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目的进行乙型肝炎病毒感染后表型不一致的单卵孪生子基因组CpG岛甲基化谱差异分析,以发现可能的差异甲基化基因。方法采用改良甲基化间区位点扩增技术,根据人基因组CpG岛甲基化位点的两种主要碱基组合,CGCG和CCGG,用3种不同的酶切组合(SmaⅠXmaⅠ、HpaⅡMspⅠ、BssHⅡPauⅠ/BsePⅠ),同时采用PersonalMolecularImagerFX分子成像系统及放射自显影相结合的方法,经QuantityOne4.4.0凝胶图像分析软件进行差异条带分析,分离回收差异甲基化条带并克隆入T载体,阳性克隆进行测序,将测序结果进行BLAST分析,初步了解与乙型肝炎病毒感染后表型不一致的单卵孪生子相关的差异甲基化基因情况。结果在一对表型一致的单卵孪生子间基因组CpG岛甲基化分析显示条带几乎相同,而在另一对表型一致以及一对表型不一致的单卵孪生子之间均存在差异甲基化条带,前者差异甲基化条带数目少于后者,将得到的差异甲基化条带克隆入T载体,目前测序分析得到4个可能与乙型肝炎病毒感染后表型不一致的单卵孪生子相关的差异甲基化基因。结论乙型肝炎病毒感染后表型不一致的单卵孪生子间基因组CpG岛甲基化水平有差异,其对疾病表型的影响及机制尚需后续深入研究。
OBJECTIVE: To analyze the difference of CpG island methylation profiles of single-egg twin genomics with inconsistent phenotypes after hepatitis B virus infection in order to find possible differentially methylated genes. Methods According to the methylation sites of CpG island in human genome, CGCG and CCGG, three different digestion combinations (SmaⅠXmaⅠ, HpaⅡMspⅠ, BssHⅡPauⅠ / BsePⅠ). At the same time, using the combination of PersonalMolecularImagerFX molecular imaging system and autoradiography, differentialOne.4.4.0 gel image analysis software was used to analyze differential bands. Differential methylation bands were recovered and cloned into T vector. The positive clones Sequencing and BLAST analysis of the sequencing results to find out the differential methylation genes associated with unicellular monozygotic twins after hepatitis B virus infection. Results Genome-wide CpG island methylation analysis of a pair of phenotypically identical monozygotic twins showed that the bands were nearly the same, while there was a difference between the other pairs of uniformed monozygotic twins with identical phenotypes Methylated bands. The number of differentially methylated bands in the former is less than the latter, and the differentially methylated bands obtained are cloned into T vector. The current 4 sequencing results may be inconsistent with the phenotype after hepatitis B virus infection Differentially methylated genes associated with monozygotic twin. Conclusion There is a difference in methylation level of genomic CpG island between single and oviparous twin with different phenotypes after hepatitis B virus infection. The effect on the phenotype of the disease and its mechanism still need further study.