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目的应用抑制性消减杂交(SSH)技术构建乙型肝炎病毒(HBV)全S蛋白反式激活基因差异表达的cDNA消减文库,克隆HBV全S蛋白反式激活相关基因。方法以HBV全S表达质粒pcDNA3.1(-)全S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,从中提取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。结果成功构建人HBV全S蛋白反式激活基因差异表达的cDNA消减文库。文库扩增后得到86个白色克隆,进行菌落PCR分析,均得到100-1000bp插入片段。挑取35个含有插入片段的阳性克隆测序分析,获得33个已知基因序列,和2个未知基因,通过生物信息学分析获得其全长序列,其中之一命名为全S蛋白反式激活基因1(CSTP1),已在GenBank中注册,注册号AY553877。未知基因的功能还正在研究中。结论应用SSH技术成功构建了HBV全S反式激活基因差异表达的cDNA消减文库。该文库的建立为进一步阐明HBV全S反式调节的靶基因及致肝病发生的分子生物学机制提供理论依据。
OBJECTIVE: To construct a cDNA subtractive library of differentially expressed transactivator genes of hepatitis B virus (HBV) and to clone the transactivation related genes of hepatitis B virus (HBV) S protein by suppression subtractive hybridization (SSH). Methods HepG2 cells were transfected with pcDNA3.1 (-) full-length HBV plasmid and pcDNA3.1 (-) vector was used as a control. Cell lysates were prepared and the mRNA was extracted and reverse transcribed into cDNA. After being digested with RsaI, the experimental group was divided into two groups. The cDNAs were ligated with two different linkers. Then the cDNAs of the control group were subtracted and subtracted twice and the two inhibitory PCRs were ligated with the T / A vector to construct the cDNA The library was subtracted and transfected into E. coli for library amplification. Randomly selected clones were amplified by PCR for sequencing and homology analysis. Results The cDNA subtractive library of differentially expressed transactivator of HBV full S protein was successfully constructed. 86 white clones were obtained after amplification of the library and colony PCR analysis was performed to obtain 100-1000 bp insert. Twenty-five positive clones containing the inserted fragment were sequenced to obtain 33 known gene sequences and 2 unknown genes. The full-length sequence was obtained by bioinformatics analysis, one of them was named the full S protein transactivation gene 1 (CSTP1), registered in GenBank with Accession Number AY553877. The function of unknown genes is still under study. Conclusion SSH subtractive cDNA library was successfully constructed. The establishment of this library will provide a theoretical basis for further elucidating the target gene of hepatitis B trans-regulation and the molecular mechanism of hepatopathy.