新型二合一PiggyBac转座系统的构建及功能验证<sup>*</sup>

doi:10.13523/j.cb.2203005

中国生物工程杂志

2022, Vol. 42

Issue (6): 39-46 DOI: 10.13523/j.cb.2203005

技术与方法

新型二合一PiggyBac转座系统的构建及功能验证^*

张尧仓¹,彭玉¹,丁可昕²,台福敏²,郑晓飞²,秦宜德¹(

),付汉江^1,²(

)

1.安徽医科大学基础医学院合肥 230032
2.军事科学院军事医学研究院辐射医学研究所放射生物学北京市重点实验室北京 100850

Construction and Functional Verification of a Novel Two-in-one PiggyBac Transposon System

ZHANG Yao-cang¹,PENG Yu¹,DING Ke-xin²,TAI Fu-min²,ZHENG Xiao-fei²,QIN Yi-de¹(

),FU Han-jiang^1,²(

)

1. School of Basic Medicine, Anhui Medical University, Hefei 230032, China
2. Beijing Key Laboratory of Radiobiology, Institute of Radiation Medicine, Academy of Military Medical Science, Beijing 100850, China

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摘要：

目的:PiggyBac(PB)转座子是一种可移动的遗传元件,采用“剪切和粘贴”机制在载体和染色体之间进行转座;通过将转座子元件和转座酶表达框整合到一个表达载体中,构建简便易用的二合一PB转座系统。方法:通过聚合酶链式反应(polymerase chain reaction,PCR)获取PiggyBac转座系统所需转座子元件和转座酶表达框,利用T4 DNA连接酶将转座酶表达框插入到pUC18载体上,再利用Gibson同源重组技术将转座子元件与重组载体结合构建二合一PB转座系统;使用该系统携带的增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)以及功能性损伤抑制蛋白(damage-suppressing protein,DSUP)检测其有效性及可靠性。结果:在所有筛选获得的嘌呤霉素抗性细胞中,EGFP都是明亮可见;利用此二合一PB转座系统成功获得了可高效表达功能性损伤抑制蛋白的稳定细胞系,证明外源基因可被有效整合到基因组DNA中并表达。结论:成功构建了新型二合一PB转座系统,使稳定表达细胞系的建立更加经济简便。

关键词： PiggyBac转座子; 稳定转染; EGFP

Abstract:

Objective: The PiggyBac (PB) transposon is a mobile genetic element that transposes between vectors and chromosomes using a “cut and paste” mechanism. Transposon elements and transposase expression cassettes were integrated into an expression vector to construct an easy-to-use two-in-one PB transposon system. Methods: The transposon elements and transposase expression cassette required for the PiggyBac transposon system were obtained by polymerase chain reaction (PCR), and the transposase expression cassette was recombined with the original pUC18 vector using T4 DNA ligase. Gibson homologous recombination technology was used to combine transposon elements with recombination vector to construct a two-in-one PB transposon system. The system was tested for its efficacy and reliability using enhanced green fluorescent protein (EGFP) and functional damage suppressor protein (DSUP). Results: EGFP was visible and bright in all puromycin-resistant cells. In addition, a cell line stably expressing functional DSUP was obtained through the two-in-one PB transposon system, demonstrating that the foreign gene can be efficiently integrated into the genomic DNA and expressed. Conclusion: A new two-in-one PB transposition system was successfully constructed, which made the establishment of stable expression cell lines more convenient and economical.

Key words: PiggyBac transposon Stable transfection EGFP

收稿日期: 2022-03-03 出版日期: 2022-07-07

ZTFLH:

Q789

基金资助: *国家自然科学基金(81773038);国家自然科学基金(32071290)

通讯作者: 秦宜德,付汉江 E-mail: fuhj75@126.com;yideqin@ahmu.edu.cn

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引用本文:

张尧仓,彭玉,丁可昕,台福敏,郑晓飞,秦宜德,付汉江. 新型二合一PiggyBac转座系统的构建及功能验证^*[J]. 中国生物工程杂志, 2022, 42(6): 39-46.

ZHANG Yao-cang,PENG Yu,DING Ke-xin,TAI Fu-min,ZHENG Xiao-fei,QIN Yi-de,FU Han-jiang. Construction and Functional Verification of a Novel Two-in-one PiggyBac Transposon System. China Biotechnology, 2022, 42(6): 39-46.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2203005 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I6/39

表1 PCR引物序列

图1 二合一PiggyBac转座系统构建

图2 载体的菌落聚合酶链式反应(PCR)验证

图3 二合一PiggyBac转座系统的可用性验证

图4 基因组DNA上插入片段检测

图5 使用二合一PiggyBac转座子系统建立稳定表达功能性DSUP蛋白的HEK-293T细胞系

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