分子生物學(xué)基因工程與基因體外表達(dá)課件

1、分子生物學(xué)基因工程與基因體外表達(dá)課件第十三章基因工程與基因體外表達(dá)Gene Engineering and Gene Expression目的與意義1.獲得感興趣的目的基因2.研究基因結(jié)構(gòu)特征3.表達(dá)基因產(chǎn)物4.研究基因功能基本要素1. 目的基因 (target genes)2. 工具酶 (tool enzymes)3. 載體 (vectors)4. 宿主細(xì)胞 (host cells)Main contents 工具酶DNA載體基因克隆過程真核細(xì)胞基因轉(zhuǎn)染基因改造克隆基因表達(dá)電子克隆基本概念及背景For Example 如何從植物中克隆法呢基焦磷酸合酶(FPS)?1. 查資料認(rèn)識FPS2. 設(shè)

2、計實驗方案3. 可行性分析For Example FPS 克?。?RT-PCR， 3-RACE， 5-RACE改進的異硫氰酸胍一步法提取三七根部總RNART-PCR擴增fps部分保守序列3-RACE 及克隆測序5-RACE 及克隆測序序列拼接mRNA: 5 AAAAAA3TTTTTTT-接頭cDNA:Oligo dT接頭基因特異性引物3 Full RACE原理圖5 Full RACE原理圖(1) P5-端磷酸化RT-Primer未知區(qū)域已知序列Target mRNAAAAAAAAA-5 AAAAAAAA-5 P未知區(qū)域逆轉(zhuǎn)錄RNA分解P未知區(qū)域未知區(qū)域S1A1S2A21st PCR prime

3、rs2nd PCR primers用RNA ligase進行環(huán)化或形成首尾連接物未知區(qū)域1stand 2nd PCR5-端磷酸化RT primer部位包含未知的5上游區(qū)域的擴增產(chǎn)物(2) (3) (4) Thank YouDNA重組 DNA recombinationSome concerned concepts:DNA重組技術(shù) DNA recombination technique分子克隆 Molecular cloning基因工程 Genetic engineering克隆 Cloning 三大理論基礎(chǔ)1953年，Watson 和Crick揭示了DNA分子的雙螺旋結(jié)構(gòu)模型和半保留復(fù)制原理，

4、解決了基因的自我復(fù)制和傳遞的過程。40年代，O.T. Avery等通過肺炎鏈球菌轉(zhuǎn)化實驗發(fā)現(xiàn)遺傳物質(zhì)的攜帶者是DNA而不是蛋白質(zhì)50年代末到60年代初，相繼提出了“中心法則”和操縱子學(xué)說，成功破譯了遺傳密碼，闡明了遺傳信息的流向和表達(dá)問題。三大技術(shù)發(fā)明 DNA分子的體外切割與連接技術(shù) 1970年 Smith, Wilcox 流感嗜血桿菌(Haemopbilus influenzae) Hind II 1972年 Boyer EcoR I “GAATTC” 1967年 世界上5個實驗室?guī)缀跬瑫r發(fā)現(xiàn)了DNA ligase 1970年 T4 DNA ligase大腸桿菌轉(zhuǎn)化體系的建立-復(fù)制工廠基因工

5、程載體的使用：plasmid, phage, viruses1978年Nobel 醫(yī)學(xué)與生理學(xué)獎The Nobel Prize in Medicine was awarded, in 1978, to Daniel Nathans, Werner Arber, and Hamilton Smith for the discovery of restriction endonucleases. Their discovery lead to the development of recombinant DNA technology that allowed, for example, the l

6、arge scale production of human insulin for diabetics using E. coli bacteria. Over 3000 restriction enzymes have been studied in detail, and more than 600 of these are available commercially and are routinely used for DNA modification and manipulation in laboratories.DNA ligase repairing chromosomal

7、damage.ligase I, DNA, ATP-dependent第一節(jié) 基因克隆是利用工具酶進行操作工具酶(Tool enzymes )-用于對DNA分子進行定點切割、連接、擴增、標(biāo)記等操作的特殊酶類，已被純化并商品化進行應(yīng)用一、 限制酶 (RE , restriction endonuclease )二、其他工具酶 (other tool enzymes)一、RE在基因克隆中用于切割DNARE (restriction enzyme, restriction endonuclease )-限制性核酸內(nèi)切酶，限制酶，能識別DNA雙鏈內(nèi)特異位點并水解磷酸二酯鍵，產(chǎn)生特定末端的酶Restri

8、ction enzyme Functions:能識別DNA內(nèi)部特異位點并且裂解磷酸二酯鍵(1) Sorting of restriction enzyme有三型，但最重要的是II型酶(2) Naming of RE 細(xì)菌屬名細(xì)菌種名細(xì)菌菌株編號Escherichia coli RY13 I屬Genus, 種-species, 菌株-strainEcoR ITwo catalytic manganese ions (one from each monomer) are shown as magenta spheres and are adjacent to the cleaved sites i

9、n the DNA made by the enzyme (depicted as gaps in the DNA backbone). Structure of the homodimeric restriction enzyme EcoRI (cyan and green cartoon diagram) bound to double stranded DNA (brown tubes) based on the PDB 1QPS X-ray crystallographic coordinates.(3) 限制酶的識別和切割位點Characters: 通常識別46個堿基，少數(shù)識別8個所

10、識別的序列一般為回文結(jié)構(gòu)(palindrome)在特異位點內(nèi)切割DNA雙鏈，產(chǎn)生兩種末端 粘性末端平端5-粘性末端3-粘性末端5-CCCGGG-33-GGGCCC55-CCC GGG-33-GGG CCC-5+平端切割(blunt end, such as Sma I )Go to 1095-GGTGAATTCAGC-33-CCACTTAAGTCG55-TTGCTGCAGAAG-33-AACGACGTCTTC55-粘性末端 (EcoR I )3-粘性末端 ( Pst I )5-GGTG AATTCAGC-33-CCACTTAA GTCG5+5-TTGCTGCA GAAG-33-AACG ACG

11、TCTTC5+(4) 同工異源酶( isoschizomers ) 來源不同，但能識別和切割同一位點的RE 例：Bam H I & Bst I ( GGATCC) Xho I & Pae R7 (CTCGAG)(5) 同尾酶( isoaudamers ) 識別序列不同，但產(chǎn)生相同粘性末端的RE 例：Bam H I ( GGATCC) Sau 3A I ( NGATCN)RE作用的條件： 一定的鹽溶度；Mg2+; 不需ATPRE識別序列長度與切點數(shù)：對同一DNA，識別序列短的，切點數(shù)多，片段較短；反之則反之二、基因克隆需要具有不同功能的工具酶其他工具酶-用于對DNA分子進行多種操作，也叫修飾酶作

12、用：剪切、補平、連接、化學(xué)修飾等常用：(1) DNA polymerase I，Klenow Fragment (2) Reverse transcriptase(3) T4 DNA ligase(4) Alkaline phosphatase(5) Terminal deoxynucleotidyl transferase, TdT(6) Taq DNA polymeraseSo on (1) DNA polymerase IActivities：53聚合活性， 53及 35外切活性Functions：在生物體內(nèi)，填補引物切除后留下的空缺，修復(fù)在生物體外，缺口平移制備探針DNA polyme

13、rase IKlenow fragment (76KD)Another small fragment 枯草桿菌蛋白酶Klenow片段35kD76kDNCE.coli DNA pol I53外切酶53聚合酶35外切酶用枯草桿菌蛋白酶裂解全酶53聚合酶35外切酶76kDCKlenow fragment 用途：1) 補齊雙鏈DNA的3-末端2) 通過補齊3端使3端標(biāo)記3) 在cDNA克隆中，第二股鏈的合成4) DNA序列分析常用的reverse transcriptase:禽類成骨細(xì)胞性白血病病毒(AMV)逆轉(zhuǎn)錄酶(2) Reverse transcriptase用途：合成cDNA，構(gòu)建cDNA文庫

14、Moloney 小鼠白血病病毒(MMLV)逆轉(zhuǎn)錄酶第二節(jié) 基因克隆需要特定的DNA載體基因克隆為什么需要載體？載體的類型-克隆載體，表達(dá)載體載體的來源細(xì)菌質(zhì)粒噬菌體DNA(DNA, M13 DNA)病毒DNA人工載體(cosmid)等DNA載體(vector)-能與DNA片段連接，構(gòu)建成新的重組DNA分子，并可攜帶該外源DNA片段進入一定宿主細(xì)胞，在宿主中擴增甚至表達(dá)，并有遺傳標(biāo)記進行篩選載體的概念Cloning vector能夠攜帶感興趣的外源DNA（target DNA）進入宿主細(xì)胞，并將外源DNA在宿主內(nèi)進行克隆和擴增，而采用的一些DNA分子稱為Cloning vector 。Cloni

15、ng vector classes Plasmid DNA (質(zhì)粒DNA)Phage DNA (噬菌體DNA)Virus DNA (病毒DNA)Expression vector 能使外源基因在宿主中表達(dá)的載體Expression vector: 含表達(dá)調(diào)控有關(guān)的元件When E coli is used as host cell, plasmid, phage, cosmid, M13 phage can usually be used as vectors載體的本質(zhì)是什么？ Vector characters (cloning vector):能在宿主細(xì)胞中自我復(fù)制、自我表達(dá)分子相對較小，

16、在宿主細(xì)胞中有高拷貝具有遺傳標(biāo)志有多個限制性酶單一酶切位點(多克隆位點)易與宿主DNA相分離一、常用克隆載體主要來自質(zhì)粒和病毒(一)質(zhì)粒是常用的克隆載體Examples:pBR322pUC Characters:Small molecular weight, high copiesMore than one genetic markMultiple cloning sites, MCSpBR322Turn to 64pUC19 (二) 噬菌體經(jīng)過重組也可以攜帶外源 DNAExamples: bacteriophage, phage; M13 phageIt is a kind of virus

17、 which can infect bacteria bacteriophages in common use:EMBL spectrum gt spectrumCharon spectrum bacteriophage, phage 噬菌體是侵犯細(xì)菌的病毒。在菌體內(nèi)為環(huán)狀DNA分子；分離產(chǎn)物為雙鏈線狀DNA分子，有天然的粘性末端（稱COS位點），進入宿主菌后可自行環(huán)合?；蚪MDNA長約為,共有66個基因，較復(fù)雜。Character：1、其生長必需的序列位于左右二臂上，中部約30%為生長非必需；2、成熟需要包裝（大小為原來的75-105%時）Two kinds of vectors：置換型載體

18、；插入型載體置換型噬菌體載體：用限制酶切除中央片段供目的基因替代，目的基因與左、右載體兩臂結(jié)合。替代片段可達(dá)923Kb。左臂中央片段右臂酶切點酶切點切除后用目的基因取代COS位點COS位點（12bp)The life period of phage(噬菌體的生活周期):Lysogenic pathway( 溶原生長途徑)Lysis pathway(溶菌生長途徑)溶原生長溶菌生長溶原轉(zhuǎn)溶菌生長以噬菌體為媒介的轉(zhuǎn)導(dǎo)作用 gt10:insertion vector; multiple cloning site: CIThe positive marks of screening:Whether th

19、e recombinant vector can be wrapped(If no extraneous DNA replaced, the vector would be too small to be wrapped)With insertion, CI activity lost, to be transparent spots; Without insertion, CI keeps intact, to be turbid spots gt11：insertion vector; MCS: lac Z；expressedWith insertion into lac z of gt1

20、1, white spotsOtherwise blue spots (四) 粘性質(zhì)粒適合克隆較大的DNA片段粘性質(zhì)粒(cosmid)由DNA的cos區(qū)與質(zhì)粒重新構(gòu)建的載體，具有與質(zhì)粒相同的結(jié)構(gòu)特點，為雙鏈環(huán)狀DNA。It is constructed with the cos region of DNA and plasmid, which is double cycle DNA with bigger content for cloning (4050kb)Characters:1) With antibiotics marks and replication element itself

21、2) With cos region of phage, can be wrapped3) With one or more cloning sites4) Small molecular weight5) non-recombination cosmid too small to be wrapped, so it is screened easily(三) M13噬菌體適合制備單鏈DNAM13 bacteriophage:It is a kind of E coli phage, the genome6.5kb, a cycle DNA containing singlestrand DN

22、ATwo forms:Wild form; Replication form (RF)The most merit:Can be released from host as single strandM13 phage Characters:In vivo, double strands DNA (can be replicated)In vitro, single strand DNA (can be used as template for DNA sequencing, or make DNA probes for hybridization )After entering host c

23、ells, it is replicated to double strands DNA and becomes replicational form DNA ( RF DNA ) which can be used as cloning vector(五) 病毒載體可將外源DNA帶入哺乳動物細(xì)胞Virus vectors in common use:逆轉(zhuǎn)錄病毒，腺病毒，腺相關(guān)病毒，EB病毒等病毒載體，可將外源基因?qū)胧荏w細(xì)胞Characters:多數(shù)均已質(zhì)?；?，由病毒啟動子、包裝元件、選擇性遺傳標(biāo)記及pBR322復(fù)制起始點組成Other vectors:YAC, yeast artifici

24、al chromosome (0.52Mb) BAC, bacterial artificial chromosome (0.4Mb)二、表達(dá)載體將外源基因帶入宿主并表達(dá)Expression vectors Concept:The conditions for expression vector:With expression structures except for the characters of cloning vectorsThe vectors which can make the extraneous DNA be expressed in host cells are ter

25、med as expression vectors.Sorts:Expression vectors of E coli (prokaryote )Expression vectors of eukaryoteExpression vectors of mammal animals (一) 原核表達(dá)載體適于原核細(xì)胞表達(dá)外源基因Expression vectors of E coli:除克隆所需成分外，還含有啟動子，核糖體結(jié)合位點，轉(zhuǎn)錄終止序列等表達(dá)元件Trp-lac promoter ( or tac promoter)Inducer: IPTGStrains: RB791, XL-1-blu

26、e, SB221, JM 1091. PromoterT7 of T7 phage It is a high effective expression promoterStrain: JM109(DE3)PL of -phage (temperature induce promoter ) Controlled by cIts857 (sensitive to temperature), clts857為溫度敏感阻抑物Strain: M5219原核生物mRNA與核蛋白體小亞基結(jié)合的分子機制AUU C C UCCACUAGG核蛋白體小亞基的16S-rRNA5A G G A PuPuUUUPuPu

27、AUG3mRNARps辨認(rèn)序列SD序列Ribosome-binding site, RBS Including AUG, SD sequence 2. 核糖體結(jié)合位點(RBS)作用： 3. 轉(zhuǎn)錄終止序列保證外源基因在宿主中的高效表達(dá)使讀碼框架能夠正確轉(zhuǎn)錄(二) 真核表達(dá)載體適于真核細(xì)胞表達(dá)外源基因Eukaryote expression vectors:含有一定的原核序列：E coli中的ori位點；antibacterial site含有一定的真核序列：真核細(xì)胞中的藥物抗性基因；真核表達(dá)組件，如真核復(fù)制起點，啟動子/ 增強子，克隆位點，加尾信號等 Expression vectors of

28、mammal animalIf a gene needs to be expressed in mammal animal cells, the eukaryote expression vector must be used.The essential elements including:Replica origin, antibiotics sites, eukaryote promoter, enhancer, cloning sites, termination signal, poly A signal第三節(jié) 基因克隆過程包括五個基本部分Gene cloning process(1

29、) 制備目的基因及相關(guān)載體(2) 將目的基因與載體進行連接(3) 將重組DNA導(dǎo)入受體細(xì)胞(4) DNA重組體的篩選與鑒定(5) DNA重組體擴增、表達(dá)及其他研究分切接篩轉(zhuǎn)一、采用不同方法獲取目的基因Methods to get a target gene:(1) To prepare genomic library(2) To prepare cDNA library or cDNA(3) To PCR(4) To synthesize the DNA fragment by chemical method(一) 從基因組文庫中獲得目的基因Genomic library :指含有一個機體基因

30、組DNA的全套重組DNA分子的克隆群體用于構(gòu)建基因組文庫的載體：-噬菌體(- phage)，粘性質(zhì)粒(cosmid)Genomic library construction:分離高分子量DNA分離回收1525 kb的DNA片段部分消化，以切成一定大小片段回收片段與適當(dāng)載體連接所有重組DNA分子轉(zhuǎn)入宿主細(xì)胞并擴增基因組文庫隨機文庫克隆數(shù)目計算隨機文庫指代表基因組各部分DNA的摩爾數(shù)相等。對于隨機文庫，有：ln(1-P)N=ln(1- )xyN: 克隆數(shù)目P: 設(shè)定的概率值(如：0.99)x: 插入片段平均大小(1520kb)y: 植物基因組大小(以kb計)如果插入片段平均大小為20kb，某植物基

31、因組大小為4x108bp，時，根據(jù)上式，N1X105。含1X105個克隆的基因文庫相當(dāng)覆蓋了5倍的基因組，在片段隨機分布時，從文庫中找到任一序列的概率不低于。植物基因組大小 及克隆文庫所需噬菌斑數(shù)植物種類 基因組大小(bp)a 重組噬菌斑數(shù)b擬南芥 74大豆 85菜豆 95碧東茄 95煙草 96玉米 96小麥 106a，基因組大小引自Rogers和Bendich; b.假設(shè)插入片段為17kb,概率為。 (二) 從cDNA文庫中獲得目的基因cDNA library :指含有一個機體某種特定細(xì)胞或特定狀態(tài)下表達(dá)基因的全部cDNA 克隆的群體cDNA library characters:不含內(nèi)含子

32、，用mRNA經(jīng)逆轉(zhuǎn)錄構(gòu)建cDNA library vectors:-gt10; -gt11; Ziplox et alcDNA library construction:分離mRNARNase H水解去掉mRNA以oligo(dT)為引物，合成cDNA第一鏈隨機引物，DNA pol I合成第二鏈修齊兩端，加人工接頭，與載體連接，得到cDNA重組體，所有cDNA 重組體均轉(zhuǎn)入宿主擴增cDNA 文庫(三) 利用PCR技術(shù)獲得目的基因采用T載體(AT克隆)：pGEM-T vector; pMD18-T Vector 合成長度有限可人工設(shè)計相應(yīng)的序列，不必使用天然模板(四) 人工合成目的基因Plasm

33、id phage cosmid M13 phageCapacity 10 kb 22 kb 4050 kb 1 kb of cloninggDNA library - + + -cDNA library + + - -Subcloning + - - +Sequencing + + - +E coli expression + + - -二、依據(jù)基因克隆的目的選擇和準(zhǔn)備載體Cloning vectors in common use:(1) The ligation by cohesive endsAdvantage: easily linked Disadvantage: easily to

34、be cycled self bidirection insertion(2) The ligation with artificial linker (3) The ligation with homopolymeric tail (4) The ligation by blunt ends High ATP con. T4 DNA ligase should be used三、選擇適當(dāng)?shù)牟呗詫NA片段進行連接Methods in common use:(一) 粘性末端的連接全同源粘性末端 最方便，但載體自身環(huán)化，并為雙向插入5G3CTTAAppAATTC3G5GCTTAAppAATTCG

35、5G3CTTAAppAATTC3G5例：用EcoR I分別切割載體和目的DNA：切割載體：切割目的DNA：GCTTAAAATTCGAATTC3G55G3CTTAA雙向插入示意圖：GCTTAAAATTCGAATTC3G55G3CTTAA粘性末端的連接(二) 用人工接頭法克隆cDNA連接酶堿性磷酸酶(三) 同聚物接尾法克隆雙鏈DNA轉(zhuǎn)化適當(dāng)?shù)乃拗魍嘶疠d體(一) 轉(zhuǎn)化(transformation ) 四、重組DNA需要導(dǎo)入宿主細(xì)胞進行擴增Methods in common use:(二) 感染(infection ) (三) 轉(zhuǎn)染(transfection ) (四) 電穿孔(electropor

36、ation) (一) 轉(zhuǎn)化是直接將DNA導(dǎo)入細(xì)菌的方法Transformation：指將質(zhì)?；蚱渌庠碊NA導(dǎo)入處于感受態(tài)的宿主菌，并使其獲得新的表型的過程。 宿主菌：E coli感受態(tài)細(xì)胞的制備：低溫CaCl2處理，使細(xì)胞通透性增加，易于攝取外源DNA，這種細(xì)胞稱為感受態(tài)細(xì)胞(competent cell)(二) 感染是利用噬菌體將外源DNA導(dǎo)入宿主Infection：指噬菌體、粘性質(zhì)?；蛘婧思?xì)胞病毒為載體的重組DNA分子，在體外經(jīng)過包裝成具有感染能力的病毒或噬菌體顆粒，然后感染適當(dāng)宿主細(xì)胞的過程 用于包裝的宿主菌：琥珀突變株Dam溶菌物：含頭部蛋白突變株Eam溶菌物：含尾部蛋白Dam溶菌物

37、 + Eam溶菌物 + DNA重組體包裝噬菌體重組有外源DNA的逆轉(zhuǎn)錄病毒載體感染輔助病毒-2細(xì)胞包裝成病毒顆粒有感染能力(三) 轉(zhuǎn)染是將外源DNA導(dǎo)入真核細(xì)胞的方法Transfection：指真核細(xì)胞主動攝取或被動導(dǎo)入外源DNA片段而獲得新的表型的過程Methods：電穿孔磷酸鈣共沉淀法脂質(zhì)體融合法進入細(xì)胞的DNA存在方式染色體外整合至宿主基因組中(一) 遺傳學(xué)方法五、重組DNA導(dǎo)入宿主后篩選與鑒定Methods in common use:(二) 免疫學(xué)方法 -檢測表達(dá)產(chǎn)物(三) 核酸雜交(四) PCR技術(shù)抗性標(biāo)記表型標(biāo)記插入失活互補(五) 酶切鑒定Go to 23Go to 103Go

38、to 104Go to 105第四節(jié) 真核細(xì)胞基因轉(zhuǎn)染Transfection：指真核細(xì)胞主動攝取或被動導(dǎo)入外源DNA片段而獲得新的表型的過程一、真核細(xì)胞轉(zhuǎn)染的方法與基本原理二、轉(zhuǎn)染細(xì)胞可利用抗性標(biāo)記進行篩選(一) 磷酸鈣共沉淀示介導(dǎo)基因轉(zhuǎn)染一、真核細(xì)胞轉(zhuǎn)染的方法與基本原理Methods in common use:(二) 電穿孔法介導(dǎo)基因轉(zhuǎn)染 (electroporation)(三) DEAE-葡聚糖法介導(dǎo)基因轉(zhuǎn)染 (DEAE-dextran)(四) 脂質(zhì)體介導(dǎo)基因轉(zhuǎn)染 (liposome )(五) 顯微注射法( microinjection )Calcium phosphate co-pr

39、ecipitation (1%) Electroporation(電穿孔法)Extraneous DNAHost cellsElectricity with high frequency (一) 利用TK-細(xì)胞突變株進行轉(zhuǎn)染細(xì)胞篩選 二、轉(zhuǎn)染細(xì)胞可利用抗性標(biāo)記進行篩選Methods in common use:(二) 藥物篩選轉(zhuǎn)染細(xì)胞穩(wěn)定轉(zhuǎn)染細(xì)胞株的篩選(一) 利用TK-細(xì)胞突變株進行轉(zhuǎn)染細(xì)胞篩選 Principle：TK (thymidine kinase) 是催化核苷酸補救合成的關(guān)鍵酶氨基喋呤(aminopterin, A )是從頭合成途徑的抑制劑，A的加入使細(xì)胞主要依賴于補救合成TK-選

40、擇系統(tǒng)Host -TK - 細(xì)胞株(TK表達(dá)缺陷)培養(yǎng)基 -HAT培養(yǎng)基H, hypoxanthine; A, aminopterin; T, thymineTk -氨基蝶呤（A）處理抑制二氫葉酸還原酶四氫葉酸逐漸耗盡dUMPdATPdCTP(-)(+)HdATPdCTPTdTTPTk +培養(yǎng)基含H、TA細(xì)胞不能存活細(xì)胞能夠存活補救合成tk基因?qū)雝k - 細(xì)胞細(xì)胞能夠存活在含HAT培養(yǎng)基中tk選擇系統(tǒng)原理圖解(越過A的抑制)The screening with antibiotics, G418 (geneticin, 新霉素衍生物)(二) 藥物篩選轉(zhuǎn)染細(xì)胞 The most used an

41、tibiotic marker: neor 新霉素抗性選擇系統(tǒng)新霉素干擾原核生物蛋白合成不影響真核生物蛋白合成新霉素類似物G418干擾原核生物蛋白合成干擾真核生物蛋白合成細(xì)菌新霉素抗性基因neor表達(dá)氨基糖苷磷酸轉(zhuǎn)移酶(APH)使G418失活表達(dá)neor 的細(xì)胞可在含G418的培養(yǎng)基中存活第五節(jié) 利用重組DNA技術(shù)進行基因改造Methods in common use:定點誘變技術(shù)寡核苷酸介導(dǎo)定點誘變技術(shù)PCR介導(dǎo)定點誘變技術(shù)目的改變基因的一級序列特征一、對特定基因可進行定點誘變What is the site-directed mutagenesis (目的基因的定點誘變)?It means

42、 the process that the one or more sites of gene be replaced or deleted by artificial method.(一) 帶突變位點的寡核苷酸可介導(dǎo)定點誘變 The site-directed mutagenesis mediated by oligonucleotideCharacters:It can cause finely the mutagenesis at the special site according to the design of researchersProcesses:1) The use of

43、Klenow fragment and a primer with an incorrect paired base, M13 as single strand template2) To extend the primer to get a heterozygous double strands DNA3) To transform the heterozygous DNA into E coli, the wild DNA and mutated DNA will be yielded4) To screen and decide the mutated DNA，further resea

44、rch the mutated DNA誘變寡核苷酸引物單鏈M13模板Klenow fragment, dNTP, T4 DNA pol雜交雙鏈DNA轉(zhuǎn)化E coli瓊脂平皿標(biāo)記誘變寡核苷酸原位分子雜交放射自顯影 寡核苷酸介導(dǎo)的定點誘變基本實驗步驟理論上最高誘變效率只有50%(二) 含U模板可以提高寡核苷介導(dǎo)定點誘變效率Principle:建立含U 單鏈模板(U取代T)-正鏈合成雜合雙鏈DNA雜合雙鏈DNA轉(zhuǎn)化野生型E coli中尿嘧啶核苷酶降解含U的正鏈帶誘變位點的負(fù)鏈能保存，合成雙鏈DNA(突變體突變率90%)誘變寡核苷酸引物Klenow fragment, dNTP, T4 DNA pol

45、轉(zhuǎn)化野生型E coli，含尿嘧啶核苷酶，可降解含U模板標(biāo)記誘變寡核苷酸原位分子雜交放射自顯影 U模板介導(dǎo)的定點誘變基本實驗步驟含突變體的負(fù)鏈保存下來并進行復(fù)制擴增，篩選鑒定突變率90%單鏈M13模板UUUUUUU雜交雙鏈DNAUUUUUUU(三) PCR技術(shù)適合進行基因的定點誘變Principle(兩對引物，三次PCR):一對常規(guī)引物a、d + 一對帶誘變點的引物b、ca + b 及d + c分別擴增出兩個帶突變點的片段上述兩個片段等量混合，變性、退火用Klenow DNA聚合酶補齊利用a、d引物對進行PCR擴增，即得所需片段5533abcd55335533555533ad5353PCR1PC

46、R2變性，退火Klenow fragment, dNTPPCR3圖8-8PCR介導(dǎo)的定點誘變法二、利用基因定點誘變技術(shù)改變基因工程蛋白特性目的使工業(yè)酶具有更好的理化特性便于應(yīng)用使臨床生物蛋白或多肽制劑療效高副作用小獲得更多對人類有益的蛋白或多肽產(chǎn)物For example: InsulinGene engineering Insulin1. 定點誘變制備長效Insulin(半衰期延至35.3h)2. 定點誘變制備快速吸收Insulin(減少六聚體形成)3. 定點誘變增加Insulin與受體的親和力B27 ThrArg, C端氨基化；A21 Asn GlyB10 HisAsp, 體外活性較野生型提

47、高5倍第六節(jié) 克隆基因在適當(dāng)宿主細(xì)胞的表達(dá)Expression systemsExpression vectors, Appropriate host cellsAim: To get a lot of proteins or polypeptidesThe expression systems in common use:E coli expression systemMammal animal expression systemInsect expression systemYeast expression system一、大腸桿菌( E coli )表達(dá)系統(tǒng)Characters：目標(biāo)基因

48、表達(dá)水平高、遺傳背景清楚、易于培養(yǎng)（培養(yǎng)方法簡單、生長快、培養(yǎng)周期短、抗污染能力強）、成本低(一)表達(dá)外源基因需要一些基本要素1. 來自真核細(xì)胞的基因需要適當(dāng)改選2. 必須選用E coli vectors3. 目的基因與載體可用不同方式連接4. 選擇適當(dāng)?shù)氖荏w菌和誘導(dǎo)條件表達(dá)非融合蛋白；表達(dá)融合蛋白If the target gene come from eukaryote, it should be cDNA. Why?1. 來自真核細(xì)胞的基因需要適當(dāng)改選The signal peptide of secretary protein has to be deleted tooThe 5-en

49、d sequence before ATG on cDNA is useless, so it has to be deleted.The vectors must be E coli expression vectors, which contain the promoters ( PL, tac, T7 ) recognized by DDRP in E coli and SD sequence.Why?2. 必須選用E coli vectorsPromoters Effectively induce transcription轉(zhuǎn)錄效應(yīng)強Can be controlled easily 能

50、被有效控制53SDTarget genea. To express the non-fusion proteins Use Nde I ( CATATG ) or Nco I ( CCATGG) to bring in ATG SDTarget geneFragment of structure gene of prokaryote3. 目的基因與載體可用不同方式連接The models of linkage:b. To express the fusion proteins Fusion protein:NThe peptide of target genepeptide of prokar

51、yoteCIt means that the peptide expressed consists of N-end of peptide coded by prokaryote DNA and C-end of peptide coded by the cloning fragment of eukaryote DNA, which is termed as fusion protein. The advantages of fusion protein:a. More stableb. With the signal peptide, the secretary product could

52、 be yieldedc. It can be purified by affinity chromatographyd. The peptide of prokaryote can be cut out by proteinase, and then the peptide of eukaryote can be released in natural form(二) 采用適當(dāng)措施可提高外源基因表達(dá)水平1. 多種策略提高翻譯水平2. 將細(xì)菌生長與外源基因表達(dá)在時間上分開3. 采用不同措施提高表達(dá)蛋白的穩(wěn)定性表達(dá)融合蛋白，采用突變菌株，表達(dá)分泌蛋白調(diào)整SD與ATG之間距離，增加mRNA穩(wěn)定性，

53、點突變改變堿基二、哺乳動物表達(dá)系統(tǒng)Characters：Eukaryote expression systems are needed The gene to be expressed can come from genome DNA or cDNA Why?How can the vectors be transformed into host cells?Plasmid vectors:Calcium phosphate co-precipitation; Electroporation; DEAE-Dextran; Microinjection; Make the extraneous

54、DNA into cells mediated by liposome Virus vectors:The virus vectors have to be introduced into wrap cells, then the pseudo-virus particles would be produced and used to infect the host cells.The screening markers:Antibiotics genes ( neor )-code the aminosaccharide phosphate transportase -make G418 l

55、ost activityAdvantages:The expression products have scarcely effect on host cells, and not easily be degraded. (1) Insect expression system1) Drosophila melanogaster (Fruit fly) expression system (DES)(果蠅表達(dá)系統(tǒng))2) Bacilliform virus expression system(桿狀病毒表達(dá)系統(tǒng))三、其他真核表達(dá)系統(tǒng)(2) Microzyme ( yeast ) expression system (啤酒酵母，Saccharomyces cerevisiae)Go to 101第七節(jié) 電子克隆輔助基因克隆In silico cloning 通過對基因數(shù)據(jù)庫進行序列搜索、對比分析、拼接整合，預(yù)測新的、假定的全長基因，然后通過分子生物學(xué)實驗方法，加以證實，并從相應(yīng)組織、細(xì)胞中獲得這種基因，稱為電子克隆In silico cloningNote: In silico is not the real gene cloningSummary The basic elements for gene cloning The basic process for ge