人巨细胞病毒基因HindⅢ F片段重组质粒pAT153的转化鉴定及其临床应用

Transformation and Identification of Recombinant Plasmid pAT153 Containing HCMV Gene HindⅢF Fragment and Its Clinical Application

作者采用常规质粒转化程序,将含人巨细胞病毒(HCMV)基因HindⅢ F大片段(20.7kb)的 质粒pAT153转化大肠杆菌HB101。转化效率24％。用同位素^(32)P标记探针筛选出阳性克隆。用改良碱 裂解法提取质粒DNA,产量和纯度高并省时,HCMV HindⅢ F片段与其他疱疹病毒DNA无同源性, 经同位素^(32)P标记作为探针,其检测灵敏度为0.25pg,适用于临床早期诊断HCMV感染,具有快速、灵 敏度和特异性高等优点。

Transformation of the recombinantplasmid pAT153 containing human cyto-megalovirus(HCMV) gene Hind Ⅲ F fragmentinto E.coli strain HB 101 was made by theCaCl_2, method with a 24% of transformationefficiency. The bacterial colonies contain-ing the recombinant plasmid were selectedin colony hybridization with ^(32)P-labelledHCMV Hind Ⅲ F fragment. The recombin-ant plasmid was isolated by the modifiedalkaline lysis method which is characteri-zed by high quality, great quantity, andtime-saving. HCMV Hind Ⅲ F fragment hasno homology to the other herpes virus.The ^(32)P-labelled HCMV Hind Ⅲ F fragmentprobe could detect 0.25 pg homologicalsequences of HCMV DNA. The use of thisprobe allows the detection of HCMV inclinical specimens with the advantages ofquickness, good sensitivity and specificity.