HU Jian—fan.ZHANG Jia—min, YANG Juan, DENG Xiao-jun, WANG Yang, HU Yuan-yang and M olecular Cloning Dendrolimus punctatus cytoplasmic polyhedrosis virus Segment 8 by Single Primer Amplification and Sequence Analysis[J]. Virologica Sinica, 2003, 18(1): 39-43.
Citation: HU Jian—fan.ZHANG Jia—min, YANG Juan, DENG Xiao-jun, WANG Yang, HU Yuan-yang, . M olecular Cloning Dendrolimus punctatus cytoplasmic polyhedrosis virus Segment 8 by Single Primer Amplification and Sequence Analysis .VIROLOGICA SINICA, 2003, 18(1) : 39-43.

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

  • 本文利用T4 RNA连接酶将5’一磷酸、3’一氨基修饰的引物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eDNA。使用单一的互补引物2进行PCR扩增。扩增产 物克隆在pMD18-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—l型末端保守序列AGTAAA’端具有保守序列G rrAGCC。起始密码子从ATG位于38-4O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。

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

  • Dendrolimus punctatus cytoplasmic polyhedrosis virus(DpCPV)was purified from infected Dendrolimus punctattts and the genomic dsRNA segments were subsequently extracted. Segment 8 dsRNA was purified by low melting-temperat agraose.A single amino-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 oligonucleotidc(primer 2).The amplified cDNA was cloned into the pMD18-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.

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

    • 1. Institutl of,Virology,Wuhan University,Wuhan 430072,China

    Abstract: Dendrolimus punctatus cytoplasmic polyhedrosis virus(DpCPV)was purified from infected Dendrolimus punctattts and the genomic dsRNA segments were subsequently extracted. Segment 8 dsRNA was purified by low melting-temperat agraose.A single amino-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 oligonucleotidc(primer 2).The amplified cDNA was cloned into the pMD18-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.

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