乙型肝炎病毒X蛋白反式激活基因XTP3的克隆化研究

Identification and cloning of human gene XTP3 transactivated by X protein of hepatitis B virus

目的 应用抑制性消减杂交 (SSH)技术筛选乙型肝炎病毒 (HBV)X蛋白 (HBxAg)反式激活基因 ,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PGEM Teasy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信号序列 ,初步确定新型基因序列。从转染了pcDNA3 .1(-) X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 发现的新基因命名为XTP3 ,在GenBank中注册 ,注册号为AF490 2 5 2。XTP3基因的编码序列全长为 10 2 0个核苷酸(nt) ,编码产物由 3 40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XTP3 。

Objective To construct a cDNA subtractive library of genes transactivated by hepatitis B virus X protein with suppression subtractive hybridization (SSH) technique and to clone genes associated with its transactivating function.Methods The mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-) X and pcDNA3.1(-)empty vector,respectively,then cDNA was synthesized. After restriction enzyme RsaI digestion,small sizes 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 PGEM-Teasy plasmid vectors to set up the subtractive library.Amplification of the library was carried out with E.coli strain JM109.The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.One colony with no homology with identified genes was primarily confirmed by use of Kozak regulation and the presentation of polyadeny1 signal sequence.The new gene was amplified by the reverse transcription PCR(RT PCR) and confirmed with sequencing.Results The new gene named XTP3 was deposited in GenBank,the registered number is AF490252 which consists of 1020 nucleotides and codes for 340 amino acids.Conclusion Success in the cloning and identification of XTP3 transactivated by hepatitis B virus X Protein by suppression subtractive hybridization provides theoretical basis and research method for the molecular biological mechanism of the transactivation effect by HBxAg.