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台成
7 years ago
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1 第九章酵母菌基因工程

2 酵母菌表达系统的优势基因表达调控机理比较清楚, 遗传操作相对简单具有真核生物蛋白质翻译后加工修饰系统安全型基因工程受体系统大规模发酵工艺简单, 成本低廉可将外源基因表达产物分泌到培养基中有助于阐明高等真核生物基因表达调控机理

3 酵母菌载体的类型整合型 ( 非自主复制型 ) 自主复制型附加体型 ( yeast episomal plasmid, YEp): 复制起点来自于酿酒酵母的天然质粒 -2μ 质粒染色体复制型 ( Yeast Replicating plasmid, YRp): 复制起点来自染色体上的自主复制序列 ARS (Autonomous replicating sequence)

4 Amp r Tc r URA3 Ori 2μ Amp r YIp Tc r Ori Amp r Ori YEp Tc r 2μ URA3 Amp r ARS YRp Tc r URA3 CEN Amp r pyac Ori TRP1 Ori Amp r Tc r YCp URA3 TEL HIS4 TEL TEL YLp TEL Amp r URA3 Tc r TEL

5 酵母菌载体的遗传标记基因大多数是以酵母菌氨基酸或核苷酸合成途径中的酶基因作为选择标记, 如 ARG4, LEU2,TRP1,HIS3,URA3 使用这些遗传标记时, 受体菌应是相应标记基因的突变型, 且是稳定的突变体 ( 缺失突变或多点突变 ), 利用遗传互补进行转化体的筛选

7 常用的酵母宿主系统酿酒酵母表达系统食品级安全性表达效率较低毕赤酵母 (Pichia pastoris) 表达系统能在以甲醇为唯一碳源和能源的培养基中生长表达效率高

8 一酿酒酵母菌的克隆载体 1. YIp (Yeast Integration plasmid ) 该类载体是在大肠杆菌质粒载体中插入酵母的标记基因构成没有酵母菌的复制子顺序, 只有依靠携带的酵母基因与酵母染色体上的同源 DNA 整合到染色体上去转化体十分稳定, 但也可能发生第二次同源重组

9 ori Amp r YIp5 Tet r 染色体 DNA URA3 Ura3

10 2. YEp 酿酒酵母中的 2μ 质粒几乎所有酿酒酵母菌株中都存在一个 6318bp 的野生型 2μ 环状质粒, 它在细胞内的拷贝数约 50~100 YIp 中可插入整个或部分 2μ 质粒 DNA 片段构成 YEp 大多数酿酒酵母菌株中都含有天然 2μ 质粒,YEp 复制所需的酶可由 2μ 质粒提供

12 3. YRp 酿酒酵母基因组中每隔 30~40kb 便有一个自主复制序列 (ARS) 含有 ARS 序列遗传标记基因及克隆位点的质粒组成 YRp Ori URA3 PstI Amp ARS YRp12 6.9kb Tc TRP1 BamHI EcoRI

13 YIp YRp

14 YRp 转化酵母的效率大幅度提高, 但转化子不稳定, 经几代培养后, 质粒丢失率高达 50%~70%, 主要原因是 YRp 质粒不能在母细胞和子细胞中均匀分配从酵母转化体中可分离到与转化前相同的质粒 DNA Yrp 也可整合到酵母染色体中去, 与 YIp 整合过程相同

15 4. YCp 载体着丝粒区域是染色体均匀分配的重要顺式作用元件将着丝粒片段插入到 YRp 质粒中, 能明显改善质粒在母细胞和子细胞中的均匀分配, 提高质粒在宿主细胞增殖过程中的稳定性 Ycp 以低拷贝存在

16 YRp YCp

17 5. YAc

18 vect or Transfomation frequency YIp 10 2 transformants per μg DNA YEp transformants per μg DNA YRp 10 4 transformants per μg DNA YCp 10 4 transformants per μg DNA Copy no./c ell Properties of the different yeast vectors 1 Much less than 1% per generation Loss in nonselective medium % per generation 1-20 Much greater than 1% per generation but can get chromosomal integration 1-2 Less than 1% per generation disadvantage 1. Low transformation frequency 2. Can only be recovered from yeast by cutting chromosomal DNA with restriction endonuclease which does not cleave original vector containing cloned gene Novel recombinants generated in vivo by recombination with endogenous 2μm plasmid Instability of transformants Low copy number makes recovery from yeast more difficult than that with YEp or YRp vectors advantage 1. Of all vectors, this kind give most stable maintenance of cloned genes. 2. An integrated YIp plasmid behaves as an ordinary genetic marker, e.g. a diploid heterozygous for an integrated plasmid segregates the plasmid in a Mendelian fashion 3. Most useful for surrogate genetics of yeast, e.g. can be used to introduce deletions, inversions and transpositions 1. Readily recovered from yeast 2. High copy number 3. High transformation frequency 4. very useful for complementation studies 1. Readily recovered from yeast 2. High copy number. Note that the copy number is usually less than that of YEp vectors but this may be useful if cloning gene whose product is deleterious to the cell if produced in excess 3. High transformation frequency 4. Very useful for complementation studies 5. Can integrate into the chromosome 1. Low copy number is useful if product of cloned gene is deleterious to cell 2. High transformation frequency 3. Very useful for complementation studies 4. At meiosis generally shows Mendelian segregation YAC 1-2 Depends on length: the longer the YAC the more stable it is Difficult to map by standard techniques 1. High-capacity cloning system permitting DNA molecules greater than 40 kb to be cloned 2. Can amplify large DNA molecules in a simple genetic background

chromosomal integration 1-2 Less than 1% per generation disadvantage 1. Low transformation frequency 2.

19 6. 酿酒酵母表达质粒启动子 : 组成型 ( 葡萄糖诱导 ) :PGK, GPD, ADH1 诱导型 :GAL 基因启动子

20 The GAL System GAL genes: all genes involved in the pathway from Galactose to G-6-P GAL genes are expressed at a high level when galactose is used as the sole carbon source The promoter from the GAL1 gene is most commonly used for expressing target protein. Following addition of galactose, GAL1 mrna is rapidly induced over 1000-fold and can reach 1% of total mrna

promoter from the GAL1 gene is most commonly used for expressing target protein.

21 Each of the GAL genes contains within its promoter at least one, and often multiple, binding sites for the transcriptional activator Gal4p Gal4p is induced by galactose Gal promoter

22 不适合工业化生产!

23 酿酒酵母整合型载体

24 酿酒酵母宿主系统已成功地用于多种重组异源蛋白的生产 Glucagen (Novo Nordisk) 甲肝乙肝疫苗 (GlaxoSmithKline ) 水蛭素 (Aventis ) 但也暴露出一些问题 : 外源基因表达水平不高重组异源蛋白超糖基化培养基复杂, 成本高昂

25 二甲醇酵母表达系统 Pichia pastoris 巴斯德毕赤酵母能在价格低廉的甲醇培养基中生长菌株可进行高密度发酵 (>400 g/l wet cell weight) 催化甲醇代谢途径第一步反应的乙醇氧化酶 (Alcohol oxidase, AOX1) 可积累到总蛋白的 30% (10 12 g/l 重组蛋白 ) AOX1 启动子是已知功能最强可诱导基因启动子没有附加体质粒, 一般是整合型载体

26 整合型分泌表达载体

29 转化方式 : 电激法氯化锂法等载体的整合 : 获得高拷贝整合转化子 :

30 毕赤酵母系统的缺陷高密度发酵的条件下, 发酵周期较长 (4-7 天 ), 易污染, 且过长时间的发酵容易使目的产物发生降解 ; 利用甲醇作为诱导原料, 不仅会给细胞带来毒害, 而且也会给生产带来安全隐患 ; 采用怎样的补料方式, 才能既能满足菌体的生长, 又能为产物表达提供足够的动力 ; 如何实现方便快捷的低成本的检测甲醇浓度的方法以实现目的产物的高效表达 ; 不是万能的蛋白表达系统, 低表达或不表达的例子也在增加

31 三酵母双杂交系统 Two-hybrid system Detect protein protein interactions.

32 Principle of yeast two-hybrid assay Many eukaryotic trans-acting transcription factors are composed of physically separable, functionally independent domains. DNA-binding domain (BD) binds to a specific enhancer-like sequence ( upstream activation site, UAS). The activation domains (AD) directs the RNA polymerase II complex to transcribe the gene downstream of the UAS

33 The domain structure of Gal4p Amino acids 1 65 interacts directly with DNA at UAS G for galactose Amino acids is responsible for the dimerization of the protein Amino acids forms the major AD Amino acids is a weak activator Amino acids is a binding site for a transcriptional repressor

34 If the DNA-BD and AD are physically separated by recombinant DNA technology and expressed in the same host cell, they do not directly interact with each other and thus cannot activate the responsive genes. However, if the DNA-BD and AD can be brought into close physical proximity in the promoter region, the transcriptional activation function will be restored. The DNA-BD and the AD are both derived from the yeast transcriptional activator GAL4P (a.a and )

36 See movie

37 酵母双杂交体系的宿主菌株及质粒载体宿主菌将酿酒酵母基因组中编码转录激活因子 GAL4 的基因剔除掉, 建立缺陷型的酵母菌株如 SFY526 和 HF7c, 它们都丧失了表达内源 GAL4P 转录激活因子的能力, 因此适于用来检测外源 GAL4P 转录激活因子的功能

38 BD 质粒载体 : 靶基因按正确的相位与 GAL4- BD 融合, 产生杂种蛋白 I AD 质粒载体 : 是用于构建 cdna 表达文库的专用载体克隆的 cdna 片段按正确的方向和相位插入载体的多克隆位点, 表达产生与 GAL4- AD 的融合蛋白, 形成杂种蛋白 II 杂种蛋白通过核定位序列 (nuclear localization sequences) 进入酵母的细胞核

40 : SV40 T-antigen nuclear localization sequence.

41 酵母双杂交体系的实验程序将已知的靶蛋白 (bait) 基因插入到 pgbt9 质粒载体的 MCS 将 cdna 片段 (prey) 克隆在 pgad424 载体上, 构成 cdna 表达文库从大肠杆菌中分别提取这两种重组质粒 DNA, 共转化酿酒酵母菌株 HF7c 将转化菌株涂布在不含 Leu 和 Trp 的基本培养基上, 筛选具有两种质粒的转化子再涂布在不含 Leu Trp 和 His (reporter gene) 的基本培养基上, 筛选能表达相互作用的杂种蛋白质的阳性克隆

42 cdna AD library Other Interaction Screens The interaction of DBD and AD is mediated through another factor.

人工抗原的鉴定

人工抗原的鉴定生物技术通报张海棠王自良刘耀东传统的理化分析方法在食品污染检测方面发挥了重要的作用但均存在一定的缺陷急需建立快速高效的检测方法食品污染免疫检测是一种新型的分析方法与理化分析方法相比具有省时省力费用低廉便