HBV感染培养人胎肝细胞后差异应答基因的初步分离和鉴定

Primary isolation and identification of differentially expressed genes of HBV-infected human fetal hepatocytes

目的：建立培养人胎肝细胞感染HBV后差异应答基因的消减文库，并从中克隆鉴定出新的应答基因．方法：分别从HBV感染及未感染的胎肝细胞中提取总RNA，用SMARTPCR技术合成全长双链cDNA，经RsaI酶切后将感染细胞的cDNA分为两组，分别衔接两个不同的接头，再与未感染细胞的cDNA进行两轮抑制性杂交及两轮抑制性PCR，将产物与T／A载体连接构建成消减cDNA文库，并转染大肠杆菌进行文库扩增，挑取克隆进行筛选排除假阳性，再行序列分析，鉴定胎肝细胞感染HBV后的差异应答基因．结果：成功构建高效消减的HBV感染胎肝细胞cDNA文库，得到158个白色的克隆，随机选择了128个克隆经PCR扩增获得含有明确单一目的片断的克隆99个，长约100～400bp，经筛选后得到70个克隆，测序表明有16个差异表达的基因，其中可能的新基因有5个．结论：用抑制性消减杂交技术成功建立了HBV感染培养人胎肝细胞的消减cDNA文库，为进一步大规模筛选HBV感染后的应答基因奠定了基础，初步筛选出的新基因片断为进一步研究宫内感染的分子机制提供了依据．

AIM： To construct a cDNA subtractive library of differential response genes of human fetal hepatocytes after HBV infection with suppression subtractive hybridization technique, and then to clone and identify the new genes that only express in HBV infected hepatocytes. METHODS： Total RNA was isolated from fetal hepatocytes with HBV infection and those without infection. Double-strand cDNAs were synthesized using SMART PCR. After Rsa I enzyme restriction, cDNA 〈 2 kb was obtained. HBV- infected hepatocyte cDNAs were then divided into 2 groups and ligated to the specific adaptor 1 and 2 respectively. After HBV- infected hepatocyte cDNAs were hybridized with cDNAs of hepatocytes without HBV infection twice and underwent nested PCR twice, they were then connected with arms of T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with IVF-α cells. After the library was screened with dot blots, the positive clones were sequenced and analyzed. RESULTS： HBV-infected hepatocyte subtractive library with high subtractive efficiency was set up successfully. The amplified library contained 158 white clones. Random analysis of 128 clones with PCR amplification showed that 99 plasmids in the clones contained 100 -400 bp inserts. After dot blot screening, 70 positive clones were obtained and the sequence analysis showed that there were 16 cDNA differentially expressed, among which 5 genes were novel genes probably. CONCLUSION： The high effective subtractive library has been successfully constructed with suppression subtractive hybridization technique, which lays a solid foundation for further screening and cloning new and specific response genes of HBV infection in fetal hepatocytes. The novel genes cloned prove to be an important clue for researching the mechanism of HBV intrauterine infection.