摘要
将含人巨细胞病毒(HCMV)DNA大片段的重组cos质粒转化入大肠杆菌DH5α。采用碱裂解法、煮沸法和SDS法提取纯化该大DNA重组cos质粒,并进行比较。通过限制性内切酶酶切分析,以HCMV DNA EcoRⅠ-Y片段作为探针,进行DNA印迹鉴定,并与33例19天～3月婴儿肝炎综合征患儿外周血白细胞DNA进行斑点杂交。结果提示:重组cos质粒转化效率为5.9×10~5转化子/μg DNA,碱裂解法可用于大质粒DNA提取,产量和纯度高,并省时。EcoRⅠ-Y片段与人白细胞DNA及其它疱疹病毒DNA无同源性,适宜予早期诊断HCMV感染,具有较血清学诊断及病毒分离培养特异性高、敏感性强和快速等优点。
Recombinant cosmid containing large human cytomegalovirus (HCMV) DNA fragments was transformed to Escherichia coli strain DH_(5α), and was extracted by alkaline lysis, boiling lysis and sodium dodecyl sulfate, respectively. Then the recombinant cosmid was identified by analysis of endonucleases and Southern hybridization with ^(32)P-labelled HCMV EcoRI-Y fragment which was also used as a probe to diagnose 33 infants with infantile hepatitis syndrome. These results showed that the transformation efficiency of recombinant cosmid was 5.9×10~5 transformants per 1μg of DNA. Alkaline lysis can be used to prepare large plasmid, because of it's high quality, quantity and time-saving. Since HCMV DNA EcoRI-Y fragment is not homologous to the human leucocytes DNA and other members of herpesvirus group, it is available for early detection of HCMV infection with the advantages of quickness, high specificity and sensitivity, compared with viral culture and serological diagnosis.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
1992年第6期335-338,T022,共5页
Chinese Journal of Medical Genetics
基金
国家自然科学基金青年基金
关键词
巨细胞病毒
重组
DNA杂交
质粒
Cytomegalovirus Re combinant cosmid DNA hybridization
