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1.
Jennifer Dumont Don Euwart Baisong Mei Scott Estes Rashmi Kshirsagar 《Critical reviews in biotechnology》2016,36(6):1110-1122
Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins. 相似文献
2.
应用PCR技术从鼠伤寒沙门氏菌基因组DNA中克隆phoQ基因片段,构建原核表达pUC18重组质粒,测定序列(GenBank登录号为DQ787014),并转入鼠伤寒沙门氏菌,经异丙基硫代半乳糖苷(IPTG)诱导,进行高效表达。对重组菌株、野生菌株进行毒力检测对比实验,通过口腔注入45日龄健康无菌KM小鼠,测定其半数致死量(LD50)。结果发现:重组菌株与野生菌株的毒力存在显著差异,其半致死量分别为3.981×107 cf u/ mL and 5.012×102 cf u/ mL,PhoQ基因重组菌株的毒力远远低于非重组菌株。说明phoQ基因是调节鼠伤寒沙门氏菌致病机制中一个重要的调节因子。 相似文献
3.
4.
目的:建立定量检测血清中重组人源化抗狂犬病毒单克隆抗体(HuMabs)NM57的间接ELISA法,为药代动力学研究提供一种简单快速的方法。方法:采用狂犬病毒糖蛋白包被酶标板、HRP标记的IgG-Fc段为标记抗体,建立定量检测HuMabsNM57的间接ELISA法,并对其特异性、灵敏度、精密度及准确度进行检测。结果:间接ELISA法检测HuMabsNM57的灵敏度为5ng/mL,组内及组间精密度分别为2.6%-6.0%、8.5%-11.3%。结论:建立了灵敏度高、特异性强的检测HuMabs NM57的间接ELISA法,精密度及准确度均符合药代动力学要求,可用于猕猴及人血清中HuMabsNM57的检测。 相似文献
5.
Joon Chul Lee Do Yun Kim Duk Jae Oh Ho Nam Chang 《Biotechnology and Bioprocess Engineering》2008,13(5):560-565
A rCHO cell line of DUKX origin 26*-320, producing recombinant antibody against the human platelet, was cultivated in a two-stage
depth filter perfusion system (DFPS) for 20 days in order to attain high recombinant antibody concentration. The productivity
of the first stage DFPS bioreactor reached 53 times that of the batch culture in a controlled stirred tank reactor and was
showed 12.1 mg/L antibody concentration at a perfusion rate of 6.0 d−1. Glucose concentration in the first DFPS was maintained at 1.5 g/L to avoid cell damage in the perfusion culture. A second
stage DFPS system was attached to the first DFPS, which resulted in a low glucose concentration of 0.02 g/L and a high antibody
concentration of 23.9 mg/L. The two-stage depth filter perfusion culture yielded 60% higher product concentration than the
batch and 49-fold higher productivity of 69.3 mg/L/d in comparison with that (1.4 mg/L/d) in a batch system. Furthermore,
antibody concentration of the second stage was 97% higher than that of the first stage, and the antibody productivities were
comparable to that of the first stage. This two-stage DFPS system also showed potential for higher titer production of recombinant
antibody and high volumetric productivity for long-term culture of bio-pharmaceutical substances. 相似文献
6.
Seungha Hwang Cha Yong Choi Eun Yeol Lee 《Biotechnology and Bioprocess Engineering》2008,13(4):453-457
An enantioconvergent biotransformation of racemic styrene oxide by using two recombinant microbial epoxide hydrolases (EHs)
in one pot has been investigated to prepare enantiopure vicinal diols. The recombinant whole cell possessing EH gene from
Aspergillus niger LK or Rhodotorula glutinis exhibited a complementary enantioselectivity and regioselectivity, compared to the recombinant cell containing Caulobacter crescentus EH gene. When two recombinant microbial EHs were used in combination, 1.3 g of enantiopure (R)-1,2-phenylethandiol with more than 90% enantiopurity and 95% overall yield was obtained from 1.2 g of racemic styrene oxide
in a preparative-scale batch enantioconvergent biotransformation. 相似文献
7.
To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen. 相似文献
8.
Chang Shin Yoon Eun Gyu Lee Young Seek Lee Il Yup Chung 《Biotechnology and Bioprocess Engineering》1997,2(2):86-89
Epidermal growth factor (EGF) known as a urgastrone is a powerful mitogen with a wide variety of possibilities for medical
usages. A mature EGF coding region was isolated from human prepro-EGF sequence by a conventional PCR and cloned into pQE vector
in which the gene product was supposed to be expressed with 6×His tag for the subsequent purification. The recombinant mature
EGF was expressed in M15[Rep4], anEscherichia coli host strain, in amount of 30–40% of total proteins present inE. coli extract by the addition of isopropylthio-β-galactopyranoside (IPTG). The recombinant EGF purified using a Ni2+-NTA affinity column chromatography was active in its ability to induce phosphorylation on tyrosine residues of several substrate
proteins when murine NIH3T3 and human MRC-5 fibroblast cells were stimulated with it. This work may provide the basic technology
and information for the production of recombinant EGF. 相似文献
9.
Jeong Seok Oh Hee Ho Park Tai Hyun Park 《Biotechnology and Bioprocess Engineering》2008,13(4):470-475
In a two-phase operation, E. coli containing λSNNU1 (Q
−
S
−) in the chromosome is typically cultured at 33°C and cloned gene expression is induced by elevating the temperature. At least
40°C is necessary for complete induction of cloned gene expression; however, temperatures above 40°C have been shown to inhibit
cloned gene expression. This suggests that a three-phase operation, which has an induction phase between the growth and production
phases, may result in higher gene expression. In this study, optimal temperature management strategies were investigated for
the three-phase operation of cloned gene expression in thermally inducible E. coli/bacteriophage systems. The optimal temperature for the induction phase was determined to be 40°C. When the temperature of
the production stage was 33°C, the optimal time period for the induction phase at 40°C was determined to be 60 min. In contrast,
when the temperature of the production phase was 37°C, the optimal period for the induction phase at 40°C was 20∼30 min. When
the three-phase temperature and temporal profile were set at a growth phase of 33°C, an induction phase at 40°C for 30 min,
and a production phase at 37°C, the highest level of cloned gene expression was achieved. 相似文献
10.
目的:分析和比较新活素(Lrh-BNP)与多巴酚丁胺(Dob)治疗急性心力衰竭(AHF)的临床效果及对其血浆半乳糖凝集素(Gal)-3、胱抑素C(CysC)、内皮素(ET)-1水平的影响。方法:选取我院2015年2月~2017年2月收治的114例AHF患者,采用随机数字表法均分为两组。Dob组给予Dob治疗,Lrh-BNP组予以Lrh-BNP治疗。比较两组治疗前后心功能参数,血浆Gal-3、CysC、ET-1水平,临床综合疗效及不良反应的发生情况。结果:与治疗前相比,两组治疗72h后FS、LVEF值均显著升高(P0.01),LVEDD、血浆Gal-3、CysC、ET-1水平均显著降低(P0.01),且Lrh-BNP组以上指标较对照组改善更显著(P0.01)。治疗72h后,Lrh-BNP组总有效率为89.5%,较Dob组明显上升(73.7%,P0.05)。两组不良反应发生率相比差异无统计学意义(P0.05)。结论:与多巴酚丁胺相比,新活素治疗急性心力衰竭的疗效更好,安全性相当,可能与其有效降低患者血浆Gal-3、CysC、ET-1水平有关。 相似文献