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151.
为建立以重组PCV2Cap蛋白为包被抗原的间接ELISA检测方法,构建了猪圆环病毒2型(PCV2)ORF2基因原核表达质粒pET28a-ORF2。SDS-PAGE显示,在0.1mmol/L IPTG和37℃条件下诱导4h,重组Cap蛋白高效表达。Western blotting证实该蛋白能够被PCV2阳性血清特异性识别。以纯化的蛋白为抗原建立了检测PCV2抗体的间接ELISA方法。结果表明,抗原最适包被质量浓度为2mg/L;血清最佳稀释度为1∶100;酶标二抗最适浓度为1∶2 000,该方法的敏感性为86.96%,特异性为100%。用该方法对河南省152份猪血清样品进行检测,与间接免疫荧光(IFA)的符合率为85.53%(130/152),与商品化的韩国金诺PCV2ELISA试剂盒的符合率为88.16%(134/152)。本试验成功建立了PCV2血清抗体间接ELISA方法,具有较高的敏感性和特异性,可用于大规模的血清学检测。 相似文献
152.
以长白猪精液为材料,采用经典的纯化方法获得N-乙酰-β-D-氨基葡萄糖苷酶(EC 3.3.1.52,NAGase)的2种同工酶,命名为:NAGaseⅠ和NAGaseⅡ。其中NAGaseⅡ的比活力为358.21U/mg,纯化倍数为6.37倍。以海藻糖、D-甘露糖、蔗糖、葡萄糖为效应物,研究其对该酶活性的影响。结果表明:在特定浓度范围内,海藻糖对酶活力基本没有影响;D-甘露糖、蔗糖、葡萄糖对该酶均有一定的抑制作用;D-甘露糖对酶的抑制作用表现为非竞争型,抑制常数KI为11.94mmo/L;蔗糖对酶的抑制作用表现为竞争型,抑制常数KI为0.56mmol/L;葡萄糖对酶的抑制作用表现为混合型,抑制常数KI和KIS分别为0.35mol/L和64.29mmol/L。 相似文献
153.
154.
[目的]研究铬胁迫下薏米对含铬污水的净化作用及铬胁迫对薏米生理生化的影响,为人工湿地净化含铬污水提供理论支持.[方法]开展桶栽模拟人工湿地试验,以薏米为材料,定期浇灌不同浓度的Cr(Ⅵ)生活污水(0、5、20、40、60mg/LK2Cr2O7),定期测定进出水的化学需氧量(COD)和总磷(TP),并检测薏米叶片过氧化物酶(POD)、超氧化物歧化酶(SOD)、丙二醛(MDA)等指标.[结果]薏米对含铬污水中COD的去除率随Cr(Ⅵ)浓度增大而降低,5mg/L的Cr(Ⅵ)处理在对总磷的去除率在90%以上,并随Cr(Ⅵ)浓度的升高而下降;薏米叶中的MDA含量随Cr(Ⅵ)浓度和处理时间的增加而呈增加趋势;叶片SOD、POD活性均随处理时间的延长而呈先上升后下降的趋势.[结论]薏米能耐受一定浓度的Cr(Ⅵ)胁迫,且对水质净化有较好的作用. 相似文献
155.
156.
Glutathione and its Related Enzymes in the Nile Fish 总被引:2,自引:0,他引:2
Ragaa R. Hamed Tahany M. Maharem Rasha A. M. Guinidi 《Fish physiology and biochemistry》2004,30(3-4):189-199
Glutathione (GSH) and related enzymes, glutathione transferase (GST), glutathione peroxidase (GPx) and glutathione reductase
(GR) form an important phase 2 biotransformation enzymes system. The objective of this study was to compare this enzymes system
in three fish species from the river Nile, Oreochromis niloticus, Claris lazera and Cyprinus carpio in order to establish the main differences and to purify and characterize GST from the liver of O. niloticus.The level of GSH and the activity of GST, GPx and GR in the liver, kidney and gills of the three fish species were examined.
A simple reproducible procedure for the purification of GST from the liver of O. niloticus to homogeneity, which includes chromatography on DEAE- cellulose followed by affinity chromatography on GSH-sepharose was
established. The molecular mass was found to be 25,460 Da by SDS-PAGE. The Michaelis-Meneten constants (Km) of the enzyme for GSH and CDNB were 0.35 mM and 0.42 mM, respectively. The affinity purified enzyme exhibited maximum pH
at pH 8.0 and increasing pH above 8.0 did not affect the observed maximum. The purified enzyme acts readily on CDNB, less
readily on some standard transferase substrates (1,2-dichloro-4-nitrobenzene and p-nitrophenethyl bromide) and not at all on others (bromosulphophthalein and p-nitrobenzyl chloride). Bromosulfophthalein, cibacron blue and hematin inhibited CDNB-conjugating activity of the purified
enzyme with IC50 0.079, 3.98 and 0.126 μM, respectively. 相似文献
157.
158.
采用热水(80℃)提取和75%乙醇沉淀、Sepharose CL-4B凝胶过滤及DEAE Sepharose CL-4B阴离子交换等方法分离纯化大杯伞(Clitocybe maxima)柄粗多糖,获得伞柄多糖纯品,制备多糖抗体;采用热水法提取大杯伞菌丝体粗多糖。用碳二亚胺法将纯化伞柄多糖与牛血清白蛋白进行共价化学偶联,并将偶联产物(拟糖蛋白)免疫兔子以制备抗血清,以酶联免疫法检测抗体滴度。依据该抗体与来自相同菌株的菌丝体粗多糖免疫反应差异对这两种多糖进行免疫识别,结果表明:经过亲和层析纯化后,第六次免疫抗血清对伞柄多糖的抗体滴度可达64K,而且对菌丝体粗多糖反应呈阴性,显示该抗体可识别这两种多糖之间的结构差异。 相似文献
159.
Stephen George David Burgess Michael Leaver Nick Frerichs 《Fish physiology and biochemistry》1992,10(1):43-54
Intraperitoneal injection of turbot with Cd induced the synthesis of a low molecular weight hepatic Cd-binding protein and a 500bp mRNA, which hybridised to a plaice metallothionein (MT) cRNA probe. The Cd-binding protein displayed cross-reactivity in a competitive ELISA with antiserum raised against rainbow trout MT and had the characteristic amino acid composition, metal stoichiometry and spectral characteristics of a Class I MT. Only one isoform was apparent on ion exchange chromatography. Southern blot analysis of DNA cleaved with four restriction enzymes suggested that only a single MT gene is present in turbot.In an established turbot fibroblast cell line, Cd induced MT mRNA and MT levels in a dose and time-dependent manner. MT was also induced by Cu, Hg and Zn but not Pb exposure. Physiological concentrations of glucocorticoids and sex hormones did not induce MT synthesis, although at high concentrations a positive response to corticosterone, dexamethasone, hydrocortisone or progesterone was observedin vitro indicating the possible presence of a functional steroid regulatory element in the fish MT gene.Abbreviations used AMP
adenosine monophosphate
- CHSE
chinook salmon embryo
- DMSO
dimethyl sulphoxide
- HPLC
high performance liquid chromatography
- KB
kenacid blue
- MT
metallothionein
- Mr
molecular weight (kDaltons)
- NR
neutral red
- PEG
polyethylene glycol
- RTH
rainbow trout hepatoma
- SDS
sodium dodecyl sulphate
- SSC
0.15M NaCl, 0.015M sodium citrate
- TF
turbot fibroblast 相似文献
160.
Pierre-Yves Le Bail Geneviève Boulard Bruno Barenton Michel Zygmunt 《Fish physiology and biochemistry》1989,7(1-6):243-251
A method for the purification of chinook Salmon (Oncorhynchus tshawytscha) GH, which retains its biological activity, is described. The biological activity was investigated with an established radioreceptor
assay using liver membranes from pregnant rabbits and bovine GH as standard and labelled hormone. The enrichment of the preparation
was checked with electrophoresis (SDS-PAGE). Extraction and further steps were carried out using low molarity alkaline buffer
(pH 8–10, M = 100 mM). Three chromatography steps were performed (Concanavalin-A sepharose, Bio-gel P60, DEAE). Ion exchange
chromatography was performed under isocratic conditions (using a 50 cm column). Two isoforms (sGH1 and sGH2) were isolated.
The purification yield is 0.7% compared to lyophilized pituitaries. The molecule is homogeneous in SDS-PAGE. Contamination
by prolactin, gonadotrophin and corticotrophin is negligible (< 0.5%). It could be demonstrated that the biological activity
of the preparation is maintained since this preparation stimulates the growth of juvenile trout (Salmo gairdneri) and binds specifically (35%) to trout liver membranes. 相似文献