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1.
用绿色荧光蛋白(GFP)作为报告分子筛选有效的siRNA 总被引:1,自引:0,他引:1
建立一种利用绿色荧光蛋白(GFP)作为报告分子筛选能有效抑制目的基因表达的siRNA的方法.以巨噬细胞移动抑制因子(MIF)基因为研究对象,筛选能有效沉默MIF表达的质粒载体介导的siRNA.构建拥有同一Kozak共有翻译启始序列、翻译启始密码子ATG的MIF-GFP融合表达载体pEGFP-MIF.分别将3个靶向MIF的siRNA表达质粒与pEGFP-MIF共转化HEK293细胞,在荧光显微镜下观察HEK293细胞中GFP的表达,并用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.同时,将MIF siRNA表达质粒分别与MIF表达载体共转化HEK293细胞,用荧光定量PCR检测HEK293细胞中MIF mRNA的表达水平.定量PCR结果显示,GFP表达低的细胞中,MIF mRNA的表达也明显降低;利用pEGFP-MIF和MIF表达载体筛选到的有效MIF siRNA的结果一致.因此,建立了目的基因与GFP融合表达,以GFP作为报告分子来筛选抑制目的基因表达siRNA的方法,并为进行多个基因的有效siRNA的筛选提供解决方案. 相似文献
2.
To screen for effective small interference RNA (siRNA), a simple and visualized method was developed using the green fluorescence protein (GFP) as a reporter. Candidate siRNAs targeting macrophage migration inhibition factor genes (MIF) were identified. By using the pEGFP-N3 vector, the MIF-GFP expression plasmid, pEGFP-MIF, was constructed with the same Kozak con-sensus translation initiation site and start code ATG for the MIF-EGFP coding sequence. Based on the siRNA expression vector pSilencer-4,1,3 candidate MIF siRNA expression plasmids were constructed and co-transfected with the pEGFP-MIF into the H EK293 cells, respectively. The GFP expression in HEK293 cells could be viewed by fluorescence microscopy and the MIF mRNA expressions were determined by real-time quantitative PCR. The 3 candidate MIF siRNA expression plasmids were also co-transfected with the MIF expression plasmid into the HEK293 cells, respectively, and the MIF mRNA expres-sions were determined by real-time quantitative PCR. The results show that the down-regulated expression of the MIF mRNA was consistent with the GFP expression and the same effective MIF siRNAs were screened by using the pEGFP-MIF or MIF expression plasmid with the candidate MIF siRNAs expression plasmids. Therefore, by using the GFP as a reporter, a useful method was provided to screen for effective siRNAs tar-geting specific genes co-expressed with the GFP. This may be a good strategy for screening for effective siRNAs tar-geting different genes. 相似文献
3.
RNA interference (RNAi) is a phenomenon of gene silence induced by a double-stranded RNA (dsRNA) homologous to a target gene.
RNAi can be used to identify the function of genes or to knock down the targeted genes. In RNAi technology, 19 bp double-stranded
short interfering RNAs (siRNA) with characteristic 39 overhangs are usually used. The effects of siRNAs are quite varied due
to the different choices in the sites of target mRNA. Moreover, there are many factors influencing siRNA activity and these
factors are usually nonlinear. To find the motif features and the effect on siRNA activity, we carried out a feature extraction
on some published experimental data and used these features to train a back-propagation neural network (BP NN). Then, we used
the trained BP NN to predict siRNA activity.
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Translated from Acta Biophysica Sinica, 2006, 22(6): 429–434 [译自: 生物物理学报] 相似文献
4.
Mutations in presenilin 1 (PS1) gene are closely associated with the early onset of familial Alzheimer’s disease (EOFAD).
The fusion genes, GFPPS1 (recombinant plasmid pEGFP-C1-PS1) and PS1-GFP (recombinant plasmid pEGFP-N2-PS1) were constructed
to study the subcellular localization of PS1 holoprotein. Recombinant plasmids were transiently transfected into two cell
lines, HEK293 and CHO, respectively, using the green fluorescence from GFP (green fluorescence protein) as the PS1 localization
signal. Then, we observed green fluorescence with a SPOT II (Olympus, BH2) and CONFOCAL microscope (Olympus, FV300) under
488 nm. The results show that PS1 located on the nuclear envelope. A few can be found on the cellular membrane and in the
cytosol in a non-homogeneous distribution.
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Translated from China Biotechnology, 2006, 26(6): 17-22 [译自: 中 国生物工程杂志] 相似文献
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Huang Guanrong Xiong Shiqin Zhao Qiuhui Wang Yinyin Reng Fangli Ye Xiongjun Chang Zhijie 《Frontiers of Biology in China》2006,1(2):104-109
Sef is a transmembrane protein inhibiting FGF signaling. To determine the correlation of Sef with human diseases, Sef expression
patterns were observed in cell lines and human cancer tissues. Western blot using anti-hSef antibodies showed that hSef, when
expressed in Cos7 cells gave a molecular mass of 100 KD as compared with 80 KD in an in vitro translation assay suggesting occurrence of glycosylation at the potential N-linked glycosylation sites in the extracellular
domain. Northern blot showed that hSef was mainly expressed in human kidney and testis. RT-PCR analysis showed a widely spread
expression pattern in several cell lines. Immunohistochemical analysis revealed a high expression level of hSef in kidney,
testis, and the corresponding carcinoma tissues. Results demonstrated that Sef might be up-regulated in the cancer tissues
suggesting a possible role of Sef in pathophysiology of human diseases.
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Translated from Chinese Journal of Biochemistry and Molecular Biology, 2005, 21 (2) [译自: 中国生物化学与分子生物学报, 2005,21(2)] 相似文献
7.
Huifang Tan Guoqing Zhang Guangyu Zheng Fumian Cui Shijun Qian 《Frontiers of Biology in China》2008,3(3):287-292
Endo-β-glucanase II (EG II) gene cDNA was isolated from the fungus Humicola insolens H31-3 by RT-PCR. It was cloned into the expression vector pGAPZαA. The resultant recombinant plasmid was introduced into
Pichia pastoris GS115 by electroporation after being linearized by BspHI digestion. The recombinant Pichia pastoris strain was obtained and SDS-PAGE showed that the molecular weight of the expression protein was about 55 kD.The cultivation
condition and the characteristics of the recombinant EG II were also explored.
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Translated from Microbiology, 2006, 33(6): 68273 [译自: 微生物学 通报] 相似文献
8.
Yan Xunyou Zhao Hongliang Zhang Weiguang Xue Chong Liu Zhimin 《Frontiers of Biology in China》2007,2(2):170-175
To obtain human tissue inhibitor of metalloproteinase-2 (TIMP-2) cDNA and the secretory expression of TIMP-2 gene in Pichia pastoris, we designed and synthesized a 618 base pairs artificial gene coding for the TIMP-2 with a computer-aided design method using
a standard chemical synthesis technique, which was composed of frequently used codons in the highly expressed Pichia pastoris genes. Then the synthetic gene encoding TIMP-2 was checked by means of dideoxynucleotide sequencing. The verified gene of
TIMP-2 was cloned to the Escherichia coli-yeast shuttle vector of pPIC9 to construct a recombinant plasmid pPIC9-T2. The plasmid was transformed into GS115 cells of
the methylotrophic yeast, Pichia pastoris by electroporation, and we got the expression cell through phenotype selection and induction with methanol. Separation, purification,
and bioactivity analysis of the expressed products were performed.
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Translated from Microbiology, 2006, 33(1): 1–6 [译自: 微生物学通报] 相似文献
9.
Yongsheng Tao Zuxin Zhang Yonglin Chen Lijia Li Yonglian Zheng 《Frontiers of Biology in China》2008,3(4):414-418
The rice BAC-DNA was used as probes and fluorescence in situ hybridization (FISH) was applied to the interphase and metaphase mitotic chromosomes of maize. To optimize the BAC-FISH technique,
we respectively assayed the effect of several factors, including maize or rice genomic C
o
t DNA used as blocking reagent of DNA, washing temperatures and FAD concentration in the washing buffer and in the hybrid
solution. The results show that C
o
t DNA of maize genome blocked the repetitive sequence of the rice BAC-DNA when the C
o
t value was below 50. Meanwhile, it was necessary to adjust the C
o
t value according to the different probes and their ratios. Decreasing the concentration of FAD in the hybridization mixtures,
adjusting the washing rate after hybridization, and most especially, blocking the ricespecific repetitive sequences of BAC-DNA
could improve the positive signals of BAC-FISH.
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Translated from Chinese Journal of Biochemistry and Molecular Biology, 2007, 23(1): 80–84 [译自: 中国生物化学与分子生物学学报] 相似文献
10.
The gene which encodes (R)-specific carbonyl reductase (rCR) from Candida parapsilosis CCTCC M203011 was cloned, sequenced and compared with genes from the GenBank. The results indicated that rCR gene was 1011
bp, encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa, and its nucleotide sequence showed 99% similarity
to those of other members of the alcohol dehydrogenase superfamily. The rCR gene could express in recombinant strain Escherichia coli JM109, and the expression plasmid could produce (R)-1-pheny-1,2-ethanediol (100% e.e., 80.14% yield) from β-hydroxyacetophenone without any additive to regenerate NAD+ from NADH.
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Translated from Microbiology, 2006, 33(4): 112–118 [译自: 微生物学通报] 相似文献
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Xiong Xia Xu Xia Li Dongling Chen Ping Liang Songping 《Frontiers of Biology in China》2007,2(1):75-79
Hainantoxin-IV (HNTX-IV) was isolated from the Chinese bird spider Ornithoctorcs hainana and identified as a novel antagonist of tetrodotoxin-sensitive (TTX-S) sodium channels. As revealed by the solution structure
of HNTX-IV solved by two-dimensional nuclear magnetic resonance (2D-NMR), HNTX-IV adopts an inhibitor cystine knot motif.
To check the role of basic residues during HNTX-IV’s interaction with TTX-S sodium channels, R26A and K27A mutants of HNTX-IV
were constructed by solid-phase chemical synthesis. The synthesized peptides were purified and refolded under optimized oxidation
conditions. Correct synthesis and folding were confirmed by MALDI-TOF mass spectrometry and NMR spectroscopy, respectively.
Using the whole-cell patch-clamp technique, Lys27 but not Arg26 was identified as a key residue for HNTX-IV’s bioactivity
against TTX-S sodium channels, because R26A-HNTX-IV showed slightly reduced activity and K27A-HNTX-IV showed almost no inhibition.
Translated from Chinese Journal of Biochemistry and Molecular Biology, 2005, 21(4): 499–503 [译自: 中国生物化学与分子生物学报] 相似文献
13.
Wang Shujing Liu Yan Lin Xuesong Fu Xue Xu Jianyong Liu Xinghan 《Frontiers of Biology in China》2007,2(3):276-283
To obtain an anti-tumor peptide of Tumstatin and detect its biological activity, the nucleotide sequence encoding 185–203
amino acids (19peptide) of Tumstatin was synthesized and inserted into the fusion protein vector pTYB2. After identification
by sequencing and restriction endonucleases, the recombined vector was transformed into BL-21 (DE3) E. coli competent cells. Transformed E. coli BL-21 (DE3) were induced by isopropyl-β-thiogalactopyranoside (IPTG), and then expressed. By 1,4-dithiothreitol (DTT) reduction,
the soluble 19peptide was obtained from a chitin affinity chromatograph. The biological activity of 19peptide was determined
by 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenytetrazolium bromide (MTT) assay, cell growth curve, the effect of the ascitic fluid
transfevent H22 hepatoma on mice and via histopathological slices. The purified 19peptide directly inhibited proliferation
and migration of murine B16 melanoma cells, SMMC-7721hepatoma carcinoma cells and human umbilical vein endothelial cells (HUVEC).
The tumor inhibition rate of mice ascitic fluid transfevent H22 hepatoma was 48.46%. Histopathological slices showed that
it could promote tumor tissue necrosis and decrease the density of blood vessels. With higher anti-tumor activity, 19peptide
has the potential to become a novel, potent anti-tumor agent.
Translated from Chinese Journal of Biochemistry and Molecular Biology, 2005, 21(3): 322–328 [译自: 中国生物化学与分子生物学学报] 相似文献
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The apoptosis of cells is one of the fields that attract increasing attention in biology today. Usually, the cells are treated
with chemicals when detecting apoptosis. It is highly desired to detect apoptosis in a real-time basis. Apoptosis of Jurkat
cells was studied using a real-time electrorotation chip. This chip allows the detection of the cell membrane capacitance
changes during the course of apoptosis and therefore facilitates the analysis of apoptosis in a real-time basis without involving
any chemical treatment.
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Translated from Acta Biophysica Sinica, 2005, 21 (1) [译自: 生物物理学报, 2005,21(1)] 相似文献
16.
A two-state hopping model was proposed to study the permeation of ion channel. The Nernst equation in equilibrium and the
Michaelis-Menten relation in steady state were derived from the two-state kinetic model. The current-voltage relationship
obtained in the symmetrical solutions case was linear when the applied potential was less than 100 mV, which met Ohm’s law.
The conductance-concentration relationship exhibited the saturation property. Moreover, the characteristic time reaching the
steady state of the KcsA channel was also discussed.
Translated from Acta Biophysica Sinica, 2005, 21(4): 289–294 [译自: 生物物理学报] 相似文献
17.
18.
To analyze the protein expression pattern of the cerebral cortex in Wistar rats using the proteomics approach, proteins were
separated by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue and digested with trypsin. Then, we
analyzed the peptide section using a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS)
and identified the protein by indexing special database (SwissProt) according to the finger printing of the peptide quality.
Eighty-four protein spots were identified, including metabolic enzymes, skeleton proteins, heat shock proteins, antioxidant
proteins, signaling proteins, proteasome related proteins, neuron and glial specific proteins and serum associated proteins.
The result of this study enriches the database of the proteome in the cerebral cortex of rats and lays a foundation for further
research of neurological disorders in rat models.
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Translated from Acta Biophysica Sinica, 2007, 23 (1): 151–156 [译自: 生物物理学报] 相似文献
19.
用Rnase Ⅲ制备的小干涉RNA降解SARS冠状病毒基因的细胞内转录物 总被引:6,自引:0,他引:6
SARS冠状病毒是引起重症急性呼吸综合症的主要原因,目前尚没有特效药物或疫苗对抗这种新病毒。RNA干涉是指双链RNA可以特异地降解细胞内同源基因的Mrna。在哺乳动物细胞中,<30bp的小双链RNA能引起RNA干涉,又可以避免干扰素反应。通过体外转录得到SARS病毒3种基因RNA依赖的RNA聚合酶、刺突蛋白及核衣壳蛋白部分片段的长双链RNA,然后用Rnase Ⅲ有限切割成长度<30bp的小干涉RNA。同时把上述3种基因片段分别连接到质粒Pgl3-Control中,得到的3个质粒Pgl-R、Pgl-S和Pgl-N可以分别在细胞内转录出荧光素酶RNA依赖的RNA聚合酶、刺突蛋白、核衣壳蛋白的杂合Mrna。上述质粒分别和相应的小干涉RNA共转染HEK293F细胞,测定荧光素酶活性,结果小干涉RNA使相应质粒表达荧光素酶的活性显著下降;用逆转录定量PCR反应测量Mrna丰度,结果表明上述小干涉RNA可以特异地降解相应的病毒基因转录物。 相似文献
20.
Zhuang Yingping Ma Wenfeng Guo Meijin Ding Mansheng Chu Ju Zhang Siliang 《Frontiers of Biology in China》2006,1(4):345-348
The effect of temperature on the formation of recombinant protein, apolipoprotein A-IMilano was investigated in the present study. The temperature of the initial growth phase was set at 30°C, while temperature variation
in induction phase was arranged in three modes. High cell-density culture of Escherichia coli and high expression of recombinant human by twice temperature-shifted induction were carried out. Experimental results showed
that ApoA-IMilano reached 4.8 g/L with the final cell density of OD600, 150. It was found that twice temperature-shifted induction could successfully avoid the effect of acetic acid on cell density
and the expression of the product. The present study provides a basic procedure for the production of recombinant ApoA-IMilano.
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Translated from Microbiology, 2005, 32(2): 54–59 [译自: 微生物学通报, 2005, 32(2): 54–59] 相似文献