玉米细菌性枯萎病菌EGase内切葡聚糖酶蛋白纯化研究初探

目的研究玉米细菌性枯萎病菌EGase内切葡聚糖酶蛋白纯化的方法。方法以玉米细菌性枯萎病菌(Pantoea stewartii subsp.stewartii)菌株为材料,分离纯化其内切葡聚糖酶Egase。制备EGase浓缩液,浓缩液经Sephradex~(TM)G-75凝胶过滤层析和DEAE-Sephorose Fast Flow阴离子交换柱层析等提纯步骤,获得了凝胶电泳均一的内切葡聚糖酶。结果经变性聚丙烯酰胺凝胶电泳检测为一条电泳带,纯化后的EGase是单体蛋白,分子量约为72.3 k Da。Egase酶反应的最适温度是60℃,最适pH为5.0。结论本研究从玉米细菌性枯萎病菌中分离得到了一种新的内切葡聚合糖酶,对其部分性质进行了表述,为后续EGase基因的克隆及表达研究提供了研究基础。     

绿茶抗氧化肽的分离纯化与抗氧化活性研究

通过饱和硫酸铵盐析的方法从干绿茶中提取得到绿茶粗蛋白,粗蛋白透析除盐后用葡聚糖凝胶Sephadex G-75层析柱进行凝胶色谱法分离纯化,收集具有抗氧化活性的蛋白样品,并冷冻干燥,同时利用SDS-PAGE的方法对凝胶分离后收集的活性样品进行纯度的鉴定及相对分子质量的测定,并采用·OH清除法及O-2·清除法研究绿茶抗氧化多肽纯品的抗氧化活性。结果表明,绿茶抗氧化多肽经葡聚糖凝胶sephadex G-75层析柱后,得到很好的分离和纯化,在SDS-PAGE电泳图谱上只有1条条带,其相对分子质量为2~15ku;绿茶抗氧化多肽对·OH具有很强的清除作用,其半清除浓度(IC50)为0.069μg/mL;对O-2·也具有较强的清除作用,其半清除浓度(IC50)为18.462μg/mL,与VC的清除作用(IC50为11.186μg/mL)相当。     

小红豆抗菌蛋白的分离纯化及其功能活性

研究旨在从小红豆中分离纯化得到一种具有抗菌活性的蛋白,对其部分生物活性进行表征和研究。通过缓冲液抽提(NaAc-HAc,20 mmol/L,pH5.4)、硫酸铵分级沉降、Affi-gel blue gel亲和色谱、Sephadex SP C-25离子交换色谱及Sephadex G-50凝胶过滤色谱进行分离纯化,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析鉴定其相对分子质量,并对其抗真菌活性进行表征。结果表明,从小红豆中分离纯化出的一种抗菌蛋白,经SDS-PAGE鉴定达到电泳纯,且其相对分子质量约为4 kDa,通过纯化的抗菌蛋白对来自茄子、甜瓜及苦瓜的尖孢镰刀菌的抑制实验,证明此抗菌蛋白具有较强的抗真菌活性。从小红豆中分离纯化出的一种分子质量约为4 kDa的抗菌蛋白,其对真菌具有抑制作用,且其抑制率对浓度的依赖性较高。     

新疆莎车1号扁桃仁2S-清蛋白的纯化及特性研究

清蛋白是扁桃种仁中主要蛋白质,为揭示扁桃仁主要蛋白质组成及结构特性,以新疆莎车1号(SC-1)扁桃仁为研究对象,对其种仁中2S-清蛋白进行分离纯化和结构特性研究,即经有机溶剂多次沉淀提取,获得扁桃仁清蛋白的粗提物,再经Q Sepharose FF分离纯化,获得扁桃仁2S-清蛋白。经高效液相色谱-质谱联用分析表明,纯化后的扁桃仁2S-清蛋白准确分子质量为31.40 k D,其在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果中显示为18.0 k D和19.5 k D 2个条带,表明其由两个不同的亚基通过二硫键结合而成。扁桃仁2S-清蛋白中谷氨酸(Glu)、天冬氨酸(Asp)和精氨酸(Arg)的含量最高;其二级结构中主要为β-折叠,占65.9%;扁桃仁2S-清蛋白羰基含量为4.45 nmol/mg、活性巯基含量为39.95?μmol/g、总巯基含量为57.11?μmol/g、表面疏水性为23.45?μg;变性温度为43.3℃。研究结果可为新疆扁桃仁的开发利用提供理论依据。     

虾类原肌球蛋白提取和活性的保持

蛋白质分离纯化的主要目的是研究其结构和理化性质,因此在分离纯化过程中应尽量保持原有的结构和性质。本文主要综述了甲壳类动物的主要过敏原-原肌球蛋白(Tropomyosin,TM)的结构,以及影响其结构稳定性的因素。同时讨论了分离纯化的方法及条件对原肌球蛋白结构和过敏原活性的影响,为提取高纯度且保留原有结构和活性的原肌球蛋白提供参考。     

热加工花生的蛋白分离及其过敏原纯化方法研究进展

花生不仅本身是一种营养丰富的食品,而且作为原料或配料广泛应用于食品加工中。然而花生及其制品是FAO/WHO认定的八大类食物过敏原之一,可导致严重的过敏反应,通常伴随终身,甚至危及生命。不同地区的人们食用花生的加工方式不同,其花生过敏的患病率也有所不同,热加工是花生的主要加工方式,因此各类热加工导致的花生致敏性变化成为研究热点。过敏原蛋白的分离作为花生热加工研究中的重要步骤,也变得十分重要。本文主要对常见的3种热加工花生(水煮、油炸和烘烤)中的花生蛋白分离及其过敏原纯化的方法研究进行综述。现有的花生热加工研究中蛋白分离技术主要是通过溶剂浸提;而过敏原纯化技术主要是借助层析法,根据各组分在物理化学性质上的差异进行纯化;此外还可以根据最终研究目的的不同采用其他的辅助方法达到分离纯化效果。通过对现有分离纯化方法进行了解和比较,可为热加工花生过敏原蛋白的分离纯化甚至进一步的分析检测提供理论参考和指导。     

低温胁迫小麦苗冰结构蛋白纯化的研究

以研究低温胁迫小麦苗冰结构蛋白(ISP)纯化为目的,以小麦苗为对象,经低温胁迫,并采用粗分离、饱和硫酸铵溶液沉淀、DEAE-Cellulose 52层析、sephadex-G75凝胶层析4个步骤对其中ISP进行纯化,分别采用差示扫描量热法(DSC)、热休克处理法和聚丙烯酰氨凝胶电泳(SDS-PAGE)测定各纯化步骤所得ISP的热滞活性(THA)、冰晶重结晶抑制能力(ICP)和分子质量(MW),并计算小麦苗存活率和各纯化步骤的得率。实验结果表明,梯度低温胁迫的小麦苗存活率与恒温低温胁迫相比提高了240%,小麦苗提取物(AWSE)中蛋白质含量与未低温胁迫小麦苗提取物(NAWSE)相比提高了7.8%;以THA表征的纯化倍数(T_1)和以ICP表征的纯化倍数(T_2)分别为451.15和137.60,得率为2.85%,THA为0.180℃(5 mg/mL),ICP为70.03%,分子质量范围为20.1~31.0 ku;与THA相比,ISP在较低浓度下即可达到ICP的峰值。小麦苗可作为制备冰结构蛋白的天然植物资源。     

骨桥蛋白的分离及其对成骨细胞黏附性的研究

目的:从新鲜牛乳中分离纯化骨桥蛋白(Osteopontin,OPN),对其进行初步鉴定,并且探讨骨桥蛋白对成骨细胞(Osteoblast,OB)的作用。采用离子交换层析、疏水层析等色谱技术从脱脂乳中分离纯化骨桥蛋白,然后对分离纯化得到的产物进行SDS-PAGE电泳、免疫印迹技术鉴定,用酶联免疫吸附及迁移分析方法测定OPN对成骨细胞的影响。分离得到相对分子量为60ku、具有活性的骨桥蛋白,该种蛋白能够促进成骨细胞发生黏附,且其促黏附功能呈浓度依赖性。黏附率在实验开始后90～120min区段达到最高值。通过本文所述方法可以得到具有活性的骨桥蛋白,并且该种蛋白具有促进成骨细胞黏附的功能,为骨桥蛋白功能性质的进一步研究奠定了基础。     

菜籽蛋白水解物的分离纯化及抗肿瘤活性研究

本研究将菜籽蛋白通过碱性蛋白酶-风味蛋白酶分步酶解制备得到菜籽蛋白水解物(RPH),在经过超滤、葡聚糖凝胶柱(Sephadex G-15)以及半制备液相(RP-HPLC)分离纯化得到各级分离组分。采用MTT比色法分析菜籽蛋白水解物各组分RPHs(MW1 ku)对人体肝癌细胞HepG2、乳腺癌细胞MBA-MD-231及人体结肠癌细胞Caco-2的体外抗增殖活性。当RPHs质量浓度为50～1 000μg/mL时,RPHs对HepG2细胞、MBA-MD-231细胞及Caco-2细胞均有一定的抑制作用,且抑制效果与样品浓度之间呈现出剂量依赖性;其中,HepG2细胞对RPHs的作用最为敏感。经Sephadex G-15凝胶柱分离后得到2个洗脱峰,收集并测定其活性,发现组分RPHs-F2具有更强的抗肿瘤细胞增殖活性;再进一步对其经半制备液相分离得到的组分进行体外抗增殖活性分析发现,除组分RPHs-F2-4外,其他3个分离组分都具有显著的抑制作用,而组分RPHs-F2-3对HepG2细胞具有最高的体外增殖抑制率,当作用质量浓度为1 000μg/mL时,抑制率为(51.53±5.59)%。因此认为菜籽蛋白酶解物分离组分RPHs-F2-3具有较好的抗肿瘤活性,可以作为功能性成分用于抗肿瘤相关的功能食品及保健品的开发。     

创伤弧菌外膜蛋白VuuA的表达纯化及复性

对创伤弧菌重组外膜蛋白VuuA进行表达纯化及复性,以获得可溶性VuuA蛋白。构建重组工程菌Escherichia coli BL21(DE3)-pET-28a-vuuA,利用IPTG诱导表达重组蛋白VuuA,利用亲和层析分离纯化蛋白并对包涵体进行复性。VuuA蛋白主要以包涵体形式存在于重组工程菌细胞内,经复性后和亲和层析纯化后,蛋白浓度达到28.2mg/L发酵液,有效提高可溶性蛋白的浓度。VuuA蛋白的纯化及复性为创伤弧菌新型疫苗的制备奠定了基础。     

Extraction and purification method of rice DNA from rice powder containing Konjak flour

Rice powder containing Konjak flour made with tuberous roots of Amorphophallus konjac is imported as a rice-processed product from China to Japan. An improved DNA purification method for the polymerase chain reaction (PCR) analysis of rice in such products is necessary, since Konjak flour constituents absorb the DNA purification buffer to form a gel, and cause problems in the subsequent purification steps. Here, we present a simple preparative system for isolation of the rice and a purification method of the rice DNA from the product. The purified DNA was confirmed to be a good template for both PCR and real-time PCR.     

乙醇冷冻法纯化分离大豆磷脂酰胆碱研究

以大豆粉末磷脂为原料,结合乙醇萃取和乙醇冷冻纯化两个步骤提取纯化磷脂酰胆碱;重点探讨乙醇冷冻纯化步骤工艺条件,考察冷冻时间、冷冻温度、料液比、乙醇浓度等因素对乙醇冷冻纯化效果影响,并在单因素试验基础上进行正交试验,确定最佳冷冻纯化工艺为:冷冻时间6h,冷冻温度-12℃,料液比1:8,乙醇浓度97.5%,在此条件下纯化分离,磷酯酰胆碱含量由45.8%提高至70.5%、得率为81.2%。     

Separation and Purification of Hydroxycinnamic Acid Derivatives in Cranberries

Hydroxycinnamic acid derivatives have been shown to be a complex mixture of closely related compounds. A procedure has been described for their isolation and purification from cranberries. The hydroxycinnamates were extracted from the plant tissue with aqueous alcohol, followed by pre-separation on a polyamide CC-6 column. For easier sample concentration, the eluted compounds were extracted in alcohol. Final separations were made on a High Performance Liquid Chromatograph. Recycling was used to purify the individual compounds. A total of 15 compounds was purified. The method can be used for the separation and purification of individual phenollc compounds from mixtures.     

碱消化法提纯大米淀粉的研究

蛋白质残留量是决定大米特性的主要因素,也是影响大米淀粉应用的关键因素。文中采用三因素正交实验设计,筛选了碱消化提纯大米淀粉的优化工艺条件:碱液浓度4g/L,提取时间4h,浆液浓度300g/L)。所得淀粉蛋白质残留量0·34%,低于Yamamoto法的0·42%;大米淀粉得率71%,比Yamamoto65%高6%。大米粉经碱消化去蛋白质提纯后,其膨润力和溶解度明显提高。     

Evaluation of an extracting method for the detection of Hepatitis A virus in shellfish by SYBR-Green real-time RT-PCR

Consumption of virus-contaminated shellfish has caused numerous outbreaks of gastroenteritis and hepatitis worldwide. In the present study, we evaluated a rapid and simple extraction method to concentrate and purify enteric viruses from shellfish tissues for their detection by real-time RT-PCR. This procedure consists of an alkaline elution with a glycine buffer, solids removal by slow speed centrifugation, purification by chloroform extraction and virus concentration by ultracentrifugation. The efficiency of this method to recover Hepatitis A virus (HAV) from oysters seeded with this virus, was assessed by real-time RT-PCR and conventional RT-nested PCR after extracting viral RNA by a commercial isolation kit. Real-time RT-PCR yielded higher detection sensitivity than the obtained by conventional RT-nested PCR. Besides the improvements in detection sensitivity, the real-time RT-PCR, by quantifying HAV RNA, allowed to check the overall extraction procedure and the recovery efficiency after each processing step. After the last phase, i.e. virus concentration by ultracentrifugation, the RNA purity was high but the estimated HAV recovery efficiency was however low, probably due to virus losses and the presence of RT-PCR inhibitors in sample concentrates. In contrast, the HAV recovery percentage was higher after the virus elution step while the RNA purity was lower. Real-time RT-PCR detection could allow to eliminate some purification and concentration steps that are required for conventional RT-nested PCR detection. The overall procedure for detecting HAV could be then simplify avoiding virus losses during manipulation.     

野生粉叶爬山虎种子等部位的原花青素提取纯化工艺及含量测定

通过正交实验研究粉叶爬山虎中原花青素提取条件。结果表明:提取时间100min,乙醇体积分数75%,提取温度65℃,料液比1:10为较佳条件。香草醛-盐酸法测得果梗、果皮、种子中含量分别为2.6317%、3.7967%、4.0934%;大孔吸附树脂分离纯化的结果表明:LSA-21树脂适合分离纯化粉叶爬山虎中原花青素,分离条件:上样液质量浓度为20mg/mL,洗脱流速为2.0BV/h,经HPLC测得纯化后果梗、果皮与种子中原花青素含量为2.39%、2.31%、3.94%;HPLC检测LSA-21树脂分离纯化后的原花青素纯度可达87%以上。     

葛根素纯化工艺及其解酒效应

探寻葛根植物中葛根素的纯化工艺并检测纯化物对醉酒小鼠的干预效果。利用葡萄糖凝胶层析法,选取洗脱次数、乙醇体积分数、液料比三个因素,纯化葛根提取物,HPLC分析检测纯化物,并采用气相色谱法(GC)检测急性醉酒小鼠模型中血液酒精含量。最佳纯化条件为:体积分数为65%乙醇,洗脱次数为6次,液料比4:2 mL/mg葛根素纯化率为70.56%±0.85%,HPLC(高效液相色谱法)检测葛根纯化物与葛根素标准品一致,GC检测显示葛根纯化物组小鼠血液酒精浓度值较酒精模型组小鼠的低,葛根纯化物组小鼠的酒精平均浓度为3.295 mg/mL(p<0.01),酒精模型组的为4.605 mg/mL。葡萄糖凝胶纯化葛根素的方法简便可行,其纯化物能降低急性醉酒小鼠血液中酒精浓度,对醉酒小鼠具有干预效果。     

Purification of α‐amylase from human saliva by superparamagnetic particles

BACKGROUND: Several studies of magnetic carrier technology have focused on the application of separation technology, because the magnetic support can separate the target from the reaction medium by application of a magnetic field and because the magnetic carrier can be easily recovered. In the present study, superparamagnetic iron oxide (SPIO) modified by epichlorohydrin was employed as a support whose surface could be coated with starch. The starch‐coated support was used for isolating human salivary amylases. RESULTS: The results showed that the starch‐SPIO support could isolate amylases from human saliva with 91.1% recovery and 3.5‐fold purification to high specific activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the purifed amylase comprised two isoamylases with estimated molecular weights of 55 and 59 kDa. The activities of crude and purified amylases showed optimal pH values of 6–7 and 7 and optimal temperatures of 40 and 30 °C respectively. The thermal stability range for both crude and purified amylases was between 20 and 40 °C. CONCLUSION: The attachment of substrates to SPIO could offer a novel and efficient method for purifying enzymes either as an initial step prior to further purification or as a final step for diagnostic usage. Copyright © 2009 Society of Chemical Industry     

大蒜绿变物质的提取及其分离

研究了提取及分离纯化大蒜绿变物质的方法。结果表明,体积分数为80%的甲醇溶液,按料液比1∶3(g:mL),室温浸提24h,提取效果最好;利用薄层层析法进行色素的分离纯化,确定展层剂为体积分数77%甲醇、3%乙酸乙酯、0.24%HCl的水溶液,最终能达到初步分离纯化的目的。     

蛹虫草多糖的纯化及其分子量的测定

以蛹虫草子实体为材料,对水提醇沉、硫酸锌盐去蛋白后获得蛹虫草粗多糖（CP）进行分离纯化。采用膜分离技术、DEAE．52柱层析及SephadexG-100柱层析对CP进行分离纯化,并使用高效凝胶渗透色谱法（HPGPC）测定各组分多糖分子量,以表征不同路径所得多糖的分子量分布。结果表明：CP经膜过滤、DEAE-52柱层析及SephadexG-100进一步柱层析得到多糖组分CP2-c2-s2,经HPGPC法鉴定,CP2-c2-s2为均一多糖组分,其平均分子量为20200Da。