High homologious nucleotide to GBV-C was amplified from DNA of MT2 and HeLa cells and PBMC of human and chimpanzee  

从MT2和HeLa细胞及人和黑猩猩PBMC的DNA扩增出与GBV-C 高同源性的核苷酸(英文)

作　　者：严杰[1] Reinhard H DENNIN 

机构地区：[1]浙江大学医学院病原生物学研究所 [2]Institute of Medical Microbiology and Hygiene, Medical University of Lbeck, Lbeck 23538, Germany

出　　处：《Acta Pharmacologica Sinica》2001年第4期34-40,共7页中国药理学报（英文版）

基　　金：Project supported by Germany Academic Exchange Fundation and completed in Medical University of Lübeck, Germany.

摘　　要：AIM: To determine whether the nucleotide sequences homologous to GBV-C genome exist in the DNA from cell lines of human origin and from peripheral blood mononuclear cells (PBMC) of human and chimpanzee. METHODS: DNA of MT2 cell, HeLa cell, and PBMC from six human and one chimpanzee were prepared by using PCR Temperate Preparation Kit. All of the DNA preparations were digested with DNase-free RNase and then were extracted by phenol-chloroform method. By using these DNA as templates, direct nested-PCR (dPCR) respectively amplified with GBV-C 5'-NCR and NS3 primers were carried out. Nucleotide sequences of the dPCR products were analyzed and positions in chromosomes of the amplification fragments were detected by using primed in situ (PRINS) sequence-specific labeling with fluorescein. RESULTS: DNA fragments amplified with GBV-C 5'- NCR primers were obtained from the DNA of MT2 and HeLa cells and the DNA in 4 of 6 human PBMC samples. DNA fragments amplified with GBV-C NS3 primers were obtained from MT2 and HeLa cells and the DNA in 5 of 6 human PBMC samples and one chimpanzee PBMC sample. The homology of the nucleotide sequences from these amplification products compared with the reported GBV-C genome sequence was from 73.80 % to 79.15 % . Fluorescence staining spots by using PRTNS were shown in the PBMC and their chro-mosomes with positive dPCR results. CONCLUSION:The nucleotide sequences with high homology to GBV-C genome at the 5'-NCR and/or NS3 region exist in the DNA of MT2 and HeLa cells and the PBMC DNA of human and chimpanzee. These sequences locate in the chromosomes of PBMC with positive dPCR results.目的:检测人源性细胞株DNA及人和黑猩猩外周血单核细胞(PBMC)DNA中是否存在与GBV-C基因组同源的核苷酸序列。方法:用PCR模板制备试剂盒提取MT2和HeLa细胞DNA及6例人和1例黑猩猩PBMC DNA,DNA经DNase的RNase消化后再用苯酚-氯仿提取。直接以上述DNA为模板,用GBV-C5’-NCR和NS3区引物进行套式PCR(dPCR)扩增。dPCR产物进行核苷酸序列分析,并用引物介导原位扩增(PRINS)DNA序列特异荧光标记技术确定扩增片段在染色体上的位置。结果:从MT2和HeLa细胞DNA及4例人PBMC DNA标本中获得GBV-C5’- NCR引物扩增片段。从MT2和HeLa细胞DNA及5例人和1例黑猩猩PBMC DNA标本中获得NS3区引物扩增片段。这些扩增产物的核苷酸序列与GBV-C基因组同源性为73.80％-79.15％.PRINS检测结果显示dPCR阳性的PBMC及其染色体上有荧光着色。结论:MT2和HeLa细胞DNA及人和黑猩猩PBMC DNA中存在与GBV-C5’-PCR和/或NS3区同源性较高的核苷酸序列,这些序列位于dPCR阳性的PBMC染色体上。

关 键 词：GB hepatitis agents monkey diseases nucleic acid sequence homology fluorescence microscopy DNA HUMAN MONOCYTES polymerase chain reaction 

分 类 号：R346[医药卫生—基础医学]