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991.
Mark R. Patrick Kerry A. Chester G. A. Pietersz 《Cancer immunology, immunotherapy : CII》1998,46(4):229-237
The major limitations of monoclonal antibody conjugates as therapeutic agents have been their poor tumour targeting, inadequate
tumour penetration and immunogenicity. More even and deeper tissue penetration has been demonstrated with smaller antibody
fragments. The smaller size and absence of an Fc segment may contribute to a lowered immunogenicity with single-chain antibodies
(scFv) and also permit their recombinant engineering and bacterial expression. We describe the successful engineering, expression
and pre-clinical characterisation of a phosphorylatable “kemptide” (Leu-Arg-Arg-Ala-Ser-Gly) anti-carcinoembryonic antigen
(anti-CEA) scFv (PKS-scFv), for use as a radioimmunotherapeutic agent. Specifically, a yield of 6 mg/l induced culture was
obtained. Site-specific phosphorylation was demonstrated without loss of specificity. In vitro assays revealed a selective
cytotoxicity of 32P-PKS-scFv for high-CEA-expressing LS-174T cells compared to the low-CEA-expressing HT-29 cells, with a rapid internalisation
rate.
Received: 20 March 1997 / Accepted: 5 February 1998 相似文献
992.
ImmunoPCRcombinesthehighsensitivityofPCRwiththespecificityofantibodyantigeninteraction.UsingimmunoPCR,asfewas600moleculesofimmobilizedantigen(bovineserumalbumin)havebeendetected[1].ThemostimportantstepinimmunoPCRistheconstructionofthegeneprobe.Sanoetal.[1,2]c… 相似文献
993.
Since the end of 2001, we have conducted a project to isolate and determine entire sequences of mouse cDNA clones which encode the polypeptides corresponding to human KIAA proteins. Towards the ultimate goal of this project to clarify the biological functions of KIAA genes, we have set production of antibodies against mouse KIAA gene products based on their sequence information as the next important stage. As the first step, we developed a high-throughput system utilizing shotgun clones generated during entire sequencing of mouse KIAA cDNAs. The system consists of the following three parts: (1) Shotgun clones encoding regions suitable for production of antigens were selected using a newly developed browser system; (2) the protein-coding sequences of the selected shotgun clones were transferred into an expression vector by in vitro recombination-assisted method in a 96-well format, and expressed as glutathione S-transferase fusion proteins in Escherichia coli; and (3) the solubility of the recombinant antigens were preliminarily assessed in a small-scale culture and then large-scale production and purification was performed using glutathione-affinity beads or retrieval from polyacrylamide gels depending on their solubility. Using these systems, we successfully produced and purified 400 antigens for production of mKIAA antibodies to date. 相似文献
994.
The gene encoding proliferating cell nuclear antigen (PCNA) was isolated from the marine coccolithophorid microalga Pleurochrysis carterae (Braarud et Fagerland) Christensen (Haptophyceae). Two mRNAs (Pcpcna1 and Pcpcna2) were identified and contained an identical coding region for 222 amino acid residues and an untranslated sequence of 302 base pair (Ut1) and 246 base pair (Ut2), respectively. Comparison between PCR‐derived genomic DNA fragments and cDNA sequences revealed five introns. The coding region of Pcpcna is similar to counterparts in other organisms and contains highly conserved functional domains. Phylogenetic analyses indicated clustering of Pcpcna with pcna in its haptophyte relative Isochrysis galbana Parke. A recombinant fusion protein of Pcpcna, overexpressed in Escherichia coli, was recognized by the PC10 antibody against rat PCNA. Using RT‐PCR and Western blotting, Pcpcna was found to be highly transcribed and translated during the exponential growth phase relative to the stationary growth phase, with a positive correlation between gene expression and growth rate. It can be concluded that the pcna is conserved in this coccolithophorid phytoplankton and that its expression is growth stage related. 相似文献
995.
William M. Hempel Celia W. Sutton Deborah Kaska David C. Ord Daniel C. Reed David R. Laur Alfred W. Ebeling Diane D. Eardley 《Journal of phycology》1989,25(1):144-149
Polyclonal rabbit antibodies to cell wall components were produced against gametophytes of the giant kelp Macrocystis pyrifera (Linnaeus) C. Agardh. These antibodies were found to react with carbohydrates extracted from M. pyrifera and Pterygophora californica Ruprecht by carbohydrate based enzyme immunoassay (EIA). The antibodies reacted with carbohydrates from both species. After affinity purification on a column with M. pyrifera carbohydrate coupled to AH-Sepharose, the eluted antibody was specific for M. pyrifera carbohydrate with little cross reactivity to P. californica carbohydrate in the EIA test. In experiments carried out to characterize the antigenic specificity of unfractionated antibody using commercially prepared carbohydrates in the EIA, the antibodies were shown to react primarily with fucoidan and to a lesser degree, alginate. The unfractionated antibody was also shown to bind to proteins from both M. pyrifera and P. californica. These results indicate that species specific carbohydrate determinants may be present in the kelp cell wall. 相似文献
996.
997.
Michael S. Munns Karen Moore Lyne Jossé Paul N. Fitchett John A. Bryant 《Experimental Biology Online》1998,3(7):1-5
A 13-residue oligopeptide corresponding to a conserved region of the MCM family of proteins was synthesised as a multiple antigen peptide in which eight copies of the peptide were conjugated to an oligo-lysine core. The multiple antigen peptide was used for raising antibodies. Western blots of the polypeptides present in the DNA polymerase–primase complex from pea (Pisum sativum L.) were challenged with the antibodies which, even at a dilution of 1:5000, clearly revealed a polypeptide of approximately 62 kDa. 相似文献
998.
Dina Tleugabulova Viviana Falc n Eduardo Pent n 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1998,720(1-2):153-163
Despite the complexity of the subject of protein–alum interactions, a valuable information can be obtained by analyzing the adsorbed and desorbed protein by common physico–chemical methods. In the present work, to approach the adsorption of hepatitis B surface antigen (HBsAg) on alum, the experimental data were supported by complementary analyses of the adsorbed protein by immunoelectron microscopy and the desorbed protein by denaturing size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. First, the depletion of HBsAg was investigated. The aspects assessed were the conditions, recovery and chromatographic performance of the desorbed protein. The results obtained strongly suggested the loss of particulate structure of HBsAg after adsorption on alum. This conclusion was further reinforced by direct immunoelectron microscopic visualization of HBsAg in the adsorbed state. 相似文献
999.
Dina Tleugabulova 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1998,707(1-2)
To investigate the factors leading to broadening of the recombinant hepatitis B surface antigen (HBsAg) peak in size-exclusion chromatography, the HBsAg particles eluting in different regions of the peak were subjected here to electrophoretic analysis. In nonreduced samples, the 24-kD band corresponding to the S monomer was detected when excessively large amounts of HBsAg were loaded onto the gel. Hence, some monomers are not disulfide-crosslinked in assembled particles. On the other hand, the results of alkylation experiments indicated the presence of free sulfhydryl group(s) in a little portion of freshly-purified HBsAg which was retarded on the size-exclusion chromatographic column and had significant antigenicity. This fraction of HBsAg was shown to be oligomeric and capable of spontaneous assembly into higher-order structures during aging. 相似文献
1000.
从猪水泡病病毒(SVDV)细胞培养物的PEG浓缩毒中提取病毒RNA,经RT-PCR和套式PCR扩增病毒主要保护性抗原蛋白基因,将扩增产物1.6kb插入pUC18载体中,经亚克隆后用双脱氧链终止法测定其序列,与已发表的SVDV分离物该区序列作比较,核苷酸同源性为96%-97%,氨基酸同源性为98%,参与构成SVDV中和性抗原位点的几个氨基酸残基均很保守;与已发表的柯萨奇B5病毒的对应序列比较,两者核苷酸序列同源性为77%,而推导的氨基酸顺序同源性竞高达92%。本文结果有助于SVDV的分子流行病学研究,并为其和柯萨奇B5病毒的相互关系提供参考数据,为SVDV新型疫苗研究提供了基础材料 相似文献