论文部分内容阅读
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。
OBJECTIVE: To detect whether there is a nucleotide sequence homologous to GBV-C genome in DNA of human cell line and human and chimpanzee peripheral blood mononuclear cell (PBMC) DNA. Methods: DNA of MT2 and HeLa cells and PBMC DNA of 6 chimpanzees and 1 chimpanzee were extracted by PCR template preparation kit. DNA was digested with DNase and then extracted with phenol - chloroform. The above DNA was directly used as a template for amplification by nested PCR (dPCR) using primers GBV-C5’-NCR and NS3. dPCR products were analyzed by nucleotide sequence. The PRINS DNA sequence-specific fluorescent labeling was used to determine the position of the amplified fragment on the chromosome. Results: The amplified fragments of GBV-C5’-NCR primer were obtained from MT2 and HeLa cell DNA and 4 human PBMC DNA samples. The amplified fragments of NS3 region primers were obtained from DNA of MT2 and HeLa cells, DNA samples from 5 human and 1 chimpanzee PBMC. The nucleotide sequence of these amplified products was 73.80% -79.15% homologous to the genome of GBV-C. The results of PRINS showed that fluorescent staining of dPCR-positive PBMCs and their chromosomes was observed. CONCLUSION: There are nucleotide sequences homologous to GBV-C5’-PCR and / or NS3 in DNA of MT2 and HeLa cells and human and chimpanzee PBMC DNA. These sequences are located on the dPCR-positive PBMC chromosomes.