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．