黄地老虎颗粒体病毒cDNA文库的构建

Construction of Agrotis segetum granulosis virus cDNA Library

以感染黄地老虎颗粒体病毒(Agrotis segetum ganulosis virus,AsGV)的黄地老虎幼虫为材料提取总RNA、分离mRNA,并反转录合成cDNA,构建了包括黄地老虎(Agrotis segetum,As)幼虫和黄地老虎颗粒体病毒的cDNA文库。用EcoR Ⅰ和Hind Ⅲ限制性内切酶酶切AsGV基因组DNA,制备地高辛探针,与上述总RNA、mR-NA、cDNA杂交,从文库中筛选出AsGV的阳性克隆1081个,经cDNA测序,cDNA编码序列与基因组编码序列相符。并根据基因组阅读框序列合成引物,PCR扩增出59个阅读框的编码基因,也完全与基因组序列相符。

A general cDNA library of A gratis segetum granulosis virus(ASGV) which was from infected A gratis segetum(As) larva has been constructed. The first and second cDNA strands were synthesized by AMV reverse transcriptase from mRNA which was isolated from total RNA of infected As larva. So the general cDNA library includes AsGV'S and AS'S, Then the library of AsGV included 1081 positive clones were screened by spot hybridization, The labeled AsGV genome DNA probe With DIG kit was cut by restriction enzyme of EcoR Ⅰ and Hind Ⅲ. The sequence of positnive clones accord with the genome of AsGV, and there are 59 coding sequence which were amplified with synthetic primer of AsGV genome according entirely with the genome of AsGV.