6第三章基因克隆载体1

Vectors that can replication both in the target host and in E. coli were used, these were called shuttled vectors.
一、载体的一般特性
Prerequisites of Vector:
Fairly small DNA molecules to facilitate isolation and handling. 载体应该很小，便于分离和操作。
从稻草中寻找针，大海捞针。
从一个重组子群体中分离一个基因必须考虑以下三个方面：
A, the individual recombinant molecules have to be physically separated from each other.---连接至载体。
B, the recombinant molecules have to be amplified to provide enough material for further analysis.---转化至宿主细胞扩 增。
插入片段将破坏lacZ´，不能产生有功能的α-肽。
β-galactosidase gene, lacZ´—This sequence contains the polylinker region, and insertion of a DNA fragment into one of the cloning sites results in a nonfunctional α-peptide.
质粒表达的α-肽 + 受体细胞表达的β一半乳糖苷 酶羧基端 = 有功能的β一半乳糖苷酶。
X-gal------蓝色产物靛蓝。
重组子---白色菌落。
This forms the basis for a powerful direct recombinant screening method using the Chromogenic substrate-----X-gal, 生色底物.
Copy number 拷贝数
Stringent control of DNA replication—严谨型复制-----low copy number—the replication of plasmid closely tied to host cell chromosomal DNA replication. 拷贝数低，接合型。
பைடு நூலகம்缺少705bp，拷贝数增加3倍。外源DNA的容纳量也 明显增加。
Insertional inactivation—外源DNA插入Apr或Tcr后 ，将引起ampicillin或tetracycline抗性基因失活。
2、改进的质粒载体 slightly more exotic plasmid vectors
非接合型不能转移，但具有mob基因时可以在 接合型质粒帮助下进行转移。
Nonconjugative—非接合型的------- are not selftransmissible, but may be mobilised by a conjugative-proficient plasmid if their mob region is functional.
Origin of replication, the DNA can be copied and thus maintained in the cell population as the host organism grows and divides. 载体应该具有复制起 始位点。
Selectable marker, enable the vector to be detected. 应该带有选择标记。
pBR322--Francisco Bolivar and his colleagues
containing DNA from three sources：
Ampr基因来自pSF2124 ColE1松驰型复制子（ori）来自pMB1 Tetr基因来自pSC101。
pAT153--------a deletion derivative of pBR322 by removal of two fragments of DNA (705bp ， necessary for mobilisation) using the restriction enzyme HaeⅡ。
origin of replication = ori ampicillin resistance gene, Apr lacI gene, lac repressor Plac, lac promoter
MCS—连接时有多种选择。 MCS provide the choice of site available for insertion of DNA during recombinant production.
Particular characteristics:
a wider range of restriction sites for cloning DNA fragments-----multiple cloning site, MCS. 多克隆位点。
specific promoters for the expression of inserted genes
vector
Basic conditions of Gene cloning: Vector---carrier molecule Host ---a living system in which the vector can be
propagated. 载体 宿主细胞---扩增和保存重组DNA分子。
Isolation of a particular sequence would seem to be a bit like looking for the proverbial needle in a haystack-----with the added complication that the needle is made of the same material as the haystack.
whilst non-conjugative plasmids are small, show relaxed DNA replication and are present at high copy numbers. 非接合型质粒小，复制为松弛型， 拷贝数高。基因工程中是使用的质粒一般为非 接合型。
Relaxed plamids—松弛型质粒------replication not dependent on host cell chromosomal DNA replication-----high copy number ，拷贝数高，非 接合型。
In general, conjugative plasmids are large, show stringent control of DNA replication, and are present at low copy numbers, 接合型质粒大，复制为严谨型，拷贝数低。
direct selection for recombinants 。用专一性启动子 表达标记基因，直接对重组体进行选择。
pUC family: pUC系列载体。 polylinker = multiple cloning site, MCS
β-galactosidase gene, lacZ´, α-peptide
Antibiotic abbreviations 抗生素缩写： Ap—ampicillin---氨苄青霉素 Cm—chloramphenicol---氯霉素 Km—kanamycin---卡那霉素 Sm—streptomycin---链霉素 Sn—sulphonamid---磺胺类药物 Tc—tetracycline---四环素 Colicin—大肠杆菌素
Have antibiotic resistance genes，带有抗性基因。
接合型质粒携带与转移有关的tra和mob基因。
Conjugative—接合型的------plasmids carried tra (transfer) and mob (mobilising)regions which mediate their own transfer between bacteria by the process of conjugation.
1、基本的克隆性质粒 Basic cloning plasmids
命名时小写p代表质粒，后面跟有构建者或其单 位的缩写。
In naming plasmids, p is used to designate plamid, and this is usually followed by the initials of the workers who isolated or constructed the plasmid.
同时具有真核细胞复制起始位点，可以在真核细胞中 表达基因。
It is often more sensible to perform the initial cloning in E. coli, isolate the required sequence, and then introduce the purified DNA into the target host for expression.
多克隆位点含多个识别序列，这样一种排列方式提供 了多种可供单独或联合使用的克隆靶位点。
便于克隆由多种限制性内切酶单独或组合切割后产生 的DNA片段。
pUC18和pUC19的差别仅在于多克隆位点的方向相反。
pUC系列质粒的优点是可以用组织化学方法鉴定重组子。
它们来自pBR322，除具有松驰型复制起点和Ampr gene外，还 具有来自大肠杆菌乳糖（Lac）操纵子的DNA片段，含β一半乳 糖苷酶基因（LacZ）的调控序列和氨基端146个氨基酸的编码 信息。