野生型p53/junB融合基因真核表达载体的构建

Construction of eukaryotic expression vector of wtp53/junB fusion gene

目的通过构建抑癌基因野生型p53(wtp53)与核转录因子junB的融合基因真核表达载体,为进一步研究联合基因疗法在肝癌中的应用奠定一定的基础。方法运用PCR(polymerase chain reaction)、RT-PCR(reverse transcription-PCR,RT-PCR)、基因重组技术构建携带增强型绿色荧光蛋白(enhanced green fluoresent protein,EGFP)报告基因的wtp53/junB融合基因真核表达载体pEGFP-C1-wtp53/junB。通过观察绿色荧光定位的方法验证wtp53/junB融合基因的真核表达载体转染人肝癌细胞株HepG2的情况。结果成功地将p53/junB融合基因克隆到pEGFP-C1质粒中,其序列与设计的完全一致;转染人肝癌细胞株HepG2后可观察到绿色荧光定位于细胞核中,证明wtp53/junB融合基因成功转染入肝癌细胞株中。结论成功构建了携带EGFP的pEGFP-C1-wtp53/junB融合基因真核表达载体,并转染入人肝癌细胞核中,为进一步研究两者在肝癌中的协同作用奠定了基础。

Objective To construct wtp53/junB fusion gene and its eukaryotic expression vector in order to provide the basis for further application of polygene union therapy in hepatocellular carcinoma. Methods Polymerase chain reaction （PCR）, reverse transcription-PCR （RT-PCR） and gene recombination techniques were used to construct the eukaryotic vector of pEGFP-Cl-wtp53/junB fusion gene, which carries the enhanced green fluorescent protein （EGFP）. The transfection of pEGFP-Cl-wtp53/junB in hepatoma HepG2 cells was detected by the location of green fluorescence. Results The DNA sequence of wtp53/junB fusion gene was successfully cloned into the pEGFP-Cl plasmid and the sequence was the same as what we expected. Green fluorescence located on cell nucleus proved that pEGFP-Cl-wtp53/junB was transfccted into HepG2 cell line successfully. Conclusion We successfully constructed the eukaryotic vector of pEGFP-Cl-wtp53/junB fusion gene, which carries the EGFP, and transfccts it into human hepatoma cell nucleus. It may lay the basis for studying the synergetic effect of wtp53 and junB in hepatocellular carcinoma.