基因工程研究技術(shù)課件

1、基因工程研究技術(shù)Preparation, analysis and manipulation of nucleic acids and proteins所有的分子生物學(xué)技術(shù)都是基于對(duì)核酸和蛋白質(zhì)性質(zhì)研究的基礎(chǔ)上發(fā)展的雜交： the base-pairing characteristics of DNA and RNAPCR： Thermophilic DNA polymeraseDNA克?。篋NA polymerase, restriction endonucleases and DNA ligase1. 核酸研究技術(shù)電泳分離：Separation by Electrophoresis 限制性

2、內(nèi)切酶切割：Cut by Restriction endonuclease雜交鑒定： Identification by HybridizationPCR擴(kuò)增： Amplification by PCR DNA克隆和基因表達(dá)：DNA Cloning and gene expressionDNA文庫(kù)的構(gòu)建和目的基因的篩選（Construction of DNA library & screening of target gene)基因組序列和分析：Genome sequence & analysis1. 凝膠電泳是根據(jù)DNA和RNA的分子量大小、拓?fù)浣Y(jié)構(gòu)等性質(zhì)進(jìn)行分離一種分子被放置到電場(chǎng)中，它就

3、會(huì)以一定的速度移向適當(dāng)?shù)碾姌O。我們把這種電泳分子在電場(chǎng)作用下的遷移速度，叫做電泳的遷移率；電泳的遷移率與電場(chǎng)強(qiáng)度和電泳分子本身所攜帶的凈電荷數(shù)成正比，與片段大小成反比。DNA、RNA由于其骨架中的磷酸基團(tuán)呈離子化狀態(tài)，帶有負(fù)電荷，在電場(chǎng)中向正電極移動(dòng)。線性DNA分子根據(jù)其分子量大小來(lái)分離。分子量大的DNA電泳速度比分子量小的DNA要慢。環(huán)狀DNA的電泳速度與其拓?fù)浣Y(jié)構(gòu)相關(guān)。不同構(gòu)型，相同大小分子量的DNA的電泳速度是：SC DNAL DNAOC DNAGel matrix (膠支持物)膠支持物是凝膠狀的多孔物質(zhì)，能允許DNA分子通過其內(nèi)部的孔隙。瓊脂糖（ Agarose）聚丙烯酰胺（ Poly

4、acrylamide）溴化乙錠（）是具有扁平分子的核酸染料，能插入或者相鄰堿基之間，在300nm波長(zhǎng)的紫外線照射下發(fā)出熒光。當(dāng)DNA和RNA用EB充分處理之后，其熒光強(qiáng)度與其分子量大小呈正比10聚丙稀酰胺 (Polyacrylamide): 分離能力高，能夠分辨相差一個(gè)堿基或者一個(gè)堿基對(duì)的DNA或者RNA分子只能分離小分子量大樣品 (1 to a few hundred bp).11DNA分子的遷移方向隨電場(chǎng)方向的周期性變化而不斷改變. 根據(jù)DNA地分子量大小、拓?fù)浣Y(jié)構(gòu)來(lái)分離樣品.用來(lái)分離大分子的樣品，分子量超過50Kb，甚至高達(dá)幾M.Pulsed-field gel electrophore

5、sis (脈沖場(chǎng)電泳)Switching between two orientations: the larger the DNA is, the longer it takes to reorient2. 限制性內(nèi)切酶在特定位點(diǎn)切割DNA分子識(shí)別位點(diǎn)粘性末端平末端同裂酶同尾酶？3. DNA雜交-確定特殊的DNA分子雜交（Hybridization):不同來(lái)源的單鏈DNA或者RNA通過堿基配對(duì)形成互補(bǔ)結(jié)構(gòu)的過程探針的標(biāo)記Radioactive labeling: display and/or magnify the signals by radioactivity. Non-radioacti

6、ve labeling: display and/or magnify the signals by antigen labeling: antibody binding enzyme binding - substrate application (signal release)Southern blotting ：將變性處理的后的DNA片段進(jìn)行凝膠電泳后，轉(zhuǎn)移至硝酸纖維素薄膜，再用放射性標(biāo)記的探針進(jìn)行雜交以顯示這些DNA，這種技術(shù)成為Southern blotting 。Northern blotting ：將RNA從瓊脂糖凝膠電泳轉(zhuǎn)移至轉(zhuǎn)移至硝酸纖維素薄膜，再用放射性標(biāo)記的探針進(jìn)行雜交的

7、技術(shù)。其原理與Southern blotting類似。探針雜交可以確認(rèn)電泳分離后的DNA和RNANorthern/Southern blot analysisgelmembraneElectrophoresisblottingHybridizationSouthern and Northern blottingDNA on blotRNA on blotGenomic DNA preparation RNA preparationRestriction digestion -Denature with alkali - Agarose gel electrophoresis DNA blott

8、ing/transfer and fixation RNA6. Probe labeling 6. Hybridization (temperature) 7. Signal detection (X-ray film or antibody) Western blottingWestern blotting：又稱為蛋白質(zhì)印跡法，用來(lái)檢測(cè)在不均一的蛋白質(zhì)樣品中是否存在目標(biāo)蛋白質(zhì)的技術(shù)。操作程序與Soutern blotting類似。先將蛋白質(zhì)經(jīng)過SDS電泳分離，然后轉(zhuǎn)移至醋酸纖維素薄膜之類的固體支持物上，通過專一性抗體探測(cè)目標(biāo)蛋白質(zhì)；隨后用標(biāo)記的第二抗體檢測(cè)在印跡中是否存在免疫復(fù)合物（一抗與抗

9、原的復(fù)合物）。22Blot typeTargetProbeApplicationsSouthern DNA DNA or RNAmapping genomic clonesestimating gene numbers, etcNorthernRNADNA or RNARNA sizes and abundance (gene expression level)WesternProteinAntibodiesprotein size and abundance (gene expression level)Comparison of Southern, Northern and Western

10、 bolt hybridizationDenaturation (變性): The target DNA (template) is separated into two stands by heating to 95Primer annealing (退火): The temperature is reduced to around 55 to allow the primers to anneal.Polymerization (elongation, extension) (延伸): The temperature is increased to 72 for optimal polym

11、erization step which uses up dNTPs and required Mg2+.The PCR cycle:Three different steps proceed in each PCR cycle. DenaturationPrimer annealing Polymerization反向PCR- reverse PCR先用一種在靶DNA區(qū)段上沒有識(shí)別位點(diǎn)的核酸內(nèi)切限制酶，從距靶DNA區(qū)段有一定距離的兩側(cè)位置切割DNA分子，然后將這些片段作分子內(nèi)連接成環(huán)形。根據(jù)已知的靶DNA序列按向外延伸的要求設(shè)計(jì)一對(duì)向外引物，保證被擴(kuò)增的是位于靶DNA區(qū)段兩側(cè)的未知DNA序列

12、。用一種在靶序列上沒有酶切位點(diǎn)的核酸限制性內(nèi)切酶消化DNA；產(chǎn)生大小不同的線性DNA片段群體，其中靶DNA區(qū)段的DNA分子長(zhǎng)度不超過23kb，經(jīng)連接后重新環(huán)化；按靶序列設(shè)計(jì)的一對(duì)引物與互補(bǔ)序列退火結(jié)合，其延伸方向如箭頭所指；反轉(zhuǎn)錄PCR：Reverse transcriptase (RT)-PCRPCR技術(shù)不僅可以擴(kuò)增DNA模板，還可以擴(kuò)增反轉(zhuǎn)錄成cDNA形式的RNA。這種在mRNA反轉(zhuǎn)錄之后進(jìn)行的PCR成為反轉(zhuǎn)錄PCR。反轉(zhuǎn)錄PCR：Reverse transcriptase (RT)-PCRAAA(A)n5-CapmRNA(dT)1218 primeranneal5-CapAAA(A)n3

13、5Reverse transcriptiondNTP, RT5-CapAAA(A)n5cDNA:mRNA hybridRegularPCRPCR誘變(PCR mutagenesis) 通過改變基因特定位點(diǎn)核苷酸序列來(lái)改變所編碼的氨基酸序列，用于研究某個(gè)氨基酸殘基對(duì)蛋白質(zhì)的結(jié)構(gòu)、催化活性以及結(jié)合配體能力的影響；也可用于改造DNA調(diào)控元件特征序列、修飾表達(dá)載體、引入新的酶切位點(diǎn)等。通過重疊延伸PCR進(jìn)行基因的定點(diǎn)突變。根據(jù)靶DNA序列設(shè)計(jì)的一對(duì)互補(bǔ)的突變引物，在相同的位點(diǎn)具有相同的堿基突變；分別以兩個(gè)突變引物進(jìn)行PCR擴(kuò)增；除去未參與擴(kuò)增的多余引物，由于具有重疊序列，經(jīng)過變性退火形成異源雙鏈分子，

14、其中一種結(jié)合方式不能延伸；正確配對(duì)的異源雙鏈分子利用靶DNA兩端的通用引物擴(kuò)增到突變位點(diǎn)位于靶DNA中間的序列5. DNA克隆和表達(dá)The ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning.34Processes of DNA cloning：Form the recombinant DNA molecules (重組DNA) by inserting your interested DNA fragments into a proper vector

15、(載體). (Require restriction enzymes and ligase)Transform (轉(zhuǎn)化) the recombinant DNA molecules into competent cells (感受態(tài)細(xì)胞).Propagation of the cells containing the recombinant DNA to form a clone (克隆), a set of identical cells containing the same recombinant DNA.Select the desired clones using the selec

16、tive marker. 用同樣的限制性內(nèi)切酶處理外源插入基因和載體；利用連接酶將切割好的插入片段和載體連接起來(lái)；將連接產(chǎn)物轉(zhuǎn)化至大腸桿菌感受態(tài)細(xì)胞中；在含有四環(huán)素的LB平板上培養(yǎng)大腸桿菌細(xì)胞質(zhì)粒和載體DNA文庫(kù)DNA文庫(kù)：由一組DNA的隨機(jī)克隆片段組成，每個(gè)單獨(dú)的片段都連接在一個(gè)載體上?；蚪M文庫(kù)：把某種生物的基因組DNA切割成適當(dāng)大小的片段，這些片段分別與載體連接，重組質(zhì)粒轉(zhuǎn)化進(jìn)宿主細(xì)胞，這樣的DNA文庫(kù)稱為基因組文庫(kù)。cDNA文庫(kù)：將mRNA經(jīng)過反轉(zhuǎn)錄形成的雙鏈DNA連接到載體上轉(zhuǎn)化進(jìn)宿主細(xì)胞，這樣的DNA文庫(kù)成為cDNA文庫(kù)。對(duì)于高等真核生物而言，基因組DNA十分龐大,基因可達(dá)數(shù)萬(wàn)個(gè)，

17、且基因組成結(jié)構(gòu)復(fù)雜，除編碼序列，還有非編碼序列及調(diào)控序列，基因間還存在大量的間隔序列和重復(fù)序列，因此，單個(gè)目的基因在整個(gè)基因組中所占的比例極其微小，除少數(shù)例外，絕大多數(shù)基因難以直接分離得到。為了解決這個(gè)難題，一種可行的方法就是將這個(gè)基因擴(kuò)增，增加成功分離目的基因的可能性。但是由于要分離的目的基因往往是未知基因，因此無(wú)法對(duì)它進(jìn)行特異性擴(kuò)增，而只能對(duì)所有的基因進(jìn)行擴(kuò)增，也就是構(gòu)建該生物材料的基因文庫(kù)，然后再根據(jù)不同的方法將所需的克隆篩選出來(lái)，最后分離得到目的基因。基因組DNA文庫(kù)的類型 根據(jù)所選用的載體可以分為： 質(zhì)粒文庫(kù)噬菌體文庫(kù)粘粒文庫(kù)人工染色體文庫(kù)（細(xì)菌人工染色體文庫(kù)、酵母人工染色體文庫(kù)等）

18、載體能夠容載的DNA片斷大小直接影響到構(gòu)建完整的基因文庫(kù)所需要的重組子的數(shù)目。載體系列： 容量為 24 kp cosmid載體： 容量為 50 kb YAC： 容量為 1 Mb BAC: 容量為 300 kb用質(zhì)粒載體構(gòu)建基因組文庫(kù)的流程該法簡(jiǎn)單、快速、易于操作，但僅適合一些低等真核生物和原核生物。用噬菌體載體構(gòu)建基因組文庫(kù)的流程用黏粒載體構(gòu)建基因組文庫(kù)的流程用酵母人工染色體構(gòu)建基因組文庫(kù)的流程基因組DNA文庫(kù)的質(zhì)量標(biāo)準(zhǔn)一個(gè)理想的基因組DNA文庫(kù)應(yīng)具備下列條件：重組克隆的總數(shù)不宜過大，以減輕篩選工作的壓力。載體的裝載量最好大于基因的長(zhǎng)度，避免基因被分隔克隆?？寺∨c克隆之間必須存在足夠長(zhǎng)度的重疊

19、區(qū)域，以利克隆排序?？寺∑我子趶妮d體分子上完整卸下。重組克隆能穩(wěn)定保存、擴(kuò)增、篩選。 基因組DNA文庫(kù)構(gòu)建的程序載體DNA的制備；高純度大相對(duì)分子質(zhì)量基因組DNA的提取和大片段的制備；高純度大相對(duì)分子質(zhì)量基因組DNA的部分酶切與脈沖電泳分級(jí)分離；載體與外源片段的連接與轉(zhuǎn)化或侵染宿主細(xì)胞；重組克隆的挑選和保存。 基因組DNA文庫(kù)構(gòu)建的程序1載體DNA片段的制備 DNA分離純化限制酶切 脫磷酸化反應(yīng)2供體DNA片段的制備總DNA分離純化物理切割法超聲波（300bp）或機(jī)械攪拌（8kb）或酶切法（內(nèi)切酶Sau3A進(jìn)行局部消化。可得到10-30kb的隨機(jī)片斷。 ）分離特定大小DNA片段 DNA的不完

20、全酶切目的片段低熔點(diǎn)瓊脂糖回收3.載體與基因組DNA大片段的連接直接連接、人工接頭或同聚物加尾。（1） 粘性末端直接連接：載體與外源DNA大片段的兩個(gè)末端都有相同的粘性末端。如：Sau3A與BamHI的酶切末端。（2）人工接頭法（adapter）人工合成的限制性內(nèi)切酶粘性末端片斷。接上人工接頭粘性末端3.載體與基因組DNA大片段的連接（3）同聚物加尾粘性末端CCCCCC末端轉(zhuǎn)移酶4.重組DNA轉(zhuǎn)化受體細(xì)胞（1）根據(jù)克隆載體的性質(zhì)選擇合適的受體細(xì)胞常見的宿主細(xì)胞：DH5:用于鋪制與培養(yǎng)質(zhì)粒平板和粘粒平板的重組缺陷型的抑制型菌株，可與pUC編碼的半乳糖苷酶氨基端實(shí)現(xiàn)互補(bǔ)。HB101: 用于大規(guī)模制

21、備質(zhì)粒的抑制型菌株，轉(zhuǎn)化效率高JM101: 可支持帶有琥珀突變載體生長(zhǎng)的宿主菌JM109: 支持帶有琥珀突變載體生長(zhǎng)的重組缺陷的抑制型菌MZ-1: 溫度敏感的溶源性菌株，用于含有噬菌體啟動(dòng)子質(zhì)粒載體的宿主菌一、基因組DNA文庫(kù)的構(gòu)建構(gòu)建DNA文庫(kù)的方法annealReverse transcriptionRegularPCRConstruction of cDNA libraryColony screening Antibiotic screening (抗生素選擇)： only the recombinant plasmids grow on the antibiotic-containin

22、g plate.Blue-white screening (藍(lán)白斑選擇): DNA insertion in the vector shuts down the LacZ gene expression, and turns the colony to white. Colony hybridization screening (菌落雜交篩選) from a library.Antibiotic screening：only intact plasmids grow on the antibiotic-containing plate.篩選方法1：抗生素選擇AmproripUC18(3 kb)

23、MCS (Multiple cloning sites,多克隆位點(diǎn)）Lac promoterlacZInsertion of a DNA fragment interrupts the ORF of lacZ gene, resulting in non-functional gene product that can not digest its substrate x-gal. 篩選方法2：藍(lán)白斑篩選Transfer to nitrocelluloseor nylon membraneDenature DNA(NaOH)Bake onto membraneProbe with 32p-la

24、bled DNA complementary to gene of interestExpose to filmSelect positive from master plateKeep master plate Screening by plaque hybridization篩選方法3：菌落原位雜交 Analysis of a clone Restriction mapping: digestion of the plasmid prepared from a clone with restriction enzymes to investigate if the interested D

25、NA is inserted the recombinant plasmid.Sequencing the cloned DNA to see if the inserted DNA maintains the correct sequence.Restriction mappingNco I & Xho ISequencing富集的混合培養(yǎng)物研究結(jié)果黏附在纖維素上的菌群研究結(jié)果一類球菌通過絲狀物質(zhì)黏附在纖維素上；細(xì)菌降解濾紙后在纖維上形成孔洞，且與菌體形態(tài)類似， 通過絲狀物質(zhì)與孔壁相連；黏附在纖維素上的球菌可能為纖維素降解菌16S rRNA基因系統(tǒng)發(fā)育樹研究結(jié)果原核生物通用引物530f/14

26、92r克隆數(shù):49個(gè)，57%為未培養(yǎng)細(xì)菌OUT: 16S rRNA97%7個(gè)OTUs: RFC1RFC797%98%98%97%95%97%99%安，木聚糖纖維素降解環(huán)境乳桿菌纖維素降解環(huán)境來(lái)自牦牛（安）產(chǎn)甲烷菌基因表達(dá)69Expression vectors: allowing the exogenous DNA to be stored and expressed in an organism.-E. coli expression vector-Yeast expression vector-Mammalian expression vectorFeatures:In addition

27、to the origin of replication, selective marker, multiple cloning site, expression vector has to contain a promoter and terminator for transcription. The inserted gene has to have a start codon and a stop codon for translation表達(dá)載體：pET28a外源基因表達(dá)策略目標(biāo)基因的PCR擴(kuò)增；連接克隆載體，轉(zhuǎn)化感受態(tài)細(xì)胞；篩選陽(yáng)性克隆子并測(cè)序鑒定；酶切陽(yáng)性克隆子和表達(dá)載體；連接目的

28、基因和表達(dá)載體；重組表達(dá)載體轉(zhuǎn)化表達(dá)菌株；IPTG誘導(dǎo)外源基因表達(dá)；SDS鑒定表達(dá)情況。外源基因表達(dá)蛋白的SDSFusion proteinsGene cloning and expression provides a very powerful way to obtain a large amount of the target protein fused with an enzyme, fluorescence protein or a tag for identification (鑒定) or purification (純化). ExamplesLac fusions (Enzyme

29、) : fuse your target gene with the LacZ coding sequence. His-tag fusions (Tag) : A sequence encodes His-tag was inserted at the N- or C- termini of the target ORF, allowing the fusion protein to be purified by binding to Ni2+ column. GFP fusions (Fluorescence protein): insert your targeted gene at t

30、he N- or C- termini of GFP (green fluorescence protein, 綠色熒光蛋白), and your fusion protein will give you green fluorescence signal. DNA 測(cè)序兩種測(cè)序方法：1. Maxam and Gilbert：化學(xué)修飾法 很少應(yīng)用；2. Sanger：雙脫氧法，鏈終止法（Chain-termination method），廣泛應(yīng)用。鏈終止法 (Chain-termination method )理論基礎(chǔ)：DNA聚合酶能利用單鏈DNA為模板，準(zhǔn)確合成DNA的互補(bǔ)鏈DNA聚合酶能夠

31、利用2,3-雙脫氧核苷酸ddNTPs作底物，使之連接到核苷酸鏈的3-末端，從而終止DNA鏈的生長(zhǎng)。雙脫氧核苷酸對(duì)DNA聚合酶的抑制機(jī)理：ddNTPs的核糖基團(tuán)上C2和C3的羥基被H取代，當(dāng)ddNTPs加到核苷酸鏈上之后，后來(lái)的核苷酸不能形成新的磷酸二酯鍵，從而終止鏈的合成。四個(gè)獨(dú)立的反應(yīng)體系： dNTP+ ddGTP, dNTP+ ddATP dNTP+ ddCTP, dNTP+ ddTTP每個(gè)ddNTP都帶有一個(gè)獨(dú)特的熒光信號(hào)，可以從電泳之后的凝膠上讀出。Automatic sequencerFluorescence Labeled ddNTP2. Polymerase catalyzed

32、技術(shù)路線與要求制備單鏈模板 將單鏈模板與一小段引物退火 加入DNA多聚酶4種脫氧核苷酸分別加入少量4種雙脫氧核苷酸 將4種反應(yīng)產(chǎn)物分別在4條泳道電泳 根據(jù)4個(gè)堿基在4條泳道的終止位置讀出基因序列 鏈終止法對(duì)DNA聚合酶的要求高酶活性：保證不會(huì)反應(yīng)提前終止；無(wú)5 3外切酶活性：保證不切除新合成鏈的5端，改變鏈長(zhǎng)度，給讀序造成誤差；無(wú)3 5外切酶活性：證不切除新合成鏈的3端，改變鏈長(zhǎng)度，給讀序造成誤差；測(cè)序的DNA聚合酶目前普遍采用的測(cè)序酶為Sequenase, 來(lái)自T7噬菌體將DNA克隆到質(zhì)粒載體這種方法是獲得測(cè)序模板DNA最常用的方法。獲得的DNA通過熱變性或者堿變性轉(zhuǎn)變?yōu)閱捂淒NA進(jìn)行測(cè)序。

33、優(yōu)點(diǎn)：可雙向測(cè)序。缺點(diǎn)：樣品可能有少量細(xì)菌的DNA或者RNA污染，會(huì)干擾測(cè)序。鏈終止反應(yīng)要求單鏈作為模板如何得到單鏈DNA ？將DNA克隆到M13噬菌體載體M13噬菌體載體是專 為得到單鏈DNA測(cè)序模板而設(shè)計(jì)的。M13噬菌體本來(lái)含有單鏈DNA基因組，感染大腸桿菌的M13可轉(zhuǎn)變?yōu)殡p鏈復(fù)制型。 M13噬菌體載體是雙鏈的，相當(dāng)于M13噬菌體的復(fù)制型。用含有待測(cè)序片段的M13噬菌體載體感染大腸桿菌，大腸桿菌分泌的噬菌體就含有單鏈DNA基因組。缺點(diǎn)：只能用于短片段DNA，大于3kb的片段在克隆過程中會(huì)發(fā)生缺失和重排。Shotgun sequencing of a bacterial genome (鳥槍

34、法測(cè)細(xì)菌的基因組)通過一定方法將細(xì)菌基因組打碎成隨機(jī)片段，每個(gè)片段的平均長(zhǎng)度為1kb左右，然后將這些隨機(jī)片段連接到載體上；隨機(jī)挑取每個(gè)含有基因組片段的重組質(zhì)粒，利用載體上的引物序列做引物，在自動(dòng)測(cè)序儀上測(cè)序。In use of shotgun sequencing strategy, multiple sequence coverage is required to obtain all genome sequence.For example: The genome of bacteria Hemophilus influenzae is 1.8 Mb, each sequence read

35、produces 600 bp of sequence. If 33,000 different clones were picked for sequencing, a total of 600 bp x 33,000= 20 Mb sequence was produced.The shotgun strategy permits a partial assembly of large genome sequenceThe core techniques for sequencing the large genomes, e.g. human, are automated shotgun

36、sequencing (obtain sequence) then the subsequent use of computer to assemble the different sequences (analyze sequence, which is the rate-limiting step).89Contigs are linked by sequencing the ends of large DNA fragments (plasmid library containing larger DNA fragments). 研究基因組時(shí)最常用的工具：BLAST: Basic Loc

37、al Alignment Search Tool可以搜索不同的編碼蛋白質(zhì)的基因之間的相似序列。Input a query sequence (詢問序列): a stretch of amino acids or the DNA sequence encoding your interested protein function. Ask the computer to search for the homologous sequences in a protein or DNA database, and you will get all the available genes that ma

38、y have the similar protein function.BLAST結(jié)果蛋白質(zhì)研究技術(shù)Protein purification (蛋白質(zhì)純化) Affinity chromatography can facilitate more rapid protein purification (親和層析純化)Protein separation by PAGE gel electrophoresis (蛋白質(zhì)分離) and identification by Western analysisProtein sequencing (蛋白質(zhì)測(cè)序)Proteomics (蛋白質(zhì)組學(xué))蛋白質(zhì)的純

39、化對(duì)于研究蛋白質(zhì)的功能非常重要。.Although there are thousands of proteins in a single cell, each protein has unique properties, such as size, charge (電荷), shape, and in many instance, function, that make its purification somewhat different from others.1. Protein purification (蛋白質(zhì)純化)柱層析法是分離蛋白質(zhì)的有效方法In this approach,

40、protein fractions are passed though glass columns filled with appropriated modified small acrylamide or agarose beads.離子交換層析根據(jù)所帶的電荷分離蛋白質(zhì).The beads are modified with either negative-charged or positive-charged chemical groups.Proteins bind more strongly requires more salt to be eluted. 凝膠過濾層析 該方法根據(jù)蛋白

41、質(zhì)的大小和形狀分離不同的蛋白質(zhì)。 The beads for it have a variety of different sized pores throughout. Small proteins can enter all of the pores, and take longer to elute; but large proteins pass quickly.2. Affinity chromatography can facilitate more rapid protein purificationIf the target protein is known to establ

42、ish a specific and high-affinity interaction with a specific protein/nucleic acids/small molecule, we can couple this specific partner of the target protein to the column and thus the target protein will be selectively bound to the column.This method is called affinity chromatography. Affinity chrom

43、atographyEnzyme-substrate bindingReceptor-ligand bindingAntibody-antigen bindingNi2+-His tag-fusion protein bindingImmunoaffinity chromatography(免疫親和層析)An antibody that is specific for the target is attached to the bead, and ideally only the target protein can bind to the column.缺點(diǎn): 有時(shí)候目標(biāo)蛋白與抗體結(jié)合力太強(qiáng)不

44、能被洗脫.Sometimes tags (epitopes, 抗原決定基) can be added to the N- or C- terminal of the target protein, using DNA cloning method, to make the fusion protein. This allows the modified fusion proteins to be purified using immunoaffinity purification and a heterologous antibody specific for the tag.Importan

45、tly, the binding affinity can change according to the condition. e.g. the concentration of the Ca2+ in the solution.Immunoprecipitation (免疫沉淀)將靶蛋白的抗體連接到固體基質(zhì)上，再將可能與靶蛋白發(fā)生相互作用的待篩選蛋白加入反應(yīng)體系中，用低離心力沉淀或微膜過濾法在固體基質(zhì)和抗體的共同作用下將蛋白復(fù)合物沉淀到試管的底部或微膜上。 Its a useful method to detect what proteins or other molecules are

46、associated with the target protein. 3. Protein separation by PAGE gel electrophoresis, followed by a western analysis天然蛋白質(zhì)沒有固定的電荷和二級(jí)結(jié)構(gòu)If we treat the protein with a strong detergent SDS, the higher structure is usually eliminated. SDS處理之后的蛋白質(zhì)都帶負(fù)電荷。106Sometimes, mercaptoethanol (巰基乙醇) is need to brea

47、k the disulphide bond.Thus, the protein molecules can be resolved by electrophoresis in the presence of SDS according to the length of individual polypeptide.After electrophoresis, the proteins can be visualized with a stain, such as Coomassie brilliant blue (考馬氏亮藍(lán)).Proteins from the PAGE gel can be

48、 transferred to a membrane, followed by a western analysis of the target protein by a corresponding antibody.1074. Protein sequencing (蛋白質(zhì)測(cè)序)Two sequence method: Edman degradation (Edman切割法)Tandem mass spectrometry (MS/MS) (串連質(zhì)譜).Due to the vast resource of complete or nearly complete genome, the de

49、termination of even a small stretch of protein sequence is sufficient to identify the gene.5. Proteomics (蛋白質(zhì)組學(xué))Proteomics is concerned with the identification of the full set of proteins produced by a cell or a tissue under a particular by a particular set of conditions. 蛋白質(zhì)組學(xué)是蛋白質(zhì)（protein）和基因組（geno

50、me）研究在形式和內(nèi)容兩方面的組合，該技術(shù)致力于研究某一物種、個(gè)體、器官、組織或細(xì)胞在特定條件、特定時(shí)間所表達(dá)的全部蛋白質(zhì)圖譜。蛋白質(zhì)組學(xué)研究的三種方法1. 雙向電泳 for protein separation (蛋白質(zhì)分離).2. 質(zhì)譜分析 for the precise determination of the molecular weight and identify of a protein (蛋白質(zhì)鑒定).3. Bioinformatics for assigning proteins and peptides to the predicted products of protei

51、n coding sequence in the genome (蛋白質(zhì)確定).雙向電泳(TwoDimensional Electrophoresis，2-D)技術(shù):等電聚焦（Isoelectric focusing）及SDS-聚丙烯酰胺凝膠雙向電泳技術(shù)。蛋白質(zhì)是兩性分子，在不同pH緩沖液中表現(xiàn)出不同的帶電性，因此，在兩性電解質(zhì)電泳體系中，不同等電點(diǎn)的蛋白質(zhì)會(huì)聚集在介質(zhì)上不同的區(qū)域（等電點(diǎn)）從而被分離。111蛋白質(zhì)與核酸相互作用的研究方法Gel retardation (凝膠阻滯)凝膠滯緩試驗(yàn)（gel retardation assay），又叫作DNA遷移率變動(dòng)試驗(yàn)（DNA mobility

52、shift assay），是用于體外研究DNA與蛋白質(zhì)相互作用的一種特殊的凝膠電泳技術(shù)。在凝膠電泳中，由于電場(chǎng)的作用，裸露的DNA朝正電極移動(dòng)的距離與其分子量的對(duì)數(shù)成反比。如果此時(shí)DNA分子與某種蛋白質(zhì)相結(jié)合，那么，由于分子量增大，它在凝膠中的遷移作用便會(huì)受到阻滯，在特定電壓和時(shí)間內(nèi)朝正電極移動(dòng)的距離也就相應(yīng)縮短了。DNA bound totwo proteinsDNA-proteincomplexBare DNAA DNA bound with more than one protein to form a larger complex.DNase I footprinting (DNase

53、 I 足跡法) 確定DNA上與蛋白質(zhì)結(jié)合的精確位點(diǎn)5*Sequence ladder is required to determine the precise positionAATAAG一種確定DNA結(jié)合蛋白在DNA分子上的結(jié)合位點(diǎn)的方法。原理：將含有特定順式元件的雙鏈DNA片段進(jìn)行單鏈單末端標(biāo)記，與初步純化的DNA結(jié)合蛋白在體外行結(jié)合作用，然后加入DNase I對(duì)DNA一蛋白質(zhì)復(fù)合物進(jìn)行隨機(jī)切割，產(chǎn)生一系列不同長(zhǎng)度的DNA片段，其相鄰片段只相差一個(gè)核苷酸。DNA結(jié)合蛋白與其特異序列結(jié)合后，由于空間位阻等多種效應(yīng)，DNase I不能對(duì)其進(jìn)行切割 .Bind proteinDNase(mild),then removeprotein and denature DNADNase footprinting （1）The protein protects DNA from attack by DNase.