Ⅰ型单纯疱疹病毒基因探针的制备及应用

本文从克隆化的HSV-1 EcoRI-H片段重组菌株中分离重组质粒DNA。经酶切、低熔点琼脂糖凝胶电泳分离出目的基因HSV-1 EcoRI-H片段DNA(13.5Kb)。用缺口翻译法制备a-~(32)P标记探针。通过斑点杂交试验检测10例临床样品及阳阴性对照病毒标准株。结果表明,该探针只与单纯疱疹病毒DNA杂交,而不与腺病毒Ⅲ型(Ad_3)、痘苗病毒(VV)、Vero细胞DNA杂交。且可检测出至少5pg的同源序列DNA。表明该探针具有良好的特异性和敏感性。检测的10例临床标本有两例出现阳性,与病毒分离培养结果一致。

The Preparation of HSV-1 DNA Probe and the Detection of Clinical Specimens

Recombinant plasmid DNA was isolated from recombinant E. coli strain which was cloned with HSV-1 EcoRI-H fragment. The recombinant plasmid was degisted with the restriction enzyme. The aim gene HSV- I EcoRI-H fragment was separated by the low melting point agarose electrophoresis. The a-~(32)p-labeled DNA probe was prepared by nick translation. DNAs obtained from HSV-1, HSV-2, Ad_(?), VV, PBR_(322), recombinent plasmid, Vero cell and ten clinical samples were detected by dot blot hybridization assay with this probe. The results showed that the probe hybridization only with HSV-1 DNA and HSV-2 DNA, but not with. Ad_(3), VV, Vero cell DNA. The probe could detect 5pg of homologous DNA sequence. Therefore, the probe was thought to be specific and sensitive. The clinical samples were examined. Two of them were positive.It agreed with virus isolation result.