Quantification of Methylation at CMV Promoter of pEGFP-C3 Vector by Gradient Touch-Down PCR Sequencing

目的改进亚硫酸氢钠测序法,并在CMV基因启动子甲基化检测中进行验证。方法提取PEgfP-C3质粒重组人肝癌细胞株HEPg2 dnA,亚硫酸氢钠化学修饰,针对修饰后质粒基因CMV启动子序列设计特异引物并结合梯度降落PCr扩增,T-A载体克隆、测序,目标区域甲基化定量。结果 PEgfP-C3质粒基因约600bP的CMV启动子区甲基化水平可以精确定量,检测结果与标准品一致,重复测量结果稳定。结论改进后亚硫酸氢钠测序法能明显减少非特异性扩增,提高PCr效率,更适于基因甲基化状态的检测。Objective To improve the sodium bisulfite sequencing method and validate it in quantification of methylation in CMV promoter.Methods DNA was extracted from recombinant HepG2 hepatoma cell line with plasmid pEGFP-C3,and chemically modified with sodium bisulfite,the gene-specific primers were designed according to the modified CMV promoter sequence and conducted PCR amplification with gradient touch-down PCR,then the DNA methylation level in the target areas was quantitated after T-A cloning and sequencing.Results The DNA methylation level of the 600 bp CMV promoter within pEGFPC3 plasmid could be quantified accurately,and be consistent with that of the methylated DNA standards,repeated measurements indicated stable quantification result with this method.Conclusion The improved sodium bisulfite sequencing method can reduce non-specific amplification significantly and improve PCR efficiency,therefore has more potential for quantitative detection of gene methylation status.国家自然科学基金资助项目(20907047);中央级公益性科研院所基本科研业务专项(2008KYYW05

Raney镍催化松相加氢反映的机理

以200号油为溶剂，Raney镍作催化剂研究了松香的加氢反应机理。由均匀设计实验方法得到松香加氢最优反应条件是：温度443 K，压力5．0 MPa，催化剂用量占松香质量分数的5．0％，溶剂质量分数为 50％，反应时间100min，搅拌转速600r／rain，据此确定了动力学的实验条件。在线跟踪测定了反应物浓度随反应时间变化的关系，使用EVIEWS软件进行参数拟合，经对Hougen-Warson的17种反应机理模型进行筛选，认为最可几的反应机理为：催化剂表面上被吸附的氢原于和液相中的枞酸型树脂酸分子进行反应，氢的解离吸附为控制步骤，其反应速度与氢压成正比和产物浓度的平方成反比。并比较了松香催化加氢的熔融法和溶剂法，结果表明溶剂法加氢反应的速度大于熔融法

Raney镍催化松香加氢反应的机理

以 2 0 0号油为溶剂 ,Raney镍作催化剂研究了松香的加氢反应机理。由均匀设计实验方法得到松香加氢最优反应条件是 :温度 4 4 3K ,压力 5 0MPa ,催化剂用量占松香质量分数的 5 0 % ,溶剂质量分数为5 0 % ,反应时间 10 0min ,搅拌转速 6 0 0r/min ,据此确定了动力学的实验条件。在线跟踪测定了反应物浓度随反应时间变化的关系 ,使用EVIEWS软件进行参数拟合 ,经对Hougen Warson的 17种反应机理模型进行筛选 ,认为最可几的反应机理为 :催化剂表面上被吸附的氢原子和液相中的枞酸型树脂酸分子进行反应 ,氢的解离吸附为控制步骤 ,其反应速度与氢压成正比和产物浓度的平方成反比。并比较了松香催化加氢的熔融法和溶剂法 ,结果表明溶剂法加氢反应的速度大于熔融

raney镍催化松相加氢反映的机理

以200号油为溶剂，Raney镍作催化剂研究了松香的加氢反应机理。由均匀设计实验方法得到松香加氢最优反应条件是：温度443 K，压力5．0 MPa，催化剂用量占松香质量分数的5．0％，溶剂质量分数为 50％，反应时间100min，搅拌转速600r／rain，据此确定了动力学的实验条件。在线跟踪测定了反应物浓度随反应时间变化的关系，使用EVIEWS软件进行参数拟合，经对Hougen-Warson的17种反应机理模型进行筛选，认为最可几的反应机理为：催化剂表面上被吸附的氢原于和液相中的枞酸型树脂酸分子进行反应，氢的解离吸附为控制步骤，其反应速度与氢压成正比和产物浓度的平方成反比。并比较了松香催化加氢的熔融法和溶剂法，结果表明溶剂法加氢反应的速度大于熔融法

A novel evaluation method for epigenetic demethylation toxicity of contaminated aquatics based on EGFP reporter

PEgfP-C3质粒经过体外人工甲基化处理后,被转染进入HEPg2细胞以构建重组细胞株.以5-AzA为阳性去甲基化毒物与重组细胞共培养,通过亚硫酸氢钠测序法定量检测EgfP基因启动子区甲基化状态,通过实时定量PCr检测EgfP基因表达,借助流式细胞术和荧光摄片定量检测共培养细胞的绿色荧光强度,在dnA甲基化、EgfP基因MrnA表达、gfP蛋白等多个层次研究5-AzA染毒处理与其去甲基化能力和荧光表达改变的响应关系.对天津污染水产的去甲基化能力进行了实际样品测试.结果表明,5-AzA与重组细胞的dnA甲基化、基因表达、蛋白产物变化之间存在显著关联,具有较低的检出浓度和良好的重复性.天津污染海域的水产去甲基化能力较强.本文初步建立了一种污染物去甲基化表观遗传毒性评价方法.To study a novel evaluation method for demethylation epigenetic toxicity of pollutants based on an artificial recombinant pEGFP-C3 plasmid, pEGFP-C3 plasmid was methylated with M.SssⅠ in vitro first and then transfected into HepG2 cells.Taking 5-AZA as positive demethylation agent, the levels of methylation of the EGFP CMV promoter region, EGFP gene expression and green fluorescence intensity of the recombinant cell lines was quantified with sodium bisulfite sequencing assay, quantitative real-time quantitative PCR and flow cytometry at the time of 24 h after the cells co-cultured with 5-AZA gradients, respectively.A dose-respond relationship was explored between the cells’ response intensity at the former three levels and the co-cultured 5-AZA.The demethylation ability of the aquatic from polluted area of Tianjin basin was tested with this novel method.Good dose-respond relationships were found between DNA methylation of CMV promoter, EGFP gene expression, green fluorescence intensity of the recombinant cells and 5-AZA.The equation for green fluorescence intensity of the test cells and 5-AZA is y = 0.640lnx + 10.284 with R2 = 0.890.This method has a detection limit of 0.00004 μM 5-AZA and good repeatability with variation of 7.5%-23.9%.Almost half of the aquatic from the Tianjin basin was found demethylation ability positively with this method.国家自然科学基金(20907047);中国环境科学研究院中央级公益性科研院所基本科研业务专项基金(2008KYYW05)资