马尾松毛虫CPV基因组第5片段的cDNA克隆及其序列分析

Molecular Cloning and Sequence Analysis of Dendrolimus punctatus cytoplasmic polyhedrosis virus Segment 5

通过差速离心从感染的马尾松毛虫幼虫虫体中提取质型多角体病毒。碱解法提纯病毒粒子,1％琼脂糖凝胶分离基因组dsRNA,回收纯化第五片段(S5)。根据同源性设计五对引物,经RT-PCR,最终获得五个亚克隆片断,测序拼接后,得到S5全长。片断全长2851个核苷酸,包括一个2643个核苷酸的开放阅读框。推测DpCPV S5基因编码了881个氨基酸长的多肽,分子量为100.3kDa,与舞毒蛾质型多角体病毒(LdCPV-1)和家蚕质型多角体病毒(BmCPV)比较,核苷酸和氨基酸的同源性都很高。进一步分析,利用几种CPV序列绘制了系统进化树,对病毒的分类和进化做了探讨研究。

Dendrolimus punctatus polyhedra were purified from infected Dendrolimus punctatus Larvae using density centrifugation. Dendrolimus punctatus cytoplasmic polyhedrosis viruses (DpCPV) were subsequently extracted from polyhedra by SDS treatment,followed by 1% agrose gel electrophoresis, and the segement dsRNA 5 was purified after being excised from the gel. On the basis of nucleotides homology, five pairs of primers were designed. After the RT-PCR, five sub-clone segments were obtained, following sequencing and splicing, the complete nucleotide sequence of the genome segment 5(S5) of DpCPV was determined. The 2851-nucleotide sequence contains a single long open reading frame which spans nucleotides 71 to 2714 and was predicted to encode a protein with a molecular mass of about 100.3kDa.Sequence analysis and deduced amino acid sequence of DpCPV S5 revealed high homology with proteins from LdCPV-1 and BmCPV. From the phylogenetic tree, we can analyse the classification and evolutionary relationship in viruses.