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1.
目的:构建两个高表达人TNFα和IL-1β细胞系,建立抗炎药物筛选细胞模型。方法:运用PCR的方法从载体pCMVSport-TNFα和p CMVSport-IL1β上扩增目的基因,以亚克隆方法将目的基因分别插入真核表达载体pcDNA3.1和pFLAG-CMV中,用单酶切、PCR扩增和基因测序的方法鉴定重组效果,然后将重组成功的质粒转入HEK293细胞系内,挑选能够稳定表达并遗传的单克隆细胞株,用蛋白免疫印迹(Western blot)法分析其表达效果。结果:三种鉴定方法均显示重组质粒构建成功。Western blot结果显示,细胞株T3、T4均能较高表达炎症因子TNF-α;细胞株I2、I3、I5均能较高表达炎症因子IL-1β。结论:成功构建了TNFα和IL-1β靶标的药物筛选细胞模型,为筛选具有抗炎作用的中药提供了一个新平台。  相似文献   
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Aim

In this study, the biological variation for improvement of the nutritive value of wheat straw by 12 Ceriporiopsis subvermispora, 10 Pleurotus eryngii and 10 Lentinula edodes strains was assessed. Screening of the best performing strains within each species was made based on the in vitro degradability of fungal‐treated wheat straw.

Methods and Results

Wheat straw was inoculated with each strain for 7 weeks of solid state fermentation. Weekly samples were evaluated for in vitro gas production (IVGP) in buffered rumen fluid for 72 h. Out of the 32 fungal strains studied, 17 strains showed a significantly higher (< 0·05) IVGP compared to the control after 7 weeks (227·7 ml g?1 OM). The three best Ceriporiopsis subvermispora strains showed a mean IVGP of 297·0 ml g?1 OM, while the three best P. eryngii and L. edodes strains showed a mean IVGP of 257·8 and 291·5 ml g?1 OM, respectively.

Conclusion

Ceriporiopsis subvermispora strains show an overall high potential to improve the ruminal degradability of wheat straw, followed by L. edodes and P. eryngii strains.

Significance and Impact of the Study

Large variation exists within and among different fungal species in the valorization of wheat straw, which offers opportunities to improve the fungal genotype by breeding.  相似文献   
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通过酸性含硒平板和摇瓶筛选出一株对低pH、高浓度硒有很好耐受性的菌株Y1,通过菌落形态特征分析和26S rDNA测序,鉴定该菌株为Pichia kudriavzevii,多抗性实验结果显示该菌是一株多重耐受性毕赤酵母。通过摇瓶实验研究了温度、接种量、摇床转速、pH对菌株除硒性能的影响,结果显示当温度为25℃,接种量为12%(v/v),摇床转速为250 r/min,pH为3.0时,菌株对硒的去除率最高为58.3%。基于不同pH发酵过程中菌体生物量及富硒量的不同表现:pH 3.0时生物量最高,pH 5.0时富硒量最高,提出两阶段pH调控策略:发酵0 h~14 h将pH控制在3.0,14 h~28 h将pH控制在5.0,最终除硒率可达78.6%,分别比pH恒定在3.0及5.0条件下提高了15.4%和21.7%。  相似文献   
5.
Stem cell–derived cardiomyocytes (CMs) hold great hopes for myocardium regeneration because of their ability to produce functional cardiac cells in large quantities. They also hold promise in dissecting the molecular principles involved in heart diseases and also in drug development, owing to their ability to model the diseases using patient‐specific human pluripotent stem cell (hPSC)–derived CMs. The CM properties essential for the desired applications are frequently evaluated through morphologic and genotypic screenings. Even though these characterizations are necessary, they cannot in principle guarantee the CM functionality and their drug response. The CM functional characteristics can be quantified by phenotype assays, including electrophysiological, optical, and/or mechanical approaches implemented in the past decades, especially when used to investigate responses of the CMs to known stimuli (eg, adrenergic stimulation). Such methods can be used to indirectly determine the electrochemomechanics of the cardiac excitation‐contraction coupling, which determines important functional properties of the hPSC‐derived CMs, such as their differentiation efficacy, their maturation level, and their functionality. In this work, we aim to systematically review the techniques and methodologies implemented in the phenotype characterization of hPSC‐derived CMs. Further, we introduce a novel approach combining atomic force microscopy, fluorescent microscopy, and external electrophysiology through microelectrode arrays. We demonstrate that this novel method can be used to gain unique information on the complex excitation‐contraction coupling dynamics of the hPSC‐derived CMs.  相似文献   
6.
The clinical application of gallium compounds as anticancer agents is hampered by development of resistance. As a potential strategy to overcome the limitation, eight series of compounds were identified through virtual screening of AXL kinase homology model. Anti-proliferative studies were carried using gallium-sensitive (S) and gallium-resistant (R) human lung adenocarcinoma (A549) cells. Compounds 5476423 and 7919469 were identified as leads. The IC50 values from treating R-cells showed compounds 5476423 and 7919469 had 80 fold and 13 fold increased potency, respectively, compared to gallium acetylacetonate (GaAcAc). The efficacy of GaAcAc against R-cells was increased 2 fold and 1.2 fold when combined with compounds 5476423 and 7919469, respectively. Compared with S-cells, R-cells showed elevated expression of AXL protein, which was significantly suppressed through treatments with the lead compounds. It is anticipated that the lead compounds could be applied in virtual screening programs to identify novel scaffolds for new therapeutic agents as well as combinatorial therapy agents in gallium resistant lung cancer.  相似文献   
7.
Five strains of Bradyrhizobium japonicum (USDA 6, 110, 122, 138, and 143) were screened in cell culture for tolerance to acidity (pH 4.2, 4.4, and 4.6) and Al (0, 3, 4, 5, and 6 mg L–1) under low P conditions. Each strain was later grown in association with seven soybean [Glycine max. (L) Merr.] cultivars which were also screened for tolerance to the same stresses in nutrient culture to determine which soybean-Bradyrhizobium combinations would establish the most effective symbiotic N2 fixing relationships. Results indicated that strains USDA 110 and 6 were more tolerant than USDA 122, 138 and 143 with USDA 110 being the most tolerant. Acidity appeared to be the more severe stress; but even when strains showed tolerance to the stresses, cell numbers were significantly reduced. This suggests that colonization of soils and soybean roots can be adversely affected under similar conditions in the field which may result in reduced nodulation. The strains found to be more tolerant to the stresses were more effective N2 fixers in symbiosis with all soybean cultivars, with USDA 110 being definitely superior. The association between the more tolerant strains and cultivars had the largest nitrogenase activity. Further studies on the inclusion of tolerant Bradyrhizobium strains in inoculum used on tolerant soybean cultivars in the field are warranted.  相似文献   
8.
Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I2 assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.  相似文献   
9.
A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples.  相似文献   
10.
【目的】筛选出一种高效、持久、安全及价廉的灭螺微生物,对其进行鉴定并观察其抑螺功效。【方法】从钉螺孳生的土壤中筛选出4株灭螺活性较强的菌株(B8、B27、B36、B59),显微镜观察菌株形态和革兰氏染色均为G+杆菌。不同分离胶浓度的SDS-PAGE分析其灭螺活性成分;优势菌株经16s rRNA扩增后,PCR产物测序,序列比对,构建系统发育树鉴定其种属。【结果】灭螺结果表明,各试验组中发酵上清液各菌株间灭螺效果差异有统计学意义(χ2=21.286,P=0.002);细菌发酵液各菌株间差异也有统计学意义(χ2=17.298,P=0.008);菌体悬液各菌株间差异无统计学意义(χ2=7.579,P=0.271);此外,B59菌株的灭螺效果优于其它菌株,尤其是其发酵上清液浸泡48 h和72 h钉螺死亡率高达73.3%和96.7%。SDS-PAGE发现在B59细菌上清液中无蛋白带出现,推测其灭螺活性物质可能是其他成分;分子系统发育分析结果显示B59菌株位于Bacillus cereus (CP001746)分支上,一致性达100%。【结论】B59菌株的发酵上清液灭螺效果最好,其灭螺活性物质可能不是蛋白质,B59菌株被鉴定为Bacillus cereus。  相似文献   
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