lishufeng高级生化实验

1、1. 注意上课时间，保证出勤率2. 实验室仪器使用规范（移液器、离心机等），损坏赔偿3. 实验：分组、试剂、实验讲义4. 实验安全，仪器、试剂、菌液5. 考试：实验40（操作报告） 闭卷606. 值日DAY 1:配试剂（DNA的提取及鉴定）高压，灭菌（培养皿、枪头、离心管、试剂）倒平皿（加抗生素Kan,不加抗生素） 每排4LK, 2LB 50mg/ml kana加到LB中（kana 1:1000稀释）连接：质粒片断目的基因 （每排2个连接反应）ddH2O 12ul ddH2O 12ul X-DNA(X-DNA(目的基因）目的基因） 3ul 3ul pET-28apET-28a（质粒片断）（质粒

2、片断） 2ul 2ul 1010buffer 2ul buffer 2ul T4 ligase 1ulT4 ligase 1ul14-1614-160C过夜过夜20ul20ul 每组接种DH5a 1支 接种BL21 1支 （10ul菌液+2ml LB）, 第二天7：30转接 1ml+20mlLB（分别准备制备感受态，两个连接反应用来转化）实验内容实验内容1.重组载体的构建及表达（6天）2.脲酶的分离纯化及活性测定（2天）总体实验安排上午下午第一天配试剂挑DH5单克隆，连接反应第二天感受态细胞的制备转化第三天观察结果讲质粒的提取鉴定原理挑单克隆过夜培养第四天质粒的提取、酶切琼脂糖凝胶电泳挑鉴定正

3、确的克隆过夜培养（3ml)第五天扩大培养（30ml)IPTG诱导，0，1，2，3小时取样SDS-PAGE第六天蛋白纯化试剂配制装柱、脲酶粗提液制备第七天层析蛋白浓度及活性测定第八天电泳检测、打扫实验室DH5aDH5a感受态细胞的制备：P159P159 转种（转种（1 1：4040） 继续振荡培养继续振荡培养2.5h 2.5h 制备感受态细胞制备感受态细胞（CaClCaCl2 2) )连接反应终止转化，涂板，3737度过夜培养 （DH5DH5：感受态一块LB,LB,感受态一块LK LK 转化菌一块LK, BL21LK, BL21同样）DAY 2:LBLB平皿平皿DH5aDH5a感受态细胞感受态细

4、胞25UL25ULLKLK平皿平皿DH5aDH5a感受态细胞感受态细胞25UL25ULLKLK平皿平皿转化菌液转化菌液50-100UL50-100ULDAY 3:看结果，配试剂（诱导表达及鉴定用） 接种：质粒提取用（2 2人/ /管）LBLB平皿平皿DH5aDH5a感受态细胞感受态细胞25UL25ULLKLK平皿平皿DH5aDH5a感受态细胞感受态细胞25UL25ULLKLK平皿平皿转化菌液转化菌液50-100UL50-100UL. . . . . . . . . .？DAY 4:质粒提取（P160P160）：碱裂解法质粒的酶切: 20ul, 37: 20ul, 370 0C,1HC,1H 琼

5、脂糖电泳检测（P161） ： 接种：诱导表达用（每一组/管），需转种单酶切：单酶切：重组质粒重组质粒DNA 5ulDNA 5ulBamH 1 1ulBamH 1 1ul1010bufferbuffer 2ul 2ulddHddH2 2O 12ulO 12ul双酶切：双酶切：重组质粒重组质粒DNA 5ulDNA 5ulBamH 1 1ulBamH 1 1ulXhol 1 1ulXhol 1 1ul1010buffer 2ulbuffer 2ulddHddH2 2O 11ulO 11ul 转种（转种（1 1：4040） 培养培养3h3h，加入，加入IPTGIPTG诱导诱导 不同时间取菌不同时间取菌

6、0h1h0h1h、2h2h、3h3h取菌取菌 电泳电泳DAY 5， DAY 6：制备PAGEPAGE凝胶（P163P163）：下层胶、上层胶诱导表达及PAGEPAGE电泳： Question：1.1.构建的载体为何需要进行酶切及琼脂糖电泳电泳的鉴定？2.2.除了以上的鉴定方法，还能知道哪些其他手段鉴定重组载体？3.3.除了选择BamH 1 BamH 1 、 Xhol 1 Xhol 1 ，还可以选择哪些酶进行鉴定？4.4.如何分析琼脂糖电泳的结果？5.5.加入IPTGIPTG诱导蛋白表达的原理？6.6.SDS-PAGESDS-PAGE电泳分离蛋白质的机理？Discovery of the Dou

7、ble Helix1953, James Watson and Francis Crick are credited with building the first model of DNA. They won the Nobel Prize in 1962The Central Dogma常用分子生物学技术 基因克隆基因克隆 DNADNA序列测定序列测定 核酸分子杂交核酸分子杂交 PCRPCR 转基因生物转基因生物 DNADNA芯片芯片Why gene Cloning?Isolation and manipulation of fragments of an organisms genome

8、Molecular analysis of proteins or other interested gene products Impossible by direct purification DNA cloning Impossible by direct isolationMake all possible ! Make all possible ! Basic procedureof DNA cloningVector 基因克隆(gene cloning):是指采用人工方法将目的基因与载体DNA在体外进行重组，并将重组后的DNA引入宿主细胞中进行扩增或表达的过程。 又称重组DNADN

9、A技术（DNA recombination DNA recombination technique)technique) 或基因工程(genetic engineering）基因工程的操作过程目的基因的分离；载体的选择和连接；重组DNA导入受体细胞；重组DNA的筛选和鉴定；克隆基因的表达。One component of the bacterial restriction-modification system.Restriction endonuclease: 1) recognize short, symmetrical DNA sequence; 2) cut DNA backbone

10、at a specific site within that sequence (kill foreign DNA).Mythylase: methylates C or A of the cellular DNA Section 1: Tool enzymes:1.Restriction endonucleaseRecognition sequencesRestriction enzymes1)Recognize 4-8 bp palindromic sequence. 2)Highly specific.1. Commercially available2. Require Mg2+ fo

11、r enzymatic activityRestriction sequences5 protruding ends3 protruding ends5-CCCGGG-33-GGGCCC-55-CCC-OH3-GGG- pp -GGG-3OH-CCC-5+SmaIblunting ends/粘性末端Cohensive ends/平末端Isoschizomers/同功异源酶:来源不同，但能识别和切割同一位点，这些酶称。：Isoaudamers/Isoaudamers/同尾酶：识别序列不同，但能产生相同的粘性末端的酶 自我复制能力； 易于从宿主细胞分离； 多克隆位点； 选择标记。载体条件：vect

12、orsCloning vectors: for cloning a gene or DNA fragmentExpression vectors: for protein expression from a geneIntegration vectors: to integration of a gene into a genome1 Plasmid vectors2 Bacteriophage vectors3 Virus4 Cosmids and YACs Cloning vectors:Multiple cloning sitesMultiple restriction sites en

13、able the convenient insertion of target DNA into a vector Bacteriophage vectorTwo examples:1. phage bacteriophage replacement vector 2.M13 phage M13 phage vector Cloning in M13 Hybrid plasmid-M13 vectors phageDNAProtein coatcoscosNonessential regionLong (left)armshort (right)armExogenous DNA(20-23 k

14、b)12bp Viruses that can infect bacteria. 48.5 kb in lengthLinear or circular genome (cos ends) phageLytic phase (Replicate and release)Lysogenic phase (integrate into host genome)5-CGGGGCGGCGACCTCG-33-GCCCCGCCGCTGGAGC-5 Cleavage Ligation(during packaging) (after infection) GGGCGGGCGACCTCG-35-CG + GC

15、-53-GCCCCGCCGCTGGAThe phage cos endsCircular form Linear form Cosmids and YACs1.Cloning large DNA fragments （ 20 kb)2.Cosmid vectors （粘粒载体）（粘粒载体）3.YAC （酵母人工染色体）（酵母人工染色体）vectors4.Selection in S. cerevisiae (啤酒酵母）啤酒酵母）Yeast Artificial ChromosomeDigestionLigationPackaging and infectFormation of a cosmi

16、d cloneEssential components of YAC vectors :Centromere (CEN着丝粒着丝粒),telomere (TEL端粒端粒) autonomous replicating sequence (ARS) ampr and markers such as TRP1 and URA3. Recognition sites of restriction enzymesYAC vectorsCan accommodate genomic DNA fragments of more than 1 MbYeast selection载体种类：载体种类：Plasm

17、idPhage and VirusCosmidYAC and BAC1.acquiring target gene 1) screening from genomic library;2) screening from cDNA library;3) by PCR4) Chemical synthesis;2. vector selection3. ligation 1) cohesive end ligation2) blunt end ligation3) through linker(人工接头）4) adding homopolymeric tail（同聚物加尾）.DNA ligatio

18、n strategy4. Introduce recombinant 4. Introduce recombinant DNA to host cellsDNA to host cellsCompetent cells: E. coli cells treated with Ca2+ solution are susceptible to take up exogenous DNA. Enzymes involved in host cell defending, such as restriction-modification system are suppressed. Transform

19、ation: a process of uptake of exogenous DNA by competent cells. Heat-shock: After the DNA is uptaken, the cells shall be put at 42oC for 1 min in order to induce the suppressed enzymes for cell defending重组DNA导入受体菌 1.1.转化（transformation）：将质粒DNADNA导入原核细胞的过程（化学法及电转化等）2.感染（infection):通过噬菌体将外源DNA导入原核细胞的过

20、程；3.转染(transfection)：将外源DNA导入真核细胞的过程。5.selection of recombinant DNA 1. antibiotics resistance（抗性） 2.insertion inactivation（插入失活）3. a-complementation（ a -互补）4.immunology methods（免疫学方法）5.southern bloting6.PCR7.restriction digestion(酶切鉴定)8.Sequencing(测序)基因工程的操作过程目的基因的分离；载体的选择和连接；重组DNA导入受体细胞；重组DNA的筛选和鉴定

21、；克隆基因的表达。pET28a连接反应 在1.5ml离心管中加入ddH2O12ulT4 DNA ligase1ulX-DNA3ulpGEX(vector)2ul10buffer2ul混匀（轻弹或反复用枪吹吸），16C过夜感受态的制备1.5菌液沉淀，加0.5ml CaCl2 混匀离心，4000rpm, 5分钟离心，4000rpm, 5分钟沉淀，加0.5ml CaCl2 混匀冰浴，30分钟离心，4000rpm, 5分钟沉淀，加100ul CaCl2 混匀立即使用4度短期保存-70度长期保存加15%-30%甘油转化10ul连接液+50-100ul感受态42度水浴热激90秒混匀，冰浴，30分钟加0.5

22、-1ml LB，混匀37度摇床，培养1小时倒上清，重悬沉淀，涂平板5000rpm, 离心3分钟10ul剩余LBLB+Kan50ul感受态LB+Kan5.selection of recombinant DNA 1. antibiotics resistance（抗性） 2.insertion inactivation（插入失活）3. a-complementation（ a -互补）4.immunology methods（免疫学方法）5.southern bloting6.PCR7.restriction digestion(酶切鉴定)8.Sequencing(测序)抗药性筛选重组体Ampi

23、cillin resistant? yes yesTetracycline resistant? No yesB X BBBXAmproriAmprTcrori2.insertional inactivationAmprTcroripBR322BReplica plating: transfer of the colonies from one plate to another using absorbent pad or velvet (绒布绒布).transfer of colonies+ampicillin+ ampicillin+ tetracyclineThese colonies

24、have bacteria with recombinant plasmidBlue white screeningAmproripUC18(3 kb)MCS (Multiple cloning sites,多克隆位点）Lac promoterlacZScreening by insertional inactivation of the lacZ geneThe insertion of the target gene interrupts the ORF of lacZ gene. .a-complementation.a-complementationlacZ encode enzyme

25、 b b-galactosidase l Plasmids encodes lacZ(N-terminal a-peptide of galactosidase). l Host strain encoding only the C-terminal portion of -galactosidase.l N-terminal + C-terminal = active -galactosidase.IPTGX-gal(substrate of the enzyme)lac promoterBlue producta-complementationa-complementationRecrea

26、ted vector: blue transformantsRecombinant plasmid containing inserted DNA: white transformantsRecreated vector (no insert)Recombinant plasmid (contain insert).Identify the protein product of an interested gene1) Protein activity2) Western blotting.用核酸杂交法进行分析鉴定：6.Restriction mapping: digestion with r

27、estriction enzymes.7.Sequencing the cloned DNA8. PCR analysis质粒 M 双 双 单 单15,00010,0007,5005,0002,5001,000250质粒提取-碱裂解法1.5菌液沉淀，加100ul溶液I离心，6000rpm, 3分钟弃上清（充分）充分混匀，冰上5分钟加200ul溶液II颠倒混匀（轻柔）冰上5分钟加150ul溶液III颠倒混匀，冰上5分钟离心，12000rpm, 10分钟用枪吸400ul上清至新管加1ml无水乙醇，混匀，冰上5-10分钟沉淀，加入1ml 70%酒精离心，12000rpm, 10分钟离心，1200rp

28、m, 5分钟用枪吸取残余液体弃上清1200rpm瞬时离心沉淀，加20ulTE+1ulRNAase开盖，晾5-10分钟质粒酶切反应 在1.5ml离心管中加入单酶切(ul) 双酶切(ul)ddH2O141310buffer2ul2质粒3ul3BamH I11Xho I/1Total 2020充分混匀后，瞬时离心，37度保温1-2小时琼脂糖凝胶电泳l溶胶（用TAE缓冲液，充分融化）l稍冷却后倒胶l加样（样品要加样品缓冲液至1）l电泳（1-10V/CM)l染色与观察拍照质粒 M 双 双 单 单15,00010,0007,5005,0002,5001,000250Lane 2: Multimeric f

29、orms of supercoiled plasmid DNA (pTZ19) which may be observed with some host strains, and should not be mistaken for genomic DNA. Lane 3: Linearized form of plasmid pTZ19 after restriction digestion with EcoRI.Lane 4: Sample contaminated with bacterial chromosomal DNA, Genomic DNA contamination can

30、easily be identified by digestion of the sample with EcoRI. A smear is observed, in contrast to the linear band seen after digestion of multimeric plasmid forms.Lane 5: EcoRI digestion of a sample contaminated with bacterial genomic DNA which gives a smear above the plasmid DNA.Lane 1: Supercoiled (

31、lower band) and open circular form (upper band) of the high-copy plasmid pUC18 with an additional band of denatured supercoiled DNA migrating just below the supercoiled form. This form may result from prolonged alkaline lysis with Buffer P2 and is resistant to restriction digestion蛋白诱导表达取样1.5ml至1.5m

32、l离心管2ml过夜培养菌液37度震荡培养3小时转移1ml至30ml培养基/Kan加IPTG至0.1-1mM37度震荡培养1小时取样0.8ml至1.5ml离心管37度震荡培养2小时取样0.5ml至1.5ml离心管SDS-PAGE样品制备弃上清再 1200rpm瞬时离心离心，4000rpm, 5分钟混匀后至沸水浴中煮3-5分钟用枪吸去上清，加50ulH2O重悬沉淀4度或-20度保存菌液加入50ul 2SDS样品缓冲液SDS-PAGE过程l 配胶l 样品制备l 加样l 电泳（200V）l 染色：考马斯亮蓝染色或银染dH2O：3.3ml30%丙烯酰胺：4.0ml1.0M Tris-Cl(pH8.8)：

33、2.5m110SDS：0.1 ml10%过硫酸铵：0.1 mlTEMED：0.005 mldH2O: 3.4ml 30%丙烯酰胺：0.85ml1M Tris-CI(pH68):0.625m110SDS：0.05ml10%过硫酸铵：0.05ml TEMED：0.005ml制备浓缩胶(5%) 制备分离胶(12) 配胶CH2CH CNH2OCH2CH CNH2OCH2CH CNH2OCH2+聚合作用催化剂CH2CHCNHCHx CH2 CH2OCOCH2NHCH2CHCHx CH2 CH2CNH2OCNH2O丙烯酰胺N,N-甲叉双丙烯酰胺聚丙烯酰胺凝胶聚丙烯酰胺凝胶电泳polyacrylamide

34、gel electrophoresis，PAGE l 单体:丙烯酰胺（Acr）+ 甲叉双丙烯酰胺（Bis） l 催化剂：过硫酸铵+四甲基乙二胺（TEMED）凝胶的聚合SDS-PAGE分离胶浓度最佳分离范围6%胶50-150kD8%胶30-90kD10%胶20-80kD12%胶12-60kD15%胶10-40kD胶浓度与有效分离范围PAGE分离效应l 浓缩效应 l 电荷效应 l 分子筛效应 故具有高分辨率 Samples must be previously boiled 5 minutes in sample buffer containing: SDS (CH3-(CH2)10-CH2OSO

35、3- Na+), 1 molecule binds every 2 amino acids residues b-Mercaptoethanol Sucrose or Glycerol Ionizable tracking dye (i.e., bromophenol blue) Separate protein according to sizeSDS-PAGE 蛋白质的变性SDS-PAGE过程l 配胶l 样品制备l 加样l 电泳（200V）l 染色：考马斯亮蓝染色或银染实验二蛋白质分离纯化脲酶的分离纯化及比活性测定l制备脲酶粗提液l装柱l凝胶过滤层析l蛋白质含量检测l蛋白质活性检测脲酶活性

36、测定原理CO(NH2)2 + H2O2NH3 + CO2脲酶NH4OH + 2(HgI22KI) + 3NaOHO NH2I + 4KI + 3NaI + 3H2OHgHg硫代双汞氨（黄色）奈氏试剂反应1反应2脲酶活性高生成氨就多黄色亦深制备脲酶粗提液称0.5克黄豆粉于小三角瓶中加2.5ml32%丙酮，震荡10min离心3000转，10分钟上清液转至新离心管，加4倍体积冷丙酮离心3000转，5分钟沉淀，待丙酮挥发后，加1.2ml蒸馏水溶解离心3000转，10分钟上清/粗提液凝胶过滤层析l装柱：保持竖直，防分层、干裂l平衡：装好后用蒸馏水平衡l加样：加0.6ml粗提液l洗脱与收集：用蒸馏水洗脱，

37、3ml/15分钟/管，收集12管l蛋白质含量检测：A280测定粗提液稀释20倍再测，测完A280，务必保留样品务必保留样品蛋白浓度（mg/ml）=A2800.75l酶活性检测蛋白含量测定空白洗脱液粗提液(1:20稀释)1239101112A280蛋白(mg/ml)1.1.硫酸铵标准曲线制作硫酸铵标准曲线制作管号 试剂12345670.001M硫酸铵(ml)0.10.20.30.40.50.6-含NH3的mol数0.20.40.60.811.2-H2O（ml)2.92.82.72.62.52.43混匀奈氏试剂0.75ml, 混匀，测A480A480脲酶活性检测-1硫酸铵标准曲线制作含NH3的mo

38、l数1.2 1.0 0.8 0.6 0.4 0.2A4800 0.2 0.4 0.6 0.8 1.0 1.2NH3的mol数 = A480 K脲酶活性检测-2空白洗脱液粗提液(1:20稀释)12391011123%尿素(ml)0.50.50.50.50.50.50.50.50.50.50.1M PBS1.01.01.01.01.01.01.01.01.01.037度保温15分钟酶液-0.50.50.50.50.50.50.50.50.5H2O0.5-混匀，37度保温15分钟向各管加入1M HCl 0.5ml,放置5分钟，加1M NaOH 0.5ml终止反应2. 酶促反应脲酶活性检测-3空白洗脱

39、液粗提液(1:20稀释)1239101112上述反应液0.50.50.50.50.50.50.50.50.50.1H2O2.52.52.52.52.52.52.52.52.52.9奈氏试剂每管加0.75ml,混匀A4803. 显色与测定酶活性计算空白洗脱液粗提液(1:20稀释)1239101112A480NH3(umol)活性/ml洗脱液活性/管蛋白(mg/ml)比活性单位：NH3(umol)数/mL洗脱液h显色用体积 活性/ml洗脱液 = NH3(umol)数 3.0取酶促反应液ml数10.56015规定1小时测15分钟测0.5ml 规定1ml酶反应液3ml 活性/管 =活性/ml洗脱液 3

40、比活性 =每毫升洗脱液的活性每毫升洗脱液的蛋白含量General principles of protein purificationlComplicated and specificl“Black art”SELECT SOURCE OF MATERIAL Concentration: choose tissue, organism with high production/concentration of target protein Developmental stage: Does level of protein change with development? Subcellula

41、r localization Use of expression systemProtein purification Pretreatment/预处理 Rough fractionation/粗分级 Fine fractionation/细分级电泳电泳 前前处处理理粗粗分分离离细细分分离离生物组织生物组织 无细胞提取液无细胞提取液破碎、差速离心、溶解膜蛋白等破碎、差速离心、溶解膜蛋白等选择性沉淀法选择性沉淀法亲和亲和层析层析离子交离子交换层析换层析精制后的蛋白质精制后的蛋白质可结晶纯化可结晶纯化凝胶凝胶过滤过滤疏水疏水层析层析吸附吸附层析层析分段盐析、有机溶剂分段盐析、有机溶剂分级沉淀、凝胶

42、层析分级沉淀、凝胶层析Methods for Protein PurificationTake advantage of the following properties of different protein.molecular weight/MWsolubilitycharge difference ligand specificity（亲和层析） selective adsorptionhydrophobic propertydialysis/透析& ultrafiltration/超滤法：Remove small molecules or ions density gradi

43、ent centrifugation密度梯度离心gel filtration/凝胶过滤法1. According to size and shape semi-permeable membrane. Pores of membrane are of a certain size. Protein stays in; small molecules pass through.Dialysis透析Gel Filtration凝胶过滤层析l Column made of porus beadsl Separates in terms of sizel Big proteins elute first

44、2. In terms of solubilityl isoelectric precipitationl Salting in and salting outl Organic solvent precipitationSalting in / Salting outlSalting INlow concentrations of salt usually increases the solubility of charged macromolecules.lSalting OUThigh concentrations of added salt lowers the solubility

45、of and they come out of solution.Effect of K2SO4 on the solubility of Hb-CO离子强度溶解度1) electrophoresis电泳 charged proteins move toward the opposite electrode.Many kinds of different electrophoresis methods3 . Based on charge difference聚丙烯酰胺凝胶电泳等电聚焦/isoelectric focusing, IEF2-dimensional electronphoresi

46、s2、 Ion-exchange chromatography 离子交换层析法Separates proteins on the basis of chargeTwo kinds:Anion/阴离子-exchangeCation/阳离子-exchangeIon-exchange chromatographyMethods for elution. changing pH and/or increasing ionic strength. Changes done by either stepwise or gradient4.Affinity Chromatography 亲和层析specif

47、ic binding of proteinenzyme substrateIgGprotein AantigenantibodyNi-matrix(His)6GSTGSHElution Mechanism shift to acidic pH (usually to pH 2-4)in the form of a step gradient. Elution with specific ligands or analog 层析的分类1）离子交换层析2）凝胶过滤层析 3）亲和层析4）吸附层析5）分配层析6）疏水层析1. Protein concentration assayTotal protein：l