柯萨奇病毒B3 cDNA生物素探针的制备研究

Preparation of Biotin-labeled cDNA Probe of Coxsackievirus

本文将柯萨奇B组3型病毒(CVB3)cDNA的重组质粒DNA(pGP51B)转化到E.coli HB101菌株中.筛选转化阳性菌株,经培养扩增后,提取重组质粒DNA,用缺口求移法制备生物素标记探针,通过原位杂交技术检测CVB1.CVB3感染的Hela细胞及正常Hela细胞对照.结果该探针只与CVB杂交,而不与细胞对照杂交,且可检测出病毒感染5h尚未出现病变细胞中的病毒核酸.表明该探针具有良好的特异性和敏感性.

pGP5lB, a recombinant plasmid of CVB3 cDNA with plasmid pUC18, was transformed into and amplified in E.coli Hl01,then isolated and purified. As a probe, the purified pGP51B was labeled with biotin in vitro by nick translation. Employed the In situ hvbridizatior procedure, the probe hybrid-izied with CVB3 & CYB1 propagated in Hela cells with cytopathic effect (CPE) (12h. after infection) and without CPE (5h. after infection), but not hybridizied with normal Hela cell control. This showed that the specificity and sensitivity of the probe was high enough to identify the CVB from infected samples.