芹菜夜蛾核型多角体病毒的空斑纯化及克隆株的生物测定

Plaque Purification of Syngrapha falcifera Multiple Nuclear Polyhedrosis Virus and Bioassay of Clone Isolate

利用空斑技术从芹菜夜蛾核型多角体病毒分离筛选了一毒力较强的SfaMNPV D克隆株。SfaMNPV D克隆株感染 5B1细胞 ,72h胞外病毒达最大滴度 ,组织培养半感染剂量为 3 .89×1 0 8TCID50 /ml,多角体产量达 4.0× 1 0 7PIBs/ml;生物测定结果表明 ,SfaMNPV D克隆株感染 3龄棉铃虫幼虫的LC50 为 7.1 4× 1 0 5PIBs/ml,原毒株的LC50 为 4.81× 1 0 6PIBs/ml。SfaMNPV D病毒DNA经几种限制性内切酶消化 ,各片段积加测得基因组DNA分子量为 1 1 3 .78kb .

We isolated a clone from Syngrapha falcifera multiple nuclear polyhedrosis virus (SfaMNPV) by plaque purification and designated it as SfaMNPV-D. The SfaMNPV-D was very sensitive to 5B1 cell line and at 72h postinfection TCID_(50) value of buded viruses was 3.89×10~8TCID_(50)/ml and at 144h p.i the production of polyhedra was 4.0×10~7PIBs/ml. Bioassay showed that LC_(50) value of SfaMNPV-D was 7.14×10~5PIBs/ml and LC_(50) value of wild type SfaMNPV was 4.81×10~6PIBs/ml when early third instar larvae of Helicoverpa armigera that infected respectively by two viruses. When the third instar larvae of H. armigera were infected with SfaMNPV-D at the concentration of 1.0×10~6 and 1.0×10~7PIBs/ml, the LT_(50) of SfaMNPV-D was 4.9 and 4.6d respectively. However, when the third instar larvae of H. armigera were infected with wild type SfaMNPV at the concentration of 1.0×10~6 and 1.0×10~7PIBs/ml, the LT_(50) of SfaMNPV was 5.5 and 5.0d respectively. SfaMNPV-D DNA was respectively cleaved with EcoRⅠ, HindⅢ, BglⅡ, PstⅠ and BamHⅠ and molecular weight of virus genome was 113.78 kb.