摘要
目的 筛选并克隆乙型肝炎病毒X蛋白 (HBxAg)反式激活新型靶基因。方法 以HBxAg表达质粒 pcDNA3.1( ) X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分析 ,应用生物信息学方法对所获基因片段序列进行分析发现 ,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。通过序列同源性搜索、比对和电子拼接 ,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 ,确定新型基因序列。从转染了pcDNA3.1( ) X的HepG2细胞提取总RNA ,以逆转录聚合酶链反应 (RT PCR)技术扩增 ,获得阳性克隆之后 ,进行鉴定并对克隆的基因及其编码产物的序列进行分析。结果 该新基因的编码序列全长为 348个核苷酸 (nt) ,编码产物由 116个氨基酸残基 (aa)组成 ,并测序证实 ,命名为XTP4 ,在GenBank中注册 ,注册号为AF4 90 2 5 3。结论 通过分子生物学技术与生物信息学技术的结合 ,发现并鉴定、克隆HBxAg反式激活作用的新型靶基因XTP4 。
Objective To screen and clone the target genes transactivated by hepatitis B virus X protein (HBxAg). Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-X and pcDNA3.1(-) empty vector, respectively. Suppression subtractive hybridization (SSH) method was employed to analyze the differentially expressed DNA sequence between the two groups. The obtained sequences were searched for homologous DNA sequence from GenBank. The new gene with no homology with known genes in this database was confirmed and electric polymerase chain reaction was conducted for the cloning of the full-length DNA of the new gene in conjunction with Kozak role and termination of polyadenyl signal sequence. The reverse transcription PCR (RT-PCR) was used to amplify the new gene from mRNA of HepG2 cell as the template. The coding sequence of the new gene was deduced according to the nucleotide sequence. Results A new gene with unknown function was named as XTP4. The nucleotide sequence of the XTP4 gene and the corresponding protein-coding amino acid had been determined, which contained 348nt and 116aa. The sequence of the XTP4 gene was deposited into GenBank, with the accession number as AF490253. Conclusions XTP4 gene transactivated by hepatitis B virus X protein was cloned and identified successfully by combining molecular biological technology and bioinformational technology. These results would pave the way for the study of the molecular mechanism of the transactivating effects of HBxAg and the development of new therapy for chronic hepatitis B.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2004年第5期380-382,共3页
Medical Journal of Chinese People's Liberation Army
基金
军队回国留学人员启动基金 (编号 98H0 38)
国家自然科学基金攻关项目 (编号C0 30 1 1 4 0 2 0
C30 0 70 689)
军队"九五"科技攻关项目 (编号98D0 63)
军队"十五"科技攻关青年基金项目 (编号 0 1Q1 38)资助课题
