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Electrophoresis(SDS-PAGE)as a Method for Screening Species of Passiflora Using Seed Proteins as Molecular Markers
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作者 Marilia Penteado Stephan Ana Maria Costa +3 位作者 Tatiana De Lima Azevedo Jeane Santos Da Rosa Alexsandro Araújo Dos Santos 《Journal of Agricultural Science and Technology(B)》 2022年第2期40-47,共8页
Brazil is the center of diversity and dispersion of species of the genus Passiflora.Two species of passion fruit Passiflora tenuifila BRS VT(passiflora garlic)and another of Passiflora setacea BRS PC were used in this... Brazil is the center of diversity and dispersion of species of the genus Passiflora.Two species of passion fruit Passiflora tenuifila BRS VT(passiflora garlic)and another of Passiflora setacea BRS PC were used in this study.The plants were grown on the Embrapa Cerrados experimental farm.The fruits were washed,minimally cut and their constituents separated.Both species differed in the proportion of wet mass between husk,seed,and pulp.As expected,both species had a higher proportion of husks and seeds and a lower amount of pulp.A semi-purification procedure for the proteins present in the seeds and husks was described and the molecular aspects were analyzed by polyacrylamide gel electrophoresis(SDS-PAGE).The quantification of soluble proteins was performed using the Bradford method.The analysis of the soluble protein extracted from the seeds and husks showed that these values are up to 70 times higher for the seeds of P.tenuifila and 28.5 times for P.setacea,in the husks.It was verified that both species present a similar protein profile,observed by the intense and diverse bands found in the polyacrylamide gel,mainly in the range of 32 to 19 kDa.This work opens up an unexplored field of tracking bioactive proteins and/or peptides including forms of nanostructure systems that protect other bioactive molecules.The objective of this work was to present an analytical procedure to semi-purify seed proteins of two Passiflora species and use electrophoresis as an analysis tool for further screening of their protein profiles and selection of a molecular marker to differentiate them. 展开更多
关键词 protein screening method bioactive peptides seeds P.setacea P.tenuifila mass distribution on fruit
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Protein stability regulators screeningassay (Pro-SRSA): protein degradation meets theCRISPR-Cas9 library 被引量:1
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作者 Yuanzhong Wu Tiebang Kang 《Chinese Journal of Cancer》 SCIE CAS CSCD 2016年第8期385-387,共3页
The regulation of protein stability is a fundamental issue for biophysical processes,but there has not previously been a convenient and unbiased method of identifying regulators of protein stability.However,as reporte... The regulation of protein stability is a fundamental issue for biophysical processes,but there has not previously been a convenient and unbiased method of identifying regulators of protein stability.However,as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25 A," recently published in Cell Discovery,our team developed a protein stability regulators screening assay(Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9(CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability.Based on our findings,we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability. 展开更多
关键词 CRISPR-Cas9 screening protein stability Cdc25A Ubiquitination ACETYLATION
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Screening proteins that interact with mutant superoxide dismutase 1 from familial amyotrophic lateral sclerosis using a yeast two-hybrid system
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作者 Guisheng Chen Shugui Shi +7 位作者 Lusi Li Kangning Chen Ju HU Zhenhua Zhou Jun WU GaoxingLuo ShunzongYuan Xu Peng 《Neural Regeneration Research》 SCIE CAS CSCD 2011年第26期2013-2017,共5页
The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 (SOD1) using a yeast two-hybrid system. Using BLAST software, 15 real proteins which ... The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 (SOD1) using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins (protein tyrosine-phosphatase non-receptor type 2, TBCl D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor a2, microtubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain), and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated protein/microtubule affinity-regulating kinase 1 and β-sarcoglycan. 展开更多
关键词 yeast two-hybrid system mutant superoxide dismutase 1 cDNA library protein-protein interaction screen amyotrophic lateral sclerosis
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The Mapping and Characterization of Cruella (Cru), a Novel Allele of Capping Protein α (Cpa), Identified from a Conditional Screen for Negative Regulators of Cell Growth and Cell Division
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作者 Ashley Cosenza Jacob D. Kagey 《Advances in Bioscience and Biotechnology》 2016年第10期373-380,共8页
A Flp/FRT EMS mutagenesis screen was conducted in the eye of Drosophila melanogaster on chromosome 2R to identify negative regulators of cell growth and cell division. In addition to the EMS mutation in the mosaic eye... A Flp/FRT EMS mutagenesis screen was conducted in the eye of Drosophila melanogaster on chromosome 2R to identify negative regulators of cell growth and cell division. In addition to the EMS mutation in the mosaic eye, an ark loss of function allele (ark<sup>82</sup>) was utilized to block apoptosis in the homozygous mutant cells, setting up a screen for conditional regulators of cell growth and cell division. In the present study, we focus on the characterization and mapping of one mutant that resulted from this screen, Cruella (cru). A cross between flies with the flippase enzyme directed to the developing eye and flies with the mutations cru, ark<sup>82</sup>, revealed an unusual phenotype that resulted in the homozygous mutant tissue appearing black, in contrast to the expected red. To map the location of this mutation, complementation tests against the Bloomington deficiency kit were conducted. Cru failed to complement previously characterized alleles of capping protein α (cpa). Thus, cpa<sup>cru</sup> is a novel allele of cpa and displays phenotypes similar to previously characterized alleles such as cpa 107E, cpa 69E, and cpa<sup>scrd</sup> . The human homolog, Cap Z, is conserved in humans and serves a similar role in act in filament regulation. 展开更多
关键词 Capping protein α APOPTOSIS Genetic screen Drosophila melanogaster
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Protein Flexibility and Multiple Docking in Ligand Docking and Virtual Screening to the BRAF(TypeⅠ1/2)Inhibitors
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作者 王路 张艳敏 +4 位作者 卢帅 唐伟方 陈亚东 陆涛 刘海春 《Chinese Journal of Structural Chemistry》 SCIE CAS CSCD 2018年第7期1057-1070,共14页
BRAF has been recognized as a promising target for cancer therapy. A number of crystal structures have been published. Molecular docking is one of the most effective techniques in the field of computer-aided drug des... BRAF has been recognized as a promising target for cancer therapy. A number of crystal structures have been published. Molecular docking is one of the most effective techniques in the field of computer-aided drug design(CADD). Appropriate protein conformation and docking method are essential for the successful virtual screening experiments. One approach considering protein flexibility and multiple docking methods was proposed in this study. Six DFG-in/αC-helix-out crystal structures of BRAF, three docking programs(Glide, GOLD and Ligand Fit) and 12 scoring functions were applied for the best combination by judging from the results of pose prediction and retrospective virtual screening(VS). The most accurate results(mean RMSD of about 0.6 A) of pose prediction were obtained with two complex structures(PDB: 3 C4 C and 3 SKC) using Glide SP. From the retrospective VS, the most active compounds were identified by using the complex structure of 3 SKC, indicated by a ROC/AUC score of 0.998 and an EF of 20.6 at 5% of the database screen with Glide-SP. On the whole, PDB 3 SKC could achieve a higher rate of correct reproduction, a better enrichment and more diverse compounds. A comparison of 3 SKC and the other X-ray crystal structures led to a rationale for the docking results. PDB 3 SKC could achieve a broad range of sulfonamide substitutions through an expanded hydrophobic pocket formed by a further shift of the αC-helix. Our study emphasized the necessity and significance of protein flexibility and scoring functions in both ligand docking and virtual screening. 展开更多
关键词 BRAF type 1/2 inhibitors protein flexibility multiple docking methods pose prediction virtual screening
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基于液基细胞学的hWAPL蛋白检验在高危型人乳头瘤病毒感染患者宫颈癌筛查中的价值研究
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作者 秦杰 张一娇 +2 位作者 路晓琳 董有伟 段爱红 《陕西医学杂志》 2025年第2期268-271,276,共5页
目的:探讨基于液基细胞学的人半翼(hWAPL)蛋白检验在高危型人乳头瘤病毒(HR-HPV)感染患者宫颈癌筛查中的价值。方法:选取300例HPV感染女性为研究对象。采用杂交捕获技术进行常规HPV-DNA检测,阴道镜下取宫颈组织标本行组织病理学检查,并... 目的:探讨基于液基细胞学的人半翼(hWAPL)蛋白检验在高危型人乳头瘤病毒(HR-HPV)感染患者宫颈癌筛查中的价值。方法:选取300例HPV感染女性为研究对象。采用杂交捕获技术进行常规HPV-DNA检测,阴道镜下取宫颈组织标本行组织病理学检查,并进行液基细胞学检查。采用免疫细胞化学技术检测液基细胞hWAPL蛋白表达。采用Kappa检验分析单独液基细胞学检查、液基细胞hWAPL蛋白检测与组织病理学检查结果的一致性,并比较单独液基细胞学检查和液基细胞hWAPL蛋白检测对HR-HPV感染患者宫颈癌的筛查效能。结果:300例宫颈液基细胞学标本中,共检测到18种HR-HPV亚型和5种低危型HPV亚型,其中HR-HPV感染患者236例,感染率为78.67%。组织病理学检查结果显示,阳性64例,阳性率为27.12%。单独液基细胞学检查结果显示,50例患者为阳性,阳性率为21.19%,与组织病理学检查一致性较高(Kappa=0.655,P<0.05)。液基细胞hWAPL蛋白检测结果显示,58例患者为阳性,阳性率为24.58%,与组织病理学检查一致性极好(Kappa=0.890,P<0.05)。与单独液基细胞学检查比较,液基细胞hWAPL蛋白检测筛查宫颈癌的敏感度、准确率、阳性预测值、阴性预测值升高,漏诊率降低(均P<0.05)。结论:基于液基细胞学的hWAPL蛋白检验对HR-HPV感染患者宫颈癌的筛查效能较高。 展开更多
关键词 宫颈癌 人半翼蛋白 人乳头瘤病毒 液基细胞学 组织病理学 筛查效能
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优良抗逆性能高蛋白酵母菌的筛选及特性研究
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作者 邓娟丽 孙长坡 +2 位作者 赵一凡 李天天 尹鹏 《粮油食品科技》 北大核心 2025年第1期192-199,共8页
我国蛋白质资源紧缺,影响饲料业和畜牧业发展,而酵母蛋白营养丰富、开发潜力强。为了筛选抗逆性能优良的高蛋白酵母菌株,实验以发面、牛瘤胃食糜、玉米浆等为筛菌对象,通过压力(pH为3.5)筛选、分离、鉴定得到不同酵母菌株,通过分析菌株... 我国蛋白质资源紧缺,影响饲料业和畜牧业发展,而酵母蛋白营养丰富、开发潜力强。为了筛选抗逆性能优良的高蛋白酵母菌株,实验以发面、牛瘤胃食糜、玉米浆等为筛菌对象,通过压力(pH为3.5)筛选、分离、鉴定得到不同酵母菌株,通过分析菌株的生长性能、菌体蛋白含量和风味确定了1株可用于酵母蛋白的潜力菌株。结果表明:从不同样品中筛选得到20株耐酸性酵母菌,鉴定出8个菌属酵母:酿酒酵母菌、毕赤酵母菌、马克斯克鲁维酵母菌、解脂耶氏酵母、热带假丝酵母、Starmerella bacillaris、鲁氏接合酵母和有孢汉逊酵母;在pH为3.5和7.0的摇瓶培养条件下,通过分析其生物量及菌体蛋白产量确定了两株性能较优的菌株,分别是毕赤酵母菌株B-1和解脂耶氏酵母菌株D-1;进一步通过风味分析,发现菌体B-1呈味核苷酸和谷氨酸含量均高于菌体D-1,且具有较高含量的酯类化合物。因此,确定B-1菌株作为潜力菌株并研究了B-1菌株的生长特征及抗逆性能,在pH≥2.0、温度≤40℃、盐(NaCl计)浓度(w/v)≤70 g/L的条件下均能较好生长。 展开更多
关键词 高蛋白酵母菌 筛选 风味 抗逆性能
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Recombinant Expression and Purification of Mouse Nectin-like 4 Glycoprotein in 293ET Cell Line
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作者 李冬冬 安泰 +3 位作者 刘枭 阴彬 彭小忠 舒鹏程 《Chinese Medical Sciences Journal》 CAS CSCD 2018年第1期1-8,共8页
Objective To screen the transient and stable cell lines with high production of Nectin-like 4(Necl-4)protein.Methods First,c DNA sequences encoding the extracellular domain of Necls were cloned into the modified vecto... Objective To screen the transient and stable cell lines with high production of Nectin-like 4(Necl-4)protein.Methods First,c DNA sequences encoding the extracellular domain of Necls were cloned into the modified vector p APtag at the N terminus of alkaline phosphatase(AP)for fusion expression.Next,293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis.Then,by adding N-glycosylation processing inhibitor kifunensine into the medium,complex glycan was inhibited to generate.The residual glycan of purified protein was removed by endoglycosidase H.Finally,AP protein was removed by using human rhinovirus protease and size exclusion chromatography.The concentration of purified Necl-4 protein was monitored by measuring the absorbance at280 nm and analyzed by SDS-PAGE.Results The transient and stable cell lines with high production of Necl-4 protein were screened by the color reaction with the AP-tag in the recombinant vector.The soluble and active form of purified Necl-4 protein was obtained after deglycosylation of native N-glycan protein with an expression level of 4 mg/L culture and purity of 95%.Conclusions By using modified AP mammalian protein expression system,we can easily screen the high productive stable cell lines by using AP activity assay.By adding mannosidase inhibitor kifunensine into the medium and cutting purified protein by using endoglycosidase H,we can obtain deglycosylated Necl-4 protein in milligram quantities.Our method might throw a light on the expression and purification of glycoprotein for structural and functional studies. 展开更多
关键词 Nectin-like IMMUNOGLOBULIN membrane protein fast and easy screening GLYCOSYLATION mammalian cell
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Synthesis and characteristic of a novel green fluorescent protein eYGFPuv 被引量:1
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作者 Shihang Fan Linbin Deng +4 位作者 Hongli Yang Liang Zhang Xingchao Sun Jinglin Liu Jing Liu 《Oil Crop Science》 2019年第2期65-74,共10页
Recently, a novel green fluorescent protein eYGFPuv has been identified in the marine organism Chiridius poppei which displays high fluorescence intensity and can be visible by eyes in dark. Although strong green fluo... Recently, a novel green fluorescent protein eYGFPuv has been identified in the marine organism Chiridius poppei which displays high fluorescence intensity and can be visible by eyes in dark. Although strong green fluorescence was achieved in transgenic petunia, 3 expression cassettes (about 8 kb) complicate its application. In this study, to confirm whether 1 expression cassette could be used as a transgenic marker in prokaryotes and eukaryotes, eYGFPuv was cloned into prokaryotic expression vector pET28α-eYGFPuv- His and plant binary expression vector 35S::eYGFPuv. Compared to EGFP, eYGFPuv protein exhibited stronger dazzling green fluorescence in E. coli under excited light at 365 nm and maintains steadily over a long period of time without degradation. When transiently expressed in tobacco leaves, eYGFPuv protein displayed strong green fluorescence. Moreover, the fluorescence of eYGFPuv protein also could be directly observed in living plant, and thus can be used easily as a marker to screen transformed lines in transgenic research. Overall, compared to previous studies on eYGFPuv tandem repeats, our data confirmed that single eYGFPuv sequence still possesses high fluorescence intensity and quenching resistance. Furthermore, because of small size of expression cassette,it is suitable for efficient transformation in both prokaryotic and eukaryotic organisms. 展开更多
关键词 eYGFPuv green FLUORESCENT protein screenING MARKER
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A High-affinity Activator of G551D-CFTR Chloride Channel Identified By High Throughput Screening 被引量:1
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作者 ZHAOLu HECheng-yan +4 位作者 LIUYan-li ZHOUHong-lan ZHOUJin-song SHANGDe-jing YANGHong 《Chemical Research in Chinese Universities》 SCIE CAS CSCD 2004年第6期738-742,共5页
A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activa... A stably transfected CHO cell line coexpressing G551D-CFTR and iodide-sensitive yellow fluorescent protein mutant EYFP-H148Q-I152L was successfully established and used as assay model to identify small-molecule activators of G551D-CFTR chloride channel from 100000 diverse combinatorial compounds by high throughput screening on a customized Beckman robotic system. A bicyclooctane compound was identified to activate G551D-CFTR chloride channel with high-affinity(K d=1.8 μmol/L). The activity of the bicyclooctane compound is G551D-CFTR-specific, reversible and non-toxic. The G551D-CFTR activator may be useful as a tool to study the mutant G551D-CFTR chloride channel structure and transport properties and as a candidate drug to cure cystic fibrosis caused by G551D-CFTR mutation. 展开更多
关键词 Cystic fibrosis Yellow fluorescent protein(YFP) High throughput screening(HTS) Small molecule
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Screening compounds against HCV based on MAVS/IFN-β pathway in a replicon model 被引量:2
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作者 Qiu-Xia Fu Li-Cui Wang +7 位作者 Shuai-Zheng Jia Bo Gao Yong Zhou Juan Du Ying-Li Wang Xiao-Hui Wang Jian-Chun Peng Lin-Sheng Zhan 《World Journal of Gastroenterology》 SCIE CAS CSCD 2010年第44期5582-5587,共6页
AIM:To develop a sensitive assay for screening compounds against hepatitis C virus (HCV).METHODS:The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling prote... AIM:To develop a sensitive assay for screening compounds against hepatitis C virus (HCV).METHODS:The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP),which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence.Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy.Cellular localization and protein levels were examined by Western blotting.RESULTS:HCV NS3/4A protease cleaved eYFP-MAVSfrom mitochondria to block the activation of interferon (IFN)-β promoter,thus resulting in downregulation of SEAP activity.The decrease in SEAP activity was proportional to the dose of active NS3/4A protease.Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A.CONCLUSION:Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors.This system will constitute a new tool to allow the efficient screening of HCV inhibitors. 展开更多
关键词 Mitochondrial antiviral signaling protein Hepatitis C virus INTERFERON-Β Drug screening
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C-Reactive Protein as a Triage Test in Guiding Who Should Get a Confirmatory Test for Pulmonary Tuberculosis Diagnosis among Adults: A Case-Control Proof-of-Concept Study from Urban Tanzania
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作者 Evarist Chiweka Thomas Maroa +8 位作者 Hosiana Temba Joseph Ponera Sharifa Athumani Lujeko Kamwela Mohamed Sasamalo Rastard Naftari Mirambi Tito Francis Mhimbira Jerry Hella 《Journal of Tuberculosis Research》 2022年第1期28-44,共17页
Background: The current screening tools for tuberculosis (TB) are inadequate resulting in insufficient TB case detection and continued community transmission of TB. As the world is geared into finding missing TB cases... Background: The current screening tools for tuberculosis (TB) are inadequate resulting in insufficient TB case detection and continued community transmission of TB. As the world is geared into finding missing TB cases, new strategies are called for to aid in rapid identification of TB cases. This study aimed to evaluate the role C-reactive protein (CRP) in triaging patients to get a definitive test for active pulmonary TB diagnosis in urban Tanzania. Methods: A case-control study was conducted among pulmonary TB (PTB) patients and contacts without active PTB. The diagnosis of PTB was performed using GeneXpert MTB/RIF assay and culture. Blood was collected from cases and controls for measuring CRP levels during recruitment. We compared socio-demographic characteristics, clinical and laboratory parameters obtained during recruitment and performed diagnostic accuracy analyses for CRP. Results: Out of all 193 study participants who were involved in final analysis, 147 (76.2%) were males. Pulmonary TB cases had significantly lower median BMI than controls (median 17.4 kg/m<sup>2</sup> [IQR: 15.8 - 19.2 kg/m<sup>2</sup>] vs., 24.9 kg/m<sup>2</sup> [IQR: 22.1 - 28.5 kg/m<sup>2</sup>), p < 0.001). There was no statistical difference in prevalence of HIV between PTB cases and controls i.e., 13.33% vs., 11.7%, p = 0.48. CRP was significantly higher in PTB cases vs., controls (median 67.8 mg/L, [IQR: 36.5 - 116.9 mg/L] vs., 1.55 mg/L, [IQR: 0.59 - 6.0 mg/L], p = 0.003). Furthermore, CRP at cut-off ≥10 mg/L was associated with best combination of sensitivity, specificity and area under the curve of 89.9%, 95% CI: 82.2 - 95.0, 80.9%, CI: 71.4 - 88.2 and 0.85, 95% CI: 0.80 - 0.90 respectively. A multivariate logistic regression model adjusted for fever, night sweats and body mass index showed that CRP above 10 mg/L was significantly associated with PTB, aOR 5.2, 95% CI 1.2 - 22.8. Conclusions: CRP at cut-off ≥10 mg/L can be used to screen pulmonary TB. These findings can be used to improve TB screening algorithm by incorporating CRP in combination with TB symptoms to identify patients who need further confirmatory TB tests. However, additional prospective studies are required to support our findings and contribute into policy recommendations on use of CRP in a screening algorithm for pulmonary TB. 展开更多
关键词 C-Reactive protein Pulmonary Tuberculosis screening Temeke Tanzania
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Establishment of Hypoglycemic Agent Screening Method Based on Human Glucokinase 被引量:1
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作者 CHOU-FEI WU,YANG XU~2,YONG TAO,AND JI-YAN YANG Key Laboratory of State Food Science and Technology,Jiangxi-OAI Joint Research Institute, Nanchang University,Nanchang 330047,Jiangxi,China 《Biomedical and Environmental Sciences》 SCIE CAS CSCD 2009年第1期62-69,共8页
Objective To establish a reliable platform for screening glucokinase activators (GKAs) in vitro. Methods Pancreatic glucokinase (PGK) protein expressed in a prokaryotic expression system as a histidine-tagged fusi... Objective To establish a reliable platform for screening glucokinase activators (GKAs) in vitro. Methods Pancreatic glucokinase (PGK) protein expressed in a prokaryotic expression system as a histidine-tagged fusion protein from Homo sapiens was produced. Then, response surface methodology (RSM) was used to optimize the microplate-based GKA screening platform. In the f'trst step of optimization with Plackett-Burman design (PBD), initial pH, reaction time and MgC12 were found to be important factors affecting the activity ratio of GKA (RO-28-1675) significantly. In the second step, a 23 full factorial central composite design (CCD) and RSM were applied to the optimal condition determination of each significant variable. A second-order polynomial was determined by a multiple regression analysis of the experimental data. Results The following optimal values for the critical factors were obtained: initial pH 0 (7.0), reaction time-0.63 (13.7 min) and MgC12 0.11 (2.11 mmol/L) with a predicted value of the maximum activity ratio of 34.1%. Conclusion Under the optimal conditions, the practical activity ratio is 34.8%. The determination coefficient (R2) is 0.9442, ensuring adequate credibility of the model. LLAE3, extracted from Folium nelumbinis in our laboratory, has prominently activated effects on PGK. 展开更多
关键词 screening mothod Human pancreatic glucokinase protein expression Glucokinase activators Response surfacemethodology
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肝癌患者外周血中抑制树突状细胞分化的蛋白成分筛选及鉴定
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作者 董晓微 郭东 +2 位作者 董育清 柳斌 樊瑞军 《宁夏医学杂志》 CAS 2024年第7期573-577,F0003,共6页
目的肝癌患者外周血中靶向抑制树突状细胞分化成熟的蛋白成分筛选及鉴定。方法利用免疫磁珠分选健康人外周血CD14^(+)单核细胞,诱导分化为未成熟的树突状细胞(iDCs),进一步诱导分化为成熟的树突状细胞(mDCs),鉴定iDCs纯度。酶切健康人... 目的肝癌患者外周血中靶向抑制树突状细胞分化成熟的蛋白成分筛选及鉴定。方法利用免疫磁珠分选健康人外周血CD14^(+)单核细胞,诱导分化为未成熟的树突状细胞(iDCs),进一步诱导分化为成熟的树突状细胞(mDCs),鉴定iDCs纯度。酶切健康人和肝癌患者血清孵育的iDCs的结合蛋白,回收酶切后的多肽,筛选对iDCs分化成熟有潜在调控作用的互作候选蛋白。靶向鉴定差异候选蛋白。构建过表达蛋白质粒,转染至正常肝细胞(HL7702),将细胞培养上清液与iDCs共同培养,诱导iDCs成熟,对特异性抑制iDCs成熟的肝癌源性血清蛋白进行特异性筛选和鉴定。结果TNF-α诱导前抗原标志物阳性细胞表达CD86^(+)比例为79.2%、黏附分子CD40^(+)比例为83.3%、MHC-Ⅱ抗原分子HLA-DR^(+)比例为67.6%、CD83^(+)比例为5.5%以及CD1a^(+)比例为13.7%,符合iDCs的抗原表型特征;TNF-α诱导后抗原标志物阳性细胞表达CD86^(+)比例为92.0%、CD40^(+)比例为89.0%、HLA-DR^(+)比例为68.3%、CD83^(+)比例为37%、CD1a^(+)比例为48.3%,提示iDCs已迅速分化成熟为mDCs,符合mDCs的抗原表型特征。初步确认40多种对iDCs分化成熟有潜在调控作用的互作候选蛋白。选择10种差异候选蛋白,对其过表达进行验证,显示AFP蛋白、AGXT蛋白、ATP1A1蛋白过表达能够明显抑制iDCs分化成熟为mDCs。结论共筛选出10种靶向抑制树突状细胞分化的肝癌外周血清蛋白,其中AFP蛋白、AGXT蛋白、ATP1A1蛋白能够显著抑制树突状细胞分化成熟。 展开更多
关键词 肝癌 外周血树突状细胞 抑制蛋白 筛选 鉴定
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山药DoWRKY40基因表达特征分析及互作蛋白筛选 被引量:1
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作者 邢丽南 张艳芳 +3 位作者 葛明然 赵令敏 陈妍 霍秀文 《生物技术通报》 CAS CSCD 北大核心 2024年第8期118-128,共11页
【目的】WRKY作为植物特异型转录因子,参与植物生长发育过程。克隆山药DoWRKY40基因,分析其表达模式,通过酵母双杂交技术筛选与DoWRKY40互作的蛋白,探究WRKY40在山药膨大过程中的调控机制。【方法】以‘大和长芋’山药为材料克隆DoWRKY4... 【目的】WRKY作为植物特异型转录因子,参与植物生长发育过程。克隆山药DoWRKY40基因,分析其表达模式,通过酵母双杂交技术筛选与DoWRKY40互作的蛋白,探究WRKY40在山药膨大过程中的调控机制。【方法】以‘大和长芋’山药为材料克隆DoWRKY40基因,并进行生物信息学分析、表达模式分析、亚细胞定位,构建山药块茎不同发育时期酵母文库,筛选与DoWRKY40互作蛋白。【结果】DoWRKY40的开放阅读框长为927 bp。DoWRKY40蛋白属于WRKY家族的第II组,含有一个典型的WRKY转录因子保守结构域,定位于细胞核。与几内亚薯蓣亲缘关系较近。DoWRKY40基因在山药块茎生长发育的120 d表达量最高,且在叶、茎和块茎中均有表达,具有组织特异性。山药cDNA文库的库容量为1.484×10^(8);pGBKT7-WRKY40诱饵载体无自激活活性。酵母双杂交法从山药文库中筛选到4类与DoWRKY40相互作用的蛋白,分别参与植物生长发育、细胞周期调节与细胞扩增、信号转导及环境胁迫应答等。【结论】克隆得到DoWRKY40基因,其响应山药的生长发育过程,筛选到的4类互作蛋白表明其在山药细胞膨大及信号转导中起调控作用。 展开更多
关键词 山药 WRKY40基因 表达特征 亚细胞定位 CDNA文库 酵母双杂交 互作蛋白筛选
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陆地棉抗黄萎病基因GhENODL6互作蛋白筛选
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作者 郑鑫鑫 张艳 +4 位作者 解美霞 张冬梅 吴立强 王省芬 杨君 《华北农学报》 CSCD 北大核心 2024年第3期173-178,共6页
研究发现陆地棉ENODL6基因具有抗黄萎病功能。为进一步揭示其在棉花抗黄萎病过程中发挥的作用,通过Nimble Cloning技术将GhENODL6插入酵母表达载体pNC-GBKT7,构建了重组质粒pNC-GBKT7-ENODL6。重组质粒转入酵母菌Y2HGold后可在DDO培养... 研究发现陆地棉ENODL6基因具有抗黄萎病功能。为进一步揭示其在棉花抗黄萎病过程中发挥的作用,通过Nimble Cloning技术将GhENODL6插入酵母表达载体pNC-GBKT7,构建了重组质粒pNC-GBKT7-ENODL6。重组质粒转入酵母菌Y2HGold后可在DDO培养基上正常生长,但不能生长于QDO/X/A培养基,表明GhENODL6蛋白对酵母宿主无毒害作用,没有自激活活性。将携带pNC-GBKT7-ENODL6诱饵载体的Y2HGold酵母菌与cDNA文库进行杂交筛选,结果获得一个能够显示蓝色的菌落。通过PCR扩增,在蓝色酵母菌中获得一段526 bp的非载体插入片段,与陆地棉基因组内基因WRKY47序列高度一致。通过同源扩增,从陆地棉中克隆到WRKY47开放读码框,全长1 587 bp,编码528个氨基酸残基。再次利用酵母双杂交技术确认了WRKY47与GhENODL6之间存在互作关系。综上,构建了重组载体pNC-GBKT7-ENODL6,并鉴定到与GhENODL6互作的蛋白WRKY47。 展开更多
关键词 陆地棉 黄萎病 酵母双杂交 互作蛋白 筛选
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木薯SAP11基因的功能分析
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作者 王文娟 李智博 +11 位作者 林晨俞 曹颖杰 李建君 郭鑫 邹良平 李文彬 赵平娟 于晓玲 张秀春 李淑霞 于晓惠 阮孟斌 《华南农业大学学报》 CAS CSCD 北大核心 2024年第4期495-504,共10页
[目的]木薯Manihot esculenta Crantz是全球热带地区重要的粮食作物和经济作物,在生长发育过程中极易遭受低温、干旱、盐碱等非生物胁迫而导致减产。胁迫相关蛋白(Stress-associated protein,SAP)是一类新型的A20/AN1锌指蛋白,在模式作... [目的]木薯Manihot esculenta Crantz是全球热带地区重要的粮食作物和经济作物,在生长发育过程中极易遭受低温、干旱、盐碱等非生物胁迫而导致减产。胁迫相关蛋白(Stress-associated protein,SAP)是一类新型的A20/AN1锌指蛋白,在模式作物应对多种非生物胁迫过程中发挥重要作用。目前,SAP基因在木薯应对非生物胁迫中的生物学功能尚不明确。本研究旨在分析木薯SAP家族成员的蛋白结构特征和表达模式,以及MeSAP11的互作蛋白,为进一步解析该家族基因在木薯抗逆中的功能提供理论支撑。[方法]利用生物信息学技术对木薯SAP家族成员的进化关系、蛋白基序信息以及时空表达模式开展系统分析。同时,通过qRT-PCR研究各基因成员在不同组织中的特异表达以及对不同非生物胁迫的响应。进一步运用酵母双杂交结合高通量测序技术获得与MeSAP11相互作用的蛋白及对应生物学通路。[结果]木薯SAP基因家族共6个大类16个成员,该家族成员在木薯根部和叶片中表达量较高,部分家族成员的表达在低温和盐胁迫中显著上调,在干旱、钾饥饿和氮饥饿显著下调。MeSAP11的表达受不同胁迫条件的显著调控,亚细胞定位结果表明MeSAP11蛋白主要定位在细胞核。利用酵母双杂交筛库技术筛选到256个与MeSAP11互作的蛋白,KEGG分析表明这些互作基因主要参与蛋白泛素化降解、内质网蛋白质加工通路等途径,暗示MeSAP11可能通过上述通路发挥功能。[结论]木薯SAP家族大部分成员显著响应低温、干旱、高盐以及缺氮、缺钾胁迫,研究结果为进一步研究MeSAP11在木薯响应非生物胁迫过程中的功能并解析其调控网络奠定了基础。下一步将把MeSAP11基因列为调控非生物逆境变化的候选基因开展深入研究。 展开更多
关键词 木薯 非生物胁迫 MeSAP基因 互作蛋白筛选
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抗氧化乳酸菌的筛选及其对夸克奶酪品质的影响
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作者 吴涵清 洪青 +3 位作者 刘振民 张灏 贾向飞 郝燕萍 《食品与发酵工业》 CAS CSCD 北大核心 2024年第22期286-293,共8页
为获得具有高蛋白水解及抗氧化能力的附属发酵剂,该文对12株乳酸菌的产酸、蛋白酶活性、蛋白水解和抗氧化能力进行综合评价,将筛选到的潜在有益菌株作为附属发酵剂制备夸克奶酪。通过测定奶酪的理化指标及贮藏期间奶酪的微生物、滴定酸... 为获得具有高蛋白水解及抗氧化能力的附属发酵剂,该文对12株乳酸菌的产酸、蛋白酶活性、蛋白水解和抗氧化能力进行综合评价,将筛选到的潜在有益菌株作为附属发酵剂制备夸克奶酪。通过测定奶酪的理化指标及贮藏期间奶酪的微生物、滴定酸度、质构和抗氧化活性的变化,评估附属发酵剂对奶酪品质的影响。结果表明,12株乳酸菌中马乳酒样乳杆菌(Lactobacillus kefiranofaciens)XZ-54产酸能力较弱,而蛋白酶活性高、蛋白水解和抗氧化能力强,具有成为夸克奶酪附属发酵剂的潜力。XZ-54的添加对夸克奶酪理化指标、硬度和涂抹性无显著影响(P>0.05),在21 d贮藏期内奶酪中XZ-54的活菌数均能维持在10^(7)CFU/g以上,且能显著提升奶酪DPPH自由基、ABTS阳离子自由基和羟自由基清除率。因此,马乳酒样乳杆菌XZ-54可以作为附属发酵剂应用于夸克奶酪,并能改善奶酪的抗氧化品质,具有开发和应用价值。 展开更多
关键词 附属发酵剂 筛选 蛋白质水解 抗氧化活性 夸克奶酪
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分子对接在筛选抗宫颈癌药物中的研究进展
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作者 王丹 张文艳 +6 位作者 罗仁洁 陈元静 韩雪 曲波 冯石芳 聂夏子 刘会玲 《中国临床药理学与治疗学》 CAS CSCD 北大核心 2024年第8期955-960,共6页
宫颈癌是最常见的妇科恶性肿瘤之一,在淋巴结转移和远处转移的情况下,五年生存率显著下降,因此研发新型抗宫颈癌药物对于治疗宫颈癌有重大的意义。分子对接技术是计算机辅助药物设计技术中最常用的研究方法之一,在筛选药物有效成分、发... 宫颈癌是最常见的妇科恶性肿瘤之一,在淋巴结转移和远处转移的情况下,五年生存率显著下降,因此研发新型抗宫颈癌药物对于治疗宫颈癌有重大的意义。分子对接技术是计算机辅助药物设计技术中最常用的研究方法之一,在筛选药物有效成分、发现药物作用于肿瘤的靶点以及探索抗肿瘤药物的作用机制中被广泛应用。本文将分子对接技术在抗宫颈癌药物筛选、抗肿瘤靶标确定方面以及抗宫颈癌作用机制等方面作一综述,为筛选抗宫颈癌药物及新药研发提供更充分的理论依据。 展开更多
关键词 宫颈癌 分子对接 虚拟筛选 靶标蛋白 作用机制
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异常糖链糖蛋白不同截断值排查肺癌的效能观察 被引量:1
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作者 农雪萍 黄建华 刘成英 《中国当代医药》 2024年第1期15-18,共4页
目的探究异常糖链糖蛋白(TAP)不同截断值排查肺癌的效能。方法选取江西省胸科医院2021年1月至2022年1月收治的66例肺癌患者与同期收治的66例非肺癌患者作为研究对象。计算肺癌与非肺癌疾病患者TAP凝聚物面积指标差异,判断TAP是否能用于... 目的探究异常糖链糖蛋白(TAP)不同截断值排查肺癌的效能。方法选取江西省胸科医院2021年1月至2022年1月收治的66例肺癌患者与同期收治的66例非肺癌患者作为研究对象。计算肺癌与非肺癌疾病患者TAP凝聚物面积指标差异,判断TAP是否能用于诊断肺癌疾病,通过绘制ROC曲线方式得到患者TAP的最佳截断值,进行肺癌诊断,判断诊断效能,并以临床常见TAP正常参考范围(<121μm^(2))与所得截断值比较诊断效能。结果肺癌组的TAP凝聚物面积大于非肺癌组,差异有统计学意义(P<0.05)。通过绘制ROC曲线,确定>170μm^(2)为最高截断值,比较其与临床常见TAP的正常参考范围(<121μm^(2))的诊断效能,结果显示二者的阳性检出率、灵敏度、特异度、阳性预测值、阴性预测值、诊断符合率比较,差异无统计学意义(P>0.05)。结论对疑似肺癌患者实行TAP不同截断值排查,以170μm^(2)截断值排查效果较佳,值得临床推广应用。 展开更多
关键词 异常糖链糖蛋白 截断值 肺癌排查 诊断效能
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