乙型肝炎病毒E抗原肝细胞作用蛋白AK026018表达谱芯片的研究

Screening and identification of genes trans-regulated by a novel HBeAg interacting protein AK026018 with microarray assay

目的应用基因芯片技术,筛选能被乙型肝炎病毒E抗原(HBeAg)肝细胞作用蛋白AK026018反式调节的靶基因,初步研究该蛋白的生物学功能。方法应用反转录聚合酶链反应(RT-PCR)技术,从HepG2细胞中扩增编码AK026018蛋白的全基因,构建真核表达载体,转染肝母细胞瘤系HepG2,提取总mRNA,逆转录为cDNA,与转染空白表达载体pcDNA3·1的HepG2细胞进行DNA芯片分析并比较。结果经限制性内切酶分析和DNA序列测定鉴定构建的重组表达载体正确。在8464个基因表达谱的筛选中,发现有122个基因有差异表达,其中78种基因表达水平显著下调,45种基因表达水平显著上调。结论成功地应用DNA芯片技术筛选出HBeAg结合蛋白新基因AK026018的反式调节蛋白,证明该基因对于肝细胞基因表达谱有显著影响。

Objective To investigate the biological functions of a novel hepatitis B virus e antigen （HBeAg） interacting protein AK026018, and to use cDNA microarray technique to screen genes regulated by the protein. Methods The AK026018 coding DNA fragment was amplified by reverse transcription polymerase chain reaction （RT-PCR） technique from HepG2 cell. The expressive vector of pcDNA3. 1-AK was constructed by routine molecular biological methods. The HepG2 cells were transfected with pcDNA3.1 and pcDNA3.1-AK, respectively by using lipofectamine. The total RNA was isolated and reverse transcribed. The cDNA of each sample was subjected to microarray screening with 8 464 cDNA probes and analyzed by bioinformatics. Results The expressive vector was constructed and confirmed by DNA sequencing analysis and restriction enzyme digestion. High quality mRNA and eDNA of transfected HepG2 cells had been prepared and successful microarray screening conducted. From the scanning results, there were 122 differential expression genes, of which 36 genes were downregulated, and 16 genes were up-regulated. Conclusion Microarry technique was successfully used to screen the genes trans-regulated by AK026018. The expression of AK026018 protein affects the expression spectrum of HepG2 cell.