基因工程的定义

基因工程的定义 1. 基因工程的定义:在体外对不同生物的遗传物质,基因,进行剪 切、重组、连接～然后插入到载体分子中,细菌质粒、病毒或噬 菌体DNA)～转入微生物、植物或动物细胞内进行无性繁殖～并表 达出基因产物。 2. 碱抽提法提取质粒DNA 原理和步骤 3、DNA的定量和纯度测定 1. 紫外光谱法 2. 琼脂糖凝胶电泳估计 原理: 3. DNA的凝胶电泳的原理 相同分子量的DNA: 闭合环状卷曲超螺旋迁移最快～ 线性分子次之～ 伸展开环状最慢。 4. 限制性核酸内切酶是一类能够识别双链DNA分子中的某种特定核苷酸序列,4—8bp,～并由此处切割DNA双链的核酸内切酶。 功能 即在核酸分子链的内部制造切口的酶。 自我保护作用。用来保护宿主不受外来DNA的感染～可降解外来的DNA～从而阻止其复制和整合到细胞中。 Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To 目前鉴定出三种不同类型的限制性内切酶。各自的特点 I 型限制性内切酶 II 类限制性内切酶 III类限制性内切酶 II I III 内切酶与甲基化三亚基双功能二亚基双酶分子 酶分子不在一起 酶 功能酶 4-6bp 大多数5-7bp 识别位点 二分非对称 非对称 为回文对称结构 无特异性 在识别位在识别位点中或 切割位点 至少在识别位点下游 靠近识别位点 点外1000bp 24-26bp 限制反应与 分开的反应 互斥 同时竞争 甲基化反应 限制反应是 No Yes Yes 否需要ATP Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To 粘性末端,sticky ends～cohensive ends,含有几个核苷酸单链的末端。分两种类型:5’端凸出,如EcoR I切点,,’端凸出,如Pst I切点, 限制性内切酶识别的序列 1. 长度:一般为4-8个碱基～最常见的为6个碱基。 • 4个碱基:sau3A? • 5 个碱基:EcoR? • 6个碱基:EcoR? • 7个碱基:BbvC? • 8个碱基:Not? 限制酶识别序列的结构 大多数为回文对称结构～切割位点在DNA两条链相对称的位置。 有些识别非对称序列～ 有些可以识别多种序列～ACC?识别序列是GT , MKAC,可以识别4中序列, 有些识别的序列呈间断对称～对称序列之间含有若干任意碱基 位点偏爱 (site preferences) 某些限制酶对同一介质中的有些位点表现出偏爱性切割～即对不同位置的同一个识别序列表现出不同的切割效率。 酶切反应条件 缓冲液～反应温度～反应时间～反应中止 Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To 星星活性(star activity) 在极端非标准条件下～限制酶能切割与识别序列相似的序列～这个改变的特殊性称星星活性。 引起星星活性的因素 ? 高浓度甘油,>5%, ? 酶过量,>100U/,g, ? 低离子强度,<25mM, ? 高pH (>pH8.0) ? 有机溶剂 PMSD(二甲基亚砜)、乙醇、乙二醇、二甲基乙酰胺,dimethylacetamide,、二甲基甲酰胺,dimethylformamide,、sulphalane ? 用其它二价阳离子代替Mg++ Mn++～Cu++～Co++, Zn++ 不同的酶对上述条件的敏感性不一样 PstI比EcoRI对高pH更敏感～但后者对甘油浓度更敏感 同裂酶,Isoschizomers) 识别相同序列的限制性内切酶～但它们的切割位点可能不同。 同尾酶(Isocaudamers, 识别的序列不同～但能切出相同的粘性末端。如BamH I、Bgl ?、Bcl I、Xho ?等 Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To 影响限制性内切酶活性的因素及使用注意事项 1. DNA的纯度 2. DNA的甲基化程度 3. 温度 4. 缓冲液(Buffer) T4DNA 连接酶的功能 主要是催化双链DNA一端的3’-OH与另一双链DNA 5’端的磷酸根形成3’,5’-磷酸二酯键～使具有相同粘末端或平端的DNA末端连接起来。 DNA聚合酶 3’,5’外5’,3’外聚合持续能 切酶活性 切酶活性 速率 力 大肠杆菌DNA聚合酶 低 有 中 低 T4DNA聚合酶 高 无 中 低 T7DNA聚合酶 无 快 高 化学修饰T7DNA聚合酶 低 无 快 高 遗传修饰T7DNA聚合酶 无 无 快 高 逆转录酶 无 无 低 中 Taq DNA聚合酶 无 有 快 高 Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To 大肠杆菌DNA聚合酶 I ?一条单链多肽。 ?5’,3’外切酶活性位于N端。 5’,3’DNA聚合酶活性 3’,5’外切酶活性 在没有dNTP的情况下～外切活性占主导地位～而当存在足够的dNTP时～外切活性和合成活性将处在动态平衡中～结果使得dsDNA成为平末端。 Klenow fragment 具有5’,3’聚合酶活性和3’,5’外切酶活性。,失去了5’,3’外切酶活性,。 在蛋白酶,枯草杆菌蛋白酶,裂解, 从全酶中除去5’—3’外切活性片段～而聚合活性和3’—5’外切活性不受影响。 逆转录酶 依赖RNA的DNA聚合酶,RNA指导的DNA聚合酶,。 来源 RNA肿瘤病毒。 AMV的性质 二链多肽(62kDa/94kDa) 具5’ ?3’DNA聚合活性 具很强的RNA酶H活性(降解与DNA杂交的RNA) Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To 碱性磷酸酶 该酶可从DNA分子的5’端去除磷酸基 碱性磷酸酶用于从DNA片段上除去5’磷酸～以防止自身连接。 核酸分子杂交定义 用标记的已知DNA或RNA片段,探针,来检测样品中未知核酸序列～通过核苷酸间碱基互补的原则发生异源性结合～再经显影或显色的～将结合核酸序列的位置或大小显示出来。 待测的核酸序列～可以是克隆的基因片段～也可以是未克隆化的基因组DNA和组织细胞的RNA。 A260值达到最大值1/2时的温度称为解链温度或熔解温度,melting temperature,Tm)～此时50%的DNA分子发生了变性。Tm与核酸的G、C含量有关。 Tm=,G+C,%×0.41+69.3 Tm也受介质中离子强度的影响～离子强度低～熔解温度也低。 核酸探针 1.定义: 一段带有检测标记的与目的基因或目的DNA特异互补的已知核苷酸序列。 为确定探针是否与相应基因组DNA杂交～有必要对探针加以标记～Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To 以便在结合部位获得可识别的信号。 探针的分类: 据制备方法及核酸性质不同～可分为: 1 基因组DNA探针 2 cDNA探针 3 RNA探针 4 寡核苷酸探针 Southern 印迹杂交 限制性内切酶酶切后,EDTA～ 65?灭活限制酶, 琼脂糖凝胶电泳分离酶切DNA片段 变性液,碱变性, 凝胶上DNA变性 预杂交 Tris缓冲液 中和 杂交 高盐下 放射自显影 DNA转印至NC膜 结果 烘干、固定 Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To Taq DNA 聚合酶 1986年 H.A. Erilish从嗜热 水生菌分离出耐高温的Taq DNA聚合酶～代替大肠杆菌DNA聚合酶Klenow片断,37 oC )～PCR技术才进入实用阶段 PCR基本原理 在模板DNA、引物和4种脱氧核苷酸存在条件下依赖于DNA聚合酶的酶促合成反应。 1、变性:通过加热使DNA双螺旋的氢键断裂～双链解离形成单链DNA 。 2、退火:当温度突然降低时～反应体系中引物和其互补的DNA模板在局部形成杂交链。 3、延伸:在DNA聚合酶和4种脱氧核苷酸,dATP、dCTP、dGTP、dTTP,及Mg2+存在条件下～5’?3’的聚合酶催化以引物为起始点的DNA链延伸反应。 PCR的特点 1,特异性强 2,敏感性高 ,3,快速 ,4,简便 ,5,可以扩增mRNA Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To Pfu DNA Polymerase 有3 ’-5’的外切酶活性～5’-3’外切酶活性。 是目前已发现的所有耐高温DNA 聚合酶中出错率最低的。 但PCR产物为平端: 引物设计: ,1)序列应位于高度保守区,与非扩增区无同源序列。 ,2,引物长度以15-40 bp为宜。 ,3,碱基尽可能随机分布,G+C占50-60%。 ,4,引物内部避免形成二级结构。 ,5,两引物间避免有互补序列。 ,6,引物3’端为关键碱基,5’端无严格限制。 重组DNA技术基本原理 基本原 理 目的基因的获克隆载体的选择和构 取 建 外源基因与载体的连DNA导入受体 接 菌 克隆基因的表达 重组体的筛 选 Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To 目的基因的获取 1. 化学合成法 要求:已知目的基因的核苷酸序列或其产物的氨基酸序列 。 2.基因组DNA文库(genomic DNA library) 3. cDNA文库(cDNA library) 4. 聚合酶链反应(polymerase chain reaction, PCR) ,见第22章, 在基因工程操作中～把能携带外源DNA进入受体细胞的DNA分子叫载体。 克隆载体(cloning vector) 为使插入的外源DNA序列被扩增而特意设计的载体称为克隆载体。 表达载体(expression vector) 为使插入的外源DNA序列可转录翻译成多肽链而特意设计的载体称为表达载体。 载体的选择标准 能自主复制, Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To 具有两个以上的遗传标记物～便于重组体的筛选和鉴定, 有克隆位点,外源DNA插入点,～常具有多个单一酶切位点～ 称为多克隆位点, 分子量小～以容纳较大的外源DNA。 外源基因与载体的连接 1. 粘性末端连接 2. 平端连接 3. 同聚物加尾连接 4. 人工接头(linker)连接 重组DNA导入受体菌 感受态大肠杆菌的制备 用CaCl2处理大肠杆菌～能增加膜的通透性 感受态细菌:是指细菌处于容易接受外源DNA的状态。 重组体的筛选 感受态细胞: • 经一定方法处理(如CaCl2处理)后具备接受外界DNA能 力的大肠杆菌。受体菌应为安全无毒菌株。,容易接受外 源DNA的状态, Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To • 方式: 转化、转染、感染 , 转化:以质粒为载体～将携带外源基因的载体DNA导入受体细 胞。 , 转染:重组DNA导入真核细胞的过程。 , 感染:以噬菌体为载体～在体外将噬菌体DNA包装成病毒颗粒～ 使其感染受体菌 重组体的筛选 1.遗传检测法 抗药性的选择 标志补救,ß-半乳糖苷酶法, 2. 电泳检测法 3.PCR检测法 4. 菌落杂交筛选法 5. 免疫化学检测法 6. DNA序列检测 E.coli表达体系优势: 一种成熟的基因克隆表达的受体细胞 繁殖迅速～培养代谢易于控制 易于进行遗传操作和高效表达 Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To 不足之处: 1,缺乏适当的转录后和翻译后加工机制。 2,缺乏表达蛋白质复性系统～表达蛋白无特异性空间结构。 3,表达产物不稳定～易被细菌蛋白酶降解。 4,表达的蛋白质常形成不溶性包涵体 (inclusion body) 5,很难表达大量可溶性蛋白 外源蛋白在大肠杆菌中的表达部位 1. 细胞质中表达 ,1,包涵体,inclusion body, 是存在于细胞质中的一种不溶性蛋白质聚集折叠而成的晶体结构物。 2. 周质中表达 3. 胞外表达 二、克隆载体 , 复制基因(replicator) , 选择性记号 , 克隆位点 Subsequently, the Anhui Army military Special Branch, in Shanghai's Central Intelligence Organization and the Communist party organization in Western Zhejiang Province, sent party backbone, respectively, went to Wu Jiangyan opened the Tomb, Tomb of heroic people, under the leadership of the Communist Party, fear no sacrifice and persevering, goal of the anti-Japanese revolutionary struggle. (A) stationed in Taoyuan area (Tomb of) 1, the new army of the Communist Party of China underground organization in September 1938, the CPC Zhuang Shaozhen (Zhuang Zhen, Nanxun people) by the Deputy Director of the political Department of the army on Deng Zihui's dispatch, operating from Jing County, Anhui Province, the Lake reached the Tomb, its tasks are: looking for reliable object, introduce them to the new, to expand the new team. Zhuang Shaozhen KMT-CPC cooperation as an opportunity, after classmates Yao Bi (Nanxun people, Ren Zhuxi force staff Director), introduced into the Kuomintang government political team, and as a political leader. He used this opportunity to invite progressive youth Hong Haiquan form a political team, at this point, the County Government has just been established, local school, unemployed youth are more quickly absorbed sheng, zhenze, Miaogang, Nanxun, huzhou, more than 20 more than required-Japanese youth. In order to raise the level of political players and the ability to work, Zhuang Shaozhen Xu Temple, is for the establishment of "youth ... Political activities will soon attract the attention of the Kuomintang. Is early Zhuang Shaozhen indicating Hong Haiquan to scoop the first elementary school, teacher's identity as a cover, carry out the party's work. However, County Director of education comes into action. To