马尾松毛虫质型多角体病毒NS5蛋白基因的cDNA克隆及序列分析

Molecular Cloning and Sequence Analysis of Dendrolimus punctatus cytoplasmic polyhedrosis virus NS5 Protein Gene

通过对马尾松毛虫质型多角体病毒的增殖、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,在使用低熔点琼脂糖凝胶电泳分离基因组dsRNA,回收纯化第九片段S9。SgRNA双链经高温变性.使用正反两种引物逆转录合成cDNA双链,使用同样的引物经PCR扩增后,克隆在pMDl8-T载体上。利用两种引物组合,获得了大小两个亚克隆片段。序列测定结果表明,较小亚克隆片段长405bp,较大亚克隆片段长677bp,经过序列拼接,得到一个977bp的序列,其中包含一个963bp大的开放阅读框(ORF)。推测DpCPVS9基因编码一个320个氨基酸的多肽,分子质量35.66kDa。和家蚕1型质型多角体病毒的I株(BmCPV-I1 strain)位于第几片段的。NS5蛋白基因相比较,核苷酸和编码氨基酸序列同源性分别为86.0％和93.4％。

For the synthesis of cDNA of Dendrolimus punctaus CPV (DpCPV)NS5 protein, the primers were designed on the basis of the RNA sequmce of BmCPV-1 (I strain) segment 9. After the RT-PCR the amplified cDNA was cloned into the pMD18-T. NS5 protein gene of DpCPV was found to be 963 nucleotides in length with a single open reading frame in one strand capable of coding a predicted protein of 320 residues (Mw of 35.66kDa). Comparison of the nucleotide sequences of the NS5 gene of DpCPV with those of BmCPV-I(1 strain) showed that nucleotides homology is 86.0%, but there are only 21 amino acid differences between the two CPVs.