Samulski R J, Chang L S, Shenk T
J Virol. 1987 Oct;61(10):3096-101. doi: 10.1128/JVI.61.10.3096-3101.1987.
A recombinant plasmid carrying an infectious adeno-associated viral genome was constructed that differs in several key respects from previously described recombinants. First, the vector is pEMBL8(+), which allows isolation of viral plus and minus strands. Second, the inserted viral sequences contain two XbaI cleavage sites that flank the viral coding domain. These inserts do not affect replication of the virus, and they allow nonviral sequences to be easily inserted between the cis-acting terminal repeats of adeno-associated virus. Third, the viral genome is flanked by PvuII cleavage sites that allow the entire, infectious viral chromosome to be excised from plasmid sequences in vitro. Viral DNA was replicated more efficiently within adenovirus-infected 293 cells if it was excised from the vector with PvuII before transfection. Presumably, the increased efficiency reflects bypass of the excision step which must normally precede replication when a recombinant plasmid enters the nucleus. The ability to bypass the excision step was exploited to search for a viral function required specifically for excision of the viral genome from the integrated state. None of the mutants tested identified a gene product required for excision that was not also essential for replication. The ability to produce pure populations of viral plus and minus strands was used to demonstrate that both strands are infectious.
构建了一种携带感染性腺相关病毒基因组的重组质粒，该质粒在几个关键方面与先前描述的重组体不同。首先，载体是pEMBL8(+)，它允许分离病毒正链和负链。其次，插入的病毒序列包含两个位于病毒编码域两侧的XbaI切割位点。这些插入片段不影响病毒的复制，并且它们允许非病毒序列轻松插入腺相关病毒的顺式作用末端重复序列之间。第三，病毒基因组两侧是PvuII切割位点，这使得完整的感染性病毒染色体能够在体外从质粒序列中切除。如果在转染前用PvuII从载体中切除病毒DNA，其在腺病毒感染的293细胞内的复制效率会更高。据推测，效率的提高反映了对切除步骤的绕过，当重组质粒进入细胞核时，切除步骤通常必须在复制之前进行。利用绕过切除步骤的能力来寻找从整合状态切除病毒基因组所特需的病毒功能。所测试的突变体均未鉴定出切除所需的基因产物，而这些产物对复制也并非必不可少。利用产生纯病毒正链和负链群体的能力来证明两条链都具有感染性。
