乙型肝炎病毒X蛋白反式激活基因XTP8的克隆

Identification and cloning of human gene XTP8 transactivated by X protein of hepatitis B virus

目的:应用抑制性消减杂交技术筛选乙型肝炎病毒 X 蛋白(HBxAg)反式激活基因差异表达的 cDNA,克隆 HBxAg 反式激活相关靶基因.方法:以 HBxAg 表达质粒 pcDNA3.1(-)-X 转染 HepG2细胞,以空载体 pcDNA3.1(-)为对照;制备转染后的细胞裂解液,提取 mRNA 并逆转录为 cDNA,经 RsaI 酶切后,将实验组 cDNA 分成两组,分别与两种不同的接头衔接,再与对照组 cDNA 进行两次消减杂交及两次抑制性 PCR,将产物与 T/A 载体连接,构建 cDNA 消减文库,并转染大肠杆菌进行文库扩增,随机挑选克隆 PCR 扩增后进行测序及同源性分析,发现其中之一为未知基因片段,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,电子拼接成功,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,初步确定新型基因序列.从转染了 pcDNA3.1(-)的 HepG2细胞提取总 RNA,以 RT-PCR 技术扩增获得该新基因的全长序列,并测序加以证实.结果:发现新基因,命名为 XTP8,在 GenBank 中注册,注册号为 AF490257.XTP8基因的编码序列全长为1590个核苷酸(nt),编码产物由530个氨基酸残基(aa)组成.结论:应用抑制性消减杂交技术成功筛选与克隆 HBxAg 反式激活新型靶基因 XTP8,为进一步阐明 HBxAg 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 construct a cDNA subtractive library of genes transactivated by hepatitis B virus X protein(HBxAg)with suppression subtractive hybridization(SSH)technique and to clone genes associated with its transactivating function. METHODS:The mRNA was isolated from HepG2 cells trans- fected pcDNA3.1(-)-X and pcDNA3.1(-)empty vector, respectively,then cDNA was synthesized.After restriction enzyme RsaI digestion,small sizes of cDNA were obtained. Then tester cDNA was subdivided into two portions and each ligated with different cDNA adaptor.After tester cDNA was hybridized with driver cDNA twice and undergone nested PCR twice and then was subcloned into T/A plasmid vectors to set up the subtractive library.Amplification of the library was carried out with E.coil strain JM109.The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.One colony with no homology with iden- tified genes was primarily confirmed by use of Kozak regu- lation and the presence 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 named XTP8 was enrolled in GenBank,the accession number is AF490256,which consists of 588 nucleotides(nt)and codes 196 amino acids. CONCLUSION:Successful cloning and identification of XTP8 transactivated by hepatitis B virus X protein by suppression subtractive hybridization provides theoretical basis and research methods for the molecular biological mechanism of the transactivation effect of HBxAg.