纯化的

Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200232,ChinaAbstract: Nine samples from swine abortus were detected by RT-PCR with a pair of primers specific to NS1 gene of Japanese encephalitis virus.

采用反转录聚合酶链式反应方法检测猪流产胎儿病料中的乙型脑炎病毒，并对阳性病料做病毒分离与纯化。

In addition, these two purified proteins could react with a specific antibody against mouse VEGF as expected. Conclusion Recombinant Xenopus laevis VEGF and its control mouse VEGF protein may provide tools for further study of anti-tumor active immunity with this xenogeneic protein vaccine in mouse tumor models.

重组非洲爪蟾和小鼠血管内皮细胞生长因子的原核表达、分离纯化及鉴定成功，为进一步研究异种VEGF蛋白质疫苗抗小鼠肿瘤模型奠定了基础。

The principal purpose of this study is to set up the isolation methods for diphtheria toxin expressed by genes, to purify the toxin with complete biological activities, and to make researches on its toxicities. In the experiments, the target gene was inserted into the cloning vectors--pET22b, then the vectors were introduced into host cells -BL21(DE3). The expression of the toxin was induced by addition of IPTG. The expression products were purified by His-6 label affinity chromatography. Then the diphtheria toxin expressed was transferred to PVC membrane using semidry blotter to determine the amino acid sequence of N-terminal. The "nicked" diphtheria toxin linked by disulfide bond was gained by protease hydrolysis.

为建立重组基因表达白喉毒素的分离纯化方法，制备出具有生物活性的蛋白质毒素，构建了白喉毒素表达载体pET22b-DT，并转入大肠杆菌BL21(DE3)中诱导表达，以融合表达的His-6作为标签通过亲和层析纯化；表达毒素经蛋白酶酶切后，形成了以二硫键连接的与天然结构相同的带"缺口"的白喉毒素，并测定白喉毒素对豚鼠的急性毒性和细胞毒性。

A basic knowledge of microbiology, will be detected with a microscope observation of micro-organisms, medium Preparation, disinfection and sterilization, separation and purification of micro-organisms, bacteria counting method A chemical analysis of the theoretical knowledge and can use chemical analysis methods and equipment of the components of material analysis Master fermentation process principles, understanding of beer, liquor, fermentation production technology Master protein related knowledge, PAGE gel electrophoresis technology, high-speed centrifugal Genetic engineering master the basic theory of knowledge, the extraction and purification of genes, PCR principle, gene recombination technology Master of the basic theoretical knowledge, the immobilized enzyme, the enzyme preparation Application Master of animal and plant tissue culture techniques of experimental steps studied experimental apparatus: Balance (scales, electronic scales, Analytical Balance), or acidity, spectrophotometer, liquid gas chromatography, acid-base titration apparatus, microscopes, centrifuges, ultrasonic cleaning, constant temperature oscillation incubator, super-clean table, steam sterilization pot, the pot constant temperature water bath, PAGE device apparatus, oven, fermenter, such as PCR instrument.

具备微生物理论基础知识，会用显微镜检测观察微生物，培养基的制备、消毒与灭菌，分离与纯化微生物，细菌的计数法具备分析化学的理论知识，能运用化学分析和仪器分析方法对物质的组分进行分析掌握发酵工艺原理，了解啤酒、白酒、发酵生产工艺掌握蛋白质相关知识，PAGE凝胶电泳技术，高速离心掌握基因工程的基本理论知识，基因的提取和纯化，PCR技术原理，基因重组技术掌握酶的基本理论知识，酶的固定化方法，酶制剂的应用掌握动植物组织培养技术的实验步骤学过的实验仪器：天平（台天平，电子天平，分析天平）、酸度计、分光光度计、液气相色谱仪，酸碱滴定仪器，显微镜，离心机，超声波清洗器、恒温振荡培养箱、超净工作台，蒸气灭菌锅、恒温水浴锅，PAGE电泳装置仪器、干燥箱、发酵罐、PCR扩增仪等。

Methods: A lysosome-targeted singal KFERQ was added to the C terminus of rRTA by DNA recombinant technology. A pKK223.3 expression system in E. coli was used to produce recombinant ricine A chain and rRTA-KFERQ. Recombinant proteins were purified by affinity chromatography using Blue-Sepharose 6B.

用DNA重组技术，將溶酶体的靶向信號肽KFERQ连接在RTA的羧基端；將构建好的重组质粒pKK223.3-RTA和pKK223.3-RTA-KFERQ转化感受態大肠桿菌JM109，经IPTG诱导表达RTA和RTA-KFERQ蛋白；重组蛋白质用Blue-Sepharose6B亲和柱纯化，以MTT法分别测定纯化后的RTA与RTA-KFERQ蛋白对体外培养的HEPG2、Hela、A5493种细胞的毒性作用。

Methods:(1) Silica column chromatography and LH-20 were used in phytochemistry study.(2) Based on the extraction ratio of arctiin, the best extraction procedure was acheived under the direction of L9(34) orthogonal experimental design. From 10 types of resins, the one with the maximal binding ability for Arctii was selected and silica-column choramatograpy was used in its seperation and purification procedure.(3) The animal diabetic experimental model in Wistar rats was established with streptozotocin and many biochemical factors were used to indicate the pharmacological effects of arctiin, including glycosylated hemoglobin, serum creatinine, seralbumin, monoxide nitrogen, glycerin trilaurate, cholesterol, HDL-ch, LDL-ch, Blood urea nitrogen, creatinine, SOD, ET, MDA, total Urine protein and albuminuria.

(1)采用硅胶柱层析、聚酰胺层析、LH-20等方法对牛蒡子的化学成分进行研究；(2)采用正交实验设计方法，以牛蒡子苷为指标，确定了牛蒡子乙醇回流提取的最佳工艺；对10种类型大孔吸附树脂进行筛选，筛选出对牛蒡子苷吸附效果最佳的树脂；采用硅胶柱层析法，对牛蒡子苷进一步分离纯化；(3)建立实验性糖尿病大鼠模型，以GLU、TC、TG、HDL-ch、LDL-ch、TB、ALB、BUN、CREA、GHbAlc、NO、SOD、ET、MDA和尿液中TB、ALB为指标，研究牛蒡子苷药理活性；(4)以差速消化法纯化Wistar大鼠的主动脉内皮细胞并进行传代培养，测定牛蒡子苷对高糖条件下RAECs活性、RAECs释放LDH、MDA和NO的变化以及RAECs表达内皮型一氧化氮合酶表达的影响。

Chicken IL-15(ChIL-15), Which was discovered in 2001, plays a main role in activating NK cells and CD8+ memory T cells, and may therefore have potential as a vaccine adjuvant.According to published ChIL-15 gene sequence, a pair of primers were designed and synthesized to clone ChIL-15 cDNA from ConA-activated chicken splenocytes by RT-PCR. Recombinant plasmid pUC19ChIL-15 carrying the ChIL-15 gene was constructed. The gene encoding mature ChIL-15 (mChIL-15) was amplified from pUC19ChIL-15 by PCR and cloned into pMD 18-T vector. After digested with Kpn I /Pst I , the mChIL-15 gene was subcloned into prokaryotic expression vector pPRoEX橦Ta and transformed into E.coli DH5a .The transformant was induced by IPTG, and the recombinant ChIL-15(rChIL-15) was expressed as a fusion protein with a histidine hexamer tag at the N-terminal end of the protein. Following expression, the protein was purified by ProBond?Nickel-Chelating Resin. The uninduced and induced protein lysates and the purified protein were separated by SDS-PAGE and transferred onto nitrocellulose membrane to identify rChIL-15 by Western blot. The rChIL-15 was used to immune the guinea pig to obtain rChIL-15 polyclonal antibody.

参考已发表的ChIL-15基因序列，设计合成引物，应用RT-PCR技术从刀豆球蛋白A活化的白来航鸡脾淋巴细胞中克隆ChIL-15 cDNA，将其与SmalⅠ酶切处理的pUC19载体连接，构建重组质粒pUC19ChIL-15；用PCR技术从pUC19ChIL-15中扩增出去信号肽的成熟ChIL-15(mChIL-15)基因，与pMD 18-T载体相连构建重组质粒pMDChIL-15；然后用KpnⅠ／PstⅠ双酶切下mChIL-15基因片段，定向亚克隆到经同样酶切处理的带有6个组氨酸标签的表达载体pP_RoEX~HTa中，构建重组质粒pP_RoChIL-15，对其测序确认读框正确后，转入大肠杆菌DH5α中诱导表达并进行纯化，用重组ChIL-15(rChIL-15)免疫豚鼠，制备多克隆抗体；再用HindⅢ／PstⅠ从质粒pP_RoChIL-15上切下mChIL-15基因，定向亚克隆到经同样酶切处理的杆状病毒转移载体pMelBacB中，经酶切、PCR和序列测定鉴定后，与杆状病毒线形化DNABac-N-Blue~(TM DNA共转染Sf9昆虫细胞，经三轮蚀斑纯化，构建重组杆状病毒rBac-ChIL-15，用该重组病毒感染处于对数生长期的Sf9细胞，按不同的时间收取样品，离心后对其上清和沉淀进行SDS-PAGE和Western blot分析。

The products were identified by IR,NMR,MS and elemental analysis. In our method,K.OH was used in place of NaOH to synthesize benzyl ester of 3,5-dimethyl-4-hydroxybenzoic acid,2',6'-dimethyl-4'-(n-succinimidyloxycarbonyl) phenyl-acridinium-9-carboxylate was purified on a silica gel column with chloroform/ethylacetate(4:l,v/v) as eluent and further purified by triturating with hexane/acetone(2:l,v/v).The luminescence produced by DMAE-NHS is a flash light with maximumemission at 0.4s and decay half-time of 0.9s. The luminescence intensity is 6.11x10 cps/mol,which is affected by the composition of trigger and surfactant.

DMAE·NHS的合成是本论文的关键和难点，我们对文献方法进行改进：文献方法用氢氧化钠与3，5-二甲基-4-羟基苯甲酸反应制得3，5-甲基-4-羟基苯甲酸钠，再用3，5-二甲基-4-羟基苯甲酸钠与苄氯作用制备3，5-二甲基-4-羟基苯甲酸苄酯，我们用氢氧化钾代替氢氧化钠，使合成取得成功；在合成2'，6'-二甲基-4'-苯基-吖啶-9-甲酸酯时，文献方法对粗产品进行两次硅胶柱层析纯化，第一次柱层析用氯仿/乙酸乙酯(4：1，Ⅴ：Ⅴ)作溶剂和淋洗剂，第二次柱层析用己烷/丙酮(2：1，Ⅴ：Ⅴ)作溶剂和淋洗剂，按照文献方法得到的不是所需要的化合物，我们只进行第一次柱层析纯化，然后用己烷/丙酮(2：1，Ⅴ：Ⅴ)进行研磨，过滤，洗涤，除去溶于己烷/丙酮(2：1，Ⅴ：Ⅴ)的部分，得到所需要的产品。

Methods All mice in the experimental groups were immunosuppressed by given different concentrations of dexamethasone phosphate added in drinking water throughout the experiment. The recovery and purity of the oocysts obtained using different purification methods was compared. The infectivity of the oocysts obtained from the same origin but different animals and different purification methods in a bovine fallopian tube epithelial cell culture system was studied.

在饮水中添加不同浓度的地塞米松对人工感染小鼠进行免疫抑制，探讨获得最多卵囊数的药物添加量；比较不同纯化方法得到的卵囊回收率和纯度；探讨来自不同动物、不同纯化方法的卵囊在牛输卵管上皮细胞培养系统的感染情况。

The results showed: when TLC solvent system was: chloroform - methanol - diethyl amine = 90:9:1, alkaloids from lotus leaves were well isolated, and five components were isolated and identified by spraying Dragendorff reagent. The optimal conditions of HPLC solvent system were: methanol- water - diethyl amine = 75:25:0.0125. Under the conditions, components of alkaloids from lotus leaves were separated very well, and ideal RP-HPLC peaks were obtained.5、Methods of High Speed Counter Current Chromatography and preparative High Performance Liquid Chromatography applied to identify and purify alkaloids from lotus leaves were set up. When solvent system of HSCCC was: chloroform - methanol - water (pH=4.00)= 4:3:2, speed of chromatogram was 700rpm, flow speed of the mobile phase was 2mL/min, four pure components relatively were attained by isolation and purification.

结果表明：薄层层析溶剂系统为氯仿：甲醇：二乙胺=90：9：1，使荷叶中的生物碱达到了较好的分离效果，并用改良的碘化铋钾试剂喷雾显色，共分离鉴别出了5种荷叶生物碱；分析型HPLC分析检测的较优溶剂系统为甲醇：水：二乙胺=75：25：0.0125，使荷叶生物碱的各个组分达到了基线分离，并获得了较好的峰形。5、建立了高速逆流色谱和制备型HPLC技术分离纯化荷叶生物碱的具体方法。H(来源：ABC46论文网www.abclunwen.com)SCCC法在溶剂系统为氯仿：甲醇：pH4.00的水=4：3：2，色谱仪转速为700rpm，流动相流速为2mL／min的条件下，分离纯化得到了4个纯度较高的化合物。

Breath, muscle contraction of the buttocks; arch body, as far as possible to hold his head, right leg straight towards the ceiling (peg-leg knee in order to avoid muscle tension).

呼气，收缩臀部肌肉；拱起身体，尽量抬起头来，右腿伸直朝向天花板（膝微屈，以避免肌肉紧张）。

The cost of moving grain food products was unchanged from May, but year over year are up 8%.

粮食产品的运输费用与5月份相比没有变化，但却比去年同期高8％。