摘要
目的:乙型肝炎病毒(HBV)X 蛋白(HBxAg)是一种具有反式激活作用的病毒蛋白质.为了探索 HBxAg 蛋白反式激活作用的新的靶基因,利用抑制性消减杂交(SSH)技术对于表达和不表达 HBxAg 蛋白的 HepG2细胞进行研究,以期发现HBxAg 蛋白反式激活作用的新靶点,为阐明 HBxAg 蛋白的反式激活作用分子生物学机制,以及 HBV 相关的肝细胞癌(HCC)形成的分子生物学机制开辟新的研究方向.方法:以 HBxAg 表达质粒 pcDNA3.1(-)-HBxAg 转染 HepG2细胞,以空载体 pcDNA3.1(-)为平行对照,提取 mRNA 并进行抑制性消减杂交(SSH)分析.对于所获基因片段序列分析表明,其中之一为新型基因片段,与 GenBank 中注册的已知功能基因序列没有同源性,利用表达序列标签(EST)序列的搜索和比对,进行电子拼接,根据基因起始密码子的Kozak 规则和终止密码子下游保守的多聚腺苷酸信号序列,克隆、鉴定新型基因.结果:在16种 HBxAg 蛋白上调的靶基因中,其中包括未知功能基因,利用生物信息学技术,获得 HBxAg 蛋白反式激活作用的新型靶基因,开放读码框架(ORF)长度为237个核苷酸(nt),编码产物由78个氨基酸残基(aa)组成,命名为XTP6,在 GenBank 中注册,注册号为 AF490255.结论:HBxAg 蛋白是一种具有反式激活作用的蛋白,应用抑制性消减杂交技术成功筛选与克隆 HBxAg 蛋白反式激活新型靶基因 XTP6,为进一步阐明 HBxAg 蛋白反式调节作用及其在 HBV 感染中的分子生物学机制提供理论依据和研究方法.
AIM:To screen and clone the target genes transactivated by HBxAg and to pave the way for elucidating the patho- genesis of hepatitis B virus(HBV)infection. METHODS:Suppression subtractive hybridization(SSH) technique and bioinformatics technique were used,and the mRNA from HepG2 cells transfected pcDNA3.1(-)-HBxAg and pcDNA3.1(-)empty vector was isolated,respectively, and SSH method was employed to analyze the differentially expressed DNA sequence between the two groups.The coding gene transactivated by HBxAg was cloned by bioinformatics and molecular biological methods. RESULTS:The obtained sequences were searched for ho- mologous DNA sequence from GenBank,one of which was a new gene with unknown function.The new gene with no homology with known genes in this database was con- firmed and electric polymerase chain reaction(PCR)was conducted for the cloning of the full-length DNA for the new gene and in conjunction with Kozak role and the exist of polyadenyl signal sequence.The reverse transcription PCR(RT-PCR)was used to amplify the new gene,named as XTP6,from the mRNA of HepG2 cells transfected.The sequence for the XTP6 gene has been deposited into GenBank.The accession number is AF490255. CONCLUSION:HBxAg is a potential transactivator.Success in the cloning and identification of human gene 6 transac- tivated by HBxAg by suppression subtractive hybridization provides theoretical basis and research methods for the molecular biological mechanism of the transactivation effect by HBxAg.
出处
《世界华人消化杂志》
CAS
2003年第12期1874-1877,共4页
World Chinese Journal of Digestology
基金
国家自然科学基金攻关项目
No.C03011402
No.C30070689
军队"九
五"科技攻关项目
No.98D063
军队回国留学人员启动基金项目
No.98H038
军队"十
五"科技攻关青年基金项目
No.01Q138
军队"十
五"科技攻关项目
No.01MB135
