绿色荧光蛋白-CCNG2表达载体的构建及鉴定

摘要

目的 为进一步探讨细胞周期蛋白G2(CCNG2)基因功能奠定基础.方法 以成人脑cDNA文库为模板,PCR扩增人CCNG2基因,In-Fusion技术定向克隆到pENTR1A中,酶切测序鉴定后重组到pcDNA6.2TM/EmGFP-DEST Gateway载体,构建pcDNA6.2-EmGFP-G2.将鉴定正确的质粒瞬时转染人舌鳞癌细胞Tca-8113,24 h后观察,定量PCR检测CCNG2 mRNA表达.结果 扩增得到1 032 bp的基因片段,经测序验证与GenBank中人CCNG2序列相符;pcDNA6.2-EmGFP-G2经酶切和测序鉴定无误.转染Tca-8113后,荧光信号除在胞质中表达外,在部分细胞的胞核DAPI染色弱信号区浓聚.转染该重组质粒后CCNG2 mRNA表达水平显著增高.结论 成功构建了以绿色荧光蛋白作为报告基因的CCNG2表达载体,为进一步研究CCNG2基因功能奠定了基础.%Objective To make a useful tool for studying the function of CCNG2 gene further. Methods Full length open reading frame of CCNG2 gene was amplified from marthon human adult brain cDNA library with polymerase chain reaction method, then cloned into pENTR1A vector through in-fusion technique and confirmed by restriction endonuclease and DNA sequencing,and was recombined with the pcDNA6.2TM/EmGFP vector after Gateway technique to construct the fusion expression plasmid pcDNA6. 2-EmGFP-G2. The plasmid was transfected into Tca-8113 and observed under fluorescence microscope. The mRNA expression of CCNG2 gene was detected by Real-time PCR. Results The amplified gene fragment was 1 032 bp and confirmed by sequencing, which was consistent with the human CCNG2 sequence in GenBank.Restriction enzyme digestion and sequencing showed that pcDNA6.2-EmGFP-G2 was constructed correctly. After transfection into Tca-8113 cell, the fluorescent signal was expressed in cytoplasma and the special area of nuclear with weak signal of DAPI. The expression level of CCNG2 mRNA increased after pcDNA6.2-E mGFP-G2 transection. Conclusions CCNG2 expression vector using green fluorescent protein as reporter gene can be constructed successfully, which provides a powerful tool for the further study of CCNG2 gene function.