一种通用型piggyBac转座子诱导细胞永生化载体的构建及其基本功能验证

Construction of a general piggyBac transposon inducible cell immortalization vector and verification of its basic properties

摘要:

为了构建一种高效、通用的细胞永生化载体pTP-hTERT，通过人工合成、PCR、酶切连接等方法，对传统piggyBac (PB) 转座子系统进行改造。改造后的载体含有转座必需元件、PB转座酶 (PBase) 表达框、共表达筛选元件和人端粒逆转录酶 (hTERT) 基因表达框。其中筛选元件中绿色荧光蛋白 (EGFP)基因和嘌呤霉素抗性 (Puror) 基因以猪捷申病毒2A自我剪切肽相连，以实现共表达。为验证载体元件功能，使用该载体转染HEK 293细胞，并对筛选出的阳性细胞进行RT-PCR、Western blotting (WB)、热不对称PCR (Tail-PCR) 和细胞克隆亚甲蓝染色与统计分析。载体测序鉴定与细胞培养结果表明，通用型永生化载体 pTP-hTERT构建成功，转染HEK293细胞后能筛选出抗嘌呤霉素细胞单克隆；WB结果显示P2A可高效切割EGFP和Puror融合蛋白，证明筛选标记基因功能正常；Tail-PCR结果表明该载体以转座整合插入宿主基因组；亚甲蓝染色统计结果显示由pTP-hTERT引发的细胞阳性克隆数与对照组相比显著提高 (P<0.01)。PB转座子永生化载体pTP-hTERT的构建为永生化细胞系的建立提供了工具，同时也为其他真核载体的构建和改造提供了参考。

Abstract:

In order to construct generally efficient cell immortalization vector, pTP-hTERT, we modified the traditional piggyBac (PB) transposon using artificial synthesis, PCR and enzyme digestion. The modified vector contained the necessary transposon elements, a PB transposase expression cassette, a co-expression selectable element and a human telomerase reverse transcriptase (hTERT) expression cassette. The co-expression selectable element had two markers, enhanced green fluorescent protein (EGFP) gene and puromycin-resistance (Puror) gene, linked by porcine teschovirus-1 2A peptide (P2A). To validate the functionality of vector elements, we transfected pTP-hTERT into HEK293 cell, selected the positive cell clones and then conducted RT-PCR, Western blotting (WB) and Tail-PCR, methylene blue staining and statistic analysis on selected cells. The results of sequencing and cell culture show that the pTP-hTERT was constructed successfully and the positive cell could be selected by puromycin. The WB results, P2A cutting EGFP and Puror fusion protein with high efficiency, reflected the selectable element worked. The sequencing result of Tail-PCR confirmed the vector integrated into the genome through transposition. The results of methylene blue staining and statistic analysis indicated the clone of positive cells triggered by pTP-hTERT significantly increased (P<0.01) compared with control group. The construction of pTP-hTERT provides an efficient tool for establishing immortalized cell lines and a demonstration for building other eukaryotic plasmids.