重组质粒转染蟑螂对黑胸大蠊浓核病毒的拯救

Rescue of Infectious Virions from Recombinant Plasmid with the Genome of Periplanta fuliginosa Densovirus

用Ｇｌａｓｓｍｉｌｋ法纯化ＰｆＤＮＶｄｓＤＮＡ，在其３′端和ＰｓｔⅠ切开的ｐＵＣ１１９质粒的３′端分别加ｄＧ和ｄＣ尾，连接、转化、筛选得到分子量为８．７ｋｂ的重组质粒。通过酶切证明插入ＤＮＡ为ＰｆＤＮＶ全基因组ＤＮＡ。利用ＤＥＡＥｄｅｘｔｒａｎ转染技术，将此重组质粒导入黑胸大蠊幼虫体内，此重组质粒能使虫体发病死亡。电镜超薄切片观察发现虫体细胞内存在大量的病毒粒子。同样，在免疫扩散实验中虫体ＰＢＳ浸出液能与抗ＰｆＤＮＶ的抗体产生沉淀线。用重组质粒感染致死的虫体喂食健康黑胸大蠊，也能使其发病死亡，通过电镜可以观察到在细胞内增殖的病毒粒子。

An infectious genome of the Periplanta fuliginosa densovirus (PfDNV) has been cloned into the bacterial plasmid pUC119. The viral genome could be rescued from the recombinant plasmid PfNDV pUC119 by transfection to sensitive Periplanta fuliginosa larvae. Virions can be found in the cells of the transfected larvaes by electron microscope, and the rescued virions were as infectious as wild type virions. In contrast, the viral genome could not be rescued from the recombinant plasmid deleted one extremity.