论文总字数：20772字

摘 要

质粒转化是基因合成中一项必要操作，它可以为后续分析目的基因结构与功能提供大量的亲本分子，从而有利于目的基因的深入研究。所以构建出所需质粒对基因工程的后续研究意义重大。
本文以人工化学合成全基因的方法，构建了质粒K19_S316A，首先进行全基因的序列合成，根据PCR原理扩增所需要的基因，将PCR产物（或酶切产物）进行凝胶回收，然后与酶切后的目的载体进行重组反应，再转化到所对应的感受态细胞中，加入LB培养基摇菌。将菌涂布在相应抗性的固体培养基上培养过夜。第二天从平板上筛选出所需的阳性克隆，而后测序、摇菌抽提得出克隆、进行双酶切验证。验证结果可以看出目的片段是否被装入目的载体中。如验证结果正确则质粒构建成功。以构建质粒K19_S316A为例，成功构建出的目的质粒可用于序列分析和转基因等重要生物技术的研究中，为其提供可以预计的分析。

关键词：亲本分子、质粒构建、筛选、测序

Transformation and extraction of recombinant plasmid

Abstract

Plasmid transformation is a necessary operation in gene synthesis, which can provide a large number of parent molecules for subsequent analysis of gene structure and function, which is useful for the further study of the target gene. So it is of great significance to construct the plasmid for the follow-up study of gene engineering.

In this paper, taking the construction of plasmid K19_S316A as an example, this paper discusses the method of the artificial modification of gene molecules. This method can provide a predictable analysis for the study of downstream genes. First full gene sequence synthesis, according to the principle of PCR amplification of the genes required, PCR product (or enzyme digestion products) gel recovery, and enzyme digestion and the target vector recombination reaction, then transformed into competent cells corresponding to the, added to the LB culture medium for shaking bacteria. The bacteria were coated on the solid medium for the night. On the second day, the positive clones were screened out from the flat plate, and then the clones were cloned and tested by double enzyme digestion. The verification results can be seen whether the target fragment is loaded into the target carrier. If the validation results are correct, the plasmid is constructed successfully. The successful construction of the plasmid can be used in the study of sequence analysis and gene transfer.

Chemical synthesis of the whole gene is the most accurate and fast method, at the same time, we can design the gene sequence and improve the expression level according to the preference of codon in different host cells and different experimental requirements.

Keywords parent、Plasmid construction 、screen、sequence 目 录

摘要 1

1前言 6

1.1基因工程技术的含义 6

1.2基因工程的应用 7

1.3基因药物产业现状及地位 8

1.4基因药物的发展前景 8

2实验原理 10

2.1.热激法 10

2.2电转化法 10

2.3实验设计 11

3实验方法 12

3.1实验试剂 12

3.2实验材料 12

3.3实验仪器 12

3.4 PCR产物的扩增 13

3.4目的载体的处理 18

3.5连接方法 18

3.6 质粒DNA的转化 19

3.7 阳性克隆的筛选与培养 19

3.8 质粒的抽提与测序 20

3.9 双酶切验证 21

4结果与分析 22

4.1 阳性克隆筛选结果 22

4.2 阳性克隆的测序结果 23

4.3 质粒DNA的双酶切验证结果 24

参考文献 25

附录 26

致谢 27

第一章 前言

1.1基因工程技术的含义

基因工程技术诞生至今已经取得了辉煌的成就，成为当今生命科学研究领域中最有生命力和最引人注目的前沿学科之一，基因工程也是当今新的产业革命的一个重要组成部分。

重组DNA技术又称基因操作技术,基因工程技术 ,主要指基因的克隆、重组和表达；严格的说基因工程还包含体外DNA突变，体内基因操作以及基因的化学合成等。

DNA重组技术是基因工程的核心内容，但不等于就是基因工程，而基因工程又可以称为基因克隆和DNA分子克隆。

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