IGF-1基因克隆及其产物的测序鉴定  

Research on the Sequence Identification of the IGF-1 Gene Cloning and Its Products

作　　者：曹尚美 邹真真 陈泊霖 杨少哲 张清伟[1] 王单单 王浩然 付秀虹[1] CAO Shangmei;ZOU Zhenzhen;CHEN Bolin

机构地区：[1]漯河市中心医院科创中心/漯河医学高等专科学校第一附属医院/河南省生育力保护与优生重点实验室,河南漯河462000

出　　处：《科技创新与应用》2024年第16期7-11,共5页Technology Innovation and Application

基　　金：河南省自然科学基金(2223000420247);2023年河南省博士后科研资助项目(312145);中央引导地方项目(Z20221343023);河南省重点研发与推广专项(212102310813、222102310361);河南省医学科技攻关项目(LHGJ20221031);漯河医学高等专科学校科技创新项目(2023ZD18)。

摘　　要：基因过表达在基础实验和基因治疗应用中都是常用的手段,而质粒的扩增在基因过表达过程中是不可或缺的技术,如何规范、高效地对质粒进行扩增和提取是基因治疗应用的前提,也是困扰很多基础研究人员的难题。利用现有的实验技术,在降低实验成本基础上,建立过表达质粒扩增、提取的最优方案。首先采用传统方法制作LB培养基和感受态细胞,对转化后的质粒进行大量扩增培养,之后使用商业试剂盒对质粒进行提取,反复试验后对商业试剂盒质粒提取过程进行优化,提高效率,最终获得高纯度和浓度的质粒溶液。经超微量分光光度计、琼脂糖凝胶电泳检测和sanger测序等多种方式检测验证后与NCBI数据库中目标基因序列一致,提示扩增、提取成功。经优化后的质粒扩增、提取方案标准、高效,成本低廉,是质粒基因克隆的最佳选择。Gene overexpression is a common method in basic experiments and gene therapy applications,and plasmid amplification is an indispensable technique in the process of gene overexpression.How to amplify and extract plasmids standardized and efficiently is the premise of gene therapy application,and it is also a difficult problem for many basic researchers.To establish the optimal scheme of amplification and extraction of overexpression plasmid on the basis of reducing the experimental cost by using the existing experimental technology.Firstly,the LB medium and competent cells were made by traditional method,and the transformed plasmid was amplified and cultured,and then the plasmid was extracted by commercial kit.After repeated experiments,the plasmid extraction process of commercial kit was optimized,the efficiency was improved,and the plasmid solution with high purity and concentration was obtained.The sequence of the target gene was verified by ultramicro spectrophotometer,agarose gel electrophoresis and sanger sequencing,which was consistent with that in NCBI database,indicating that the amplification and extraction were successful.The optimized plasmid amplification and extraction scheme is standard,efficient and low cost,which is the best choice for plasmid gene cloning.

关 键 词：IGF-1基因 质粒克隆 基因过表达 质粒的扩增 基因治疗应用 

分 类 号：Q811.4[生物学—生物工程]