向下转膜法在乙肝病毒Southern杂交中的应用

The Application of Downward Capillary Transfer in the Southern Blotting of HBV

目的评价向下转膜法在乙肝病毒(HBV)Southern杂交中的应用效果。方法用带有1.3倍HBV基因的真核表达载体转染HepG2细胞,使其产生瞬时HBV表达,72小时收集细胞并提取细胞总DNA,琼脂糖凝胶电泳后,以向上和向下两种方式转移在尼龙膜上;用化学发光标记和检测试剂盒将乙肝病毒基因片段标记成探针,再与向上和向下转膜后的尼龙膜进行杂交,判断产生明显HBV杂交条带所需的细胞总DNA量,从而验证向下转膜法在乙肝病毒Southern杂交中的应用效果。结果发现向下转膜后,检测HBV表达所需的细胞总DNA量极少,灵敏度高,0.5μg细胞总DNA电泳后转膜,即可检测出HBVDNA的表达,凝胶中仅有8%的DNA残留;而向上转膜所需的总DNA量大,灵敏度低,25μg的细胞总DNA电泳后转膜,仅能检测出弱的条带。结论向下转膜法是一种较好的转膜方式,适于对乙肝病毒的Southern杂交,值得推广应用。

Objective To investigate the effect of downward capillary transfer in the Southern blotting of HBV. Methods Transfecting the plasmid which contains 1.3 fold overlength genome of HBV to HepG2 cells so as to get the expression of HBV, extracting total genome DNA of cells by common protocol and loading on agarose gel electrophoresis, transfering the DNA of the agarose gel to Nylon membranes by both the downward and upward capillary transfer;labelling HBV DNA probe with the Gene Image Alkphos Direct labelling system and hybridizing with the total DNA of cells on the Nylon membranes, comparing the sensitivity of upward with downward capillary transfers in the Southern blotting of HBV. Results We found that the sensitivity of downward capillary transfer is higher than that of upward in the Southern blotting of HBV, 0.5μg of total DNA of cells can be detected the DNA expression of HBV, there are only 8 percent of total DNA left in the agarose gel after transferred by downward capillary transfer;conversely,25μg of total DNA of cells can only be detected low DNA expression of HBV in upward capillary transfer. Conclusion The downward capillary transfer is better than that of upward in the Southern blotting of HBV, and should be applied broadly.