全文获取类型
收费全文 | 4685篇 |
免费 | 343篇 |
国内免费 | 464篇 |
出版年
2024年 | 4篇 |
2023年 | 72篇 |
2022年 | 83篇 |
2021年 | 158篇 |
2020年 | 132篇 |
2019年 | 134篇 |
2018年 | 143篇 |
2017年 | 128篇 |
2016年 | 125篇 |
2015年 | 155篇 |
2014年 | 228篇 |
2013年 | 329篇 |
2012年 | 191篇 |
2011年 | 206篇 |
2010年 | 200篇 |
2009年 | 224篇 |
2008年 | 244篇 |
2007年 | 265篇 |
2006年 | 245篇 |
2005年 | 255篇 |
2004年 | 178篇 |
2003年 | 179篇 |
2002年 | 207篇 |
2001年 | 184篇 |
2000年 | 137篇 |
1999年 | 115篇 |
1998年 | 82篇 |
1997年 | 78篇 |
1996年 | 70篇 |
1995年 | 69篇 |
1994年 | 71篇 |
1993年 | 69篇 |
1992年 | 74篇 |
1991年 | 44篇 |
1990年 | 32篇 |
1989年 | 24篇 |
1988年 | 33篇 |
1987年 | 30篇 |
1986年 | 21篇 |
1985年 | 55篇 |
1984年 | 50篇 |
1983年 | 26篇 |
1982年 | 46篇 |
1981年 | 30篇 |
1980年 | 16篇 |
1979年 | 32篇 |
1978年 | 7篇 |
1976年 | 4篇 |
1975年 | 3篇 |
1973年 | 3篇 |
排序方式: 共有5492条查询结果,搜索用时 15 毫秒
71.
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
72.
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
73.
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
74.
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
75.
Barnett YA Eger K Eriksson S Folkers G Hansen PE Hofbauer R Komitowsky D Milon A Munch-Petersen B;European Thymidine Kinase Study Group 《Biotechnology advances》1994,12(4):663-668
A precondition for the chemotherapeutic treatment of a variety of virally-induced human diseases and malignant conditions is a highly selective interaction of the drug molecule to be used with it's biological target. To ensure the development of novel, effective drugs, it is essential that the biological target is well characterised with regard to it's structure and activity. Such characterisation relies upon adequate amounts of pure target being available. One of the most important enzymatic importers for antimetabolites is the enzyme thymidine kinase. In this article an in vitro protein expression system is described which facilitates the production of milligram amounts of pure and biologically active thymidine kinase, from a number of important biological sources. Results have shown that the in vitro produced enzyme has the exact biochemical propeties of the in vivo enzyme. Thus the in vitro protein expression system is an ideal vechicle to facilitate an in depth investigation of the enzyme's biological properties. 相似文献
76.
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
77.
The effects of lipids on the glycosylation of recombinant human interferon- expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon- deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon- glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.Abbreviations BSA
bovine serum albumin
- CHO
chinese hamster ovary
- DHFR
dihydrofolate reductase
- FCS
foetal calf serum
- IFN-
human interferon-gamma
-
q
IFN
specific interferon production rate
-
specific growth rate
- 2N
doubly-gycosylated
- 1N
singly-glycosylated
- ON
non-glycosylated 相似文献
78.
79.
精子作载体的转基因鱼研究 总被引:6,自引:0,他引:6
本文报道了以精子为载体将美洲拟蝶抗冻蛋白基因导入罗非鱼卵,构建转基因鱼的方法,此法简单易行。斑点杂文和SouthernBlot杂交结果表明,外源基因的整合率为18.1%,与其它方法构建转基因鱼的外源基因整合率相近。 相似文献
80.
A two-stage culture strategy was studied for continuous high-level production of a foreign protein in the chemically inducible T7 expression system. The first stage is dedicated to the maintenance of plasmid-bearing cells and the second stage to the target protein synthesis by induction of cells coming from the first stage. On entering the second stage, recombinant cells undergo a gradual induction of the target gene expression. These plasmid-bearing cells experience dynamic changes in intracellular compositions and specific growth rates with their individual residence times. Therefore, the overall cultural characteristics in the production stage are really averages of the contributions from the various cells with different residence times. The behavior of the two-stage culture is described by a model, which accounts for dynamic variations of cell growth and protein synthesis rates with cell residence times. Model simulations were compared with experimental results at a variety of operating conditions such as inducer concentration and dilution rate. This model is useful for understanding the behavior of two-stage continuous cultures. (c) 1993 John Wiley & Sons, Inc. 相似文献