三氧化二砷反式激活基因1的克隆化

Cloning of gene AsTP1 transactivated by arsenic trioxide

目的克隆三氧化二砷反式激活靶基因1(AsTP1)。方法从构建的三氧化二砷差异表达的肝HepG2细胞、T淋巴细胞cDNA消减文库筛选,利用RT-PCR和生物信息学分析相结合的方法获得新基因AsTP1的编码序列,对其氨基酸序列进行分析比较,并对其进行克隆化研究。结果AsTP1基因编码区为243个核苷酸(nt),编码产物为80个氨基酸残基(aa)。经核苷酸序列数据库(GenBank)和蛋白质一级结构序列数据库(SwissProt)同源序列的搜寻,与已知基因序列之间没有显著同源性,说明克隆的AsTP1基因属于未知功能新基因。结论发现三氧化二砷反式激活靶基因AsTP1,为进一步研究三氧化二砷的反式激活作用和揭示慢性砷中毒的分子机制提供新线索。

Objective 10 clone new gene-AsTH1 transactivated by arsenic trioxide. Methods Suppression subtractive hypriclization（SSH）technique was used. The mRNA was isolated from Jurkat cells treated with arsenic trioxide（5μmol/L） and 0.9 percent sodium chloride, respectively. Then cDNA was synthesized. SSH method was employed to analyze the differentially expressed DNA sequence between the two groups. On the base of subtractive cDNA library of genes transactivated by arsenic trioxide, the coding sequence of new gene was obtained by bioinformatics methods. The reverse transcription PCR （RT-PCR）was used to amplify the new gene from the mRNA of Jurkat cells and HepG2 cells; this new gene named as AsTP1. The sequece for the AsTP1 gene was deposited into GenBank, and the accession number was AY605064. Results The coding sequence of new gene was cloned and identified successfully. Conclusion A gene is recognized as the new target gene transactivated by arsenic trioxide. The results will pave the way for the study of the molecular mechanism of the transactivating effects of arsenic trioxide and the development of new clue for carcinogenic mechanism of chronic arsenisim.