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91.
Soraia A. C. Jorge Alexandra S. Santos Ângela Spina Carlos A. Pereira 《Cytotechnology》2008,57(1):51-59
Drosophila melanogaster S2 cells were transfected with a plasmid vector (pAcHBsAgHy) containing the S gene, coding for the hepatitis B virus surface
antigen (HBsAg), under control of the constitutive drosophila actin promoter (pAc), and the hygromycin B (Hy) selection gene.
The vector was introduced into Schneider 2 (S2) Drosophila cells by DNA transfection and a cell population (S2AcHBsAgHy) was
selected by its resistance to hygromycin B. The pAcHBsAgHy vector integrated in transfected S2 cell genome and approximately
1,000 copies per cell were found in a higher HBsAg producer cell subpopulation. The HBsAg production varied in different subpopulations,
but did not when a given subpopulation was cultivated in different culture flasks. Higher HBsAg expression was found in S2AcHBsAgHy
cells cultivated in Insect Xpress medium (13.5 μg/1E7 cells) and SFX medium (7 μg/1E7 cells) in comparison to SF900II medium
(0.6 μg/1E7 cells). An increase of HBsAg was observed in culture maintained under hygromycin selection pressure. Data presented
in the paper show that S2AcHBsAgHy cells produce efficiently the HBsAg which is mainly found in the cell supernatant, suggesting
that HBsAg is secreted from the cells. The data also show that our approach using the Drosophila expression system is suitable
for the preparation of other viral protein preparation. 相似文献
92.
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94.
目的评价初治、单药使用替比夫定治疗HBeAg阳性的慢性乙型肝炎(CHB)患者48周的e抗原血清学转换的基线预测因素。方法97例HBeAg阳性CHB患者分别以基线HBsAg、ALT和HBVDNA水平高低分组,对比两组治疗48周时生化学、病毒学和血清学应答情况。结果基线HBsAg-101500IU/mL组e抗原阴转率和血清学转换率均为42.3%,基线HBsAg〉1500IU/mL组分别为20%和17.8%,两组比较差异均有统计学意义(P〈0.05);基线ALT〉5ULN组e抗原阴转率和血清学转换率均为45.1%,基线ALT05ULN组分别17.4%和15.2%,两组比较差异均有统计学意义(P〈0.01);基线HBVDNA〈8.0log-10copies/mL组和基线HBVDNA≥8.0log-10copies/mL组e抗原阴转率和血清学转换率相比,差异无统计学意义(P〉0.05);基线水平HBsAg≤1500IU/mL且ALT〉5ULN的CHB患者共40例作为观察组,其余57例患者作为对照组,治疗48周时观察组e抗原阴转率和血清学转换率均为45%,对照组分别为22.8%和21.1%,两组比较差异均有统计学意义(P〈0.05)。结论基线HBsAg水平≤1500IU/mL和ALT水平〉5ULN的HBeAg阳性慢性乙型肝炎患者,在接受替比夫定治疗48周时,有较高的e抗原转阴率和血清学转换率;基线HBsAg和ALT水平是替比夫定治疗e抗原血清学转换的重要预测因素。 相似文献
95.
Yong Liu Rui Huang Yali Xiong Qi Zhao Guangmei Chen Juan Xia Chao Wu 《Biochemical and biophysical research communications》2014
Chronic hepatitis B virus (HBV) infection is the result of an inadequate antiviral immune response to the virus. In this study, we aimed to investigate whether the soluble CD40 ligand-activated B (CD40-B) cells could present antigen and induce specific cytotoxic T lymphocytes (CTLs) in patients with chronic HBV infection. We observed that after activated by sCD40L, the expression of CD80, CD86, major histocompatibility complex (MHC) I and II molecules on the CD40-B cells was significantly increased. Cytometry and fluorescence microscopy showed that more than 41.34% CD40-B cells were loaded by the HBcAg peptide. Furthermore, after been activated and HBcAg18–27 antigen peptide pulsed, B cells obtained from patients with chronic HBV infection could induce HBcAg18–27 specific CTLs in vitro. Taken together, our results show that B cells from patients with chronic HBV infection can be activated by sCD40L and may function as antigen presenting cells and induce HBV-specific CTLs. 相似文献
96.
The murine monoclonal antibody 125E11 is an IgG which recognizes PreS1(21-47) fragment of large hepatitis B surface antigen. It has been successfully used for clinical detection of HBV virion in serum of hepatitis B patients. In present study, the genes of variable region in heavy chain (VH) and light chain (VL) of 125E11 have been cloned. Sequence analysis of cloned VH gene and VL gene showed that they had general characterization of immunoglobin variable region genes. According to Kabat classification, VH gene and VL gene belong to VH10 family, subgroup IIID and Vkappa family subgroup I, respectively. An expression vector of 125E11 single-chain Fv antibody fusion protein, in which VH and VL peptide were connected by a flexible linker (Gly(4)Ser)(3), was constructed. The scFv fusion protein was highly expressed in Escherichia coli mainly in inclusion body form. Using urea and pH gradient gel filtration method, the refolding of scFv was efficiently achieved. The refolding efficiency reached about 11% and 2.7 mg refolded scFv was obtained from 1L of culture. The binding activity and specificity of 125E11 scFv against PreS1(21-47)-containing antigen were also analyzed. 相似文献
97.
Expression of a single-chain Fv antibody fragment specific for the Hepatitis B surface antigen in transgenic tobacco plants 总被引:10,自引:0,他引:10
Ramírez N Ayala M Lorenzo D Palenzuela D Herrera L Doreste V Pérez M Gavilond JV Oramas P 《Transgenic research》2002,11(1):61-64
An anti-Hepatitis B virus surface antigen (HBsAg) single chain Fv (scFv) antibody fragment was expressed in Nicotiana tabacum transgenic plants. The 6-histidine tagged scFv was targeted to either the cytosol, apoplast, and vacuole, or for retention in the endoplasmic reticulum. Expression of active scFv was detected by ELISA in fresh leaf material from F1 transgenic plant lines representative of the genetic constructs targeting the antibody fragment to the apoplastic fluid (AF-12, 0.031% of the total soluble protein), vacuole (V-20, 0.032% of the total soluble protein), and endoplasmic reticulum (ER-52, 0.22% of the total soluble protein). No scFv was detected by ELISA or western blot in the plants transformed with the cytosol construct. The biologically active scFv was easily purified (to 94–95% purity) from ER-52 and AF-12 plant material using immobilized metal ion affinity chromatography. Recovery estimated from the ER-52 plant line indicates that 15–20g of pure active scFv can be obtained per gram of fresh leaf material, on a laboratory scale. 相似文献
98.
99.
Lam WY Leung KT Law PT Lee SM Chan HL Fung KP Ooi VE Waye MM 《Journal of cellular biochemistry》2006,97(4):795-812
Ethanolic extract of Phyllanthus nanus (P. nanus) treatment exhibited potent antiviral activity against Hepatitis B virus (HBV). The effects of these extracts on HBV in the HBV genome integrated cell lines--Alexander cells and HepG2 2.2.15 cells were examined. Experimental results showed that the ethanolic extract of P. nanus produced suppressive effect on HBsAg secretion and HBsAg mRNA expression. The extract also inhibited HBV replication as measured by HBV DNA level in vitro. In addition, using a duck HBV (DHBV) primary culture model, the P. nanus ethanolic extract suppressed viral replication of DHBV in DHBV infected primary duck hepatocytes. The gene expression pattern in Alexander cells that had been treated with the ethanolic extract of P. nanus was also revealed by microarray techniques. The microarray results indicated that there was up-regulation of expression of several genes, including annexin A7 (Axn7). The subcellular localization of Axn7 and anti-HBV effect of Axn7 over-expression in Alexander cells were also investigated. Results showed that expression of Axn7-GFP fusion protein are localized around the secretory vesicles and could cause a decrease in HBsAg secretion in Alexander cells. Axn7 protein might play an important role in the medicinal effect of the active principle(s) of P. nanus. 相似文献
100.
Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression 总被引:4,自引:0,他引:4
To assess the utility of the tomato fruit-specific E8 gene's promoter for driving vaccine antigen expression in plant, the 2.2 kb and 1.1 kb E8 promoters were isolated and sequenced from Lycopersicon esculentum cv. Jinfeng #1. The 1.1 kb promoter was fused to vaccine antigen HBsAg M gene for the transfer to Nicotiana tabacum, and the CaMV 35S promoter was used for comparison. Cholera toxin B (ctb) gene under the control of the 1.1 kb promoter was transformed into both N. tabacum and L. esculentum. Southern blot hybridization confirmed the stable integration of the target genes into the tomato and tobacco genomes. ELISA assay showed that the expression product of HBsAg M gene under the control of the 1.1 kb E8 promoter could not be detected in transgenic tobacco tissues such as leaves, flowers, and seeds. In contrast, the expression of HBsAg M gene driven by CaMV 35S promoter could be detected in transgenic tobacco. ELISA assay for CTB proved that the 1.1 kb E8 promoter was able to direct the expression of exotic gene in ripe fruits of transgenic tomato, but expression was absent in leaf, flower, and unripe fruit of tomato, and CTB protein was not detected in transgenic tobacco tissues such as leaves, flowers, and seeds when the gene was under the control of the 1.1 kb E8 promoter. The results indicated that the E8 promoter acted not only in an organ-specific, but also in a species-specific fashion in plant transformation. 相似文献