1、全國(guó)高等醫(yī)藥教材建設(shè)研究會(huì) 衛(wèi)生部規(guī)劃教材 全國(guó)高等學(xué)校教材 供七、八年制臨床醫(yī)學(xué)等專業(yè)用 醫(yī)學(xué)分子生物學(xué) Medical Molecular Biology 主編 馮作化 人民衛(wèi)生出版社 2005年8月第一版 課件制作：吳耀生(版權(quán)所有) 廣西醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院 生物化學(xué)與分子生物學(xué)教研室 2009.12,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 en

2、zymes),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(guò)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(shí)FPS,2. 設(shè)計(jì)實(shí)驗(yàn)方案,3. 可行性分析,For Example,FPS 克?。?RT-PCR， 3-RACE， 5-RACE,改進(jìn)的異硫氰酸胍一步法提取三七根部總RNA,RT-PCR擴(kuò)增fps部分保守序列,3-RACE 及克隆測(cè)序,5-RACE 及克隆測(cè)序,序列拼接,mRNA: 5 AAA

3、AAA3,TTTTTTT-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),5-端磷酸化RT-Primer,逆轉(zhuǎn)錄,RNA分解,用RNA ligase進(jìn)行環(huán)化或形成首尾連接物,1stand 2nd PCR,5-端磷酸化RT primer部位,包含未知的5上游區(qū)域的擴(kuò)增產(chǎn)物,(2),(3),(4),Thank You,DNA重組 DNA recombination,Some concerned concepts:,DNA重組技術(shù) DNA recombination technique,分子克隆 Molecular clon

4、ing,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，解決了基因的自我復(fù)制和傳遞的過(guò)程。,40年代，O.T. Avery等通過(guò)肺炎鏈球菌轉(zhuǎn)化實(shí)驗(yàn)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(shuō)，成功破譯了遺傳密碼，闡明了遺傳信息的流向和表達(dá)問(wèn)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

5、年 Boyer EcoR I “GAATTC” 1967年 世界上5個(gè)實(shí)驗(yàn)室?guī)缀跬瑫r(shí)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,基因工程載體的使用：plasmid, phage, viruses,1978年Nobel 醫(yī)學(xué)與生理學(xué)獎(jiǎng),The Nobel Prize in Medicine was awarded, in 1978, to Daniel Nathans, Werner Arber, and Hamilton Smith for the discovery of restriction endonucleases. Thei

6、r discovery lead to the development of recombinant DNA technology that allowed, for example, the large 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 rout

7、inely used for DNA modification and manipulation in laboratories.,DNA ligase repairing chromosomal damage. ligase I, DNA, ATP-dependent,第一節(jié) 基因克隆是利用工具酶進(jìn)行操作,工具酶(Tool enzymes )-,用于對(duì)DNA分子進(jìn)行定點(diǎn)切割、連接、擴(kuò)增、 標(biāo)記等操作的特殊酶類，已被純化并商品化進(jìn) 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

8、中用于切割DNA,RE (restriction enzyme, restriction endonuclease )-限制性核酸內(nèi)切酶，限制酶，能識(shí)別DNA雙鏈內(nèi)特異位點(diǎn)并水解磷酸二酯鍵，產(chǎn)生特定末端的酶,Restriction enzyme Functions: 能識(shí)別DNA內(nèi)部特異位點(diǎn)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(hào),Escherichia coli RY13 I,屬Genus, 種-species, 菌株-strain,EcoR I,

9、Two catalytic manganese ions (one from each monomer) are shown as magenta spheres and are adjacent to the cleaved sites in 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 strande

10、d DNA (brown tubes) based on the PDB 1QPS X-ray crystallographic coordinates.,(3) 限制酶的識(shí)別和切割位點(diǎn) Characters: 通常識(shí)別46個(gè)堿基，少數(shù)識(shí)別8個(gè) 所識(shí)別的序列一般為回文結(jié)構(gòu)(palindrome) 在特異位點(diǎn)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-3 3-GGGCCC5,5-CCC GGG-3 3-GGG CCC-5,+,平端切割(blunt end, such as Sma I ),Go to 109,5-GGTGAATTCAGC-3 3-

11、CCACTTAAGTCG5,5-TTGCTGCAGAAG-3 3-AACGACGTCTTC5,5-粘性末端 (EcoR I ),3-粘性末端 ( Pst I ),5-GGTG AATTCAGC-3 3-CCACTTAA GTCG5,+,5-TTGCTGCA GAAG-3 3-AACG ACGTCTTC5,+,(4) 同工異源酶( isoschizomers ) 來(lái)源不同，但能識(shí)別和切割同一位點(diǎn)的RE 例：Bam H I 不需ATP RE識(shí)別序列長(zhǎng)度與切點(diǎn)數(shù)： 對(duì)同一DNA，識(shí)別序列短的，切點(diǎn)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(duì)DNA分子進(jìn)行多種操

12、作，也叫修飾酶,作用：剪切、補(bǔ)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 polymerase So on,(1) DNA polymerase I Activities： 53聚合活性， 53及 35外切活性 Functions： 在生物體內(nèi)，填補(bǔ)引物切除后留下的空缺，修復(fù) 在生物

13、體外，缺口平移制備探針,DNA polymerase I,Klenow fragment (76KD),Another small fragment,枯草桿菌 蛋白酶,Klenow片段,E.coli DNA pol I,53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,Klenow fragment 用途：,1) 補(bǔ)齊雙鏈DNA的3-末端 2) 通過(guò)補(bǔ)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 trans

14、criptase,用途：合成cDNA，構(gòu)建cDNA文庫(kù),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(lái)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(jìn)入一定宿主細(xì)胞，在宿主中擴(kuò)增甚至表達(dá)，并有遺傳標(biāo)記進(jìn)行篩選,載體的概念,Cloning vector能夠攜帶感興趣的外源DNA（target DNA）進(jìn)入宿主細(xì)胞，并將外源DNA在宿主內(nèi)進(jìn)行克

15、隆和擴(kuò)增，而采用的一些DNA分子稱為Cloning vector 。,Cloning 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 chara

16、cters (cloning vector): 能在宿主細(xì)胞中自我復(fù)制、自我表達(dá) 分子相對(duì)較小，在宿主細(xì)胞中有高拷貝 具有遺傳標(biāo)志 有多個(gè)限制性酶單一酶切位點(diǎn)(多克隆位點(diǎn)) 易與宿主DNA相分離,一、常用克隆載體主要來(lái)自質(zhì)粒和病毒,(一)質(zhì)粒是常用的克隆載體,Examples: pBR322 pUC,Characters: Small molecular weight, high copies More than one genetic mark Multiple cloning sites, MCS,pBR322,Turn to 64,pUC19,(二) 噬菌體經(jīng)過(guò)重組也可以攜帶外源 DNA

17、,Examples: bacteriophage, phage; M13 phage It is a kind of virus which can infect bacteria, bacteriophages in common use: EMBL spectrum gt spectrum Charon 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(diǎn)），進(jìn)入宿主菌后可自行環(huán)合。基因組DNA長(zhǎng)約為48.5Kb,共有66個(gè)基因，較復(fù)雜。,Character：1、其生長(zhǎng)必

18、需的序列位于左右二臂上，中部約30%為生長(zhǎng)非必需；2、成熟需要包裝（大小為原來(lái)的75-105%時(shí)）,Two kinds of vectors： 置換型載體；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(diǎn),酶切點(diǎn),切除后用目的基因取代,COS位點(diǎn),COS位點(diǎn)（12bp),The life period of phage(噬菌體的生活周期):,Lysogenic pathway( 溶原生長(zhǎng)途徑),Lysis pathway(溶菌生長(zhǎng)途徑),溶原生長(zhǎng),溶菌生長(zhǎng),溶原轉(zhuǎn)溶菌生長(zhǎng),以噬菌體為媒介的轉(zhuǎn)

19、導(dǎo)作用, gt10:insertion vector; multiple cloning site: CI,The positive marks of screening: Whether the 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 int

20、act, to be turbid spots, gt11：insertion vector; MCS: lac Z；expressed,With insertion into lac z of gt11, white spots Otherwise 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(diǎn)，為雙鏈環(huán)狀DNA。It is constructed with the cos region of DNA and plasmid, which is double cycle DNA wit

21、h bigger content for cloning (4050kb),Characters: 1) With antibiotics marks and replication element itself 2) With cos region of phage, can be wrapped 3) With one or more cloning sites 4) Small molecular weight 5) non-recombination cosmid too small to be wrapped, so it is screened easily,(三) M13噬菌體適

22、合制備單鏈DNA,M13 bacteriophage:,It is a kind of E coli phage, the genome 6.5kb, a cycle DNA containing single strand DNA,Two forms:,Wild form; Replication form (RF),The most merit:,Can be released from host as single strand,M13 phage Characters:,In vivo, double strands DNA (can be replicated),In vitro,

23、single strand DNA (can be used as template for DNA sequencing, or make DNA probes for hybridization ),After entering host cells, 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(dòng)物細(xì)胞,Virus vectors in common us

24、e:,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)粒化，由病毒啟動(dòng)子、包裝元件、選 擇性遺傳標(biāo)記及pBR322復(fù)制起始點(diǎn)組成,Other vectors:,YAC, yeast artificial 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

25、 structures except for the characters of cloning vectors,The vectors which can make the extraneous DNA be expressed in host cells are termed as expression vectors.,Sorts:,Expression vectors of E coli (prokaryote ),Expression vectors of eukaryote,Expression vectors of mammal animals,(一) 原核表達(dá)載體適于原核細(xì)胞表

26、達(dá)外源基因,Expression vectors of E coli:,除克隆所需成分外，還含有啟動(dòng)子，核糖體結(jié)合位點(diǎn)，轉(zhuǎn)錄終止序列等表達(dá)元件,Trp-lac promoter ( or tac promoter) Inducer: IPTG Strains: RB791, XL-1-blue, SB221, JM 109,1. Promoter,T7 of T7 phage It is a high effective expression promoter Strain: JM109(DE3),PL of -phage (temperature induce promoter ) Cont

27、rolled 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(jī)制,A,U,U C C U,C,CACUAGG,核蛋白體小亞基的16S-rRNA,5,A G G A PuPuUUUPuPuAUG,3,mRNA,Rps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(diǎn)(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)

28、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(diǎn)；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(diǎn)，啟動(dòng)子/ 增強(qiáng)子，克隆位點(diǎn)，加 尾信號(hào)等,Expression vectors of mammal animal If a gene needs to be expressed in mammal animal cells, the eukaryote expression vector must be used.,The esse

29、ntial 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(guò)程包括五個(gè)基本部分,Gene cloning process,(1) 制備目的基因及相關(guān)載體,(2) 將目的基因與載體進(jìn)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(kuò)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

30、 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(kù)中獲得目的基因,Genomic library :,指含有一個(gè)機(jī)體基因組DNA的全套重組DNA分 子的克隆群體,用于構(gòu)建基因組文庫(kù)的載體：,-噬菌體(- phage)，粘性質(zhì)粒(cosmid),Genomic library construction:,分離高分子量DN

31、A,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(kuò)增,基因組文庫(kù),隨機(jī)文庫(kù)克隆數(shù)目計(jì)算,隨機(jī)文庫(kù)指代表基因組各部分DNA的摩爾數(shù)相等。對(duì)于隨機(jī)文庫(kù)，有：,N: 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(jì)),如果插入片段平均大小為20kb，某植物基因組大小為4x108bp，P=0.99時(shí)，根據(jù)上式，N1X105。含1X105個(gè)克隆的基因文庫(kù)相當(dāng)覆蓋了5倍的基因組，在片段隨機(jī)分布時(shí)，從文庫(kù)中找到任一序列的概率不低于0.99。,植物基因組大小 及克隆

32、文庫(kù)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 7.7X107 2.1X104,大豆 8.7X108 2.4X105,菜豆 1.8X109 4.9X105,碧?hào)|茄 1.9X109 5.1X105,煙草 3.8X109 1.0X106,玉米 3.9X109 1.1X106,小麥 1.5X1010 4.1X106,a，基因組大小引自Rogers和Bendich; b.假設(shè)插入片段為17kb,概率為0.99。,(二) 從cDNA文庫(kù)中獲得目的基因,cDNA library :,指含有一個(gè)機(jī)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 librar

33、y characters:,不含內(nèi)含子，用mRNA經(jīng)逆轉(zhuǎn)錄構(gòu)建,cDNA library vectors:,-gt10; -gt11; Ziplox et al,cDNA library construction:,分離mRNA,RNase H水解去掉mRNA,以oligo(dT)為引物，合成cDNA第一鏈,隨機(jī)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(kuò)增,cDNA 文庫(kù),(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(zhǎng)度有限,可人

34、工設(shè)計(jì)相應(yīng)的序列，不必使用天然模板,(四) 人工合成目的基因,Plasmid phage cosmid M13 phage,Capacity 10 kb 22 kb 4050 kb 1 kb of cloning gDNA 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 ends Adv

35、antage: easily linked Disadvantage: easily to 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(jìn)行連接,Methods in common use:,(一) 粘性末端的連接,全同源粘性末端

36、 最方便，但載體自身環(huán)化，并為雙向插入,例：用EcoR I分別切割載體和目的DNA：,切割載體：,切割目的DNA：,雙向插入示意圖：,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?退火,載體,(一) 轉(zhuǎn)化(transformation ),四、重組DNA需要導(dǎo)入宿主細(xì)胞進(jìn)行擴(kuò)增,Methods in common use:,(二) 感染(infection ),(三) 轉(zhuǎn)染(transfection ),(四) 電穿孔(electroporation),(一) 轉(zhuǎn)化是直接將DNA導(dǎo)入細(xì)菌的方法,Transformati

37、on：,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(guò)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(guò)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(guò)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 + Eam溶菌物 + DNA重組體,包裝噬菌體,

38、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(dòng)攝取或被動(dòng)導(dǎo)入外源DNA片段 而獲得新的表型的過(guò)程,Methods：,電穿孔,磷酸鈣共沉淀法,脂質(zhì)體融合法,進(jìn)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(cè)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(bǔ),(五) 酶切鑒定,Go to 23,Go

39、to 103,Go to 104,Go 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(dòng)攝取或被動(dòng)導(dǎo)入外源DNA片段 而獲得新的表型的過(guò)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(jìn)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

40、),Calcium phosphate co-precipitation (1%),Electroporation(電穿孔法),Extraneous DNA,Host cells,Electricity with high frequency,(一) 利用TK-細(xì)胞突變株進(jìn)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(jìn)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(jìn)行轉(zhuǎn)染細(xì)胞篩選,Principle：,TK (thymidine kinase) 是催化核苷酸補(bǔ)救合 成的關(guān)鍵酶,氨基喋呤(aminopterin,

41、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(bǔ)救合成,TK-選擇系統(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, thymine,Tk -,氨基蝶呤（A）處理,抑制二氫葉 酸還原酶,四氫葉酸 逐漸耗盡,dUMP,dATP dCTP,(-),(+),H,dATP dCTP,T,dTTP,Tk +,培養(yǎng)基含H、T,A,細(xì)胞不 能存活,細(xì)胞能 夠存活,補(bǔ)救合成,tk基因?qū)?入tk - 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(guò)A的抑制),The screenin

42、g with antibiotics, G418 (geneticin, 新霉素衍生物),(二) 藥物篩選轉(zhuǎn)染細(xì)胞,The most used antibiotic 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(jìn)行基因改造,Methods in common use:,定點(diǎn)誘變技術(shù),寡核苷酸介導(dǎo)定點(diǎn)誘變技術(shù),PCR介

43、導(dǎo)定點(diǎn)誘變技術(shù),目的,改變基因的一級(jí)序列特征,一、對(duì)特定基因可進(jìn)行定點(diǎn)誘變,What is the site-directed mutagenesis (目的基因的定點(diǎn)誘變)?,It means the process that the one or more sites of gene be replaced or deleted by artificial method.,(一) 帶突變位點(diǎn)的寡核苷酸可介導(dǎo)定點(diǎn)誘變,The site-directed mutagenesis mediated by oligonucleotide,Characters: It can cause finel

44、y the mutagenesis at the special site according to the design of researchers,Processes:,1) The use of Klenow fragment and a primer with an incorrect paired base, M13 as single strand template,2) To extend the primer to get a heterozygous double strands DNA,3) To transform the heterozygous DNA into E

45、 coli, the wild DNA and mutated DNA will be yielded,4) To screen and decide the mutated DNA，further research 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(diǎn)誘變基本實(shí)驗(yàn)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(diǎn)誘變效率,Principle:,建立含U 單鏈模板(U取代T

46、)-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(diǎn)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,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(diǎn)誘變基本實(shí)驗(yàn)步驟,含突變體的負(fù)鏈保存下來(lái)并進(jìn)行復(fù)制擴(kuò)增，篩選鑒定,突變率90%,(三) PCR技術(shù)適合進(jìn)行基因的定點(diǎn)誘變,Principle(兩對(duì)引物，三次PCR):,一對(duì)常規(guī)引物a、d + 一對(duì)帶誘變點(diǎn)的引物b、c,a + b 及d + c

47、分別擴(kuò)增出兩個(gè)帶突變點(diǎn)的片段,上述兩個(gè)片段等量混合，變性、退火,用Klenow DNA聚合酶補(bǔ)齊,利用a、d引物對(duì)進(jìn)行PCR擴(kuò)增，即得所需片段,5,5,3,3,a,b,c,d,5,5,3,3,5,5,3,3,5,5,5,5,3,3,a,d,5,3,5,3,PCR1,PCR2,變性，退火,Klenow fragment, dNTP,PCR3,圖8-8PCR介導(dǎo)的定點(diǎn)誘變法,二、利用基因定點(diǎn)誘變技術(shù)改造基因工程蛋白,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(duì)人類有益的蛋白或多肽產(chǎn)物,For example: Insulin,Gene enginee

48、ring Insulin,1. 定點(diǎn)誘變制備長(zhǎng)效Insulin(半衰期延至35.3h),2. 定點(diǎn)誘變制備快速吸收Insulin(減少六聚體形成),3. 定點(diǎn)誘變?cè)黾覫nsulin與受體的親和力,B27 ThrArg, C端氨基化；A21 Asn Gly,B10 HisAsp, 體外活性較野生型提高5倍,第六節(jié) 克隆基因在適當(dāng)宿主細(xì)胞的表達(dá),Expression systems,Expression vectors, Appropriate host cells,Aim: To get a lot of proteins or polypeptides,The expression syste

49、ms in common use:,E coli expression system,Mammal animal expression system,Insect expression system,Yeast expression system,一、大腸桿菌( E coli )表達(dá)系統(tǒng),Characters： 目標(biāo)基因表達(dá)水平高、遺傳背景清楚、易于培 養(yǎng)（培養(yǎng)方法簡(jiǎn)單、生長(zhǎng)快、培養(yǎng)周期短、抗 污染能力強(qiáng)）、成本低,(一)表達(dá)外源基因需要一些基本要素,1. 來(lái)自真核細(xì)胞的基因需要適當(dāng)改選,2. 必須選用E coli vectors,3. 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

50、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(lái)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 too,The 5-end sequence before ATG on cDNA is useless, so it has to be deleted.,The vectors must be E coli expression vectors, which contain

51、 the promoters ( PL, tac, T7 ) recognized by DDRP in E coli and SD sequence. Why?,2. 必須選用E coli vectors,Promoters,Effectively induce transcription轉(zhuǎn)錄效應(yīng)強(qiáng),Can be controlled easily 能被有效控制,5,3,SD,Target gene,a. To express the non-fusion proteins Use Nde I ( CATATG ) or Nco I ( CCATGG) to bring in ATG,SD,

52、Target gene,Fragment of structure gene of prokaryote,3. 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:,N,The peptide of target gene,peptide of prokaryote,C,It means that the peptide expressed consists of N-end of peptide coded by prokaryote DNA and C-end of

53、peptide coded by the cloning fragment of eukaryote DNA, which is termed as fusion protein.,The advantages of fusion protein:,a. More stable,b. With the signal peptide, the secretary product could be yielded,c. It can be purified by affinity chromatography,d. The peptide of prokaryote can be cut out

54、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(zhǎng)與外源基因表達(dá)在時(shí)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， 點(diǎn)突變改變堿基,二、哺乳動(dòng)物表達(dá)系統(tǒng),Characters： Eukaryote expression systems are needed The gene to be expressed

55、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 DNA into cells mediated by liposome,Virus vectors: The virus vectors have to be introduced

56、 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 lost activity,Advantages: The expression products have scarcely effect on host cells, an

57、d not easily be degraded.,(1) Insect expression system 1) 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(guò)對(duì)基因數(shù)據(jù)庫(kù)進(jìn)行序列搜索、對(duì)比分析、 拼接整合，預(yù)測(cè)新的、假定的全長(zhǎng)基因，然后 通過(guò)分子生物學(xué)實(shí)驗(yàn)方法，加以證實(shí)，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 cloning,Note: In silico is not the real gene cloning,Summary,The basic elements for gene cloning,The basic process for gene cloning,The gene