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

Engineering practice of the treatment of wastewater from flexible printed circuit board factories

介绍了厦门某电子科技有限公司废水处理工程实例。针对该线路板厂废水来源的不同,依据分类收集、先预处理再综合处理的原则对废水进行合理的细化分类,对不同的水质进行不同的物化处理。工程试车结果表明,采用本工艺路线处理后的柔性印制电路板废水出水水质达到或优于GB8978—1996一级排放标准。An example of the engineering practice of the wastewater treatment at an electronic technology company in Xiamen is introduced. Considering the different characteristics of wastewater sources at the circuit board factory, and based on classifying the collections, the principle of classifying the wastewater is followed and the wastewater is treated properly and detailedly by pre-treatment firstly and then by comprehensive treatment. Wastewater with different water quality is treated by different physicochemical treatment. The practice results show that when flexible printed circuit board wastewater is treated by this process,the effluent water quality meets or superior to the requirement of the first class of the standard,GB 8978—1996

一种有孔载玻片

本实用新型提供了一种有孔载玻片，其由上玻片和下玻片组成;该上玻片的下表面与下玻片的上表面均为平面结构，并且二者呈上下密闭连接状;该上玻片设有透孔，透孔自上玻片的上表面穿透至下玻片的上表面，与下玻片的上表面形成凹槽。与现有的凹形载玻片相比，该有孔载玻片的凹槽制作简单，底面呈平面结构，避免了现有凹形载玻片的凹面难加工与底面不均一的问题，有利于降低检测误差、提高检测精度，因此是一种实用、有效的载玻片，能广泛应用于生物、医学、化学等领域

声子玻璃物理建模的探索研究

声子玻璃(Phononic glass)是一种新型的水下吸声材料,具有复杂的内部结构。如果要探明其吸声机制与影响因素就要建立数值分析模型,模拟不同影响条件与结构下该材料的声吸收特性。由于声子玻璃结构的特殊性,需要建立有针对性地物理模型进行数值分析,本文将在一维物理模型的基础上建立声子玻璃的二维物理模型,并通过模拟泡沫铜基声子玻璃,探索二维模型中横向单元与其他角度振动模式对带隙的影响

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

一种具有低分子量分布的丙烯腈共聚物纺丝液的制备方法

本发明公开了一种具有低分子量分布的丙烯腈共聚物纺丝液的制备方法。本发明通过在丙烯腈单体与共聚单体的溶液聚合过程中，采用连续或间歇降温的方式来调节共聚物的重均分子量和数均分子量，从而使共聚物的分子量分布指数降低。与其它方法相比，该方法简单易行，工艺可操作性较强，利用该方法制得的丙烯腈共聚物的分子量分布指数可控制在2.3～2.8之间，这种丙烯腈共聚物溶液经过脱除残余单体和气泡后可用作高性能聚丙烯腈基碳纤维的纺丝液