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排序方式: 共有29条查询结果,搜索用时 15 毫秒
1.
目的:制备人源性抗核抗体Fab片段.方法:通过4轮淘筛,富集已构建的人源性抗核抗体Fab片段噬菌体抗体库,间接ELISA法鉴定4轮淘筛后抗核抗体Fab片段噬菌体抗体;提取阳性克隆的噬菌粒DNA,切除gⅢ基因片段,自连接后转化大肠杆菌XL1-Blue,以IPTG诱导表达可溶性人源性抗核抗体Fab片段;应用间接ELISA法及荧光免疫法对表达上清进行鉴定.结果:第4轮洗脱的噬菌体滴度较第1轮增加200条倍,从抗核抗体Fab片段噬菌体库中筛选出2株阳性克隆,切除gⅢ基因后自连的噬菌粒DNA经XhoⅠ单酶切证实连接成功.间接ELISA法检测结果显示:制备的可溶性人源性抗核抗体Fab片段均呈现抗dsDNA阳性,具有抗原特异性;免疫荧光法结果显示:Hep2细胞和猴肝脏组织细胞核显示均质型荧光,绿蝇短膜虫的动基体显示均质型荧光.结论:成功制备具有抗原特异性的可溶性、人源性抗核抗体Fab片段,为高亲和力抗核抗体Fab片段的制备奠定了基础.  相似文献   
2.
目的优化大规模噬菌体DNA测序样品制备的方法,提高筛选效率。方法通过PCR扩增噬菌体环七肽库第一轮、第二轮和第三轮淘选产物中含插入目的片段噬菌体DNA,经琼脂糖凝胶电泳和DNA测序鉴定,并与经典的噬菌体DNA提取方法进行比较。结果PCR扩增法和经典法制备的噬菌体DNA的测序成功率分别为92.73%、93.75%,差异无统计学意义(P〉0.05);两种方法制备的同一个噬菌体克隆的DNA经测序鉴定显示测序结果一致;第一轮、第二轮和第三轮淘选产物的噬菌体克隆经PCR扩增和电泳鉴定,发现无插入目的片段的噬菌体克隆检出率逐轮增加分别为1.04%、4.17%和22.69%(P〈0.01)。结论PCR扩增含插入目的片段噬菌体DNA的方法可使大规模噬菌体筛选和DNA测序更加方便、快捷、实用,值得推广。  相似文献   
3.
A cell separation method using immunoglobulin (Ig)-coated plates, originally devised for murine spleen cells, was modified and adapted for enrichment (and depletion) of cellular subpopulations from human peripheral blood. For the direct separation of B and T cells, F(ab')2 fragments of anti-human Ig were used to coat the plates. For indirect separation, the cells were first incubated with monoclonal antibodies to cell surface antigens and then separated in plates coated with anti-mouse Ig. Plates were first coated with poly-L-lysine to facilitate the adherence of anti-Ig antibodies, and finally with bovine serum albumin to mask free poly-L-lysine. Cells which did not react with the anti-Ig antibodies or which were nonadherent to the plate were pipetted off; cells which reacted with the anti-Ig antibodies or which were adherent were eluted after incubation with excess serum. T, non-T, T4+, T4-, T8+, and T8- lymphocytes were separated with high viability, purity, and yield. The method was used to study suppressor activity of a patient who was treated by bone marrow transplantation for myelofibrosis. Strong suppressor activity was associated with unfractionated peripheral blood mononuclear leukocytes, monocytes, T, T8+, and T4- cells but not with B, T8-, and T4+ cells.  相似文献   
4.
A simple method is described for labelling cells with fluorescein and using them in artificial mixtures to assess cell separation procedures. The method facilitates the examination of the variables in a separation procedure. It is thus possible to tailor a separation procedure (for example panning with monoclonal antibody) to suit the specific requirements of the experiment.  相似文献   
5.
To target CD30 on Hodgkin's disease and anaplastic large-cell lymphoma, anti-CD30 single-chain antibodies were obtained by DNA immunization of mice with the complete human CD30 cDNA. Spleens were isolated from mice with high anti-CD30 titer, and the RNA was used for the production of an scFv-displaying phage library. Specific phages were enriched by 3 rounds of panning on soluble CD30 or CD30+ K562 cells. Recombinant immunotoxins (rITs) were made from 3 ELISA-positive scFv phages by fusion to a 38 kDa truncated mutant of Pseudomonas exotoxin (PE38) with or without a KDEL mutant sequence at the C terminus. In vitro cytotoxicity of purified anti-CD30 rITs was measured on CD30-transfected A431 cells. IC50 values ranged from 3 to 7 ng/ml (50-110 pM) for PE38 rITs and 0.1 ng/ml (2 pM) for the PE38-KDEL IT on A431-CD30 cells. The parental A431 cells were resistant, indicating that the cytotoxicity was specific and CD30-mediated. rITs were tested for anti-tumor activity in a nude mouse model. A431-CD30 cells were injected s.c. on day 0; then, mice bearing measurable tumors were treated beginning on day 4 with 3 alternate daily doses i.v. Anti-tumor activity was dose-dependent and not found when irrelevant ITs were administered or when CD30- tumors were treated. Our data show that DNA immunization and antibody phage display may be useful in producing new rITs against hematologic malignancies. Published 2001 Wiley-Liss, Inc.  相似文献   
6.
噬菌体改组库的构建及高活性水蛭素变异体的筛选   总被引:1,自引:0,他引:1  
目的: 运用DNA家族改组方法提高水蛭素的抗凝血酶活性.方法: 将水蛭素HV1、 HV2、 HV3基因等量混合, 用DNase I消化, 回收50 bp左右的片段, 再经无引物和有引物PCR扩增获得与原基因大小相同的改组分子, 构建噬菌体展示的水蛭素改组文库, 经凝血酶亲和淘选, ELISA筛选高活性的水蛭素变异体.结果: 经过DNA改组和噬菌体亲和筛选, 成功获得抗凝血酶活性提高的水蛭素变异体HV2-N47K.结论: 用DNA家族改组及亲和淘选技术能够筛选出高活性的水蛭素变异体蛋白, 这为其他分子的改组提供了借鉴.  相似文献   
7.
神经细胞粘附分子(N-CAM)是一种神经元的特异性细胞表面标志蛋白或抗原.本研究利用抗原与抗体特异性结合的原理,先用抗N-CAM的抗血清包被培养皿,继而用之吸附大鼠延髓细胞悬液,经缓冲液洗涤后收集吸附的细胞,将其种植于含10%~20%小牛血清的DMEM全培养液中进行常规培养.于24、48和72h观察培养神经元的生长状况,最后用神经元特异的烯醇化酶(NSE)来鉴定纯化效果.结果表明,用此方法可获得高纯度的(92.3%)神经元类群,培养的神经无形态好,突起生长的速度、数目和长度不亚于未经纯化培养的神经元.于培养72h时镜检可见培养的神经元之间存在轴-树、轴-体、轴-轴和体-体等多种形式的终扣样接触,形成复杂的环路样联系.说明,使用本法培养的神经元纯度高、密度适当,是单细胞膜通道电生理研究等方面的理想研究材料.  相似文献   
8.
Traditional strategies for the identification of cell-surface cancer targets often fall short of their objective. For example, whole-cell panning of antibody libraries to isolate a diverse panel of antibodies directed against targets on cancer cells often identifies all immunogenic and/or abundant cell-surface antigens, not simply tumor-specific or tumor-associated antigens. Here we describe the use of stringent negative selection in combination with positive panning to increase tumor specificity and clinical relevance of selected antibodies. Sera from cancer cell-immunized mice showed strong binding to immunizing cancer cell lines but also cross-reacted strongly with human blood cells. Antisera blood cell binding was considerably decreased after stringent subtraction with human red blood cells (RBCs) and white blood cells (WBCs), yet cancer cell specificity was retained. In order to select for a higher percentage of clinically relevant antibodies for potential therapeutic use, stringent negative selection by RBC subtraction was employed in whole-cell panning of a disease-specific phage displayed antibody library on the prostate cancer cell line, PC-3. Isolated antibodies were found to bind to target antigens implicated in tumorigenicity and cancer cell migration and/or invasion, and included CD26, CDCP1, and the integrin complexes 2/β1, 3/β1, 5/β1, and 6/β4. Compared with traditional cell panning, this method considerably increased the selectivity of antibodies to tumor-associated antigens.  相似文献   
9.
目的 构建大肠癌病人自然致敏淋巴结抗体Fab段噬菌体呈现库,初步筛选相关抗大肠癌抗体。方法取大肠癌病 人转移淋巴结,提取淋巴结总RNA,逆转录PCR扩增重链Fd和K轻链cDNA。依次将PCR产物插入载体pComb3的 相应部位,构建人源性大肠癌噬菌体Fab基因库,并应用噬菌体表面呈现技术对该抗体库进行淘选及鉴定。结果所选 2种Ig亚类的重链 Fd片段、2种κ轻链cDNA得到扩增。 Fd片段和κ轻链均插入pComb3的重组率为40%,Fab噬菌 体表达库容达1.48×106。经3轮淘选,抗体库得到约120倍的富集。3轮抗体库的点印记免疫染色均显示有Fab表达;酶 联免疫吸附实验显示其与大肠癌抗原有结合活性。结论成功构建了大肠癌病人自然致敏淋巴结抗体Fab段噬菌体呈 现库,利用噬菌体抗体库技术,可筛选相关抗大肠癌抗体。  相似文献   
10.
The anti-sperm activities of a series of monoclonal antibodies to human sperm have been compared using agglutination, immunofluorescence, ELISA and 'panning' assays. The antibodies fell into two categories, those that could be detected by agglutination but not immunofluorescence assays and those that could be detected by immunofluorescence but not agglutination. Antibodies positive in the agglutination assays were also positive in the 'panning' assay. None of the antibodies tested was positive in the ELISA assays. These results, and others, are discussed in relation to the problems associated with the detection of anti-sperm antibodies in sub-fertile human populations.  相似文献   
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