摘要
以家蚕胚胎细胞系(Bm E-SWU1)为病毒培养载体,利用中性红染色的方法对家蚕核型多角体病毒(Bm NPV)GD株进行了空斑克隆,经扩大培养,获得了GD株空斑克隆株系。根据Bm NPV CQ1株基因组序列的单核苷酸多态性(SNP)分析,选出其中SNP位点较多的gcn 2(General control nonderepressible 2)基因,用于验证BmNPVGD株空斑克隆株系的纯粹性。分别从BmNPVGD株和空斑克隆株的基因组中扩增gcn2基因,经pMD18-T克隆后测序;将两株病毒的gcn2基因分别进行SNP位点分析,发现GD株gcn2基因的潜在SNP位点有2个,而空斑克隆后的病毒株5条gcn2基因序列比较后均未发现多态性位点,说明空斑克隆株经纯化后,病原基因组的纯粹性获得了提高,病原得到了纯化。
With neutral red staining, Bm NPV GD was plaque-purified using Bombyx mori-SWU1（Bm E-SWU1） as carrier. According to the candidate SNP analysis of Bm NPV CQ1 sequences, gcn2 （General control nonderepressible 2） with quite a few SNP sites in CQ1 genome was selected to verify the purity of the cloned strain through sequence comparison between the pre-cloned GD isolate gcn2 and cloned GD strain gcn2. gcn2 gene was amplified from Bm NPV GD strain and cloned strain respectively, and then both cloned into pMD18-T vector for sequencing. Candidate SNP sites of gcn2 were analyzed between cloned strain and GD strain. There were two candidate SNP sites in GD strain, meanwhile no SNP site was found in the clone-purified strain. It indicated that the clone-purified GD strain was homozygous at genome level.
出处
《湖北农业科学》
北大核心
2010年第4期785-787,共3页
Hubei Agricultural Sciences
基金
重庆市自然科学基金项目(CSTC-2005BB1138)
