Molecular cloning and expression of key gene encoding hypothetical DNA polymerase from B. mori parvo-like virus

• Main Authors: Junhong Zhang, Guohui Li, Huiqing Chen, Xiaogang Li, Meng Lv, Keping Chen, Qin Yao
• Format: Article
• Language: English
• Published: Sociedade Brasileira de Genética 2010-01-01
• Series: Genetics and Molecular Biology
• Subjects: Bombyx mori parvo-like virus; DNA polymerase; recombinant protein; Sf-9
• Online Access: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572010000400021

BmPLV-Z is the abbreviation for Bombyx mori parvo-like virus (China isolate). This is a novel virus with two single-stranded linear DNA molecules, viz., VD1 (6543 bp) and VD2 (6022 bp), which are encapsidated respectively into separate virions. Analysis of the deduced amino acid sequence of VD1-ORF4 indicated the existence of a putative DNA-polymerase with exonuclease activity, possibly involved in the replication of BmPLV-Z. In the present study, a recombinant baculovirus was constructed to express the full length of the protein encoded by the VD1-ORF4 gene (3318 bp). In addition, a 2163-bp fragment amplified from the very same gene was cloned into prokaryotic expression vector pET-30a and expressed in E.coli Rosetta 2 (DE3) pLysS. The expressed fusion protein was employed to immunize New Zealand white rabbits for the production of an antiserum, afterwards used for examining the expression of the protein encoded by VD1-ORF4 gene in Sf-9 cells infected with recombinant baculovirus. Western blot analysis of extracts from thus cells infected revealed a specific band of about 120 kDa, thereby indicating that the full length protein encoded by the VD1-ORF4 gene had been successfully and stably expressed in Sf-9 cells.