HBV X蛋白反式调节基因XTP11的克隆化研究

Cloning of Gene Transactivated by Hepatitis B Virus XTP11

目的:应用寡核苷酸基因芯片技术及生物信息学分析,筛选并克隆乙肝病毒X蛋白(HBxAg)反式激活新型靶基因。方法:以HBxAg表达质粒pcDNA 3 1(-) X转染HepG2细胞,以空载体pcDNA 3. 1(-)为平行对照,提取mRNA并进行寡核苷酸基因芯片分析。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。通过序列同源性搜索和比对和电子拼接,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pcDNA 3 . 1(-) X的HepG2细胞提取总RNA ,以逆转录聚合酶链反应(RT- PCR)技术,扩增获得该新基因的全长序列,并测序证实。结果:该新基因命名为XTP11,在GenBank中注册,注册号为AY740 5 2 0。该基因的编码序列全长为13 44个核苷酸(nt) ,编码产物由44 8个氨基酸残基(aa)组成。结论:HBxAg反式激活新型靶基因XTP11的筛选与克隆,为进一步研究HBxAg反式激活作用的分子生物学机制奠定基础。

Objective:To clone and identify genes transactivated by hepatitis B virus X protein(HBxAg) using oligonucleotide DNA chip technology and bioinformatics analysis.Methods:The mRNA was isolated from HepG2 cells transfected with pcDNA 3.1(-)-X and pcDNA 3.1(-)empty vector,respectively,and oligonucleotide DNA chip technology was employed to analyze the differentially expressed DNA sequence between the two groups. The obtained sequences were searched for homologous DNA sequence from GenBank,one of which was a new gene with unknown frunction. 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 for the new gene and in conjunction with Kozak role and exist of polyadenyl signal sequence. The new gene was amplified by the reverse transcription PCR (RT-PCR) and confirmed with sequencing assay.Results:The new gene was named as XTP11,which consists of 1 344 nucleotides(nt) and encodes 448 amino acid (aa). The sequence for the XTP11 gene was enrolled in GenBank,the accession number is AY740520. Conclusion: Cloning and identification of XTP11 transactivated by HBxAg using oligonucleotide DNA chip technology provides theoretical basis and research method for the molecular biological mechanism of the transactivation effect by HBxAg.