再纯化

When the first purification alcohol extract first enrichment, then add water to dissolve acid, then the acid fluid, finally using organic solvent basification extraction, the organic solvent extraction layer to get concentration, total alkaloid.

纯化时首先把醇提取液先浓缩，然后加入酸水进行溶解，接着再把得到的酸水液碱化，最后用有机溶剂萃取，萃取得到的有机溶剂层再进行浓缩，就得到总生物碱。

Three different antiserum, rabbitantiserum against formalin-killed-cells of V. parahaemolyticus zj2003 (apathogenic strain isolated from diseased large yellow croaker, from Xiangshan bay,Zhejiang province), rabbit antiserum against OMP of the bacteria and large yellowcroaker convalescent antiserum were prepared for western blotting of the OMPpreparation.

以饱和硫酸铵梯度盐析法结合琼脂糖柱sephadex G200凝胶过滤法初步纯化了大黄鱼血清免疫球蛋白，再经割胶回收法得到进一步纯化的重链片段，分子量约为75kDa，免疫新西兰兔后获得兔抗大黄鱼免疫球蛋白血清，为ELISA和Western blotting检测技术奠定了基础。

Methods: Biological technique, including centrifugalization, ion-exchange chromatography, hydrophobic chromatography and dialysis was used. Osteopontin from bovine milk were identified by SDS-PAGE and Western Blot.

运用生物化学技术，通过离心脱脂后，再经过离子交换层析、疏水层析、透析等技术达到分离纯化目的；随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。

Primary culture of goat mammary gland cells could be derived by outgrowth of migrating cells from fragment of tissue. Fibroblast and epithelial cells could be pured according to their different sensibility to trypsin. Complex cultures treated by 0.25% trypsin in Hanks' for 5~7min at 37℃, the dispersed cells were mainly fibroblasts,then the rest treated by 0.15% trypsin-0.02% EDTA in Hanks'for 5~8min at 37℃, the majority cells harvested were mammary epithelial cells.

用组织块培养法可获得良好的山羊乳腺细胞原代培养物；培养的山羊乳腺成纤维细胞比上皮细胞对胰蛋白酶更敏感，据此可在传代过程中将二者分离纯化，获得纯细胞系；混合培养物先用0.25%胰蛋白酶在37℃消化5~7min 所收集的细胞主要为成纤维细胞；再加0.15%胰蛋白酶-0.02鞹A 消化液在37℃继续消化5~6min 所回收的细胞绝大多数为上皮细胞，经过2~3 代，即可得到纯化的乳腺上皮细胞系。

Primary culture of goat mammary gland cells could be derived by outgrowth of migrating cells from fragment of tissue. Fibroblast and epithelial cells could be pured according to their different sensibility to trypsin. Complex cultures treated by 0.25% trypsin in Hanks' for 5~7min at 37℃, the dispersed cells were mainly fibroblasts,then the rest treated by 0.15% trypsin-0.02% EDTA in Hanks'for 5~8min at 37℃, the majority cells harvested were mammary epithelial cells.

用组织块培养法可获得良好的山羊乳腺细胞原代培养物；培养的山羊乳腺成纤维细胞比上皮细胞对胰蛋白酶更敏感，据此可在传代过程中将二者分离纯化，获得纯细胞系；混合培养物先用0.25%胰蛋白酶在37℃消化5~7min 所收集的细胞主要为成纤维细胞；再加0.15%胰蛋白酶-0.02%EDTA 消化液在37℃继续消化5~6min 所回收的细胞绝大多数为上皮细胞，经过2~3 代，即可得到纯化的乳腺上皮细胞系。

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。

The results showed that plenty of the purified DPV virions were obtained after the cell lysate was condensed by 5-fold by ultrafiltration,and the concentrated DPV fluid was ultracentrifuged with 300g/L sucrose cushion method and 300-600g/kg sucrose density gradient centrifugation methods.

结果显示，用切向超滤法将病毒培养物浓缩为原体积的1/5后，再采用300g/L蔗糖垫底超速离心和300～600g/kg蔗糖密度梯度离心的方法进行纯化，可获得大量的纯化病毒粒子。

At first, we investigated the optimal components of culture medium for thereafter purification. We found that the optimal culture medium could produce better yield of red-pigmented antibiotic when 1.5% fructose and 1.5% malt extract included, and incubated at 30℃ under shaking conditions for 6 days.

我们逐步由探讨培养基条件为始，以利后续的生产纯化的进行，发现以1.5%果糖与1.5%麦芽抽出物之液态培养条件下，以30℃震荡培养6天后，有最适红色抗菌物质之产率，并且可减少其他色素的产生，再试著将其纯化。

METHODS Using heparin sepharose Cl-6B chromatography,the osteoinduction protein in reconstituted bone xenograftwas pu-rified and fractions obtained from purification were bioassayed for bone-induction activity by implantation into the thigh muscles of mouse.

采用肝素亲和层析技术纯化RBX中诱骨活性蛋白即骨形态发生蛋白，利用小鼠肌袋实验检测其骨诱导活性，再用聚丙烯酰胺电泳测量RBX中诱骨活性蛋白纯化后的相对分子质量。

As she looked at Warrington's manly face, and dark, melancholy eyes, she had settled in her mind that he must have been the victim of an unhappy attachment.

每逢看到沃林顿那刚毅的脸，那乌黑、忧郁的眼睛，她便会相信，他一定作过不幸的爱情的受害者。

Maybe they'll disappear into a pothole.

也许他们将在壶穴里消失