家蚕浓核病毒中国(镇江)株基因组的克隆及重组质粒转染家蚕对浓核病毒的拯救

Cloning of the Genome of Bombyx mori Densovirus Zhenjiang Strain and Rescue of Infectious Virions From Recombinant Plasmids in the Silkworm

家蚕浓核病毒中国(镇江)株(BombyxmoriDensovirus Zhenjiang Strain,BmDNV-ZJ)包含VD1和VD2共2种基因组DNA。以BmDNV-ZJ感染家蚕中肠总DNA为模板,采用PCR方法扩增得到2种DNA,并将其克隆到质粒pGEM-T构建了重组质粒pGEM-VD1和pGEM-VD2。采用DEAE-dextran转染技术,将2种重组质粒分别导入家蚕幼虫体内,能使家蚕幼虫发病,免疫双向扩散和免疫酶组化法检测都能检出阳性反应,但转染pGEM-VD2重组质粒的家蚕幼虫发病率较低。通过重组质粒转染方法,可以使BmDNV-ZJ感受性品种和抵抗性品种都发病,但感受性品种的发病率较高。将重组质粒转染发病蚕的中肠匀浆,取上清液经口接种健康蚕幼虫,也能使其发病。上述结果表明,携带BmDNV-ZJ DNA的重组质粒在家蚕幼虫体内拯救出了感染性的病毒粒子。

The genome of Bombyx mori Densovirus Zhenjiang Strain （BmDNV-ZJ） consists of two kinds of DNA molecules, VD1 and VD2. With the total DNA extracted from the midgut of BmDNV-ZJ infected silkworm larvae as template, VD1 and VD2 were amplified by polymerase chain reaction （PCR） and cloned into the plasmid pGEM-T to construct recombinant plasmids pGEM-VD1 and pGEM-VD2. With the mediation of DEAE- dextran, the recombinant plasmids were transfected into silkworm larvae of susceptible and non-susceptible varieties respectively, which resulted in the occurrence of the viral disease to the silkworm, while with lower infection rate in the non-susceptible variety. The disease was confirmed by detection of virions with immunodiffusion and immunoenzyme histochemical method. However, the transfection of pGEM-VD2 resulted in only lower morbidity to the silkworm than that of pGEM-VD1 did. The virions extracted from transfected larvae were as infectious to silkworm as wild-type ones. These results revealed that the viral genome could be rescued from the recombinant plasmids pGEM-VD1 and pGEM-VD2 by transfection of the plasmid DNA to silkworm larvae.