目的 应用抑制性消减杂交(SSH)技术及生物信息学技术筛选肝细胞内人类次要组织相容性抗原-HAS(HLA-HA8)的反式激活基因.方法 以HLA-HA8基因表达质粒pcDNA3.1(-)-HLA-HAB和空载体pcDNA3.1(-)转染HepG2细胞,提取mRNA并反转录合成双链cDNA(dscDNA),分别标记为Tester和Driver;经Rsa I酶切后,将Tester cDNA分成两组,分别与两种不同的接头adapter 1和adapter 2衔接,再与Driver cDNA进行两次消减杂交及两次抑制性聚合酶链反应,将产物与pGEM-T easy载体连接,构建cDNA消减文库,并转染大肠埃希菌进行文库扩增,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HLA-HA8反式激活基因差异表达cDNA消减文库.扩增后得到101个阳性克隆,菌落PCR分析均得到200～1000bp的插入片段.随机挑选其中28个阳性片段测序,同源分析结果显示,共获得16种编码基因,其中4个功能未知.结论 HLA-HA8基因反式调节基因与细胞结构和生长、物质及能量代谢、物质转运和信号转导密切相关,为HLA-HA8的功能以及HBV感染慢性化机制研究提供了新的思路.%Objective To clone and identify the target genes trans-activated by human minor histocompatibility antigen HLA-HA8 in hepatocytes with suppression subtractive hybridization (SSH) and bioinfomatics technique. Methods mRNA was isolated from HepG2 cells transfected by pcDNA3.1(-)-HLA-HA8 and pcDNA3.1(-) empty vector, and then used to synthesize the double-stranded cDNA (marked as Tester and Driver, respectively) by reverse transcription. After being digested with restriction enzyme Rsa I, the tester cDNA was divided into two parts and ligated to the specific adaptor 1 and adaptor 2 , respectively, and then hybridized with driver cDNA twice and underwent PCR twice. The production was subcloned into pEGM-Teasy plasmid vectors to set up the subtractive library. The library was then amplified bv transfection into E. coli strain DH5α. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR amplification. Results The subtractive library of genes trans-activated by HLA-HA8 was constructed successfully. The amplified library contained 101 positive clones. Colony PCR showed that all these clones contained 200-1000bp inserts. Twenty eight clones were selected randomly to analyze the sequences. The result of homologous analysis showed that altogether 16 coding sequences were gotten, of which 4 sequences were with unknown function. Conclusions The obtained sequences trans-activated by HLA-HA8 may code different proteins and play important roles in cell growth and metabolism, energy synthesis and metabolism, material transport and signal transduction. This finding will bring some new clues for the studies not only on the biological functions of HLA-HA8, but also on the HBV infection mechanism.
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