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.

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

  • Available online: 02 February 2003
  • 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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