摘要
目的构建羧基末端截短的乙型肝炎病毒表面抗原中蛋白(MHBst)反式激活基因的消减文库,并观察其对细胞原癌基因c-myc的反式激活作用。方法以重组表达质粒pcDNA3.1(-)-MHBst和空载体pcDNA3.1(-)瞬时转染HepG2细胞,提取细胞mRNA并逆转录为cDNA,分别标记为实验组与对照组,应用抑制性消减杂交技术,构建MHBst反式激活的cDNA消减文库。针对其中的原癌基因c-myc,进一步克隆其启动子序列并构建报告基因表达载体pCAT3-c-myc,与pcDNA3.1(-)-MHBst共转染HepG2细胞,ELISA法检测报告基因氯霉素乙酰转移酶CAT的表达活性。同时,应用反转录聚合酶链反应和免疫印迹方法检测表达MHBst的细胞中c-myc基因的mRNA转录和蛋白表达水平。结果随机挑选消减文库中的50个阳性克隆进行测序和生物信息学分析,显示19种已知功能基因,涉及细胞代谢、信号转导、细胞凋亡和肿瘤发生等生物学过程。细胞内瞬时表达的MHBst蛋白能够反式激活细胞原癌基因c-myc启动子的转录活性,并且RT-PCR和Western blotting结果也证明了表达MHBst蛋白的细胞中原癌基因c-myc的表达水平明显增强。结论成功构建MHBst反式激活基因的消减文库,MHBst可以反式激活细胞原癌基因c-myc的表达,为深入了解乙型肝炎病毒致癌的分子生物学机制提供理论基础。
Objective To construct a subtracted cDNA library transactivated by C-terminally truncated middle surface protein of hepatitis B virus （MHBst） , and to investigate the transactivating effect of MHBst on cell proto-oncogene c-myc. Methods The recombinant expression plasmid containing pcDNA3. 1 （-）-MHBst and pcDNA3. 1 （-） empty vector were transiently transfected into HepG2 cells, respectively. The mRNAs were extracted and the cDNAs were synthesized, which were named as tester and control group. Suppression subtractive hybridization （SSH） technique.was used to construct the subtracted cDNA library. The bioinformatics analysis showed some sequences might be involved in tumor development such as human proto-oncogene c-myc. To elucidate the relationship of the proto-oncogene c-myc and the MHBst167 protein, the rep＇orter plasmid pCAT3-c-myc containing the chloramphenicol acetyhransferase （CAT） gene under the control of c-myc promoter was generated and was co-transfected into HepG2 cells with pcDNA3.1 （-）-MHBst. The activity of CAT was detected by ELISA. Simultaneously, the expression of the proto-oncogene c-myc in HepG2 cells expressing MHBst167 protein was detected by reverse transcription-polymerase chain reaction （RT-PCR） and western blotting assays. Results Sequence analysis was performed in fifty positive clones randomly and searched for homologous DNA sequence in GenBank by bioinformatics method. Altogether 19 kinds of coding sequences with known functions were obtained , among which some gene coding proteins were involved in cell growth regulation, metabolism, signal transduction, cell apoptosis and tumor development. In addition, the transcriptional activity of proto-oncogene c-myc promoter was up-regulated by MHBst167 protein, and the up-regulated expression of proto-oncogene c-myc mRNA and protein were also verified. Conclusion The subtracted cDNA library transactivated by MHBst was successfully generated and MHBst protein could transcriptionally transactivate the expression of hu
出处
《胃肠病学和肝病学杂志》
CAS
2007年第1期28-32,共5页
Chinese Journal of Gastroenterology and Hepatology
