lishufeng-高級生化實(shí)驗(yàn).ppt

1、高級生化實(shí)驗(yàn),實(shí)驗(yàn)部分 1.實(shí)驗(yàn)17：脲酶的分離純化及比活性測定 2.實(shí)驗(yàn)19：重組pGEX-X載體的構(gòu)建及表達(dá) 理論部分 第2章 電泳技術(shù) 影響電泳的因素和PAGE，SDS-PAGE的原理 第3章 層析技術(shù) 層析的概念及種類 離子交換層析、凝膠層析和親和層析的原理 第4章 蛋白質(zhì)的分離純化 第7章 DNA的制備與分析 質(zhì)粒DNA的制備和DNA的瓊脂糖凝膠電泳 第8章 聚合酶鏈反應(yīng) 第1、2節(jié)-基本原理及反應(yīng)體系 第10章 基因工程（重點(diǎn)）,李淑鋒，Ph.D, associate professor E-mail: ,注意上課時(shí)間，保證出勤率 實(shí)驗(yàn)室儀器使用規(guī)范（移液器、離心機(jī)等），損壞賠償

2、實(shí)驗(yàn)：分組、試劑、實(shí)驗(yàn)講義 實(shí)驗(yàn)安全，儀器、試劑、菌液 5. 考試：實(shí)驗(yàn)40（操作報(bào)告） 閉卷60 6. 值日,DAY 1:,配試劑（DNA的提取及鑒定） 高壓，滅菌（培養(yǎng)皿、槍頭、離心管、試劑） 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稀釋） 連接：質(zhì)粒片斷目的基因 （每排2個連接反應(yīng)）,每組接種DH5a 1支 接種BL21 1支 （10ul菌液+2ml LB）, 第二天7：30轉(zhuǎn)接 1ml+20mlLB（分別準(zhǔn)備制備感受態(tài)，兩個連接反應(yīng)用來轉(zhuǎn)化）,實(shí)驗(yàn)內(nèi)容,1.重組載體的構(gòu)建及表達(dá)（6天） 2.脲酶的分離純化及活

3、性測定（2天）,總體實(shí)驗(yàn)安排,DH5a感受態(tài)細(xì)胞的制備：P159 轉(zhuǎn)種（1：40） 繼續(xù)振蕩培養(yǎng)2.5h 制備感受態(tài)細(xì)胞（CaCl2) 連接反應(yīng)終止 轉(zhuǎn)化，涂板，37度過夜培養(yǎng) （DH5：感受態(tài)一塊LB,感受態(tài)一塊LK 轉(zhuǎn)化菌一塊LK, BL21同樣）,DAY 2:,LB平皿 DH5a感受態(tài)細(xì)胞 25UL,LK平皿 DH5a感受態(tài)細(xì)胞 25UL,LK平皿 轉(zhuǎn)化菌液 50-100UL,DAY 3:,看結(jié)果，配試劑（誘導(dǎo)表達(dá)及鑒定用）,接種：質(zhì)粒提取用（2人/管）,LB平皿 DH5a感受態(tài)細(xì)胞 25UL,LK平皿 DH5a感受態(tài)細(xì)胞 25UL,LK平皿 轉(zhuǎn)化菌液 50-100UL,. . . .

4、. . .,？,DAY 4:,質(zhì)粒提取（P160）：堿裂解法 質(zhì)粒的酶切: 20ul, 370C,1H,瓊脂糖電泳檢測（P161） ： 接種：誘導(dǎo)表達(dá)用（每一組/管），需轉(zhuǎn)種,單酶切： 重組質(zhì)粒DNA 5ul BamH 1 1ul 10buffer 2ul ddH2O 12ul,雙酶切： 重組質(zhì)粒DNA 5ul BamH 1 1ul Xhol 1 1ul 10buffer 2ul ddH2O 11ul,轉(zhuǎn)種（1：40） 培養(yǎng)3h，加入IPTG誘導(dǎo) 不同時(shí)間取菌0h1h、2h、3h取菌 電泳,DAY 5， DAY 6：,制備PAGE凝膠（P163）：下層膠、上層膠 誘導(dǎo)表達(dá)及PAGE電泳：,Qu

5、estion：,構(gòu)建的載體為何需要進(jìn)行酶切及瓊脂糖電泳電泳的鑒定？ 除了以上的鑒定方法，還能知道哪些其他手段鑒定重組載體？ 除了選擇BamH 1 、 Xhol 1 ，還可以選擇哪些酶進(jìn)行鑒定？ 如何分析瓊脂糖電泳的結(jié)果？ 加入IPTG誘導(dǎo)蛋白表達(dá)的原理？ SDS-PAGE電泳分離蛋白質(zhì)的機(jī)理？,Discovery of the Double Helix,1953, James Watson and Francis Crick are credited with building the first model of DNA. They won the Nobel Prize in 1962,T

6、he Central Dogma,常用分子生物學(xué)技術(shù),基因克隆 DNA序列測定 核酸分子雜交 PCR 轉(zhuǎn)基因生物 DNA芯片,Why gene Cloning?,Isolation and manipulation of fragments of an organisms genome,Molecular analysis of proteins or other interested gene products,Impossible by direct purification,DNA cloning,Impossible by direct isolation,Make all possi

7、ble !,Basic procedure of DNA cloning,Vector,基因克隆(gene cloning):是指采用人工方法將目的基因與載體DNA在體外進(jìn)行重組，并將重組后的DNA引入宿主細(xì)胞中進(jìn)行擴(kuò)增或表達(dá)的過程。,又稱重組DNA技術(shù)（DNA recombination technique) 或 基因工程(genetic engineering）,基因工程的操作過程,目的基因的分離； 載體的選擇和連接； 重組DNA導(dǎo)入受體細(xì)胞； 重組DNA的篩選和鑒定； 克隆基因的表達(dá)。,One component of the bacterial restriction-modifica

8、tion system. Restriction endonuclease: 1) recognize short, symmetrical DNA sequence; 2) cut DNA backbone 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 endonuclease,Recognition sequences,Restriction

9、enzymes,1)Recognize 4-8 bp palindromic sequence. 2)Highly specific.,Commercially available Require Mg2+ for enzymatic activity,Restriction sequences,5 protruding ends,3 protruding ends,5-CCCGGG-3 3-GGGCCC-5,5-CCC-OH 3-GGG- p,+,SmaI,blunting ends/粘性末端,Cohensive ends/平末端,Isoschizomers/同功異源酶:來源不同，但能識別和

10、切割同一位點(diǎn)，這些酶稱。,：,Isoaudamers/同尾酶：識別序列不同，但能產(chǎn)生相同的粘性末端的酶,自我復(fù)制能力； 易于從宿主細(xì)胞分離； 多克隆位點(diǎn)； 選擇標(biāo)記。,載體條件：,vectors,Cloning vectors: for cloning a gene or DNA fragment Expression vectors: for protein expression from a gene Integration vectors: to integration of a gene into a genome,1 Plasmid vectors 2 Bacteriophage v

11、ectors 3 Virus 4 Cosmids and YACs,Cloning vectors:,Multiple cloning sites,Multiple restriction sites enable the convenient insertion of target DNA into a vector,Bacteriophage vector,Two examples: phage bacteriophage replacement vector M13 phage M13 phage vector Cloning in M13 Hybrid plasmid-M13 vect

12、ors, phage,Viruses that can infect bacteria. 48.5 kb in length Linear or circular genome (cos ends), phage,Lytic phase (Replicate and release),Lysogenic phase (integrate into host genome),5-CGGGGCGGCGACCTCG-3 3-GCCCCGCCGCTGGAGC-5 Cleavage Ligation (during packaging) (after infection) GGGCGGGCGACCTCG

13、-3 5-CG + GC-5 3-GCCCCGCCGCTGGA,The phage cos ends,Circular form,Linear form,Cosmids and YACs,Cloning large DNA fragments （ 20 kb) Cosmid vectors （粘粒載體） YAC （酵母人工染色體）vectors Selection in S. cerevisiae (啤酒酵母）,Yeast Artificial Chromosome,Packaging and infect,Formation of a cosmid clone,Essential compo

14、nents of YAC vectors : Centromere (CEN著絲粒), telomere (TEL端粒) autonomous replicating sequence (ARS) ampr and markers such as TRP1 and URA3. Recognition sites of restriction enzymes,YAC vectors,Can accommodate genomic DNA fragments of more than 1 Mb,Yeast selection,載體種類：,Plasmid Phage and Virus Cosmid

15、 YAC and BAC,1.acquiring target gene 1) screening from genomic library; 2) screening from cDNA library; 3) by PCR 4) Chemical synthesis; 2. vector selection 3. ligation,1) cohesive end ligation 2) blunt end ligation 3) through linker(人工接頭） 4) adding homopolymeric tail（同聚物加尾）.,DNA ligation strategy,4

16、. Introduce recombinant DNA to host cells,Competent 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. Transformation: a process of uptake of exogenous DNA by compe

17、tent 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,重組DNA導(dǎo)入受體菌,1.轉(zhuǎn)化（transformation）：將質(zhì)粒DNA導(dǎo)入原核細(xì)胞的過程（化學(xué)法及電轉(zhuǎn)化等） 2.感染（infection):通過噬菌體將外源DNA導(dǎo)入原核細(xì)胞的過程； 3.轉(zhuǎn)染(transfection)：將外源DNA導(dǎo)入真核細(xì)胞的過程。,5.selection of r

18、ecombinant DNA,1. antibiotics resistance（抗性） 2.insertion inactivation（插入失活） 3. a-complementation（ a -互補(bǔ)） 4.immunology methods（免疫學(xué)方法） 5.southern bloting 6.PCR 7.restriction digestion(酶切鑒定) 8.Sequencing(測序),基因工程的操作過程,目的基因的分離； 載體的選擇和連接； 重組DNA導(dǎo)入受體細(xì)胞； 重組DNA的篩選和鑒定； 克隆基因的表達(dá)。,pET28a,連接反應(yīng),在1.5ml離心管中加入 ddH2O1

19、2ul T4 DNA ligase1ul X-DNA3ul pGEX(vector)2ul 10buffer2ul 混勻（輕彈或反復(fù)用槍吹吸），16C過夜,感受態(tài)的制備,轉(zhuǎn)化,10ul連接液+50-100ul感受態(tài),42度水浴熱激90秒,混勻，冰浴，30分鐘,加0.5-1ml LB，混勻,37度搖床，培養(yǎng)1小時(shí),倒上清，重懸沉淀，涂平板,5000rpm, 離心3分鐘,10ul,剩余,LB,LB+Kan,50ul感受態(tài),LB+Kan,5.selection of recombinant DNA,1. antibiotics resistance（抗性） 2.insertion inactivat

20、ion（插入失活） 3. a-complementation（ a -互補(bǔ)） 4.immunology methods（免疫學(xué)方法） 5.southern bloting 6.PCR 7.restriction digestion(酶切鑒定) 8.Sequencing(測序),抗藥性篩選重組體,Ampicillin resistant? yes yes Tetracycline resistant? No yes,B X B,B,B,X,Ampr,ori,Ampr,Tcr,ori,2.insertional inactivation,Ampr,Tcr,ori,pBR322,B,Replica

21、plating: transfer of the colonies from one plate to another using absorbent pad or velvet (絨布).,transfer of colonies,+ampicillin,+ ampicillin + tetracycline,These colonies have bacteria with recombinant plasmid,Blue white screening,Screening by insertional inactivation of the lacZ gene,The insertion

22、 of the target gene interrupts the ORF of lacZ gene.,.a-complementation,lacZ encode enzyme b-galactosidase,Plasmids encodes lacZ(N-terminal a-peptide of galactosidase). Host strain encoding only the C-terminal portion of -galactosidase. N-terminal + C-terminal = active -galactosidase.,IPTG,X-gal,(su

23、bstrate of the enzyme),lac promoter,Blue product,a-complementation,Recreated vector: blue transformants Recombinant plasmid containing inserted DNA: white transformants,Recreated vector (no insert),Recombinant plasmid (contain insert),.Identify the protein product of an interested gene 1) Protein ac

24、tivity 2) Western blotting,.用核酸雜交法進(jìn)行分析鑒定：,6.Restriction mapping: digestion with restriction enzymes. 7.Sequencing the cloned DNA 8. PCR analysis,質(zhì)粒提取-堿裂解法,1.5菌液,沉淀，加100ul溶液I,離心，6000rpm, 3分鐘 棄上清（充分）,充分混勻，冰上5分鐘,加200ul溶液II,顛倒混勻（輕柔）冰上5分鐘,加150ul溶液III,顛倒混勻，冰上5分鐘 離心，12000rpm, 10分鐘,用槍吸400ul上清至新管,加1ml無水乙醇，混勻

25、，冰上5-10分鐘,沉淀，加入1ml 70%酒精,離心，12000rpm, 10分鐘,離心，1200rpm, 5分鐘,用槍吸取殘余液體,棄上清,1200rpm瞬時(shí)離心,沉淀，加20ulTE+1ulRNAase,開蓋，晾5-10分鐘,質(zhì)粒酶切反應(yīng),在1.5ml離心管中加入,充分混勻后，瞬時(shí)離心，37度保溫1-2小時(shí),瓊脂糖凝膠電泳,溶膠（用TAE緩沖液，充分融化） 稍冷卻后倒膠 加樣（樣品要加樣品緩沖液至1） 電泳（1-10V/CM) 染色與觀察拍照,Lane 2: Multimeric forms of supercoiled plasmid DNA (pTZ19) which may be

26、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 easily be identified by digestion of the sample with

27、 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 (lower band) and open circular form (upper band) of

28、 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,蛋白誘導(dǎo)表達(dá),SDS-PAGE樣品制備,棄上清再 1200rpm瞬時(shí)離心,離心，4000rpm, 5分鐘,混勻后至沸水浴中煮3-5分鐘

29、,用槍吸去上清，加50ulH2O重懸沉淀,4度或-20度保存,菌液,加入50ul 2SDS樣品緩沖液,SDS-PAGE過程,配膠 樣品制備 加樣 電泳（200V） 染色：考馬斯亮藍(lán)染色或銀染,dH2O：3.3ml 30%丙烯酰胺：4.0ml 1.0M Tris-Cl(pH8.8)：2.5m1 10SDS：0.1 ml 10%過硫酸銨：0.1 ml TEMED：0.005 ml,dH2O: 3.4ml 30%丙烯酰胺：0.85ml 1M Tris-CI(pH68):0.625m1 10SDS：0.05ml 10%過硫酸銨：0.05ml TEMED：0.005ml,制備濃縮膠(5%),制備分離膠(

30、12),配膠,聚丙烯酰胺凝膠電泳polyacrylamide gel electrophoresis，PAGE,單體:丙烯酰胺（Acr）+ 甲叉雙丙烯酰胺（Bis） 催化劑：過硫酸銨+四甲基乙二胺（TEMED）,凝膠的聚合,膠濃度與有效分離范圍,PAGE分離效應(yīng),濃縮效應(yīng) 電荷效應(yīng) 分子篩效應(yīng),故具有高分辨率,Samples must be previously boiled 5 minutes in sample buffer containing: SDS (CH3-(CH2)10-CH2OSO3- Na+), 1 molecule binds every 2 amino acids re

31、sidues b-Mercaptoethanol Sucrose or Glycerol Ionizable tracking dye (i.e., bromophenol blue) Separate protein according to size,SDS-PAGE,蛋白質(zhì)的變性,SDS-PAGE過程,配膠 樣品制備 加樣 電泳（200V） 染色：考馬斯亮藍(lán)染色或銀染,實(shí)驗(yàn)二蛋白質(zhì)分離純化,脲酶的分離純化及比活性測定,制備脲酶粗提液 裝柱 凝膠過濾層析 蛋白質(zhì)含量檢測 蛋白質(zhì)活性檢測,脲酶活性測定原理,反應(yīng)1,反應(yīng)2,制備脲酶粗提液,稱0.5克黃豆粉于小三角瓶中,加2.5ml32%丙酮，

32、震蕩10min,離心3000轉(zhuǎn)，10分鐘,上清液轉(zhuǎn)至新離心管，加4倍體積冷丙酮,離心3000轉(zhuǎn)，5分鐘,沉淀，待丙酮揮發(fā)后，加1.2ml蒸餾水溶解,離心3000轉(zhuǎn)，10分鐘,上清/粗提液,凝膠過濾層析,裝柱：保持豎直，防分層、干裂 平衡：裝好后用蒸餾水平衡 加樣：加0.6ml粗提液 洗脫與收集：用蒸餾水洗脫，3ml/15分鐘/管，收集12管 蛋白質(zhì)含量檢測：A280測定 粗提液稀釋20倍再測，測完A280，務(wù)必保留樣品 蛋白濃度（mg/ml）=A2800.75 酶活性檢測,蛋白含量測定,1.硫酸銨標(biāo)準(zhǔn)曲線制作,脲酶活性檢測-1,硫酸銨標(biāo)準(zhǔn)曲線制作,NH3的mol數(shù) = A480 K,脲酶活性檢

33、測-2,2. 酶促反應(yīng),脲酶活性檢測-3,3. 顯色與測定,酶活性計(jì)算,單位：NH3(umol)數(shù)/mL洗脫液h,General principles of protein purification,Complicated and specific “Black art”,SELECT SOURCE OF MATERIAL,Concentration: choose tissue, organism with high production/concentration of target protein Developmental stage: Does level of protein ch

34、ange with development? Subcellular localization Use of expression system,Protein purification,Pretreatment/預(yù)處理 Rough fractionation/粗分級 Fine fractionation/細(xì)分級,Methods for Protein Purification,Take advantage of the following properties of different protein. molecular weight/MW solubility charge differ

35、ence ligand specificity（親和層析） selective adsorption hydrophobic property,dialysis/透析 small molecules pass through.,Dialysis透析,Gel Filtration凝膠過濾層析,Column made of porus beads Separates in terms of size Big proteins elute first,2. In terms of solubility isoelectric precipitation Salting in and salting

36、out Organic solvent precipitation,Salting in / Salting out,Salting IN low concentrations of salt usually increases the solubility of charged macromolecules. Salting OUT high concentrations of added salt lowers the solubility of and they come out of solution.,Effect of K2SO4 on the solubility of Hb-CO,1) electrophoresis電泳 charged proteins move toward the opposite electrode. Many kinds of different electrophoresis methods,3 . Based on charge difference,聚丙烯酰胺凝膠電泳,等電聚焦/isoelectric focusing, IEF,2-dimensional electronphoresis,2、 Ion-exchange chromatography 離子交換層析法,Separates protei