文山松毛虫质型多角体病毒(DpwCPV)NS5蛋白基因的cDNA克隆及序列分析  

Molecular Cloning and Sequencing of Dendrolimus punctatus Wenshanensis Cytoplasmic Polyhedrosis Virus (DpwCPV) NS5 Protein Gene

作　　者：王琼[1] 张珈敏[1] 文力[1] 杨娟[1] 胡远扬[1] 

机构地区：[1]武汉大学病毒研究所,武汉430072

出　　处：《微生物学报》2003年第1期21-28,共8页Acta Microbiologica Sinica

摘　　要：通过对文山松毛虫质型多角体病毒 (DendrolimuspunctatusWenshanensiscytoplasmicpoly hedrosisvirus,DpwCPV)的增殖、纯化 ,获得一株单一类型的质型多角体病毒。提纯的病毒粒子经SDS 热酚法抽提得到基因组dSRNA ,使用低熔点琼脂糖凝胶电泳分离并回收纯化第九片段S9。S9RNA双链经高温变性 ,逆转录合成cDNA双链。根据DpwCPV与BmCPV 1的同源性设计引物 ,将S9进行PCR扩增后 ,克隆到PMD1 8 T载体上。最终获得一个 977bp的序列 ,其中包含一个 963bp的开放阅读框 (ORF)。推测DpwCPVS9基因编码一个 32 0个氨基酸的蛋白 ,分子量约为 35 5 60。DpwCPV were purified from infected Dendrolimus punctatus Wenshanensis larvae by density gradient centrifugation. The genomic dsRNA segments were subsequently extracted directly from the purified polyhedra by SDS treatmen, phenol/chloroform extraction and ethanol precipitation. Defferent segments of genomic dsRNA were separated by low melting point agarose electrophoresis, and S9 dsRNA was purified after being excised from the gel. S9 RNA was denaturalized in high temperature. The primers was designed on the basis of the RNA sequence of S9 in BmCPV-1 (H strain). After RT-PCR, the amplified cDNA was cloned into the pMD18-T Vector. The NS5 protein gene of DpwCPV is found to be 977 nucleotides in length with one open reading frame in one strand. It can code a predicted protein of 320 residues (35560).

关 键 词：文山松毛虫 质型多角体病毒 NS5蛋白基因 CDNA克隆 序列分析 

分 类 号：S763.12[农业科学—森林保护学]