第十三讲-2006基因工程.ppt

1、Genetic engineering and biotechnology,生物技术-在生产过程中使用了某种有机体或有机体来源的物质. Brewing and wine-making，酿酒 Food processing and manufacture，食品加工 The production of pharmaceuticals，药物生产 Biotechnology-the use of an organism (usually a microorganism) or a biologically derived substance (usually an enzyme) in a produ

2、ction or conversion process.,现代生物技术通常与遗传修饰体系有关。 生产重组蛋白（recombinant protein）已经成为一种成熟的技术. Modern biotechnology is often associated with the use of genetically modified systems. there is a bewildering range of different vector/host combinations.,第一节 生产蛋白质 利用克隆的基因生产重组蛋白（尤其是贵重的药物蛋白）是基因工程最为重要的研究方向之一。 Many

3、 such proteins have already been produced by recombinant DNA techniques, and are already in widespread use.,利用重组DNA技术生产蛋白质时需要考虑优化两个方面： 1，体系的生物学特征-细菌宿主细胞或真核宿主细胞。 2，生产过程本身-从实验室进入大规模生产。 In protein production there are two aspects which require optimization:the biology of the systerm-bacterial host cell

4、 or eukaryotic host。the production process itself-large-scale production-scale-up from laboratory.,一、天然或融合蛋白 为了有效表达克隆的基因，必须使用含有启动子的载体，并导入合适的宿主细胞，如大肠杆菌。 Native and fusion proteins. For efficient expression of cloned DNA, the gene must be inserted into a vector that has a suitable promoter and which c

5、an be introduced into an appropriate host such as E. coli.,在大肠杆菌中表达真核蛋白时，要使用大肠杆菌细胞能够识别的表达信号，包括: promoter, terminator-for transcription ribosome binding sites (Shine-Dalgarno sequence, SD序列)-for translation,啤酒酵母或培养的哺乳动物细胞可能更加适合真核基因的表达。 alternatively, a eukaryotic host such as the yeast S. cerevisiae,

6、 or mammalian cells in tissue culture, may be more suitable for certain proteins.,在原核细胞中表达真核蛋白时，编码序列应该使用来自mRNA的cDNA克隆。 当一个真核基因含有内含子（intron）时这一点非常重要，因为原核细胞不能进行RNA加工。 eukaryotic protein, coding sequence, prokaryotic host can not process out of the primary transcript.,表达载体分为两类：产生天然蛋白或融合蛋白。 天然蛋白由cDNA编码序

7、列直接转录、翻译产生。 There are two main categories of expression vectors: 产生native protein or fusion protein. Native proteins are synthesized directly from the N terminus of the cDNA.,融合蛋白N末端的一段短序列由载体编码。 有些情况下，这些序列对蛋白质的稳定性和分泌十分重要。 Fusion proteins contain short, N-terminal amino acid sequences encoded by the

8、vector. stability or secretion.,当融合蛋白在融合位点含有甲硫氨酸（methionine residue）时，可用溴化氰（cyanogen bromide, CNBr）断开，从而得到期望的多肽。 但当目的蛋白内部含有甲硫氨酸时，该方法无法使用。 也可以在融合位点插入特异性蛋白酶的识别位点，表达产生的融合蛋白可通过蛋白酶水解，最终获得目的蛋白质。,生产重组融合蛋白时，在构建表达载体过程中要考虑读码框的正确性. 在融合位点需要增加或删除一个或两个碱基对，以保证阅读框不会改变。 The addition or deletion of one or two base-pa

9、irs at the vector/insert junction may be necessary to ensure correct reading frame. correct reading frame.,有些系列载体已经考虑了三种可能的阅读框，在与插入片段连接时可以选择使用。 there are vectors that have been constructed so that all three potential reading frames are represented for a particular vector/insert combination. Thus by

10、using the three variants of the vector, the correct in-frame fusion can be obtained.,二、酵母表达体系-yeast expression systems 啤酒酵母-Saccharomyces cerevisiae 裂殖酵母-Schizosaccharomyces pombe 多型汉逊酵母-Hansela polymorphia 乳酸克鲁维酵母-Kluyveromyces lactis 耶鲁酵母-Yarrowia lipolytica,酵母细胞可以在相对便宜的培养基中生长。 酵母中有很多不同的突变株和载体可供选择

11、。 酵母可以进行规模化发酵。 Grow rapidly on relatively inexpensive media. There are a range of different mutant strains and vectors can be used for various applications. Scale-up fermentations,与大肠杆菌相比，酵母的优点包括： 1，可以进行翻译后修饰，如糖基化。 2，生产的重组蛋白在三维构象和免疫原性方面的真实性更高，更接近于原来的天然蛋白质。 post-translational modifications such as gl

12、ycosylation. there is usually a higher degree of authenticity with respect to 3D conformation and the immunogenic properties of the protein.,毕赤酵母的表达水平是啤酒酵母的10-100倍。 在使用甲醇为唯一碳源时，每升培养的毕赤酵母细胞可以产生约12克外源蛋白。 Yields of heterologous proteins of around 12g/L have been obtained using Pichia. pastoris (grown o

13、n methanol as sole carbon source), 10-100 times more than in Saccharomyces cerevisiae.,毕赤酵母在以甲醇作为唯一碳源时，生长受醇氧化酶调节。 甲醇诱导时，醇氧化酶基因会过表达，该蛋白可以占到细胞内可溶性蛋白的30%。 P. pastoris can grown on methanol as sole carbon source, in this situation growth is regulated by alcohol oxidase, which has a low specific activity

14、 and is consequently overexpressed in these cells, making up around 30% of total soluble protein.,将外源基因置于醇氧化酶基因启动子AOX1之后，可以实现高水平表达。 By placing heterologous genes downstream from the alcohol oxidase promoter (AOX1), high levels of expression could be achieved.,三、杆状病毒表达体系 杆状病毒感染昆虫， 正常情况下感染昆虫细胞时，病毒颗粒被包

15、裹在多面体中。 多面体是一种核内包含体，主要由多角体蛋白构成。 the baculovirus expression system. baculovirus infect insects. During normal infection of insect cells, virus particles are packaged within polyhedra, which are nuclear inclusion bodies composed mostly of the protein polyhedrin。,多角体蛋白在病毒感染循环的后期合成，充分表达时可以占到被感染细胞蛋白质总量的5

16、0%。 多角体为昆虫感染所需要，但感染培养细胞时并不需要。 所以多角体蛋白基因是一种非必需的基因，可以用其启动子驱动外源基因表达。 polyhedrin gene is an obvious candidate for construction of an expression vector, because it encodes a late-expressed dispensable protein that is synthesized in large amounts.,杆状病毒基因组为环状双链DNA分子，88 to 200 kb 。 不同杆状病毒的基因组大小也不同，它们的基因组太大，

17、不能直接进行操作。 所以外源DNA的插入必须借助于转移载体 Baculovirus genome-a circular double-stranded DNA molecule-transfer vector.,转移载体为大肠杆菌质粒，带有多角体蛋白基因启动子和其他基本的表达信号，两侧具有两段杆状病毒同源序列。 Transfer vectors are based on E. coli plasmids, and carry the promoter for the polyhedrin gene (or for another viral gene) and any other essent

18、ial expression signals.,将目的基因连接到多角体蛋白基因启动子下游。 然后与杆状病毒DNA一起共转染昆虫细胞。 The cloned gene for expression is inserted into the transfer vector, and the recombinant is used to co-transfect insect cells with non-recombinant viral DNA.,病毒DNA与转移载体之间的同源重组可以产生重组的病毒DNA，可以选择出来用于目的蛋白的生产。 Homologous recombination bet

19、ween the viral DNA and the transfer vector results in the generation of recombinant viral genomes, which can be selected for and used to produce the protein of interest.,四、哺乳动物细胞系 表达重组的人类蛋白时，哺乳动物宿主细胞要优于细菌酵母. 但哺乳动物细胞的培养基十分复杂和昂贵。 同时哺乳动物细胞的生长能力不如微生物细胞，很难进行大规模发酵. 下游的表达产物纯化也很难进行. mammalian cell lines. Ba

20、cteria or eukaryotic microbes. large scale fermentation. downstream processing.,尽管如此，现在已有很多载体可以用于在哺乳动物细胞中表达蛋白质. 通常使用一个病毒作为载体，以抗药性标记进行选择，载体中应该包含启动子。 猿猴病毒和巨细胞病毒的启动子 They often based on a viral system, vectors utilize selectable markers (often drug-resistance markers) and have promoters that enable exp

21、ression of the cloned gene sequence. Common promoters: Simian virus (SV40), Cytomegalovirus (CMV).,第二节 蛋白质工程 protein engineering 蛋白质工程：通过改变基因序列来改变蛋白质结构。 主要使用离体突变技术。 离体突变技术可以在基因序列中引入特定的突变。 Altering the structure of proteins via alteration to the gene sequence, and has become possible due to the techn

22、ique of mutagenesis in vitro. Mutagenesis in vitro enables specific mutations to be introduced into a gene sequence.,定点突变，也称寡核苷酸介导的突变： 1，用M13噬菌体制备单链模板，含需要改变的基因。 2，合成15-30个核苷酸长度的寡核苷酸片段，该片段与待突变的模板DNA区域互补，中间含突变碱基。 Oligonucleotide-directed or site-directed mutagenesis: Single-stranded template (ss DNA)-

23、containing the gene to be altered.-produced via M13 cloning system. An oligonucleotide (usually 15-30 nucleotides in length)，complementary to the region on interest, and has desired mutation in the middle.,3，模板与寡核苷酸退火。 4，突变位点错配，但两侧的互补序列可以起到稳定作用。 The template and oligonucleotide are annealed. the mut

24、ation site will mismatch, but the flanking sequences will confer stability.,5，用DNA聚合酶和DNA连接酶合成和连接产生双链DNA分子。 6，导入大肠杆菌复制，产生两个子代分子，其中一个的靶位点已经发生改变。 the template is then copyed using DNA polymerase. This gives rise to a ds DNA which, on replication (when introduced into E. coli), will yield two daughter

25、molecules, one of which will contain the desired mutation.,定点突变技术的用途： 1，准确定位(pinpoint)蛋白质序列中的关键氨基酸。 2，改变这些部位的氨基酸组成，研究它们在蛋白质功能中的作用。 3，修饰活性位点周围的氨基酸，优化酶的催化活性。 alteration of the catalytic activity of an enzyme by modification of the residues around the active site.,4，改善贮藏蛋白的营养价值。 5，改善工业和医药用蛋白的稳定性. Impro

26、vement in the nutritional status of a storage protein.Improvement in the stability of a protein used in industry or medicine.,突变DNA的鉴定可以通过与带有放射性标记的突变寡核甘酸序列在高严谨条件下杂交来进行。 突变克隆将产生杂交信号，未突变克隆没有信号。 放射自显影。 high stringency. Radiolabelled. Autoradiography. Even a single base-pair change can be picked up usin

27、g this technique. The mutant can then be sequenced to confirm its identity.,突变以后的基因在表达时可以用以下的载体进行： 1，lac promoter-通过添加IPTG（异丙基硫代半乳糖苷）诱导转录。 2，PL promoter-使用可以产生温度敏感型cI蛋白的宿主细胞，30目的基因不表达，42可以表达。 can be used with a temperature-sensitive cI repressor ，so that expression of the mutant gene is repressed at

28、 30 but is permitted at 42.,比较突变蛋白与原来的蛋白质在性能上的差别。 通过这种方法，可以使蛋白质结构发生细微变化并优化其功能。 Analysis of the mutant protein can be carried out by comparison with the wild-type protein. In this way, proteins can be engineered by incorporating subtle structural changes that alter their functional characteristics.,第

29、三节 重组DNA技术应用举例 rDNA，recombinant DNA，重组DNA。 指在离体条件下将不同来源的DNA连接组合产生的DNA分子。 Examples of biotechnological applications of rDNA technology.,一、酶的生产 production of enzymes 酶已被用于酿酒、食品加工、纺织品生产、皮革工业、洗衣粉生产、药品以及基础研究。 brewing, food processing, textile manufacture, leather industry, washing powder, medical applica

30、tions, basic scientific research.,很多情况下，酶的来源为从自然资源提取。 现在，可以通过重组DNA方法产生很多种酶。 In many cases, the enzymes are prepared from natural sources. But in recent years, many enzymes were produced by recombinant DNA methods. Cost-benefit analysis-成本与收益分析。,例如用于PCR等反应体系的聚合酶（polymerase）已通过重组DNA技术进行生产。 重组酶可能更能满足特殊

31、加工过程的需要（criteria = criterion），使得反应过程的保真度和有效性大大增加。 Recombinant enzymes can sometimes be engineered so that their characteristics fit the criteria for a particular process better than the natural enzyme, which increases the fidelity and efficiency of the process.,（一）重组凝乳酶 食品工业利用重组凝乳酶来生产奶酪。 粗制凝乳酶Rennet

32、 = rennin = chymase = chymosin。 Food industry use recombinant enzyme to produce cheese.,Chymosin作为一种蛋白酶参与乳酸菌发酵后牛奶中酪蛋白的凝结。 Chymosn is a protease that is involved in the coagulation of milk casein following fermentation by lactic acid bacteria.,该酶的传统制备方法为从牛、猪或真菌中提取。 1960s, 联合国粮农组织（the Food and Agricul

33、ture Organisation of the United Nations）预言: 由于小牛被饲养到成熟阶段以提供肉类产品，牛粗制凝乳酶Rennet将会短缺。,chymosin有六种自然（natural）来源： 食用小牛-veal calves 成年牛-adult cow 猪-pigs 真菌Rhizomucor miehei-根毛霉菌 真菌 Endothia parasitica-藓霉菌属栗疫菌 真菌Rhizomucor pusillus-微小根毛霉菌,重组凝乳酶于1981开发成功，1988年获准生产。 在英国用重组凝乳酶生产的奶酪目前占到90%。 Recombinant chymosin

34、 is now used to prepare around 90% of hard cheeses in the UK.,重组凝乳酶的生产体系包括：大肠杆菌、乳酸克鲁维酵母、黑曲霉。 chymosin is now also available as a recombinant-derived preparation from E. coli, Kluyveromyces lactis and Aspergillus niger。,基因操作奶酪在三个方面遭到质疑： 1，产奶的牛可能用重组生长激素处理过； 2，产奶的牛可能吃过含有转基因大豆或玉米的饲料-fed with animal feed

35、s containing GM soya（大豆，soybean）or maize. 3，奶酪生产时使用了重组凝乳酶。 GM cheese-gene manipulation cheese。,尽管如此，很多消费者同意奶酪本身不是一种遗传修饰的有机体，而是一种GMO产品的产品。 GMO = Genetically Modified Organism，经过遗传修饰的有机体。,（二）重组脂酶，recombinant proteases and lipases 洗衣粉中使用重组的蛋白酶和脂酶，帮助清洗蛋白质和脂类污迹。 to assist cleaning by degradation of prote

36、in and lipid-based staining.,1988年， Novozymes 公司(原名Novo Nordisk A/V)开发成功重组脂酶，商品名为lipolase. 该公司是世界上最大的酶制剂供应商。 lipolase-a recombinant lipase was developed in 1988 by Novozymes the company is the largest supplier of enzymes for commercial use in cleaning applications.,Lipolase第一种用重组DNA技术开发成功的商业用酶，也是去污剂

37、中添加的第一种脂酶。 Lipolase Ultra-超强Lipolase，由Lipolase修饰产生，在低温下去除油污的能力更强。 Lipolase was the first commercial enzyme developed using rDNA technology, and the first lipase used in detergents. Lipolase Ultra-an engineered variant of Lipolase, which gives enhanced fat removal at low wash temperatures.,二、the BST

38、story rBST-recombinant bovine somatotropin-重组牛促生长素，牛生长激素 基础研究-技术转化-获准生产-市场接受。 basic science-technology transfer-approval by regulatory bodiesmarket acceptance,In the early 1980s, 科学家已经开始研究rBST。 BST基因是最早被克隆并在细菌细胞中表达的哺乳动物基因之一。,1，克隆BST基因cDNA. 2，连接至质粒表达载体。 3，导入大肠杆菌，获得工程菌。 4，大规模发酵培养。 5，提取纯化rBST.,牛生长激素可以促

39、进牛的生长，进而提高牛奶产量。 BST is also known as bovine growth hormone and can promote the growth of cattle. Administering（给药）BST-increase milk production. Dairy industry-牛奶业,1994年，美国食品和药物管理局（the Food and Drug Administration of USA, FDA）批准生产rBST。 由孟山都公司（Monsanto）经营，商业名称（trade name）为Posilac。 当时，欧盟由于不需要增加牛奶产量没有同意

40、生产该产品。,rBST的效果需要从三个不同的方面来考虑： 1，对牛奶产量的影响； 2，对动物的影响； 3，对消费者（consumer）的影响。,用rBST处理后，牛的奶产量通常增加15%，这对牧场经营者来说是有利可图的。 但注射rBST可以引起局部肿胀，加重脚部感染、乳腺炎，并给牛的繁殖能力带来影响。 Administering rBST can produce localized swelling at the site of injection, and can exacerbate problems with foot infections, mastitis and reproduct

41、ion.,Counter-argument-赞成rBST的观点认为上述问题在没有注射rBST的牛群中也存在 天然的或重组的生长激素可以增加胰岛素样生长因子（insulin-like growth factor, IGF-1）的水平，进而增加牛奶产量。,有证据表明IGF-1可以促进癌细胞的生长。所以使用rBST可能对健康带来危害。 但赞成rBST的人认为哺乳期早期阶段的IGF-1水平比哺乳期开始100天后注射rBST后牛的IGF-1水平要高。 There is evidence that IGF-1 can stimulate the growth of cancer cells.,反对者认为：

42、 rBST与治疗性蛋白不同，后者只给少数病人使用。 而牛奶是大部分人的消费品，不管rBST引起的危险多么微不足道，都是不能接受的。 Those who oppose the use of rBST point out that, unlike a therapeutic protein that would be used for a limited number of patients, milk is consumed by most people, and any inherent risk, no matter how small, is therefore unacceptable.

43、,三、治疗性药物 重组DNA技术也可以用于： 开发治疗性药物。 医学诊断。 开发医学诊断试剂。 therapeutic products for use in human healthcare ，medical diagnostics with recombinant DNA technology,用于医学治疗的重组蛋白质药物可以分为三类： 1，用于替代或补充病人体内没有功能或缺少的蛋白质（R/S）。 Recombinant DNA products for use in medical therapy can be divided into three main categories: Us

44、ed for replacement or supplementation of human proteins that may be absent or ineffective in patients with a particular illness,2，用于治疗特殊疾病，通过干扰来缓解病情（SDT）. 3，生产重组疫苗（V）. 该研究方向发展很快. Used in specific disease therapy, to alleviate a disease state by intervention. The production of recombinant vaccines is

45、 an area that is developing rapidly and which offers great promise.,糖尿病-由胰腺郎氏小岛-细胞不能产生足够的胰岛素引起。 世界卫生组织估计2025年将会加倍。 DM （diabetes mellitus）is usually caused by -cells in the islets of Langerhans in the pancreas failing to produce adequate amounts of the hormone insulin. World Health Organization estum

46、ated in 2025 will double.,有两种糖尿病患者： 1，依赖胰岛素，insulin-dependent DM，IDDM; 2，不依赖胰岛素， non-insulin dependent DM，NIDDB.) IDDM需要胰岛素，而很多NIDDB也通过注射胰岛素来控制病情。 另外一种服用方法为吸入胰岛素粉末，inhalation of insulin powder.,胰岛素由两条多肽链组成。 Insulin is composed of two amino acid chains. The A-chain-acidic, 21 amino acids. B-chain-bas

47、ic, 30 amino acids.,新翻译产生的前胰岛素(proinsulin)在A链和B链之间有一段30个氨基酸长的连接肽链，称为C-chain. 81个氨基酸的前胰岛素分子经蛋白酶断裂形成有活性的胰岛素。 A链和B链之间由二硫键相连-linked together by disulphide bonds between cysteine residues.,胰岛素是第一个被测序的蛋白质，二十世纪五十年代由Frederick Sanger完成。 Insulin was the first protein to be sequenced by Frederick Sanger in the

48、 mid-1950s.,由于DM由正常的体内组分胰岛素引起，所以对它的治疗属于置换或补充疗法. Banting and Best 1921年发现和纯化出胰岛素并用于治疗糖尿病患者，一直依赖自然提纯的胰岛素，来源有限，质量也有问题。 replacement or supplementation. developed the use of insulin therapy in 1921.,七十年代后期和八十年代早期，重组DNA技术使得科学家能够在细菌中翻译合成胰岛素, 并于1982年获准生产。 重组胰岛素现在有几种成品，其中商业名称为Humulin的制剂由Eli Lilly company生产。

49、In the late 1970s and early 1980s, recombinant DNA technology enabled scientists to synthesise insulin in bacteria。,生产重组胰岛素的早期方法中，A链和B链在两个细菌菌株中被单独合成。 两段编码序列均由lac启动子驱动，可以用乳糖诱导表达。 The insulin A- and B-genes were placed under the control of the lac promoter, so that expression of the cloned genes could

50、 be switched on by using lactose as inducer.,纯化的A链和B链通过化学处理成为完整的胰岛素分子。 Following purification of the A- and B-chains, they were linked together by a chemical process to produce the final insulin molecule.,改进的方法：从全序列表达产生完整前胰岛素。 然后通过酶的作用转化成胰岛素。 A development of this method involves the synthesis of th

51、e entire proinsulin polypeptide from a single gene sequence. the product is converted to insulin enzymatically.,胰岛素，insulin 治疗糖尿病 生长激素，growth hormone 治疗儿童生长激素缺乏 -干扰素， - interferon 治疗多毛细胞白血病 乙肝疫苗，Hepatitis B vaccine 预防乙肝 组织纤溶酶原激活剂，tPA 治疗心肌梗塞，溶血栓 凝血因子8，factor 8 治疗血友病 DNA酶，DNase 治疗囊性纤维化,特殊疾病疗法-specific disease therapy 组织（血）纤（蛋白）溶酶原激活剂（TPA），该蛋白作用