ProteomeLab XL-A XL-I Protein Characterization System _BR-9462A

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中性粒细胞明胶酶相关脂质运载蛋(NGAL)

中性粒细胞明胶酶相关脂质运载蛋白一、临床适应症背景情况中性粒细胞明胶酶相关脂质运载蛋白（Neutrophil gelatinase-associated lipocalin,NGAL）又称人脂质运载蛋白2(lipocalin2,Ln2)或噬铁蛋白(siderocalin)，是人脂质运载蛋白家族中的一个新成员。

近年来，NGAL 作为一种新的肾损伤标志物倍受关注。

早期急性肾损伤、慢性肾病、心肾综合症等疾病血清和尿液中NGAL会显著升高，且其水平监测对病情轻重、疗效及预后判断具有较好的诊断价值。

临床检测NGAL主要有免疫层析法、免疫比浊法、速率色散比浊法、乳胶增强免疫比浊法等。

胶乳增强免疫比浊法是近年来出现的一种较为稳定、准确的体液蛋白均相免疫比浊检测方法。

肾脏损伤约占所有泌尿生殖道损伤的65%左右。

急性肾损伤是一组临床综合征，是指突发(1-7d内)和持续(>24h)的肾功能突然下降，定义为血清肌酐至少上升0.5mg/dl 1，表现为氮质血症、水电解质和酸碱平衡以及全身各系统症状，可伴有少尿（<400ml/24h或17ml/h）或无尿（<100ml/24h）。

肾病的发病迁延难愈，时间超过三个月，病人尿液和相关的血液指标出现异常，肾脏病理学、影像学发现异常，或肾脏的肾小球有效滤过率低于60%，都可统称为“慢性肾病”。

慢性肾病如未能及时有效救治，导致病情恶化进展，则随病程迁延，慢性肾病患者将发展成为慢性肾功能不全、肾衰竭，最终形成尿毒症。

心肾综合征（cardio-renalsyndrome，CRS)即心脏或肾脏对另一器官的损害不能代偿时，互为因果,形成恶性循环，最终加速心脏和肾脏功能的共同损害和衰竭。

其发病率、死亡率高，给社会和患者带来沉重的负担。

目前对心肾综合征的认识有限，缺乏有效的治疗措施，是临床处理的难题。

早期诊断急性肾功能损伤时血、尿NGAL浓度通常会迅速升高，2h最为明显( 比临界值升十至几百倍)，血清肌酐、尿酶等传统指标往往要在24～72h才后明显升高，因而NGAL可用于急性肾功能损伤的早期诊断。

小鼠Ⅰ型胶原α2 (COL1a2)-ELISA试剂盒说明书

小鼠Ⅰ型胶原α2 (COL1a2)酶联免疫吸附测定试剂盒使用说明书产品编号：E-EL-M0318（本试剂盒仅供体外研究使用、不用于临床诊断!）声明：尊敬的客户，感谢您选用本公司的产品。

本产品选用世界著名生产厂家的原料，采用专业ELISA kit生产技术制造。

适用于体外定量检测小鼠血清、血浆、组织匀浆或细胞培养上清液中天然和重组COL1a2浓度。

使用前请仔细阅读说明书并检查试剂组分！如有疑问，请及时联系伊莱瑞特生物科技有限公司。

*: [96T/48T]（打开包装后请及时检查所有物品是否齐全完整）检测原理:本试剂盒采用双抗体夹心ELISA法。

用抗小鼠COL1a2抗体包被于酶标板上，实验时标本或标准品中的COL1a2会与包被抗体结合，游离的成分被洗去。

依次加入生物素化的抗小鼠COL1a2抗体和辣根过氧化物酶标记的亲和素。

抗小鼠COL1a2抗体与结合在包被抗体上的小鼠COL1a2结合、生物素与亲和素特异性结合而形成免疫复合物，游离的成分被洗去。

加入显色底物(TMB)，TMB 在辣根过氧化物酶的催化下现蓝色，加终止液后变黄。

用酶标仪在450nm波长处测OD值，COL1a2浓度与OD450值之间呈正比，通过绘制标准曲线求出标本中COL1a2的浓度。

标本收集:1.血清：全血标本于室温放置2小时或4℃过夜后于1000×g离心20分钟，取上清即可检测，收集血液的试管应为一次性的无热原，无内毒素试管。

2.血浆：抗凝剂推荐使用EDTA.Na2，标本采集后30分钟内于1000×g离心15分钟，取上清即可检测。

避免使用溶血，高血脂标本。

3.组织匀浆：用预冷的PBS (0.01M, pH=7.4)冲洗组织，以去除残留血液（匀浆中裂解的红细胞会影响测量结果），称重后将组织剪碎。

将剪碎的组织与对应体积的PBS（一般按1:9的重量体积比，比如1g的组织样本对应9mL的PBS，具体体积可根据实验需要适当调整，并做好记录。

常用pGEX载体图谱

常用pGEX载体图谱Rosetta系列的表达菌株可以提供T7 RNA聚合酶，它能表达PET系列载体上的外源基因。

pGEX系列载体上的外源基因不需要T7 RNA 聚合酶，普通的大肠杆菌经IPTG诱导即可表达Tac启动子是一组由Lac和trp启动子人工构建的杂合启动子，受Lac阻遏蛋白的负调节，它的启动能力比Lac和trp都强。

其中Tac 1是由Trp启动子的－35区加上一个合成的46 bp DNA片段(包括Pribnow 盒)和Lac操纵基因构成，Tac 12是由Trp的启动子-35区和Lac 启动子的-10区，加上Lac操纵子中的操纵基因部分和SD序列融合而成蛋白标签：A myc tag is a polypeptide proteintag derived from the c-myc gene product that can be added to a proteinusing recombinant DNA technology. It can be used for affinity chromatography, then used to separate recombinant, overexpressed protein from wild type protein expressed by the host organism. Itcan also be used in the isolation of protein complexes with multiple subunits.A myc tag can be used in many different assays that require recognition byan antibody. If there is no antibody against the studied protein, adding a myc-tag allows one to follow the protein with an antibody against the Myc epitope. Examples are cellular localization studies by immunofluorescence or detectionby Western blotting.The peptide sequence of the myc-tag is (in 1- and 3-letter codes, respectively):N-EQKLISEEDL-C,N-Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu -C, where N stands for Amino-terminus and C stands for Carboxy terminus. The tag is approximately 1202 Daltons in atomic mass and has 10 amino acids.It can be fused to the C-terminus andthe N-terminus of a protein. It is advisablenot to fuse the tag directly behind the signal peptide of a secretory protein, since it can interfere with translocation intothe secretory pathway.A monoclonal antibody against themyc epitope, named 9E10, is available from the non-commercial Developmental Studies Hybridoma BankpGEX4T1载体基本信息出品公司: GE别名: pGEX-4T-1, pGEX4T1, pGEX 4T 1质粒类型: 大肠杆菌蛋白表达载体表达水平: 高拷贝启动子: Tac克隆方法: 多克隆位点，限制性内切酶载体大小: 4969 bp5' 测序引物及序列: pGEX5': GGGCTGGCAAGCCACGTTTGGTG3' 测序引物及序列: pGEX3': CCGGGAGCTGCATGTGTCAGAGG载体标签: N-GST载体抗性: Ampicillin 氨苄备注: 复制子是pMB1产品目录号: 27-4580-01稳定性: 瞬时表达 Transient组成型: 诱导表达病毒/非病毒: 非病毒pGEX4T1载体质粒图谱和多克隆位点信息原核生物DNA复制起始点，是DNA链上独特的具有起始DNA复制功能的碱基序列。

高迁移率族蛋白1——新的“晚期”促炎性细胞因子

·热点快讯·高迁移率族蛋白1——新的“晚期”促炎性细胞因子刘峰姚咏明*（解放军总医院第304临床部全军烧伤研究所，北京100037）通讯作者简介：姚咏明(1965-)，男，博士后(奥地利)，教授，博士生导师。

现任中国微生物学会微生物毒素分会副主任委员、中国中西医结合急救学会常委、国家自然科学基金重点项目评委等职。

《中华外科杂志》等14种杂志副主编或编委，Crit Care Med等9种杂志英文编审或特约审稿人等。

主持国际合作、国家、军队和省部级科研项目共计17项，包括国家杰出青年基金、国家“973”项目等。

在国内外期刊发表论文312篇(SCI收录40篇，ISTP收录6篇，被SCI引用530余次)，主编及参编专著18部，获国际希拉格奖1项，中国青年科技奖1项，国家科技进步二等奖2项、三等奖1项，北京市和军队科技进步一、二等奖8项。

主要研究方向为细菌内/外毒素血症、休克、脓毒症和多器官功能障碍综合征发病机制、诊断方法与防治策略。

摘要：高迁移率族蛋白1（HMGB1）是一种高度保守的核蛋白，普遍存在于哺乳动物细胞。

最近发现可由激活的单核/巨噬细胞等主动分泌或由坏死细胞被动释放到细胞外，在感染及炎症时血浆HMGB1水平显著升高，起到重要的促炎效应，包括激活单核/巨噬细胞产生多种细胞因子，诱导内皮黏附分子的表达及损伤上皮细胞屏障功能等；可引起发热和厌食，参与多种疾病尤其是脓毒症、关节炎、结肠炎等发病过程。

在“早期”炎症介质释放后，给予抗HMGB1抗体，仍可对致命性内毒素血症、脓毒症及急性肺损伤动物发挥保护作用，为临床提供了更加宽广的治疗时机与新的干预途径。

关键词高迁移率族蛋白1；细胞因子；炎症；脓毒症；晚期糖基化终末产物受体中图分类号：R364.5 文献标识码：A文章编号：1673－2588（2005）05－0377－04收稿日期：2005－10－08*通讯作者，E－mail：c-ff@基金项目：国家重点基础研究发展规划项目(G1999054203，2005CB522602)、国家杰出青年基金(30125020)脓毒症（sepsis）作为感染引起的致命性炎症反应，其较高的发病率与病死率一直无法得到有效控制。

酵母表达载体pPICZ手册

pPICZ A, B, and CPichia expression vectors for selection onZeocin™ and purification of recombinant proteins Catalog no. V190-20Rev. Date: 7 July 2010Manual part no. 25-0148MAN00000034User ManualiiTable of ContentsKit Contents and Storage (iv)Accessory Products (v)Introduction (1)Overview (1)Methods (3)Cloning into pPICZ A, B, and C (3)Pichia Transformation (9)Expression in Pichia (13)Purification (15)Appendix (17)Recipes (17)Zeocin™ (19)Map and Features of pPICZ A, B, and C (21)Lithium Chloride Transformation Method (23)Construction of In Vitro Multimers (24)Technical Support (32)Purchaser Notification (33)References (34)iiiKit Contents and StorageContents The following components are included with Catalog no. V190–20. Note that thepPICZ expression vectors are supplied in suspension.Component QuantityCompositionpPICZ A Expression Vector 20 μg 40 μl of 0.5 μg/μl vector in10 mM Tris–HCl, 1 mM EDTA,pH 8.0pPICZ B Expression Vector 20 μg 40 μl of 0.5 μg/μl vector in10 mM Tris–HCl, 1 mM EDTA,pH 8.0pPICZ C Expression Vector 20 μg 40 μl of 0.5 μg/μl vector in10 mM Tris–HCl, 1 mM EDTA,pH 8.0GS115/pPICZ/lacZ Positive1 stab --Control strainShipping/Storage The components included with Catalog no. V190–20 are shipped on wet ice.Upon receipt, store as directed below.For long-term storage of your positive control stab strain, we recommendpreparing a glycerol stock immediately upon receipt and storing at –80°C.Component ShippingStorage pPICZ A Expression Vector Wet ice Store at –20°CpPICZ B Expression Vector Wet ice Store at –20°CpPICZ C Expression Vector Wet ice Store at –20°CGS115/pPICZ/lacZ positive control strain Wet ice Store at 4°CivAccessory ProductsAdditional ProductsThe products listed in this section are intended for use with the pPICZ vectors.For more information, visit our web site at or contactTechnical Support (page 32).Product QuantityCatalogno. X-33 Pichia strain 1 stab C180-00GS115 Pichia strain 1 stab C181-00KM71H Pichia strain 1 stab C182-00SMD1168H Pichia strain 1 stab C184-00pPICZα A, B, and C 20 μg each V195-20pPIC6α A,B, and C 20 μg each V215-20pPIC6 A, B, and C 20 μg each V210-20pPIC6 Starter Kit 1 kit K210-01Original Pichia Expression Kit 1 kit K1710-01EasySelect™Pichia Expression Kit 1 kit K1740-01Pichia EasyComp™ Transformation Kit 1 kit K1730-01Pichia Protocols 1 book G100-01PureLink™ Gel Extraction Kit 50 preps250 prepsK2100–12K2100–25S.N.A.P ™ Gel Purification Kit 25 preps K1999–25PureLink™ Quick Plasmid Miniprep Kit 50 preps250 prepsK2100–10K2100–11PureLink™ HiPure Plasmid Midiprep Kit 25 preps50 prepsK2100–04K2100–13One Shot® TOP10 (chemically competent E. coli) 10 reactions20 reactionsC4040–10C4040–03One Shot® TOP10 Electrocompetent E. Coli 10 reactions20 reactionsC4040-50C4040-52TOP10 Electrocomp™ Kits 20 reactions C664–55Positope™ Control Protein 5 μg R900-50CIAP (Calf Intestinal Alkaline Phosphatase) 1,000 units 18009–019T4 DNA Ligase 100 units500 units15224–01715224–025Zeocin™ 1g5 gR250-01R250-05β-Gal Assay Kit 1 kit K1455-01β-Gal Staining Kit 1 kit K1465-01E-Gel® Agarose Gels E-Gel® Agarose Gels are bufferless, pre-cast agarose gels designed for fast, convenient electrophoresis of DNA samples. E-Gel® agarose gels are available in different agarose percentage and well format for your convenience.For more details on these products, visit our web site at or contact Technical Support (page 32).Continued on next pagevAccessory Products, ContinuedZeocin™Zeocin™ may be obtained from Invitrogen (see above). For your convenience, the drug is prepared in autoclaved, deionized water and available in 1.25 ml aliquotsat a concentration of 100 mg/ml. The stability of Zeocin™ is guaranteed for sixmonths if stored at –20°C.Detection of Fusion Protein A number of antibodies are available from Invitrogen to detect expression ofyour fusion protein from the pPICZ vector. Horseradish peroxidase (HRP)-conjugated antibodies allow one-step detection in Western blots usingcolorimetric or chemiluminescent detection methods. The amount of antibodysupplied is sufficient for 25 Western Blots.Antibody Epitope Catalogno.Anti-myc R950–25 Anti-myc-HRPDetects the 10 amino acid epitopederived from c-myc (Evans et al., 1985):EQKLISEEDLR951–25Anti-His(C-term) R930–25Anti-His(C-term)-HRPDetects the C-terminal polyhistidine(6xHis) tag (requires the free carboxylgroup for detection) (Lindner et al., 1997):HHHHHH-COOHR931–25Purification of Fusion Protein The polyhistidine (6xHis) tag allows purification of the recombinant fusionprotein using metal-chelating resins such as ProBond™. Ordering information forProBond™ resin is provided below.Product QuantityCatalogno. ProBond™ Purification System 6 purifications K850–01ProBond ™ Purification System with Anti-myc-HRP Antibody1 Kit K852–01ProBond ™ Purification System with Anti-His(C-term)-HRP Antibody1 Kit K853–01ProBond™ Nickel-Chelating Resin 50 ml150 mlR801–01R801–15Purification Columns 50 each R640–50viIntroductionOverviewIntroduction pPICZ A, B, and C are 3.3 kb expression vectors used to express recombinantproteins in Pichia pastoris. Recombinant proteins are expressed as fusions to aC-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag.The vector allows high-level, methanol inducible expression of the gene ofinterest in Pichia, and can be used in any Pichia strain including X33, GS115,SMD1168H, and KM71H. pPICZ contains the following elements:•5′ fragment containing the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest (Ellis et al., 1985; Koutz et al., 1989;Tschopp et al., 1987a)•Zeocin™ resistance gene for selection in both E. coli and Pichia (Baron et al.,1992; Drocourt et al., 1990)•C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis)tag for detection and purification of a recombinant fusion protein (if desired)•Three reading frames to facilitate in-frame cloning with the C-terminalpeptideReference Sources The pPICZ A, B, and C expression vectors may be used with the Original Pichia Expression Kit, and are included in the EasySelect™Pichia Expression Kit (see page v for ordering information). Additional general information about recombinant protein expression in Pichia pastoris is provided in the manuals for the Original Pichia Expression Kit and the EasySelect™Pichia Expression Kit. For more information about the Original Pichia Expression Kit, the EasySelect™Pichia Expression Kit, or their manuals, visit our web site at or contact Technical Support (page 32).More detailed information and protocols dealing with Pichia pastoris may also be found in the following general reference:Higgins, D. R., and Cregg, J. M. (1998) Pichia Protocols. In Methods in Molecular Biology, Vol. 103. (J. M. Walker, ed. Humana Press, Totowa, NJ) (see page v for ordering information).Recommended Pichia Host Strain We recommend using the X-33 Pichia strain as the host for expression of recombinant proteins from pPICZ. Other Pichia strains including GS115, KM71H, and SMD1168H are suitable. The X-33 Pichia strain and other strains are available from Invitrogen (see page v for ordering information). The X-33 Pichia strain has the following genotype and phenotype:Genotype: Wild-typePhenotype: Mut+1Overview, ContinuedExperimental Overview The following table describes the basic steps needed to clone and express your gene of interest in pPICZ.Step Action1 Propagate pPICZ A, B, and C by transformation into a rec A, end A1E. coli strain such as TOP10, DH5 , or JM109.2 Develop a cloning strategy and ligate your gene into one of the pPICZvectors in frame with the C-terminal tag.3 TransformintoE. coli and select transformants on Low Salt LB platescontaining 25 μg/ml Zeocin™.4 Analyze 10–20 transformants by restriction mapping or sequencing toconfirm in-frame fusion of your gene with the C-terminal tag.5 Purify and linearize the recombinant plasmid for transformation intoPichia pastoris.6 TransformyourPichia strain and plate onto YPDS plates containing the appropriate concentration of Zeocin™.7 Select for Zeocin™-resistant transformants.8 Optimize expression of your gene.9 Purify your fusion protein on metal-chelating resin (i.e. ProBond™).Continued on next page2MethodsCloning into pPICZ A, B, and CIntroduction The pPICZ vector is supplied with the multiple cloning site in three readingframes (A, B, and C) to facilitate cloning your gene of interest in frame with theC-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag.Use the diagrams provided on pages 5–7 to help you design a strategy to cloneyour gene of interest in frame with the C-terminal peptide. Generalconsiderations for cloning and transformation are discussed in this section.General Molecular Biology Techniques For assistance with E. coli transformations, restriction enzyme analysis, DNA biochemistry, and plasmid preparation, refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).E. coli Strain Many E. coli strains are suitable for the propagation of the pPICZ vectorsincluding TOP10, JM109, and DH5 . We recommend that you propagate thepPICZ vectors in E. coli strains that are recombination deficient (rec A) andendonuclease A deficient (end A).For your convenience, TOP10 E. coli are available as chemically competent orelectrocompetent cells from Invitrogen (page v).Transformation Method You may use any method of choice for transformation. Chemical transformation is the most convenient for many researchers. Electroporation is the most efficient and the method of choice for large plasmids.Maintenance of Plasmids The pPICZ vectors contain the Zeocin™ resistance (Sh ble) gene to allow selection of the plasmid using Zeocin™. To propagate and maintain the pPICZ plasmids, we recommend using the following procedure:e 10 ng of your vector to transform a rec A, end A E. coli strain like TOP10,DH5 , JM109, or equivalent (see above).2.Select transformants on Low Salt LB plates containing 25 μg/ml Zeocin™ (seepage 17 for a recipe).3.Prepare a glycerol stock from each transformant containing plasmid forlong-term storage (see page 8).Continued on next page3Cloning into pPICZ A, B, and C, ContinuedGeneral Considerations The following are some general points to consider when using pPICZ to express your gene of interest in Pichia:•The codon usage in Pichia is believed to be similar to Saccharomyces cerevisiae.•Many Saccharomyces genes have proven to be functional in Pichia.•The premature termination of transcripts because of "AT rich regions" has been observed in Pichia and other eukaryotic systems (Henikoff & Cohen, 1984; Irniger et al., 1991; Scorer et al., 1993; Zaret & Sherman, 1984). If you have problems expressing your gene, check for premature termination by northern analysis and check your sequence for AT rich regions. It may be necessary to change the sequence in order to express your gene (Scorer et al., 1993).•The native 5´ end of the AOX1 mRNA is noted in the diagram for each multiple cloning site. This information is needed to calculate the size of the expressed mRNA of the gene of interest if you need to analyze mRNA for any reason.Cloning Considerations For proper initiation of translation, your insert should contain an initiation ATG codon as part of a yeast consensus sequence (Romanos et al., 1992). An example of a yeast consensus sequence is provided below. The ATG initiation codon is shown underlined.(G/A)NNATG GTo express your gene as a recombinant fusion protein, you must clone your gene in frame with the C-terminal peptide containing the c-myc epitope and the polyhistidine tag. The vector is supplied in three reading frames to facilitate cloning. Refer to the diagrams on pages 5–7 to develop a cloning strategy.If you wish to express your protein without the C-terminal peptide, be sure to include a stop codon.Construction of Multimeric Plasmids pPICZ A, B, and C contain unique Bgl II and Bam H I sites to allow construction of plasmids containing multiple copies of your gene. For information on how to construct multimers, refer to pages 24–31.Continued on next page4Multiple CloningSite of pPICZ A Below is the multiple cloning site for pPICZ A. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region.The multiple cloning site has been confirmed by sequencing and functionaltesting.You can download the complete sequence of pPICZ A from our web site at or by contacting Technical Support (see page 32).For a map and a description of the features of pPICZ, refer to the Appendix(pages 21–22). AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGTTCCTCAG TTCAAGTTGG GCACTTACGA GAAGACCGGT CTTGCTAGAT TCTAATCAAG AGGATGTCAG AATGCCATTT GCCTGAGAGA TGCAGGCTTC ATTTTTGATA CTTTTTTATTTGTAACCTAT ATAGTATAGG ATTTTTTTTG TCATTTTGTT 1218Asn Ser Ala Val Asp His His His His His His ***3´ AOX1 priming site TGA GTTTTAGCCT TAGACATGAC AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC GGCGGCCGCC AGCTT GGGCCC GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG 811Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 5´ AOX1 priming sitemyc epitope3´polyadenylation sitePolyhistidine tag5´ end of AOX1 mRNA Sfu I Eco R I Pml I Sfi I Bsm B I Asp 718 I Kpn I Xho ISac II Not I Apa I 104210981158871931991Continued on next pageMultiple CloningSite of pPICZ B Below is the multiple cloning site for pPICZ B. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region.The multiple cloning site has been confirmed by sequencing and functionaltesting.You can download the complete sequence of pPICZ B from our web site at or by contacting Technical Support (see page 32).For a map and a description of the features of pPICZ, refer to the Appendix(pages 21–22). AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC TTAGACATGA CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC 1216Asn Ser Ala Val Asp His His His His His His ***3´ AOX1 priming site TGA GTTTGTAGCC TTAGACATGA AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATTATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC GGCGGCCGCC AGCTT TCTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG 811Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 5´ AOX1 priming sitemyc epitope3´ polyadenylation site Polyhistidine tag5´ end of AOX1 mRNA Sfu I Eco R I Pml I Sfi I Bsm B I Asp 718 I Kpn I Xho ISac II Not I Xba I 104010961156871931991Continued on next pageMultiple CloningSite of pPICZ C Below is the multiple cloning site for pPICZ C. Restriction sites are labeled to indicate the cleavage site. The boxed nucleotides indicate the variable region.The multiple cloning site has been confirmed by sequencing and functionaltesting.You can download the complete sequence of pPICZ C from our web site at or by contacting Technical Support (see page 32).For a map and a description of the features of pPICZ, refer to the Appendix(pages 21–22). AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTGTAGCC TTAGACATGA CTGTTCCTCA GTTCAAGTTG GGCACTTACG AGAAGACCGG TCTTGCTAGA TTCTAATCAA GAGGATGTCA GAATGCCATT TGCCTGAGAG ATGCAGGCTT CATTTTTGAT ACTTTTTTAT TTGTAACCTA TATAGTATAG GATTTTTTTT GTCATTTTGT TTC 1217Asn Ser Ala Val Asp His His His His His His ***3´ AOX1 priming siteTGA GTTTGTAGCC TTAGACATGA AACCTTTTTT TTTATCATCA TTATTAGCTT ACTTTCATAA TTGCGACTGG TTCCAATTGA CAAGCTTTTG ATTTTAACGA CTTTTAACGA CAACTTGAGA AGATCAAAAA ACAACTAATT ATTCGAAACG AGGAATTCAC GTGGCCCAGC CGGCCGTCTC GGATCGGTAC CTCGAGCCGC GGCGGCCGCC AGCTT ACGTA GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG 811Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 5´ AOX1 priming sitemyc epitope3´ polyadenylation site Polyhistidine tag5´ end of AOX1 mRNA Sfu I Eco R I Pml I Sfi I Bsm B I Asp 718 I Kpn I Xho I Sac II Not I Sna B I 104110971157871931991Continued on next pageE. coli Transformation Transform your ligation mixtures into a competent rec A, end A E. coli strain(e.g. TOP10, DH5, JM109) and select on Low Salt LB agar plates containing25 μg/ml Zeocin™ (see below). Note that there is no blue/white screening for the presence of insert with pPICZ A, B, or C. Once you have obtained Zeocin™-resistant colonies, pick 10 transformants and screen for the presence and orientation of your insert.Important To facilitate selection of Zeocin™-resistant E. coli, the salt concentration of the medium must remain low (<90 mM) and the pH must be 7.5. Prepare Low Salt LB broth and plates using the recipe in the Appendix, page 17.Failure to lower the salt content of your LB medium will result in non-selection due to inhibition of the drug.C We recommend that you sequence your construct to confirm that your gene is in the correct orientation for expression and cloned in frame with the C-terminal peptide (if desired). Refer to the diagrams on pages 5–7 for the sequences and location of the priming sites.Preparing a Glycerol Stock Once you have identified the correct clone, be sure to purify the colony and make a glycerol stock for long-term storage. It is also a good idea to keep a DNA stock of your plasmid at –20°C.1.Streak the original colony out on an Low Salt LB plate containing 25 μg/mlZeocin™. Incubate the plate at 37°C overnight.2.Isolate a single colony and inoculate into 1–2 ml of Low Salt LB containing25 μg/ml Zeocin™.3.Grow the culture to mid-log phase (OD600 = 0.5–0.7).4.Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to acryovial.5.Store at –80°C.Plasmid Preparation Once you have cloned and sequenced your insert, generate enough plasmid DNA to transform Pichia (5–10 μg of each plasmid per transformation). We recommend isolating plasmid DNA using the PureLink™ Quick Plasmid Miniprep Kit or the PureLink™ HiPure Plasmid Midiprep Kit (page v), or CsCl gradient centrifugation.Once you have purified plasmid DNA, proceed to Pichia Transformation, next page.Pichia TransformationIntroduction You should now have your gene cloned into one of the pPICZ vectors. Yourconstruct should be correctly fused to the C-terminal peptide (if desired). Thissection provides general guidelines to prepare plasmid DNA, transform yourPichia strain, and select for Zeocin™-resistant clones.Zeocin™ Selection We generally use 100 μg/ml Zeocin™ to select for transformants when using the X-33 Pichia strain. If you are transforming your pPICZ construct into anotherPichia strain, note that selection conditions may vary. We recommendperforming a dose response curve to determine the appropriate concentration ofZeocin™ to use for selection of transformants in your strain.Method of Transformation We do not recommend spheroplasting for transformation of Pichia with plasmids containing the Zeocin™ resistance marker. Spheroplasting involves removal of the cell wall to allow DNA to enter the cell. Cells must first regenerate the cell wall before they are able to express the Zeocin™ resistance gene. For this reason, plating spheroplasts directly onto selective medium containing Zeocin™ does not yield any transformants.We recommend electroporation for transformation of Pichia with pPICZ A, B, or C. Electroporation yields 103 to 104 transformants per μg of linearized DNA and does not destroy the cell wall of Pichia. If you do not have access to an electroporation device, use the LiCl protocol on page 23 or the Pichia EasyComp™Transformation Kit available from Invitrogen (see below).PichiaEasyComp™Transformation Kit If you wish to perform chemical transformation of your Pichia strain with pPICZ A, B, or C, the Pichia EasyComp™ Transformation Kit is available from Invitrogen (see page v for ordering information). The Pichia EasyComp™ Transformation Kit provides reagents to prepare 6 preparations of competent cells. Each preparation will yield enough competent cells for 20 transformations. Competent cells may be used immediately or frozen and stored for future use. For more information, visit our web site at or contact Technical Support (page 32).Important Since pPICZ does not contain the HIS4 gene, integration can only occur at the AOX1 locus. Vector linearized within the 5´ AOX1 region will integrate by gene insertion into the host 5´ AOX1 region. Therefore, the Pichia host that you use will determine whether the recombinant strain is able to metabolize methanol (Mut+) or not (Mut S). To generate a Mut+ recombinant strain, you must use a Pichia host that contains the native AOX1 gene (e.g. X-33, GS115, SMD1168H). If you wish to generate a Mut S recombinant strain, then use a Pichia host that has a disrupted AOX1 gene (i.e. KM71H).Continued on next pageHis4 Host Strains Host strains containing the his4 allele (e.g. GS115) and transformed with thepPICZ vectors require histidine when grown in minimal media. Add histidine toa final concentration of 0.004% to ensure growth of your transformants.The pPICZ vectors do not contain a yeast origin of replication. Transformantscan only be isolated if recombination occurs between the plasmid and the Pichiagenome.Materials Needed You will need the following items:Note: Inclusion of sorbitol in YPD plates stabilizes electroporated cells as they appear tobe somewhat osmotically sensitive.•5–10 μg pure pPICZ containing your insert•YPD Medium•50 ml conical polypropylene tubes• 1 liter cold (4°C) sterile water (place on ice the day of the experiment)•25 ml cold (4°C) sterile 1 M sorbitol (place on ice the day of the experiment)•30°C incubator•Electroporation device and 0.2 cm cuvettes•YPDS plates containing the appropriate concentration of Zeocin™ (seepage 18 for recipe)Linearizing YourpPICZ ConstructTo promote integration, we recommend that you linearize your pPICZ constructwithin the 5′ AOX1 region. The table below lists unique sites that may be used tolinearize pPICZ prior to transformation. Other restriction sites are possible.Note that for the enzymes listed below, the cleavage site is the same for versionsA, B, and C of pPICZ. Be sure that your insert does not contain the restriction siteyou wish to use to linearize your vector.Enzyme Restriction Site (bp) SupplierSac I 209 ManyPme I 414 New England BiolabsBst X I 707 ManyRestriction Digest 1.Digest ~5–10 μg of plasmid DNA with one of the enzymes listed above.2.Check a small aliquot of your digest by agarose gel electrophoresis forcomplete linearization.3.If the vector is completely linearized, heat inactivate or add EDTA to stopthe reaction, phenol/chloroform extract once, and ethanol precipitate using1/10 volume 3 M sodium acetate and 2.5 volumes of 100% ethanol.4.Centrifuge the solution to pellet the DNA, wash the pellet with 80% ethanol,air-dry, and resuspend in 10 μl sterile, deionized water. Use immediately orstore at –20°C.Continued on next pagePreparation of Pichia for Electroporation Follow the procedure below to prepare your Pichia pastoris strain for electroporation.1. Grow 5 ml of your Pichia pastoris strain in YPD in a 50 ml conical tube at30°C overnight.2. Inoculate 500 ml of fresh medium in a 2 liter flask with 0.1–0.5 ml of theovernight culture. Grow overnight again to an OD600 = 1.3–1.5.3. Centrifuge the cells at 1500 × g for 5 minutes at 4°C. Resuspend the pelletwith 500 ml of ice-cold (0–4°C), sterile water.4. Centrifuge the cells as in Step 3, then resuspend the pellet with 250 ml ofice-cold (0–4°C), sterile water.5. Centrifuge the cells as in Step 3, then resuspend the pellet in 20 ml of ice-cold (0–4°C) 1 M sorbitol.6. Centrifuge the cells as in Step 3, then resuspend the pellet in 1 ml of ice-cold(0–4°C) 1 M sorbitol for a final volume of approximately 1.5 ml. Keep the cells on ice and use that day. Do not store cells.Transformation by Electroporation 1.Mix 80 μl of the cells from Step 6 (above) with 5–10 μg of linearized pPICZDNA (in 5–10 μl sterile water) and transfer them to an ice-cold (0–4°C)0.2 cm electroporation cuvette.2.Incubate the cuvette with the cells on ice for 5 minutes.3.Pulse the cells according to the parameters for yeast (Saccharomycescerevisiae) as suggested by the manufacturer of the specific electroporation device being used.4.Immediately add 1 ml of ice-cold 1 M sorbitol to the cuvette. Transfer thecuvette contents to a sterile 15 ml tube.5.Let the tube incubate at 30°C without shaking for 1 to 2 hours.6.Spread 50-200 μl each on separate, labeled YPDS plates containing theappropriate concentration of Zeocin™.7.Incubate plates for 2–3 days at 30°C until colonies form.8.Pick 10–20 colonies and purify (streak for single colonies) on fresh YPD orYPDS plates containing the appropriate concentration of Zeocin™.Continued on next pageGenerally, several hundred Zeocin™-resistant colonies are generated using theprotocol on the previous page. If more colonies are needed, the protocol may bemodified as described below. Note that you will need ~20, 150 mm plates withYPDS agar containing the appropriate concentration of Zeocin™.1. Set up two transformations per construct and follow Steps 1 through 5 ofthe Transformation by Electroporation protocol, page 11.2. After 1 hour in 1 M sorbitol at 30°C (Step 5, previous page), add 1 ml YPDmedium to each tube.3. Shake (~200 rpm) the cultures at 30°C.4. After 1 hour, take one of the tubes and plate out all of the cells by spreading200 μl on 150 mm plates containing the appropriate concentration ofZeocin™.5. Optional: Continue incubating the other culture for three more hours (for atotal of four hours) and then plate out all of the cells by spreading 200 μl on150 mm plates containing the appropriate concentration of Zeocin™.6. Incubate plates for 2–4 days at 30°C until colonies form.Mut Phenotype If you used a Pichia strain containing a native AOX1 gene (e.g. X-33, GS115,SMD1168H) as the host for your pPICZ construct, your Zeocin™-resistanttransformants will be Mut+. If you used a strain containing a deletion in theAOX1 gene (e.g. KM71H), your transformants will be Mut S.If you wish to verify the Mut phenotype of your Zeocin™-resistant transformants,you may refer to the general guidelines provided in the EasySelect™PichiaExpression Kit manual or the Original Pichia Expression Kit manual or topublished reference sources (Higgins & Cregg, 1998).You are now ready to test your transformants for expression of your gene ofinterest. See Expression in Pichia, next page.。

人天然免疫蛋白载脂蛋白B mRNA编辑酶催化亚基3A(APOBEC3A)的克隆表

ＣＨＥＮＧＳｈａｎ，ＣＡＯＬｉｙａｎ，ＤＵＪｕａｎ，ＷＡＮＧＷｅｎｙｏｎｇ，ＧＵＯＹａｎｈａｉ
ＤｅｐａｒｔｍｅｎｔｏｆＰｈａｒｍａｃｙ，ＦｏｕｒｔｈＭｉｌｉｔａｒｙＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｘｉ ’ ａｎ７１００３２；ＤｅｐａｒｔｍｅｎｔｆｏＰａｔｈｏｌｏｇｙ，ＦｏｕｒｔｈＭｉｌｉｔａｒｙＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｘｉ ’ ａｌｌ７１００３２，ＣｈｉｎａＣｏｒｒｅｓｐｏｎｄｉｎｇａｕｔｈｏｒ，Ｅ・ｍａｉｌ：ｇｙａｒｄａａｉ＠ｆｍｍｕ．ｅｄｕ．ｃｎ
［Ａｂｓｔｒａｃｔ］ＯｂｊｅｃｔｉｖｅＴｏｃｏｎｓｔｒｕｃｔｔｈｅｅｘｐｒｅｓｓｉｏｎｖｅｃｔｏｒｏｆａｐｏｌｉｐｏｐｒｏｔｅｉｎＢｍＲＮＡｅｄｉｔｉｎｇｅｎｚｙｍｅｃａｔａｌｙｔｉｃｓｕｂｕｎｉｔ３Ａ
［摘要】目的构建载脂蛋白ＢｍＲＮＡ编辑酶催化亚基３Ａ（ＡＰＯＢＥＣ３Ａ）表达载体，真核细胞表达ＡＰＯＢＥＣ３Ａ并鉴定其胞嘧构建ＡＰＯＢＥＣ３Ａ真核表达载体ｐｃＤＮＡ３．０一ＡＰＯＢＥＣ３Ａ，转染ＨＥＫ２９３Ｔ细胞和ＨｅｐＧ２细胞。ＷｅｓｔｅｒｎｂｌｏｔＤＮＡ测序证实

重组人抗血栓蛋白在大肠杆菌中的自诱导表达及纯化

重组人抗血栓蛋白在大肠杆菌中的自诱导表达及纯化龚志飞;杨翔;颜法宝;陈强;孙小强;华子春【摘要】旨在优化重组人抗血栓蛋白(rHAP)工程菌的自诱导培养基,以提高菌体产量及可溶性的重组rHAP蛋白含量.选取蛋白胨、酵母提取物、甘油、葡萄糖为因素,各取4个水平,采用正交表L16(45)进行试验设计,时影响菌体生长和可溶性rHAP表达水平的乳糖浓度进行了优化.结果表明自诱导培养基碳氮源的最优配比:2％蛋白胨,1.5％酵母,0.5％甘油,0.03％葡萄糖,0.2％乳糖.此时表达菌密度OD600和可溶性rHAP目标蛋白表达量分别是未优化前的2.04倍和2.85倍.在20 L发酵表达时,rHAP工程菌OD600值高迭93,菌体温量为1 620 g/20L.利用Q-Sepharose和SP-Sepharose纯化,Western blotting结果表明表达蛋白为目的融合蛋白.【期刊名称】《生物技术通报》【年(卷),期】2011(000)012【总页数】7页(P192-198)【关键词】抗血栓蛋白;优化培养;发酵;纯化;鉴定【作者】龚志飞;杨翔;颜法宝;陈强;孙小强;华子春【作者单位】常州大学石油化工学院,常州213164;常州靶标生物医药研究所有限公司,常州213164;南京大学常州高新技术研究院,常州213164;常州靶标生物医药研究所有限公司,常州213164;南京大学常州高新技术研究院,常州213164;常州靶标生物医药研究所有限公司,常州213164;南京大学常州高新技术研究院,常州213164;常州大学石油化工学院,常州213164;南京大学常州高新技术研究院,常州213164【正文语种】中文重组人抗血栓蛋白（rHAP）[1]正是利用蛋白质工程和基因工程技术，将人胎盘抗凝蛋白与水蛭素C端结构域结合在一起的一种新的抗凝蛋白，分子量大小约为38 kD。

初步研究表明，重组人胎盘抗凝蛋白变体保留了Annexin V和血小板结合的性质，同时又具有明显的凝血酶抑制活性，而且其凝血酶抑制活性具有明显的剂量依赖关系[2]。

酪啡肽及其酪蛋白水解肽对早期断奶仔猪分泌型免疫球蛋白A和细胞因子水平的影响

０５）ｇｇ．４ｎ／。与对照组相比，验 Ⅱ组差异极显著（００）胃食糜中未检测到Ｓｇ试Ｐ＜．５；ＩＡ。提示乳源活性肽对早期断
奶仔猪细胞免疫和黏膜免疫活性具有一定的促进作用。关键词：．啡肽．；蛋白水解肽；奶仔猪；ｐ酪７酪断细胞因子；ＩＡＳｇ
蛋白：胃蛋白酶＝３：１（量分数）３￣，０质，７Ｃ
ｌ材料与方法
１１试验动物．
ｐ１４消化１０ｍｉ，１ｌＬＮａＨ．，２ｎ用０ｍｏ／ＯＨ终止反应，ｐ至中性。１０， ℃离心１ｎ取上调Ｈ６００ｇ４５ｍｉ，清液用微量凯氏定氮法测定该酪蛋白酶解产物的含
张源淑邓艳部重点开放动物生理生化研究室，京２０９）南南１０５摘要：文旨在研究酪啡肽及其酪蛋白水解肽对早期断奶仔猪分泌型免疫球蛋白Ａ（ＩＡ）细胞因子水平的本Ｓｇ和
疫和黏膜免疫之间的关系，以期为酪啡肽在断奶期
新生动物免疫系统功能发育中的作用及其机制提供
依据。
酪蛋白酶解肽制备参考潘翠玲等所建方法。称取于酪素６７３（酪蛋白６００）酪９．０ｍｇ含０．３ｍｇ，
酪啡肽（ａｏｒｈｎＣＭ）一个具有多种生ｃｓｍｏｐｉ，是

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Main Peak (Monomer) 45.5%
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1
0 0 2 4 6 8 10 12 14 16 18 20 22 24
Sedimentation Coefficient (Svedbergs)
Source: J. S. Philo Ph. D., Alliance Protein Laboratories
g(s*) distribution 100KDa, 50%
0.05
0.04
0.03
0.02
0.01
0.00 0 10 20 30 40
Sedimentation Coefficient (Svedbergs)
800KDa, 40%
1600KDa, 10%
Normalized c(s) [Total Area=1]
+
Because the ProteomeLab XL-A/XL-I’s measurements are based
on the first principles of thermodynamics and hydrodynamics,
no standards or calibrations are required. As a result, you spend
? Free Light Chain 1.4% ? HL Half Molecule 0.8%
Dimer 30.6% Trimer 14.6% Tetramer 5.3% Pentamer 1.4% Hexamer 0.4% Heptamer 0.1%
Protein heterogeneity.
The ability to characterize sample heterogeneity is an important part of process development, formulation and QA/QC. The accompanying graphs are distribution plots with heterogeneity visually indicated by peaks. The position of each peak indicates the sedimentation coefficient, while the area gives the amount of that species. The graph (left) uses the g(s)-distribution method to resolve three non-interacting proteins. Levels of <10% by weight can be quantified. For more in-depth analysis, third party software is available for enhanced application versatility and sensitivity. The graph (right) is a distribution plot of a highly stressed monoclonal antibody sample, with heterogeneity visually indicated by higher resolution peaks. Use of the more sophisticated c(s) distribution method provides enhanced resolution by removing the effects of diffusion. Aggregates or other variants can be quantified at levels well below 1% by weight.
BR-9462A
Native-condition protein characterization. For better lead optimization.
PROTEOMELABTM XL-A/XL-I
PROTEIN CHARACTERIZATION SYSTEM
ProteomeLab XL-A/XL-I.
An important component of lead optimization is the ability to evaluate a drug candidate under concentrated “shelf-like”conditions before it moves into expensive preclinical or clinical trials. The ProteomeLab XL-A/XL-I functions over a wide range of concentration, temperature, ionic strength and pH, delivering more thorough characterization than other methods.
less time on setup, and more time on discovery.
To ensure compatibility with the many additional capabilities and product support provided by third party software, all data is saved in standard ASCII format.
A critical step in the proteomics process, characterizatiቤተ መጻሕፍቲ ባይዱn involves putting a protein’s structure and function into context with all other interactions in a particular biological pathway.
By analyzing proteins as interacting elements instead of in isolation, the ProteomeLab XL-A/XL-I more closely approximates true physiological conditions by considering the protein’s conformation (folded or unfolded), assembly reversibility (interacting systems), stoichiometry (associative state), and heterogeneity (aggregation).
An integrated suite of efficient proteomics solutions.
All of Beckman Coulter’s ProteomeLab offerings are designed to serve as an extension of your thinking, making it easier for you to do your best science, your way. As a result, you get a complete solution that puts meaningful, actionable information into your hands, quickly and easily.
By measuring proteins as interacting elements instead of in isolation, the ProteomeLab XL-A/XL-I accelerates the development of diagnostic markers and therapeutic targets, and delivers a unique system for protein characterization in solution.
“For monitoring protein aggregation and heterogeneity in many different projects, my XL-I is invaluable.”
Kurt Drickamer, Ph.D., Department of Biochemistry, University of Oxford, UK
Interacting systems.
The ProteomeLab XL-A/XL-I is adept at helping determine yes/no binding of a small molecule to a target protein. In the top-most example, a small-molecule inhibitor (the green square) of a hormone-receptor interaction could bind to either the receptor (A) or the hormone (B). The inhibitor uniquely absorbs at 321nm and has a low molecular weight and therefore does not sediment. If it binds to a protein, however, the bound molecule will sediment with the apparent molecular weight of the protein at 321nm. The left-hand graph represents sedimentation equilibrium of an inhibitor, receptor mixture, with the absence of curvature for the inhibitor (red) indicating that the compound does not bind to the receptor. The right-hand graph shows a pronounced curve, indicating that the inhibitor binds to the hormone.