摘要
目的:构建调控β-半乳糖苷酶(β-gal)基因表达的四环素基因表达调控载体,验证其在肝癌细胞内调控β-半乳糖苷酶基因的表达,为进一步利用该调控系统调控治疗基因治疗肝癌奠定实验基础。方法:根据2个四环素调控子结合位点(TetO2)基因与pcDNA3.1载体的基因核苷酸序列,设计引物,以pcDNA3.1载体为模板进行PCR。将具有串联2个四环素调控子结合位点(TetO2)基因的PCR产物连接入pcDNA3.1载体CMV启动子下游,构建成pcDNA3.1-TetO2载体,应用ABI3130测序仪利用pcDNA3.1-TetO2载体对TetO2PCR产物进行测序分析。再将β-gal克隆入pcDNA3.1-TetO2载体,构建成受四环素调控表达的β-半乳糖苷酶基因的表达载体pcDNA3.1-TetO2-β-gal。利用脂质体将携带四环素调控子基因(TR)的pcDNA6/TR载体转染到肝癌细胞HepG2中,利用杀稻瘟菌素进行细胞转染的稳定筛选,采用逆转录聚合酶链反应(RT-PCR)法检测TR基因在HepG2细胞内的表达。将pcDNA3.1-TetO2-β-gal载体转染到稳定表达pcDNA6/TR的HepG2细胞中,转染3～4d后,给予强力霉素(4mg.L-1),利用β半乳糖苷酶原位染色试剂盒染色,检测β-半乳糖苷酶基因的表达。结果:构建的pcDNA3.1-TetO2载体测序结果表明,串联2个四环素调控子结合位点(TetO2)基因片段插入到pcDNA3.1载体CMV启动子基因下游。pcDNA3.1-TetO2-β-gal载体酶切鉴定结果表明,β-gal成功地克隆入pcDNA3.1-TetO2载体。RT-PCR结果显示,TR基因在pcDNA6/TR转染的经杀稻瘟菌素筛选的HepG2细胞内得到稳定表达。给予强力霉素后,β-半乳糖苷酶基因在稳定转染TR基因的HepG2细胞内得以表达;而未给予强力霉素,β-半乳糖苷酶基因在稳定转染TR基因的HepG2细胞内表达受到抑制。结论:成功地构建了调控β-半乳糖苷酶基因表达的四环素基因表达调控载体,利用该载体成功地调控了β-半乳糖苷酶基因在HepG2细胞内的表达。
Objective To construct the vector for β-galactosidase（β-gal） gene expression regulation with tetracycline-regulated gene expression system, and to control β-gatactosidase gene expression in hepG2 cells with the vector. So that offer a experimental base for regulating gene expression for liver cancer gene therpy with this system. Methods The primers for explanding two tetracycline operator （TetO2） gene according to the gene nueleotide sequences of TetO2 gene and pcDNA3.1 vector were designed. TetO2 gene was amplified by PCR with pcDNA3.1 plasmid as template. The TetO2 PCR products were cloned into pcDNA3. 1 and the vector was named as peDNA3.1-TetO2. The pcDNA3.1-Teto2 with TetO2 PCR products sequence was analyzed by ABI3130 sequencing analysis. Then β-gal gene was cloned into pcDNA3. 1-TetO2, the vector was named as pcDNA3.1-TetO2-β-gal. The pcDNA6/TR plasmid vector with tetracycline repressor （TR） was transfected into HepG2 cells by Lipotap, and the stable transfection cells were screened by hlasticidin. TR gene expression was detected by RT-PCR in HepG-2 cells with and without pcDNA6/TR plasmid transfection. The pcDNA3.1-TetO2-β-gal plasmid vector was transfected into HepG2 cells with and without pcDNA6/TR plasmid transfection, and 3 to 4 d later, these transfected gene cells were treated with doxycline （4 mg·L^-1）. After 48 h, β-gal gene expression was detected with β-gal cell staining. Results The pcDNA3.1-TetO2 sequencing analysis results showed that a cassette was made for a cytomegalovirus-type 2 tetracycline operator （TetO2） -TetO2 promoter in pcDNA3.1-Teto2. The double digestion reslut of pcDNA3.1-TetO2-β-gal plasmid vector demonstrated that β-gal gene was successfully cloned into pcDNA3.1-TetO2 vector. The RT-PCR result of TR gene showed TR gene could be expressed in HepG2 cells with pcDNA6/TR plasmid transfection and TR gene didn＇ t express in HepG2 cells without pcDNA6/TR plasmid transfection. β-gal gene expressed in HepG2 cells without pcDNA6/TR plasmid transfection,
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2009年第2期304-308,共5页
Journal of Jilin University：Medicine Edition
基金
吉林省发展与改革委员会高科技项目资助课题(2004987)
吉林省科技厅科技发展计划项目资助课题(20050405-7)
吉林省科技厅应用基础项目资助课题(200705335)
