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991.
旨在研究蛋白G IgG Fc段结合域(PGFB)的克隆、表达及其抗体结合功能,用于抗体的纯化.根据PGFB的氨基酸序列,选择大肠杆菌偏爱的密码子,设计并合成了4个寡核苷酸片段.通过重叠延伸PCR方法合成了PGFB DNA片段,测序鉴定后克隆至原核表达系统pET-28a-c(+)上,转化大肠杆菌,获得表达菌株;IPTG诱导表达PGFB,经Ni+-NTA琼脂糖凝胶层析纯化后偶联到琼脂糖凝胶6B上,用其纯化多克隆抗体.结果显示,PGFB在大肠杆菌BL21(DE3)中获得高效表达,纯化后纯度达到90%以上,相对分子量为12.25 kD,与预期值相符.此外,偶联产物纯化多克隆抗体达到了良好的效果,每毫升基质可结合20 mg抗体.本研究克隆构建并高效表达了具有较好抗体亲和能力的PGFB,为多克隆抗体的快速纯化提供了方便. 相似文献
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Huan Zhang Yong-an Zhang Qilian Qin Xuan Li Lin Miao Yuzhu Wang Liangjian Qu Aijun Zhang Qing Yang 《In vitro cellular & developmental biology. Animal》2009,45(5-6):201-204
A cell strain (IOZCAS-Spex-II-A) cloned from IOZCAS-Spex-II, a cell line established from the fat body of Spodoptera exigua (Lepidoptera: Noctuidae) larva, was characterized, and its capability to produce S. exigua nucleopolyhedrovirus was high with infection rate exceeding 90% compared with its parental cell line IOZCAS-Spex-II that scored only 50%. Growth curve of budded virus (BV) in the strain was analyzed and the titer of BV reached the highest of 3.7?×?104 pfu/mL by 96 h after inoculation. Concentration of occlusion bodies (OBs) produced by the cloned cell strain (IOZCAS-Spex-II-A) was 7.1?×?107 OBs/mL, while the parental cell line produced 2.4?×?107 OBs/mL. The average yield of the virus was 176 OBs/cell of IOZCAS-Spex-II-A compared with 211 OBs/cell that of the parental cell line. Significant differences were observed in virus production, growth characters, cell shape, between the parental cell line, and its clone. The cell lines (IOZCAS-Spex-II and IOZCAS-Spex-II-A) were also susceptible to Autographa californica multiple nucleopolyhedrovirus infection. In addition, they were characterized with regard to their growth rates and DNA amplification fingerprinting technique employing polymerase chain reaction. 相似文献
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996.
Wei Qu Yabin Zhou Chunhong Shao Yundong Sun Qunye Zhang Chunyan Chen Jihui Jia 《Journal of microbiology (Seoul, Korea)》2009,47(4):486-493
Helicobacter pylori is a highly pathogenic microorganism with various strategies to evade human immune responses. Nitric oxide (NO) and reactive nitrogen species (RNS) generated via nitric oxide synthase pathway are important effectors during the innate immune response. However, the mechanisms of H. pylori to survive the nitrosative stress are not clear. Here the proteomic approach has been used to define the adaptive response of H. pylori to nitrosative stress. Proteomic analysis showed that 38 protein spots were regulated by NO donor, sodium nitroprusside (SNP). These proteins were involved in protein processing, anti-oxidation, general stress response, and virulence, as well as some unknown functions. Particularly, some of them were participated in iron metabolism, potentially under the control of ferric uptake regulator (Fur). Real time PCR revealed that fur was induced under nitrosative stress, consistent with our deduction. One stress-related protein up-regulated under nitrosative conditions was thioredoxin reductase (TrxR). Inactiva-tion of fur or trxR can lead to increased susceptivity to nitrosative stress respectively. These studies described the adaptive response of H. pylori to nitric oxide stress, and analyzed the relevant role of Fur regulon and TrxR in nitrosative stress management. 相似文献
997.
P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways 下载免费PDF全文
Activation of the P2X7 receptor (P2X7R) triggers a remarkably diverse array of membrane trafficking responses in leukocytes
and epithelial cells. These responses result in altered profiles of cell surface lipid and protein composition that can modulate
the direct interactions of P2X7R-expressing cells with other cell types in the circulation, in blood vessels, at epithelial
barriers, or within sites of immune and inflammatory activation. Additionally, these responses can result in the release of
bioactive proteins, lipids, and large membrane complexes into extracellular compartments for remote communication between
P2X7R-expressing cells and other cells that amplify or modulate inflammation, immunity, and responses to tissue damages. This
review will discuss P2X7R-mediated effects on membrane composition and trafficking in the plasma membrane (PM) and intracellular
organelles, as well as actions of P2X7R in controlling various modes of non-classical secretion. It will review P2X7R regulation
of: (1) phosphatidylserine distribution in the PM outer leaflet; (2) shedding of PM surface proteins; (3) release of PM-derived
microvesicles or microparticles; (4) PM blebbing; (5) cell–cell fusion resulting in formation of multinucleate cells; (6)
phagosome maturation and fusion with lysosomes; (7) permeability of endosomes with internalized pathogen-associated molecular
patterns; (8) permeability/integrity of mitochondria; (9) exocytosis of secretory lysosomes; and (10) release of exosomes
from multivesicular bodies.
This work was supported by NIH grants R01-GM36387 and P01-HLHL18708 (G.R.D.). 相似文献
998.
Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes. 相似文献
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Expression and functional analysis of the rice plasma-membrane intrinsic protein gene family 总被引:6,自引:2,他引:4
Plasma membrane intrinsic proteins (PIPs) are a subfamily ofaquaporins that enable fast and controlled translocation of water across the membrane. In this study, we systematically identified and cloned ten PIP genes from rice. Based on the similarity of the amino acid sequences they encoded, these rice PIP genes were classified into two groups and designated as OsPIP1-1 to OsPIP1-3 and OsPIP2-1 to OsPIP2-7 following the nomenclature of PIP genes in maize. Quantitative RT-PCR analysis identified three root-specific and one leaf-specific OsPIP genes. Furthermore, the expression profile of each OsPIP gene in response to salt, drought and ABA treatment was examined in detail. Analysis on transgenic plants over-expressing of either OsPIP1 (OsPIP1-1) or OsPIP2 (OsPIP2-2) in wild-type Arabidopsis, showed enhanced tolerance to salt (100 mM of NaCl) and drought (200 mM ofmannitol), but not to salt treatment of higher concentration (150 mM of NaCl). Taken together, these data suggest a distinct role of each OsPIP gene in response to different stresses, and should add a new layer to the understanding of the physiological function of rice PIP genes. 相似文献