全文获取类型
收费全文 | 413篇 |
免费 | 48篇 |
国内免费 | 32篇 |
出版年
2024年 | 1篇 |
2023年 | 7篇 |
2022年 | 12篇 |
2021年 | 24篇 |
2020年 | 4篇 |
2019年 | 16篇 |
2018年 | 21篇 |
2017年 | 8篇 |
2016年 | 11篇 |
2015年 | 24篇 |
2014年 | 24篇 |
2013年 | 34篇 |
2012年 | 39篇 |
2011年 | 25篇 |
2010年 | 24篇 |
2009年 | 21篇 |
2008年 | 25篇 |
2007年 | 19篇 |
2006年 | 25篇 |
2005年 | 14篇 |
2004年 | 27篇 |
2003年 | 16篇 |
2002年 | 20篇 |
2001年 | 7篇 |
2000年 | 7篇 |
1999年 | 11篇 |
1998年 | 3篇 |
1997年 | 4篇 |
1996年 | 2篇 |
1995年 | 2篇 |
1994年 | 2篇 |
1993年 | 4篇 |
1992年 | 2篇 |
1991年 | 1篇 |
1989年 | 1篇 |
1987年 | 1篇 |
1985年 | 1篇 |
1984年 | 1篇 |
1983年 | 2篇 |
1982年 | 1篇 |
排序方式: 共有493条查询结果,搜索用时 15 毫秒
41.
Kewei Zhang Juan Wang Xiaorui Hu Aifang Yang Juren Zhang 《Plant Cell, Tissue and Organ Culture》2010,102(2):135-143
Apical meristems of multiple shoots produced from axenic seedlings of Kentucky bluegrass (Poa pratensis L.) were used for Agrobacterium tumefaciens-mediated transformation. Transformation parameters were optimized for concentration of bacterial cells, duration of infection,
and vacuum infiltration. The highest transformation frequency (1.42%) was obtained by infection with Agrobacterium suspension of OD600 = 0.6 for 5 min, under a negative pressure of 0.5 × 105 Pa. After co-cultivation, the herbicide-resistant plants were rooted and transplanted into flowerpots. Transgenic plants
were confirmed by polymerase chain reaction (PCR) assay and Southern blot analysis. Using this transformation system, the
betA gene encoding choline dehydrogenase and mutant als gene encoding the enzyme acetolactate synthase were introduced into three Kentucky bluegrass cultivars. 相似文献
42.
野油菜黄单胞菌野油菜致病变种(Xanthomonas campestris pv.campestris,Xcc)是十字花科植物黑腐病的病原细菌。我们建立了Xcc的蛋白质组学研究平台,用于分离、鉴定该菌的致病相关蛋白。为了减少胞外多糖(exopolysaccharide,EPS)对蛋白材料质量的影响,我们构建了EPS缺陷的Xcc8004ΔgumB突变体。本研究以Xcc8004ΔgumB出发菌株,用诱导培养液培养,取细胞上清通过超滤浓缩得到蛋白质粗提物,分别采用丙酮沉淀法、试剂盒纯化法和丙酮沉淀-试剂盒联用法来纯化蛋白质粗提物,通过比较双向电泳的结果优选最佳的样品制备方法。结果证明丙酮沉淀-试剂盒联用法较为理想,所得双向电泳图片清晰,分辨率高。因此,此方法可以用于制备野油菜黄单胞菌分泌组双向电泳样品,并可以满足进一步研究的需要。 相似文献
43.
Sixteen kinds of human immunodeficiency virus (HIV) target genes were cloned by polymerase chain reaction (PCR) amplification,
and specific plasmids were constructed as the templates for the expression of these genes in the cell-free system. Similarly,
the linear PCR templates of these genes for cell-free protein expression were also constructed by using two PCR amplification
process. These different templates can be employed to biosynthesize HIV proteins in the cell-free system simultaneously and
can be adapted for some high-throughput processes. HIV protease (P10) was performed as a target protein, and two different
templates (plasmid and PCR product) were prepared and used for P10 expression in the Escherichia coli cell-free system. The target protein P10 was detected in sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels either
by using a plasmid template or by a PCR template. These results are promising and helpful to develop a high throughput process
for drug discovery. 相似文献
44.
Functional expression and purification of bovine enterokinase light chain in recombinant Escherichia coli 总被引:2,自引:0,他引:2
Enterokinase (EC 3.4.21.9) is a serine proteinase of the intestinal brush border that exhibits specificity for the sequence (Asp)(4)-Lys and converts trypsinogen into its active form, trypsin. A codon optimized sequence coding light chain (catalytic subunit) of bovine enterokinase gene (sBEKLC) was synthesized, and it was fused with DsbA to construct the expression vector (pET39-sBEKLC). Then, the plasmid was transformed into E. coli BL21 (DE3) for expression. Under optimal conditions, the volumetric productivity of fusion protein reached 151.2 mg L(-1), i.e., 80.6 mg sBEKLC L(-1). The cold osmotic shock technique was successfully used to extract sBEKLC from periplasmic space, and nickel affinity chromatography was employed to obtain mature sBEKLC. Finally, about 6.8 mg of bioactive sBEKLC was purified from 1 liter fermentation broth and could be used to cleave one tested fusion protein with an inter-domain enteropeptidase recognition site. This work will be helpful for large-scale production of this increasingly demanded enterokinase. 相似文献
45.
Hu Xiao-Kun Lin Zhuo-Ru Zhang Qing-Chun Kong Fan-Zhou Cen Jing-Yi Zeng Yu-Lan Yu Ren-Cheng 《Journal of applied phycology》2022,34(3):1483-1496
Journal of Applied Phycology - The dinoflagellates Karenia mikimotoi and Prorocentrum donghaiense are both important causative species of harmful algal blooms (HABs) in the East China Sea. The... 相似文献
46.
Xiao Xiaolan Sun Shuangxi Li Yingbin Cen Xuecheng Wu Shibiao Lu Aili Cai Jun Zhao Junjie Li Shaoxue 《Molecular biology reports》2022,49(7):6303-6311
Molecular Biology Reports - Oxidative stress and neurocyte apoptosis are crucial pathophysiological process in early brain injury (EBI) after subarachnoid hemorrhage (SAH). Geniposide (GNP) has... 相似文献
47.
Danfeng?Dong Zhen?Li Lihua?Zhang Cen?Jiang Enqiang?Mao Xuefeng?WangEmail author Yibing?PengEmail author 《Mycopathologia》2015,180(5-6):407-419
In this study, fungemia cases from four tertiary hospitals located in Shanghai and Anhui province in China from March 2012 to December 2013 were enrolled to investigate clinical features, species distribution, antifungal susceptibility and strain relatedness. During the study period, 137 non-duplicate cases and their corresponding isolates were collected. Six different genera of fungi were identified, of which Candida spp. were the most common (126/137, 91.97 %), with C. albicans predominating (48/137, 35.03 %). The non-Candida fungi rate reached 8.03 % (11/137), and Pichia spp. was the most common (5/137, 3.65 %). Compared with C. albicans, non-C. albicans fungi-associated fungemia was more likely in younger (p = 0.004) and male patients (χ 2 = 6.2618, p = 0.0123) and patients from ICUs (χ 2 = 6.3783, p = 0.0116). Overall, the susceptible/WT rates of common Candida spp. to fluconazole, itraconazole, voriconazole, flucytosine, amphotericin B and caspofungin were 74.63, 92.31, 93.16, 96.58, 100 and 95.69 %, respectively. C. tropicalis and C. guilliermondii had a low susceptibility to fluconazole: 79.95 and 77.78 %, respectively. No isolates were resistant/WT to caspofungin, but C. parapsilosis and C. guilliermondii had high MIC90 values; 1 and 2 mg/L, respectively. In terms of genotyping, MLST was taken for C. glabrata and C. tropicalis, while microsatellite marker analysis was used for C. albicans and C. parapsilosis. C. glabrata was predominantly clone ST7, accounting for 75 %, while the other isolates showed genetic diversity. Considering the increased proportion of non-C. albicans fungi and the presence of endemic clones of C. glabrata, it is essential to undertake additional surveillance of fungemia. 相似文献
48.
BST-2是最近发现的可以抑制成熟HIV-1(human immunodeficiency virus,HIV)病毒颗粒从哺乳动物细胞表面释放的宿主因子,随之发现其也可以抑制多种包膜病毒的释放。本研究采用密码子优化的表达HIV-1 gag和gag-pol蛋白的质粒所形成的病毒样颗粒作为研究对象,观测BST-2对这两种病毒样颗粒(Virus-like particle,VLP)的释放抑制情况及其作用机制。结果发现,瞬时表达和稳定表达的BST-2均可以显著抑制病毒样颗粒从哺乳动物细胞释放,同时发现这两种病毒样颗粒(gag/gag-pol)的释放都可以被BST-2抑制;而且,HIV-1中Vpu蛋白可以拮抗BST-2抑制HIV病毒样颗粒释放的作用,另外,通过化学试剂和酶学方法处理,确证BST-2可以被包装进病毒样颗粒中。 相似文献
49.
50.
Xie C Yang S Zhong D Dai X Chen X 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2011,879(28):3071-3075
Dronedarone is a derivative of amiodarone--a popular antiarrhythmic drug. It was developed to overcome the limiting iodine-associated toxicities of amiodarone. Debutyldronedarone is a major circulating active metabolite of dronedarone in humans. To investigate the pharmacokinetics of dronedarone, a rapid, simple, and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously determine dronedarone and debutyldronedarone in human plasma using amiodarone as internal standard (IS). Acetonitrile with IS was used to precipitate proteins from a 50-μL aliquot of plasma. Effective chromatographic separation was performed on a CAPCELL PAK C(18) MG (100 mm × 4.6 mm, 5 μm) column with gradient elution (5 mmol/L ammonium acetate-acetonitrile, with each phase containing 0.2% acetic acid) at a flow rate of 0.7 mL/min. Complete separation was achieved within 5.5 min. Detection was carried out on an tandem mass spectrometer in multiple reaction monitoring mode using a positive atmospheric pressure chemical ionization interface. A lower limit of quantification of 0.200 ng/mL was achieved for both dronedarone and debutyldronedarone, with acceptable precision and accuracy. The linear range of the method was from 0.200 to 200 ng/mL for each analyte. Intra- and inter-day precisions were lower than 7.2% in relation to relative standard deviation, while accuracy was within ±5.1% in terms of relative error for analytes. Our findings demonstrate the successful application of the validated LC-MS/MS method to a pharmacokinetic study after a single oral administration of 400mg dronedarone to six healthy volunteers. 相似文献