珙桐干种子总DNA提取方法的比较

以珙桐干种子为材料,采用简易CTAB法、SDS法、高盐沉淀法与高盐低pH值法分别对珙桐干种子进行了总DNA提取效果的比较分析。结果表明,四种方法均能有效地提取较高质量的珙桐种子DNA,其中SDS法效果最好,获得的DNA纯度最高,产量最大,CTAB法次之,高盐沉淀与高盐低pH值法获得DNA质量相对较低,低于其它两种方法。获得最佳的DNA提取方法,可为PCR扩增,引物设计,筛选珙桐种子中的相关基因奠定基础。     

Optimization of low-abundance protein extraction and abundant protein removal from defatted soybean meal

The aim of this study was to optimize the conditions for the extraction of low-abundance proteins (LAPs) and the removal of abundant proteins (APs; β-conglycinin and glycinin) from soybean meal. Single factor and orthogonal experiments were designed to determine the effects of four factors (isopropanol concentration, total extraction time, ultrasonic power, and ultrasonic time) on protein concentration in isopropanol extracts. Proteins in the isopropanol supernatant and the cold acetone precipitate of isopropanol were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). The results showed that the optimal conditions were 50% isopropanol, ultrasonic pretreatment for 15 min at 350 W, and a total extraction time of 1 h. Under these conditions, the protein concentration in the isopropanol extracts reached 0.8081 g/L. Many LAPs were detected, including β-amylase, soybean agglutinin, soybean trypsin inhibitor, fumarylacetoacetase-like, phospholipase D alpha 1-like, oleosin, and even some unknown soybean proteins. The soybean APs (β-conglycinin and glycinin) were not found. The method may be useful for discovering new soybean proteins and extracting enough LAPs of soybean to allow further studies of their physiological effects on animals without the influence of APs.     

基于磁珠处理的MALDI-TOF实验技术优化

目的:利用基质辅助激光解析离子化飞行时间质谱(matrix-assisted laser desorption/ionization time-offlight mass spectrometry,MALDI-TOF MS)技术检测血清中蛋白,目前常用磁珠来分离提纯,本研究目的是寻找一套有效的血清样品收集、处理、存储方法,应用于蛋白组学分析。方法:采用磁珠分离提纯人体血清,利用MALDI-TOF技术检测1000~10000 Da范围蛋白或多肽,考察其重复性及血清不同的冻融次数和基质与样品搭配比例对结果的影响。结果:人类血清的质谱图在1000~10000 Da范围内可检测到230多个峰,重复性极好。样品多次冻融对小分子量的多肽有一定影响。在适当范围内改变样品与基质比例对结果无影响。结论:基于磁珠分离提纯的MALDI-TOF技术在应用于人体血清检测时应用标准化程序可以减少变异。     

Review：Proteomic technology for biomarker profiling in cancer： an update

CANCER BIOMARKERS: CURRENT STATUS Despite advances in diagnostic imaging technol-ogy, surgical management, and therapeutic modalities,cancer remains a major cause of mortality worldwide.Currently, it is estimated that cancer kills over 6 mill-ion people per year worldwide, with over 10 millionnew cases being diagnosed every year. Mortality ismainly attributed to dissemination of primary cancerto distant organs, on which no effective treatment isavailable. The dilemma in oncology p…     

变色酸2B光度法测定血清蛋白质的研究

在pH1.62的Clark-Lubs(C-L)缓冲介质中,变色酸2B能与血清蛋白质形成有色复合物,其λmax为579nm,比试剂本身红移约65nm,表观摩尔吸光系数为2.09×105L·mol-1·cm-1(HSA),线性范围为0-100mg·L-1.研究了该反应体系的影响因素,确定了最佳反应条件,应用于人血清样品总蛋白的测定,结果与经典的考马斯亮蓝G-250法一致,而且线性范围宽,基本无干扰,操作与重现性都优于考马斯亮蓝法.     

紫色马铃薯Bora valley花青素的提取与含量的测定

用马铃薯的全薯、薯皮、薯肉作为实验材料,采用溶剂浸提（95％乙醇,0．1mol／L盐酸）法、超声波破碎提取法以及液氮-丙酮提取法这三种不同的实验方法对其花青素进行了提取,并使用分光光度计比色及pH示差法计算了花青素的含量．结果显示,不同的提取方法所测得的花青素相对浓度存在着一定的差异．其中,通过溶液浸提法得到的Bora的花青素含量为全薯为0．041mg·g^-1鲜重,薯皮0．109mg·g。鲜重,薯肉为0．030mg·g^-1鲜重．通过超声波破碎提取法得到的花青素的含量为全薯0．055mg·g^-1鲜重,薯皮0．156mg·g^-1鲜重,薯肉为0．038mg·g^-1鲜重．而通过液氮-丙酮提取法得到的花青素的含量为全薯0．028mg·g^-1鲜重,薯皮0．064mg·g^-1鲜重,薯肉为O．028mg·g^-1鲜重．其中,通过超声波破碎法得到的花青素的量较高．实验结果表明,Bora中较高的花青素含量可使其在天然抗氧化剂的提取及制备方面拥有更广阔的前景和应用价值．     

Fusion expression of pedA gene to obtain biologically active pediocin PA-1 in Escherichia coli

Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1. Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR), then cloned into vector pET32a(+), and expressed as thioredoxin-PedA fusion protein in the host strain E. coli BL21 (DE3). The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column. Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase. Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E. coli (pPA003PED1) using Listeria monocytogenes as the indicator strain. Thioredoxin-PedA fusion gene was further cloned into pET20b(+). Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces. The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E. coli (pPA003PED2). Therefore, biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.     

四种药用蕨类DNA提取的比较研究

以四种药用蕨类的叶片为材料,采用改进的CTAB法、SDS法分别对四种蕨类进行了总DNA提取效果的比较分析,结果表明,通过实验获得最佳的DNA提取方法,为PCR扩增,引物设计,为筛选四种药用蕨类中的活性成份的相关基因奠定理论和实践基础。两种方法均能有效的提取较高质量的DNA,其中CTAB法效果最好,获得的DNA纯度较高、产量较大;SDS法提取的量次之且多糖等杂质含量更多。     

茶废料中茶单宁提取工艺对比研究

以丙酮浓度、料液比、提取时间和提取温度为考察因素,分别采用微波法和超声波法进行正交试验,探讨了茶叶废料中提取茶单宁的最佳工艺参数.结果表明,采用微波提取法效果较好,其最佳提取工艺为：料液比为1∶60（g/g）,提取温度为40℃,提取时间为5 min,丙酮浓度为70%,茶单宁的提取量达215.04 mg/g.     

黑糯玉米多糖的提取及脱蛋白工艺的比较

本文研究了黑糯玉米多糖的提取及两种脱蛋白分离工艺的比较.采用热水浸提、醇沉法提取黑糯玉米多糖,用两种改进的方法(氯仿-正丁醇法和三氯乙酸-正丁醇法)脱除蛋白质,以脱蛋白率为指标,通过正交实验L9(34)确定最佳脱蛋白分离工艺后,对比两种方法的最佳组合粗多糖得率及510nm处吸光值的差异.结果表明:三氯乙酸-正丁醇法比氯仿-正丁醇法的脱蛋白效果好,最佳水平脱蛋白率为70.69%,多糖得率仅为67.87%;氯仿-正丁醇法最佳水平脱蛋白率为51.39%,多糖得率为80.31%.本文建立的优化工艺可为黑糯玉米多糖的分离制备提供参考.     

Comparison of volatile and semivolatile compounds fromcommercial cigarette by supercritical fluid extractionand simultaneous distillation extraction

Supercritical carbon dioxide fluid extraction (SFE) was studied as a rapid method for extraction of volatile and semivolatile compounds of Chinese commercial cigarettes. The method was compared with simultaneous distillation and extraction (SDE). Temperature and pressure for the SFE were optimized. The extracts obtained by the two methods showed different characters in composition and represented differently the flavor characteristics of tobacco; compared to SDE, SFE can extract compounds within a shorter time and avoid the thermal degradation and solvent contamination of samples. The extracts by the two extraction methods are complementary for investigating the flavor characteristic of tobacco products.     

A data-mining approach to biomarker identification from protein profiles using discrete stationary wavelet transform

Objective: To develop a new bioinformatic tool based on a data-mining approach for extraction of the most infor-mative proteins that could be used to fred the potential biomarkers for the detection of cancer. Methods: Two independent datasets from serum samples of 253 ovarian cancer and 167 breast cancer patients were used. The samples were examined by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). The datasets were used to extract the informative proteins using a data-mining method in the discrete stationary wavelet transform domain. As a dimensionality re-duction procedure, the hard threshoiding method was applied to reduce the number of wavelet coefficients. Also, a distance measure was used to select the most discriminative coefficients. To find the potential biomarkers using the selected wavelet coefficients, we applied the inverse discrete stationary wavelet transform combined with a two-sided t-test. Results: From the ovarian cancer dataset, a set of five proteins were detected as potential biomarkers that could be used to identify the cancer patients from the healthy cases with accuracy, sensitivity, and specificity of 100%. Also, from the breast cancer dataset, a set of eight proteins were found as the potential biomarkers that could separate the healthy cases from the cancer patients with accuracy of 98.26%, sensitivity of 100%, and specificity of 95.6%. Conclusion: The results have shown that the new bioinformatic tool can be used in combination with the high-throughput proteomic data such as SELDI-TOF MS to find the potential biomarkers with high discriminative power.     

入侵植物五爪金龙全蛋白质提取及双向电泳的优化

五爪金龙是粤东及整个华南地区危害最为严重的入侵杂草之一.探索五爪金龙的全蛋白质提取方法及优化其二维电泳的条件,可以为将来深入研究五爪金龙的蛋白质组学奠定基础.采用TCA-丙酮法、Trizol法以及酚抽提法提取五爪金龙全蛋白质,测定各种方法提取蛋白质的浓度,等量上样于二维电泳11 cm胶条上,结果显示TCA-丙酮法提取五爪金龙全蛋白质的二维电泳图片最好.优化二维电泳条件显示,在用TCA-丙酮法提取五爪金龙全蛋白质,上样量为40μg,等电聚焦总电压30 000 Vh时二维电泳图谱效果最好,重现性最高.     

Comparison of volatile and semivolatile compounds from commercial cigarette by supercritical fluid extraction and simultaneous distillation extraction

Supercritical carbon dioxide fluid extraction (SFE) was studied as a rapid method for extraction of volatile and semivolatile compounds of Chinese commercial cigarettes. The method was compared with simultaneous distillation and extraction (SDE). Temperature and pressure for the SFE were optimized. The extracts obtained by the two methods showed different characters in composition and represented differently the flavor characteristics of tobacco; compared to SDE, SFE can extract compounds within a shorter time and avoid the thermal degradation and solvent contamination of samples. The extracts by the two extraction methods are complementary for investigating the flavor characteristic of tobacco products.     

四羧基铝酞菁与蛋白质结合反应及其在测定蛋白质中的应用

在pH=5．32的Bhtton—Robinson缓冲介质中,四羧基铝酞菁（AlC4Pc）与牛血清白蛋白（BSA）发生结合反应,引起AlC4Pc吸光度降低,降低的吸光度值△A与蛋白质的浓度在一定范围内成正比。基于此,建立了AlC4Pc为光谱探针,分光光度法测定蛋白质含量的新方法。该方法灵敏度较高,线性关系好,BSA的含量在3．7～32．5μg.mL^-1范围内与体系的吸光度降低值呈现良好的线性关系;表观摩尔吸收系数ε691=4．55×10^5L.mol^-1．cm^-1,检测限为3．0μgmL^-1。方法用于人血清样品中总蛋白的测定,实验结果与考马斯亮蓝G-250法一致。     

狗血浆中哌唑嗪的含量测定方法及其在药代动力学中的应用

建立并评价了一种简单灵敏的方法测定狗血浆中PZS的含量.该方法利用HPLC-FLD结合乙醚萃取的前处理方法,采用C18色谱柱(150 mm×4.6 mm,5 μm),流动相为30％乙腈和70％乙酸-乙酸钠缓冲液(pH=3.6),荧光检测器的激发波长为258 nm,发射波长为387 nm,在流动相流速为1.0 mL/mi...     

简便快捷提取大蒜总DNA的方法

目的:探讨简便快速而高效的提取大蒜总DNA的方法。方法:采用十六烷基三甲基溴化胺(CTAB)法、简化CTAB法、改良十二烷基硫酸钠(SDS)法分别以大蒜的根、茎、叶为材料提取细胞总DNA,并进行紫外分光检测和琼脂糖凝胶电泳分析。结果:三种方法的提取率和纯度明显不同:CTAB法>SDS法>简化CTAB法,三种材料组织中以茎的提取效果最佳。结论:采用CTAB法或SDS法从茎中提取大蒜总DNA可获得产率与纯度符合要求的DNA样品。     

Progress in Chemical Synthesis of Peptides and Proteins

For the proteins that cannot be expressed exactly by cell expression technology(e.g., proteins with multiple posttranslational modifications or toxic proteins), chemical synthesis is an important substitute. Given the limited peptide length offered by solid-phase peptide synthesis invented by Professor Merrifield, peptide ligation plays a key role in long peptide or protein synthesis by ligating two small peptides to a long one. Moreover, high-molecularweight proteins must be synthesized using two or more peptide ligation steps, and sequential peptide ligation is such an efficient way. In this paper, we reviewed the development of chemical protein synthesis, including solid-phase peptide synthesis, chemical ligation, and sequential chemical ligation.     

赣南脐橙果园土壤重金属形态分布特征研究

在赣南18个脐橙产区县(市),根据不同土壤类型,选择具有代表性的果园约90个作为取样果园,采用BCR法对土壤样品的土壤重金属的形态进行分析,研究果园土壤中不同重金属元素的形态分布特征.结果表明:赣南脐橙果园土壤中生物有效性重金属形态含量所占比例从高到低依次为:Pb>Cu>Zn>Ni>Cr,同时,重金属元素Ni、Cu、Cr、Zn和Pb在不同类型土壤中的含量和形态分布规律基本一致,且均以残渣态为主,五种重金属元素的潜在环境风险都较小.     

Science Letters: Proteomic analysis of the serum in patients with idiopathic pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a rare disease of unknown etiology. The exact pathogenesis of pulmonary arterial hypertension is still not well known. In the past decades, many protein molecules have been found to be involved in the development of IPAH. With proteomic techniques, profiling of human plasma proteome becomes more feasible in searching for disease-related markers. In present study, we showed the protein expression profiles of the serum of IPAH and healthy controls after depleting a few high-abundant proteins in serum. Thirteen spots had changed significantly in IPAH compared with healthy controls and were identified by LC-MS/MS. Alpha-1-antitrypsin and vitronectin were down-regulated in IPAH and may be valuable candidates for further explorations of their roles in the development of IPAH.