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1.
目的:构建及筛选高效表达原创性全人源抗人Ig E单克隆抗体的重组工程细胞株。方法:将采用核糖体展示技术筛选到的原创性全人源抗人Ig E单链抗体(sc Fv)基因改构设计为Ig G1κ型全长抗体,构建重组真核表达质粒并电转染CHO-S细胞,Dot-blot法选取多株高表达克隆进行40ml摇瓶批次培养,再据细胞生长特征及抗体表达量选取高表达克隆进行40ml摇瓶及3L摇瓶流加培养研究,选取候选细胞株并对改构前后抗体的生物学活性进行比较研究。结果:成功构建了p MH3-H、p MH3-L、p CApuro-H、p CApuro-L四种重组真核表达质粒并成功共转染CHO-S细胞。完成了4次电转染8轮细胞克隆筛选,获得两株表达量较高的候选克隆Mab1#和Mab2#,在3L摇瓶流加培养中抗体表达量分别达到470mg/L及499mg/L。生物膜光干涉技术(Bio-Layer Interferometry,BLI)亲和力结果显示Mab1#及Mab2#两株单抗亲和力均达到nmol/L级(10-9),与现有唯一上市的抗人Ig E单抗药物奥马珠单抗(Omalizumab)的亲和力相当。选取Mab1#全长抗体与其改构前的母本单链抗体的表面等离子共振技术(surface plasmon resonance,SPR)中和活性比较结果显示Mab1#抑制h Ig E与FcεRI结合的EC50为3nmol/L,EC90为9nmol/L,较改造前亲和力提高了4.3倍,中和活性(EC50)提高了23.7倍,中和活性(EC90)提高了41.3倍。结论:成功将表达原创性全人源抗人Ig E的单链抗体(约25k Da)改造为亲和力及中和活性均大幅提升的全长抗体(约150k Da),获得2个候选细胞株。  相似文献   
2.
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   
3.
Multiple sclerosis (MS) is a chronic auto‐immune disease characterized by a damage to the myelin component of the central nervous system. Self‐antigens created by aberrant glycosylation have been described to be a key component in the formation of auto‐antibodies. CSF114(Glc) is a synthetic glucopeptide detecting in vitro MS‐specific auto‐antibodies, and it is actively used in diagnostics and research to monitor and quantify MS‐associated Ig levels. We reasoned that antibodies raised against this probe could have been relevant for MS. We therefore screened a human Domain Antibody library against CSF114(Glc) using magnetic separation as a panning method. We obtained and described several clones, and the one with the highest signals was produced as a 6×His‐tagged protein to properly study the binding properties as a soluble antibody. By surface plasmon resonance measurements, we evidenced that our clone recognized CSF114(Glc) with high affinity and specific for the glucosylated peptide. Kinetic parameters of peptide–clone interaction were calculated obtaining a value of KD in the nanomolar range. Harboring a human framework, this antibody should be very well tolerated by human immune system and may represent a valuable tool for MS diagnosis and therapy, paving the way to new research strategies. Copyright © 2014 John Wiley & Sons, Ltd.  相似文献   
4.
采用Rotofor等电聚焦和分子筛技术纯化大肠杆菌表达的重组链激酶(r—SK),其纯度为97%。比活性1×106IU/mg,回收率41%。分析所纯化的r—SK N端氨基酸顺序证实其1—15个氨基酸顺序与SK基因的核苷酸顺序所示3联密码子一致。以纯化r—SK为抗原,通过杂交瘤技术构建了分泌抗r—SK单克隆抗体(McAb)杂交瘤细胞(4D11),该McAb属IgG1亚型.特异地作用于r-SK和C组口溶血性链球菌分泌的SK。用底物显色法测定该McAb可抑制SK对纤溶酶原的激活。Western blot法证实该McAb能识别分子量为16 250Da的r—SK的CNBr裂解片段。推测SK蛋白中第71残基至第237残基间的氨基酸顺序参与SK与纤溶酶原的结合。  相似文献   
5.
正常细胞转化成癌细胞后,其表型发生了一系列不同于正常细胞的变化,成为肿瘤细胞的标志。Gold和Freeman(1965)用人结肠癌组织的抽提物免疫兔,发现有些用人正常结肠组织吸收后的抗血清能够与肿瘤组织和胚胎肠道抽提物起反应,但不与正常组织抽提物起反应,由于这种抗原最初被发现在胚胎组织,故名为癌胚抗原(embryonic carcinoma antigen,简称CEA)。用敏感的放射免疫或免疫酶标方  相似文献   
6.
Cytosolic superoxide dismutase (SOD) of the onion maggot, Delia antiqua, was purified to apparent homogeneity by ammonium sulfate fractionation followed by anion exchange, hydrophobic interaction, and gel filtration chromatographies. Native molecular mass was estimated as 32,000 daltons. SDS-PAGE revealed only one subunit of 16,000 daltons, indicating that SOD is a homodimer. Isoelectric focusing revealed 3 charge isomers of pls 5.3, 5.5, and 5.7. The specific activity of purified SOD was 4,250 U/mg protein. A monoclonal antibody (MAb, aSOD2B7) raised against Delia SOD recognized only SOD of the same genus, but another MAb (aSOD1H11) recognized SOD of Drosophila melanogaster as well. © 1995 Wiley-Liss, Inc.  相似文献   
7.
Summary A monoclonal antibody (designated SF25), which recognizes a protein antigen expressed on a large number of human colon carcinomas, was used for drug targeting. Daunomycin-antibody conjugates were prepared by two previously described procedures. In one, the drug was bound to the antibody through a spacer of small molecular mass (cis-aconitic acid), while in the other a dextran bridge served as the link between drug and antibody. High substitution rates of drug to antibody were obtained using the latter binding procedure. Both conjugates were tested in vitro against two human colon carcinoma cell lines, LS180 and KM-12. The efficacy of a daunomycin-dextran-SF25 antibody conjugate was tested against colon carcinoma LS180 tumors transplanted at different sites into athymic mice. The specific conjugate was significantly more inhibitory to a subcutaneous tumor growth than its components or their mixture. SF25 antibody alone showed antitumoral effects against all three forms of transplanted tumor tested, namely, local, metastatic or intrahepatic, whereas daunomycin, on its own, was effective only against the subcutaneous tumor. Binding of daunomycin to dextran partially improved its inhibitory activity against the metastatic tumor. The conjugate, daunomycin-dextran-SF25 antibody reduced the number of metastatic foci, increased the survival rate and delayed death. Yet against lymph node metastases it was not significantly better than a mixture of both constituents. However, results obtained with an intrahepatic tumor, a model that mimics the natural progression of the disease, resembled those described with the subcutaneous tumor. Daunomycin-dextran-SF25 antibody was significantly more effective than all components separately and than a mixture of drug and antibody, provided a highly drug-substituted conjugate was used.  相似文献   
8.
建立一种靶点蛋白质快速定量检测方法。在原有侧向流动免疫层析技术的基础上,通过优化层析材料和纳米微球的均一性、改进检测区的检测方法,经逐点扫描技术,建立标准浓度曲线,以达到对临床靶点蛋白质的定量检测。以乳腺癌组织中的Her2表达为例,通过对已知浓度样品的检测,验证本技术方法的准确度大于96%。另外,以蛋白质免疫印迹作为组织中特定蛋白质检测金标准,分析临床肿瘤组织中Her2蛋白的含量,其准确率也达到95.5%,而免疫组织化学方法检测准确率仅为69.58%。新型免疫层析法检测结果与靶向治疗患者的愈后密切相关(P<0.01)。改进后的新型免疫层析方法能够准确地对临床靶点蛋白质进行定量检测,而且结合侧向流动技术的简单、快速和易用性,这种新型检测方法可以广泛应用于临床组织标本、血液标本和体液标本中靶点蛋白质的临场定量检测,在一定程度上可以替代免疫组化技术。  相似文献   
9.
目的了解阳江市健康人群乙型肝炎表面抗体(抗-HBs)水平,评价乙型肝炎疫苗免疫效果。方法采用整群随机抽样方法,在阳江市辖区内随机抽取20个乡镇的常住健康人群为调查对象。采用电化学发光免疫法定量检测抗-HBs水平,运用SPSS18.0和Excel 2007软件进行数据整理和分析。结果共采集1 054人份血清进行检测,人群中乙肝疫苗接种率为94.88%,抗-HBs阳性率为63.47%、抗-HBs高应答率及GMT分别为29.51%和2 3.47 m IU/m L。乙肝疫苗接种率显示,随着年龄的增长疫苗接种率越低;20岁以下和20岁以上人群疫苗接种率分别为98.35%和54.22%,且男性高于女性;各地区间疫苗接种率差异无统计学意义(χ2=5.99,P=0.11)。抗-HBs阳性率显示,1~14岁儿童抗-HBs阳性率为63.00%。其中,1~岁组儿童最高为87.20%,7~岁组最低为54.56%;江城区抗-HBs阳性率最高(70.61%),阳西县最低(56.60%)。1岁以下儿童的抗-HBs应答率和GMT均最高,分别为61.22%和(91.56±2.15)m IU/m L。5~岁组最低,分别为13.74%和(12.44±1.46)m IU/m L,差异均有显著的统计学意义(χ2=57.31,P=0.00,F=5.77,P=0.00)。结论阳江市健康人群乙肝疫苗接种率高,初步形成免疫屏障。积极开展20岁以下人群的加强免疫和成人的乙肝疫苗的接种工作。  相似文献   
10.
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