生物素交联补骨脂素标记HPV基因探针的制备

Preparation of HPV DNA probe labeled with biotinlated psoralen

对含ＨＰＶ１６和ＨＰＶ１８ＤＮＡ质粒的大肠杆菌进行培养、扩增，提取大量质粒ＤＮＡ，纯化、酶切、回收ＨＰＶＤＮＡ片段，用国产生物素交联补骨脂素标记，再经纯化鉴定。制备的ＨＰＶＤＮＡ探针用亲合素辣根过氧化物酶显色，敏感性可达ｌｐｇ。检测靶核苷酸的敏感性可达５ｐｇ。该探针用于原位杂交检测１０例食管癌组织及相应癌旁粘膜组织中的ＨＰＶＤＮＡ，也取得良好结果。结果提示：用生物素交联补骨脂素标记基因探针是一种简单的方法，且效果良好，适于研究和临床检测。

Escherichia coli contained the recombined plasmids inserted in human papivillary virus(HPV) DNA fragments 16 or 18 subtype were cultured and amplified for large scale preparation of the recombined plasmids .The plasmids were then purified and digested with restriction endonucleases to recover desired DNA fragments.The DNA fragments were labeled with home produced biotinlated psoralen,then purified and identified.The results showed that the sensitivity of color development with horseradish peroxidase could get the level of l pg.The sensitivity to detect the target sequence could get the level of 5 pg.By using the probe,good and effective results can be obtained in investigating the HPV DNA in esophageal carcinoma.It was indicated that the preparation of the probe labeled with biotinlated psoralen was a handy and effective method.