Alu-PCR检测人肝癌细胞株PLC/PRF/5基因组中乙型肝炎病毒DNA的整合

目的 应用Alu-PCR方法检测人肝癌细胞株PLC/PRF/5 基因组中乙型肝炎病毒(HBV)DNA的整合。 方法 提取经培养扩增的人肝癌细胞株PLC/PRF/5 基因组DNA,根据Alu-PCR方法经过3轮PCR反应扩增潜在的HBV DNA和人基因组DNA整合片段。琼脂糖凝胶电泳观察PCR 扩增产物片段,切取并纯化整合阳性的电泳条带,对纯化产物进行核酸测序,得到整合片段的核苷酸序列。 结果 经琼脂糖凝胶电泳检测,用Alu-PCR方法能够从PLC/PRF/5 细胞株中扩增得到4条HBV DNA整合序列,经测序后与比对其中3条整合序列能够定位于人染色体03p21.31、05p15.33、12q13.12~q14.1。 结论 Alu-PCR可以准确测定肝细胞中HBV DNA的整合,为研究HBV DNA在肝细胞中的整合研究提供了一个简单、经济的方法。

Detection of Human Hepatocarcinoma Cell Line PLC/PRF/5 Genome Hepatitis b Virus DNA Integration with Alu-PCR

Objective In this research with the method of Alu-PCR we investigate the integration of hepatitis B virus (HBV) DNA in human hepatocarcinoma cell line (PLC/PRF/5) genome DNA. Methods We at first extracted the genome DNA from PLC/PRF/5 cells, and then the potential integration fragments of HBV DNA and human genome DNA were amplified with according to the Alu-PCR after three rounds PCR. The Alu-PCR amplification products were observated with agarose gel electrophoresis, then integration positive electrophoresis bandings were chipped and purified for nucleic acid sequencing. At last he bioinformatics information was acquired by blast online. Results Through agarose gel electrophoresis after Alu-PCR amplification, we got four potential integration bindings, among which we got three integration sequences of HBV DNA in human genome DNA. These integration sequences could be individually located in the human chromosome of 03p21.31, 05p15.33, 12q13 and 12-q14.1. Conclusion With Alu-PCR we can accurately measure the integration of HBV DNA in human genome DNA, and Alu-PCR can be a a convenience and economic method in the study of HBV DNA's integration in human genome DNA.