单引物法扩增马尾松毛虫CPV基因组第8片段及其序列分析

Molecular Cloning Dendrolimus punctatus cytoplasmic polyhedrosis virus Segment 8 by Single Primer Amplification and Sequence Analysis

本文利用T4 RNA连接酶将5’-磷酸、3’-氨基修饰的引物1连接到马尾松毛虫质型多角体病毒第8片段dsRNA的3’-OH端。经逆转录、退火、补齐形成全长双链cDNA。使用单一的互补引物2进行PCR扩增。扩增产物克隆在pMDl8-T载体上。对重组子进行限制性内切酶分析及序列测定,结果表明,克隆片段全长330bp,S’端具有CPV-1型末端保守序列AGTAAA’端具有保守序列GTTAGCC。起始密码子从ATG位于38-40残基,终止密码子TAA位于1208～1210残基。推测S8片段编码390个氨基酸多肽。分子量为44kDa。与舞毒蛾质型多角体病(LdCPV)第8片段相比较,核苷酸和氨基酸同源性分别为97％和98％。与家蚕质型多角体病毒(BmCPV)第8片段相比较,核苷酸和氨基酸的同源性分别为83％和85％。与人的呼肠孤病毒第8片段比较没有明显的同源性。

Dendrolimus punctatus cytoplasmic polyhedrosis virus (DpCPV) was purified from infected Dendrolimus punctatus and the genomic dsRNA segments were subsequently extracted. Segment 8 dsRNA was purified by low melting-temperat agraose. A single ammo-linked modified oligonucleotide (primer 1) was ligated to either 3'end of dsRNA genome segment 8 by Using T4 RNA Ligase. Following reverse transcription, annealing and repair of cDNA strand, amplification of S8 dsRNA genome was accomplished by PCR using a single complementary oligonucleotide (primer 2). The amplified cDNA was cloned into the pMDIS-T vector.Nucleotide sequence analysis of cDNA derived from the segment 8 revealed that it consists of 1 330 nucleotides encoding putative protein of 390 amino acids with molecular weith of 44KD. Comparison of the nucleolide sequence of the segment 8 of DpCPV with that of BmCPV and LdCPV showed that nucleotides homology is 83% and 97%, respectively.