Abstract:

In order to preparate Prunus dwarf virus’ special antiserum, the coat protein gene of PDV was cloned, the prokaryotic expression vector was constructed and the expression condition was optimized. The sweet cherry leaves which is infected with PDV is the test material and the total RNA was extracted from them. According to the coat protein gene of PDV in Genbank (L28145.1), specific primers were designed to amplify the coding region of coat protein by RT-PCR, the PCR products were cloned and sequenced, the prokaryotic expression vector pET30a -PDVCP was constructed and the recombinant protein was expressed in E.coli BL21(DE3). Sequence analysis showed that the amplicons (NCBI: JF333587.1) included 657 bp nucleotides, encoding 217 amino acids and shared the similarity of 89.2%-93.9% at nucleotide acid level and the identity of 97%-99% at amino acid level with other PDV isolates. The phylogenetic tree constructed with the complete nucleotide sequences of the CP gene showed that PDV isolates were divided into 3 groups, Taian, Brazil, Turkey et al isolates belonged to groupⅠ. The prokaryotic expression vector pET30a-PDVCP was constructed successfully and the fusion protein was expressed by inducing in vitro. A specific recombinant protein of approximately 24 kDa was induced in the BL21 (DE3) which the prokaryotic expression vector pET30a-PDVCP was transformed into and the optimized expression condition was 30℃, 1.0 mmol/L IPTG for 4 hours.