单纯疱疹病毒2型特异性DNA片段的克隆和鉴定

Cloning and ldentification of the Type-specific DNA Fragment of Herpes Simplex Virus Type 2

用核酸限制性内切酶BamHI对单纯疱疹病毒2型(HSV—2)的DNA进行酶解,回收位于基因组中的反向重复序列区的Bam HIG片段,然后将其克隆在载体质粒PUC 8的Bam HI切点上,进一步用核酸限制性内切酶Eco RI和KPNI对这一重组质粒联合酶解,移去EcoRI—KPNI小片段,经末端修饰后,将其连接得到新的重组质粒pRC102,它含有一小段HSV—2的DNA序列。以此质粒为探针,分别与HSV—1、HSV—2及细胞DNA进行斑点杂交;与HSV—1和HSV—2酶解后的DNA片段进行Southern转印系交。两组实验结果显示,pRC102质粒DNA只与HSV—2 DNA特异性杂交,其HSV—2的型特异性良好。

The DNA of Herpes Simplex Virus type 2 (HSV-2)digested with restrictionenzyme BamHI and the BamHI G fragment was isolated, which locates in thereverse repeat sequence and spans the junction site of L and S component.Then the BamHI G was inserted on the BamHI cut-site of the vector plasmidpUC 8. In order to modify this recombinant plasmid, the EcoRI--KPN I fra-gment of the recombinant plasmid was removed by restriction enzyme digestionwith EcoRI and KPN 1. After modification of the ends and ligation of them,a new recombinant plasmid pRC 102 was constructed which still contains asmall HSV-2 DNA fragment. Labeled with ^(32)P, the recombinant plasmid pRC102 was utilized to hybridize with HSV-1, HSV-2 DNA and other DNA. Theresults of the dot blot and the Southern blot hybridization showed that the probepRC 102 DNA hybridized only with HSV-2 DNA. The plasmid pRC 102 canbe used to type HSV by DNA hybridization.