摘要
目的克隆Nucleophosmin(NPM)的编码序列,构建含Nucleophosmin基因的真核细胞表达载体并在肝癌细胞系HepG2中表达,为进一步研究该基因在肝癌多药耐药中的作用奠定基础。方法根据已发表的Nucleophosmin基因的核苷酸序列设计合成一对引物,以人乳腺癌组织抽提的RNA为模板进行反转录-聚合酶链反应(RT-PCR),扩增产物用BamHI和XhoI双酶酶切后定向克隆到真核细胞表达载体pEGFP-N1中,用限制性内切酶酶切重组质粒pEGFP/NPM和DNA序列测定进行鉴定。用脂质体法将pEGFP/NPM导入肝癌细胞系HepG2中,G418选择培养,经免疫组织化学法和RT-PCR鉴定其表达。结果 RT-PCR扩增出长894bp的特异性片段,经克隆至pEGFP-N1后酶切鉴定证实,并测序表明序列与GenBank报道完全一致。pEGFP/NPM在HepG2细胞中有稳定表达。结论成功克隆了NPM的编码序列,构建了其真核细胞表达载体pEGFP/NPM/1,有助于对NPM基因在肝癌多药耐药中的机制做进一步研究。
Objective To clone the coding sequence of Nucleophosmin（NPM） gene,establish NPM-containing eukaryotic cell expression vector and further express NPM in hepatocarcinoma cell strain HepG2,so as to provide a basis for exploring the roles of NPM gene in hepatocellular carcinoma multiple-drug resistance.Methods A pair of prime were designed and synthesized according to the published nucleotide sequence of Nucleophosmin gene,and reverse transcription polymerase chain reaction（RT-PCR） was performed based on the RNA module extracted from human breast carcinoma tissues.The product for amplification was cloned to the oriented eukaryocyte expression vector pEGFP-N1 after being cleaved by BamH1 and Xhol,and was identified according to both the recombinant plasmid pEGFP/NPM cleaved by restriction endonuclease and DNA sequencing.PEGFP/NPM was transfected into the hepatocarcinoma cell strain HepG2 using the lysosomal method,selected by G418 for culture,and was identified using immunohistochemistry and RT-PCR.Results An 894 bp specific DNA fragment,amplified by RT-PCR,was identified and verified after being cloned to pEGFG-N1 and further cleaved by endonuclease.Gene sequencing demonstrated sequence identical to as reported in GenBank.pEGFP/NPM was constantly expressed in HepG2 cells.Conclusion The coding sequence of human NPM gene was successfully cloned in this study.The establishment of eukaryocyte vector pEGFP/NPM/1 may be helpful to the understanding of mechanism by which it influences hepatocarcinoma multiple-drug resistance in further studies.
出处
《中国药物与临床》
CAS
2010年第8期868-870,共3页
Chinese Remedies & Clinics
基金
山西省卫生厅科技攻关项目(200910)
太原市大学生创业创新专项(09122043)
