一种构建载体的新方法:一步式混合胶回收连接法

A novel method for vector construction: Gel extraction of mixed target DNA fragments before ligation

以构建原核表达载体重组质粒G4FPAGB1为例,介绍了一种新的载体构建方法。棉铃虫单核衣壳核多角体病毒G4毒株基因组上的Ha–FP–A片段用Hin dⅢ和Eco RⅤ双酶切,Ha–FP–B片段用XhoⅠ和Bam HⅠ双酶切,绿色荧光蛋白(Green fluorescent protein,GFP)基因片段用Eco RⅤ和XhoⅠ双酶切,载体质粒p Bluescript SK(+)用Hin dⅢ和Bam HⅠ双酶切。酶切产物经电泳检测后,将所有含目的片段的胶块切下,放在同一个离心管中进行DNA回收,得到的混合DNA产物直接加入连接酶进行连接、转化。结果表明,3个目的片段均成功连接到载体质粒上。本方法可高效、简单地构建包含多个目的片段的重组质粒。

A simple vector construction procedure, allowing rapidly and efficiently clone DNA fragments into cloning vector, was recommended in this study by taken target DNA fragments, Ha-FP-A and Ha-FP-B from Heliocoverpa armigera single nucleocapsid nucleopolyhedro virus （HaSNPV） of G4, green fluorescent protein gene （GFP） and an appropriate cloning vector of G4FPAGB1 as examples. Fragment of them were digested as follows： Ha-FP-A by Hin d Ⅲ and Eco RV, Ha-FP-B by Xho Ⅰ and Bam H Ⅰ, GFP by Eco RVand Xho Ⅰ, and the vector by Hin d Ⅲ and Bam H Ⅰ, respectively. All these fragments and vectors were determined with electrophoresis first, and then extracted from the gel at the same tube. Following the ligation system developed, the mixture of target DNA fragments was automatically ligated and transformed. The Results showed that the 3 target DNA fragments were all successfully ligated to the plasmid vector. Therefore, this method was simple, efficient and universal for the recombination of plasmid vector production.