疯牛病和羊痒病Western blotting检测方法的建立

以朊蛋白单抗AH6和碱性磷酸酶标记的马抗鼠酶标二抗建立了疯牛病和羊痒病的Western blotting检测方法。对Western blotting各种反应条件进行摸索,并确定了最佳工作条件,结果表明:匀浆缓冲液为RIPA时的最佳反应条件包括浓缩胶电泳电压为恒压90V,分离胶电泳电压为恒压160V,转印的最佳电压和时间为恒压100V1.5h;封闭液为3%BSA时,封闭15min,封闭效果最好;AH6的最佳稀释度为1∶4000,4℃下孵育过夜,马抗鼠二抗的最佳稀释度1∶1000,室温下孵育30min。采用已确立的反应条件对样品进行检测并与Prionics-Check WEST-ERN进口试剂盒的检测结果比较,发现其敏感性为100%,特异性为99.4%,与进口试剂盒(100%,100%)无显著差异,这为国产试剂盒研制提供了条件。

拟南芥DBB1a(double B-box 1a)蛋白的表达、纯化及Western blotting检测

PCR扩增拟南芥(Arabidopsis thaliana)DBB1a cDNA的保守区段(GenBank登录号:AT2G21320),转化到冷诱导表达载体pCold TF上,构建pCold-DBB1a重组质粒,转化大肠杆菌DH5a。15℃下IPTG诱导表达融合蛋白,并通过SDS-PAGE检测。证实目的蛋白以可溶形式在约20 kD处高效表达,与预期蛋白大小相吻合。表达蛋白经Ni琼脂糖凝胶亲和层析纯化,SDS-PAGE及Western blotting检测证实纯化后获得高纯度融合蛋白,这为进一步研究DBBl a功能奠定了基础。

医学检验专业开设Western Blotting综合性实验初探

分子生物学是当前生命科学发展的主流，其理论与技术已广泛应用于各个领域。Western Blottlng作为分子生物学的常规技术，在医学领域具有不可忽视的地位。因此，在医学检验专业开设Western Blotting实验课程，已成为一项非常必要的实验教学内容。其一，Western Blotting实验是分子生物学与免疫学的有效结合体，有助于学生对前沿的、跨学科知识加深了解；

用western blotting方法检测乙醇固定细胞中细胞周期素的表达

用 western blotting法测定乙醇固定细胞中细胞周期素 (cyclin)的表达情况 ,探索 western blotting和流式细胞仪对固定细胞在流式细胞术分选前或分选后对同批标本进行蛋白同步分析的可行性。采用对数生长期的 Molt- 4细胞 ,经两种常规固定方法固定后 ,用 western blotting方法检测 cyclin A、B1 、D和 E的表达。另取乙醇固定经流式细胞仪分选的 G1 期和 G2 / M期细胞裂解后 ,用 western blotting方法检测不同时相细胞中 cyclin B1 的表达。结果显示从固定细胞中提取的蛋白经 western blotting检测可得到清晰且分子量正确的条带 ,两种不同方法固定的细胞中检测到的 cyclins表达未见明显差异。乙醇固定细胞经分选后提取蛋白行 western blotting检测可见 G2 / M期细胞的 cyclin B1 有明显表达而 G1 期细胞不明显。细胞进行固定、洗涤、染色和分选等处理后不影响 western blotting对其 cyclins表达的分析 ,说明用 western

高内涵分析及Western blotting法在蛋白质核质分布研究中的应用及比较研究

目的使用不同方法对蛋白质的核质分布进行检测并比较差异。方法细胞经免疫荧光染色后使用高内涵分析(high content analysis,HCA)对蛋白质的细胞质或细胞核总荧光强度进行分析;分别提取胞质及胞核蛋白,使用Western blotting法对核质组分中的蛋白质含量进行鉴定。结果两种方法均可检测到蛋白质的出核转运;经定量分析,HCA法检测到的HuR蛋白质/核比升高倍数小于Western blotting法。结论使用HCA法检测蛋白质核质定位具有准确、客观、成本低、快捷、多参数等特点,不失为Western blotting的有效补充及替代方法。

肝脏特异性表达载体的构建及Western blotting检测

研究靶向于肝细胞的组织特异性siRNA,实现siRNA基因治疗的组织特异性,可用于特异性治疗乙肝,突破RNA干扰技术在临床应用的一大障碍。用靶向于EGFP的siRNA(以下简称siEGFP)替代靶向于乙肝病毒保守区的siRNA,构建可以在肝细胞中特异性表达的载体,用来表达siRNA。将目标载体分别转染肝癌细胞HepG2、乳腺癌细胞MDB-MB-231、人胚肾细胞293,在蛋白质水平上检验siRNA抑制的组织特异性。Western blotting结果证实,siEGFP在肝组织来源的细胞系HepG2中抑制效率明显高于其他两组细胞。构建的载体可以在肝癌细胞系中特异性的表达siRNA,而在其他组织细胞中不表达,实现了组织特异性。进一步将siEGFP替换为抑制HBV表达的siRNA,同样可以实现肝脏特异性的表达。

快慢转法及不同滤膜和显色检测法在Western blotting中的应用分析

目的探讨快、慢转法及不同滤膜和显色检测法在Western blotting中的应用及各实验环节分析。方法采用MDA-MB-231细胞制备细胞蛋白,应用不同印迹膜[硝酸纤维素膜、聚偏乙烯二氟(PVDF)膜和阳离子尼龙膜]分别采用快转法和慢转法进行增强化学发光法(ECL)和DAB化学显色法检测,并对Western blotting的各实验环节进行了分析。结果(1)在快、慢转法中硝纤膜的蛋白预染marker条带略强于尼龙膜,而尼龙膜又略强于PVDF膜;PVDF膜和尼龙膜的正反面易于混淆,而硝纤膜的正反面不易混淆。(2)在快、慢转法中,PVDF膜的DAB化学显色法图像略强于尼龙膜和硝纤膜,而尼龙膜和硝纤膜无明显差异;与慢转法相比,快转的硝纤膜和尼龙膜中的蛋白条带略呈波浪状。(3)在慢转法中三种膜化学发光法的图像无明显背景,但在快转法中尼龙膜的背景较明显,而硝纤膜和PVDF膜亦无明显背景。结论应根据实验需要选用不同的印迹膜;慢转法通常优于快转法,增强化学发光法优于DAB化学显色法;增强化学发光法特异胜强、灵敏度高,是分析蛋白质表达较为理想的方法。

Western blotting中低丰度蛋白转膜方法改进

目的:改进Western blotting转膜方法,提高低丰度蛋白转膜效率。方法:在Western blotting转膜过程中,采用不同孔径的PVDF膜、不同的转膜时间及不同的甲醇含量进行转膜。转膜后,考马斯亮蓝染色观察PAGE胶上蛋白残留情况,ECL发光法检测FGFR1曝光强度。结果:通过改变膜孔径大小和转膜时间所得实验结果可见,孔径为0.22μm的PVDF膜转移2h的转膜效率更高。通过改变转膜液中甲醇含量所得实验结果可见,甲醇含量为10%转膜效率更高。结论:本实验对Western blotting转膜过程中低丰度蛋白的转膜方法进行了成功改进,改进后能提高蛋白显影强度,具有广泛的应用前景,是一种方便可行的操作方法。

In Search of Regulators of <i>LeSPL-CNR</i>by South-Western Blotting and Yeast One-Hybrid Library Screening System

LeSPL-CNR is a crucial transcription factor for fruit ripening of Solanum lycopersicum. The cnr (colorless non-ripening) epimutation resulted from hypermethylation in a 286 bp region of LeSPL-CNR promoter inhibits normal fruit ripening. In present study, potential regulators of LeSPL-CNR, which could bind to the specific 286 bp region, were screened via south-western blotting and yeast one-hybrid (Y1H) library screening system. Results indicated that a total of 13 and 19 candidate proteins were acquired respectively, and both ribulose-1,5-bisphosphate carboxylase/oxygenase and 40S ribosomal protein were identified by two methods. These would provide some information for revealing roles of DNA methylation and the regulatory mechanism for LeSPL-CNR.

采用Western blotting的方法分析蚕蛹致敏原成份

目的 应用Western blotting的方法检测蚕蛹（silkworm chrysalis）浸出液中致敏原的成份，为蚕蛹致敏原的研究和临床诊断提供依据。方法取蚕蛹浸出液，经SDS-聚丙烯酰胺凝胶分离，再转印到PVDF膜上，封闭后将血清与膜条共同孵育，后与辣根过氧化物酶标记的羊抗人IgE抗体反应，显色底物用对氨基联苯胺。结果SDS-PAGE显示蚕蛹可辨条带有12条，分子量在14～94kD之间，其中主带有8条，分子量依次为80kD、68kD、62kD、50kD、29kD、23KD、18kD、16kD。Western blotting结果表明，22例蚕蛹过敏患者血清全部呈阳性反应，浸出液中共有6条致敏条带，其中分子量68kD和29kD是主要致敏组份，阳性反应率均为100％。结论蚕蛹分子量68KD和29kD的组份为主要致敏组份，结果可为开发适舍我国特色的蚕蛹变应原提供依据。

利用生物素标记法和 Western blotting技术检测血小板膜糖蛋白

为了建立一种简便的血小板膜糖蛋白检测方法,用生物素标记血小板膜糖蛋白,然后用westernblotting法进行检测。结果显示生物素可标记十几种血小板膜糖蛋白。用以标记的生物素浓度以5mmol/L较为适宜。在Western blotting图谱上,血小板无力症患者的膜糖蛋白Ⅱb、Ⅲa与正常对照相比明显减少。结果提示该方法可用于血小板膜糖蛋白减少性疾病的检测。

Western blotting检测survivin封闭载体转染大肠癌SW620细胞的实验研究

【目的】探讨KNA interferenece(RNAi)对survivin在大肠癌细胞SW620表达的影响及siRNA干扰后诱导survivin蛋白表达降低的机制。【方法】构建survivin特异性的RNA封闭性载体survivin,通过培养大肠癌细胞SW620,survivin封闭性载体转染SW620细胞;Hoechest染色大肠癌细胞,通过荧光显微镜观察细胞形态变化;Western blotting分析肿瘤细胞的蛋白表达情况。【结果】DNA测序证实表达质粒构建成功;免疫荧光显微镜可见细胞凋亡小体;Western blotting显示:大肠癌survivin的蛋白表达明显弱于对照组。【结论】Survivin与大肠癌的发生存在一定的关系,RNAi能够下调survivin的表达。

A Physics-informed Deep-learning Intensity Prediction Scheme for Tropical Cyclones over the Western North Pacific

Accurate prediction of tropical cyclone(TC)intensity is challenging due to the complex physical processes involved.Here,we introduce a new TC intensity prediction scheme for the western North Pacific(WNP)based on a time-dependent theory of TC intensification,termed the energetically based dynamical system(EBDS)model,together with the use of a long short-term memory(LSTM)neural network.In time-dependent theory,TC intensity change is controlled by both the internal dynamics of the TC system and various environmental factors,expressed as environmental dynamical efficiency.The LSTM neural network is used to predict the environmental dynamical efficiency in the EBDS model trained using besttrack TC data and global reanalysis data during 1982–2017.The transfer learning and ensemble methods are used to retrain the scheme using the environmental factors predicted by the Global Forecast System(GFS)of the National Centers for Environmental Prediction during 2017–21.The predicted environmental dynamical efficiency is finally iterated into the EBDS equations to predict TC intensity.The new scheme is evaluated for TC intensity prediction using both reanalysis data and the GFS prediction data.The intensity prediction by the new scheme shows better skill than the official prediction from the China Meteorological Administration(CMA)and those by other state-of-art statistical and dynamical forecast systems,except for the 72-h forecast.Particularly at the longer lead times of 96 h and 120 h,the new scheme has smaller forecast errors,with a more than 30%improvement over the official forecasts.

Quantitative effect of kerogen type on the hydrocarbon generation potential of Paleogene lacustrine source rocks,Liaohe Western Depression,China

Kerogen types exert a decisive effect on the onset and capacity of hydrocarbon generation of source rocks.Lacustrine source rocks in the Liaohe Western Depression are characterized by thick deposition,high total organic carbon(TOC)content,various kerogen types,and a wide range of thermal maturity.Consequently,their hydrocarbon generation potential and resource estimation can be misinterpreted.In this study,geochemical tests,numerical analysis,hydrocarbon generation kinetics,and basin modeling were integrated to investigate the differential effects of kerogen types on the hydrocarbon generation potential of lacustrine source rocks.Optimized hydrocarbon generation and expulsion(HGE)models of different kerogen types were established quantitatively upon abundant Rock-Eval/TOC/vitrinite reflectance(R_(o))datasets.Three sets of good-excellent source rocks deposited in the fourth(Es4),third(Es3),and first(Es1)members of Paleogene Shahejie Formation,are predominantly types I-II_(1),II_(1)-II_(2),and II-III,respectively.The activation energy of types I-II_(2)kerogen is concentrated(180-230 kcal/mol),whereas that of type III kerogen is widely distributed(150-280 kcal/mol).The original hydrocarbon generation potentials of types I,II_(1),II_(2),and III kerogens are 790,510,270,and 85 mg/g TOC,respectively.The Ro values of the hydrocarbon generation threshold for type I-III source rocks gradually increase from 0.42%to 0.74%,and Ro values of the hydrocarbon expulsion threshold increase from 0.49%to 0.87%.Types I and II_(1)source rocks are characterized by earlier hydrocarbon generation,more rapid hydrocarbon expulsion,and narrower hydrocarbon generation windows than types II_(2)and III source rocks.The kerogen types also affect the HGE history and resource potential.Three types(conventional,tight,and shale oil/gas)and three levels(realistic,expected,and prospective)of hydrocarbon resources of different members in the Liaohe Western Depression are evaluated.Findings suggest that the Es3 member has considerable conventional and unconventional hydrocarbon resources.This study can quantitatively characterize the hydrocarbon generation potential of source rocks with different kerogen types,and facilitate a quick and accurate assessment of hydrocarbon resources,providing strategies for future oil and gas exploration.

应用western blotting检测结直肠癌中FHIT蛋白的表达

目的探讨免疫印迹(western blotting)技术在分析结直肠癌脆性组氨酸三联体(FHIT)基因蛋白表达中的应用。方法应用western blotting检测30例结直肠癌及其癌旁正常组织(≥5 cm)脆性组氨酸三联体(FHIT)基因蛋白情况,并分析FHIT蛋白表达与各临床病理指标的相互关系。结果癌组织FHIT表达强度较正常黏膜高或者相等者为13例(43.3%),较正常黏膜低者为17例(56.7%)。FHIT蛋白低表达癌在癌组织学分级中的分布为Ⅰ级癌3/11,Ⅱ级癌8/12,Ⅲ级癌6/7,各级癌组间比较,有显著性差异(P<0.05)。FHIT蛋白低表达癌在Dukes分期中的分布为A期+B期癌6/17,C期+D期癌11/13,已伴淋巴结转移组的C、D期癌与未转移组的A、B期癌比较,有显著性差异(P<0.05)。结论Western blotting法特异性强、灵敏度高,是分析FHIT蛋白质表达较为理想的方法;FHIT蛋白表达状况可能与组织学分级、Dukes分期及淋巴结转移相关,提示FHIT蛋白表达降低可能对结直肠癌发生、发展具有重要作用。

Natural gas exploration potential and favorable targets of Permian Fengcheng Formation in the western Central Depression of Junggar Basin,NW China

Based on the organic geochemical data and the molecular and stable carbon isotopic compositions of natural gas of the Lower Permian Fengcheng Formation in the western Central Depression of Junggar Basin,combined with sedimentary environment analysis and hydrocarbon-generating simulation,the gas-generating potential of the Fengcheng source rock is evaluated,the distribution of large-scale effective source kitchen is described,the genetic types of natural gas are clarified,and four types of favorable exploration targets are selected.The results show that:(1)The Fengcheng Formation is a set of oil-prone source rocks,and the retained liquid hydrocarbon is conducive to late cracking into gas,with characteristics of high gas-generating potential and late accumulation;(2)The maximum thickness of Fengcheng source rock reaches 900 m.The source rock has entered the main gas-generating stage in Penyijingxi and Shawan sags,and the area with gas-generating intensity greater than 20×10^(8) m^(3)/km^(2) is approximately 6500 km^(2).(3)Around the western Central Depression,highly mature oil-type gas with light carbon isotope composition was identified to be derived from the Fengcheng source rocks mainly,while the rest was coal-derived gas from the Carboniferous source rock;(4)Four types of favorable exploration targets with exploration potential were developed in the western Central Depression which are structural traps neighboring to the source,stratigraphic traps neighboring to the source,shale-gas type within the source,and structural traps within the source.Great attention should be paid to these targets.

Geomorphic signatures and active tectonics in western Saurashtra,Gujarat,India

Active tectonics in an area includes ongoing or recent geologic events.This paper investigates the tectonic influence on the subsidence,uplift and tilt of western Saurashtra through morphotectonic analysis of ten watersheds along with characteristics of relief and drainage orientation.Watersheds 7-9 to the north(N)are tectonically active,which can be linked with the North Kathiawar Fault System(NKFS)and followed by watersheds 6,10,1,4 and 5.Stream-length gradient index and sinuosity index indicate the effect of tectonic events along the master streams in watersheds 6-9.Higher R^(2)values of the linear curve fit for watershed 7 indicate its master stream is much more tectonically active than the others.The R^(2)curve fitting model and earthquake magnitude/depth analysis confirm the region to be active.The reactivation of the NKFS most likely led to the vertical movement of western Saurashtra.

Geophysical and Crustal Movement Characteristic of Western Sichuan

Analysis and research of large complex phenomena before and after the devastating earthquake to reduce the threat of natural disasters to human survival environment is of great significance.This paper analyzes the Wenchuan earthquake zone characteristics of gravity anomaly distributions,and then use edge detection and Euler deconvolution method to inverse Longmenshan gravity anomaly before earthquake.Fault distribution features and the general depth about the fault top of Longmenshan and its adjacent area before earthquake has been obtained.Morphology difference and possible earthquake formation have been analyzed through the Euler deconvolution result of gravity anomaly profile before and after the earthquake.

Food Hygiene and Safety Practices amongst Food Vendors in the Western Area, Sierra Leone

Food safety and hygiene practices require a multisectoral approach including food, water, sanitation, waste management, transport, education, trade, policies and programs that enable safe food preparation, storage and service. Unsafe food can cause illness keeping people from achieving their full potential and death. This was a descriptive study that uses a mixed method approach to derive insights into the characteristics of food vendors related to demography, knowledge, practices, infrastructure, compliance and recommendation for a policymaking framework. Using the Lemeshows’ sample size formula, 473 vendors from formal (restaurants) and informal (cookri-baffa/table top) sites were interviewed and observed. We found from discussions that respondents had a good understanding on how to keep food safe. However, observed practices were poor 93% handled food with their bare hands, 83% did not cover their hair, and 76% did not wear an apron whilst handling, preparing or serving food, 61% did not keep their finger nails clean or short and 57% did not wash their hand before preparing or serving food. Over half (51%) had access to a toilet but 32% reported their use required payment and emphasized their poor condition/inadequate management. Nearly half (47%) of the vending sites did not have a handwashing facility, with soap and water available. Only 7% reported having any authority oversight of food safety. Food safety and hygiene practices in most cookri shops and restaurants was ‘poor’ with very limited surveillance system in place by competent authorities for compliance of food operators. Hand washing, clean surroundings, and covered food were the most common and emphasized practices to mitigate the risks associated with unsafe food.

Climate warming is significantly influenced by rising summer maximum temperatures: insights from tree-ring evidence of the Western Tianshan Mountains, China

As one of the regions most affected by global cli-mate warming,the Tianshan mountains has experienced sev-eral ecological crises,including retreating glaciers and water deficits.Climate warming in these mountains is considered mainly to be caused by increases in minimum temperatures and winter temperatures,while the influence of maximum temperatures is unclear.In this study,a 300-year tree-ring chronology developed from the Western Tianshan Moun-tains was used to reconstruct the summer(June-August)maximum temperature(T_(max6-8))variations from 1718 to 2017.The reconstruction explained 53.1% of the variance in the observed T_(max6-8).Over the past 300 years,the T_(max6-8)reconstruction showed clear interannual and decadal vari-abilities.There was a significant warming trend(0.18°C/decade)after the 1950s,which was close to the increasing rates of the minimum and mean temperatures.The increase in maximum temperature was also present over the whole Tianshan mountains and its impact on climate warming has increased.The T_(max6-8) variations in the Western Tianshan mountains were influenced by frequent volcanic eruptions combined with the influence of solar activity and the sum-mer North Atlantic Oscillation.This study reveals that cli-mate warming is significantly influenced by the increase in maximum temperatures and clarifies possible driving mech-anisms of temperature variations in the Western Tianshan mountains which should aid climate predictions.