cell)
- q
specific consumption rate (mole/cell.h)
- pfu
plaque forming unit (# of infectious viral particle)
- Y
yield (g/E6 cells or mole/cell) 相似文献