摘要
目的应用抑制性消减杂交(SSH)技术筛选乙型肝炎病毒(HBV)核糖核酸酶RNaseH蛋白反式激活基因差异表达的cDNA,克隆RNaseH反式激活相关靶基因。方法以RNaseH表达质粒pcDNA31(-)RNaseH转染HepG2细胞,以空载体pcDNA31(-)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,其未知基因片段通过Kozak规则和多聚腺苷酸信号序列,初步确定。以逆转录聚合酶链反应(RTPCR)技术扩增获得该新基因的全长序列,并测序加以证实。结果该新基因被命名为DNAPTP4,在GenBank中注册,注册号为AY450392。DNAPTP4基因的编码序列全长为828个核苷酸(nt),编码产物由276个氨基酸残基(aa)组成。结论应用SSH技术成功筛选与克隆RNaseH反式激活新型靶基因DNAPTP4,为进一步阐明RNaseH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。
Objective:To construct a cDNA subtractive library of genes transactivated by hepatitis B virus(HBV) RNase H 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 pcDNA3.1(-)-RNase H 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 was 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.coli strain JM109.The cDNA was sequenced and analyzed in GenBank with Blast search after PCR.One clony with no homology with identified genes was primarily confirmed by use of Kozak regulation and the presentation of polyadenyl signal sequence.The new gene was amplified by the reverse transcription PCR(RT-PCR) technique and confirmed with sequencing assay.Results:The new gene was named as DNAPTP4 and enrolled in GenBank,the registered number is AY450392 which consists of 828 nucleotides and codes 276 amino acids.Conclusion:Success in the cloning and identification of DNAPTP4 transactivated by hepatitis B virus RNase H protein by suppression subtractive hybridization provides theoretical basis and research method for the molecular biological mechanism of the transactivation effect by RNase H protein.
出处
《中西医结合肝病杂志》
CAS
2004年第6期339-342,共4页
Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases
基金
国家自然科学基金资助(NoC39970674
NoC3011402
NoC39900130
NoC30070689)
