Construction, Expression and Purification of Prokaryotic Expression Vector of Hyphantria cunea Nuclear Polyhedrosis Virus ORF72

Objective The ORF72 gene of Hyphantria cunea nuclear polyhedrosis virus belongs to the GIY-YIG endonuclease family, it has a high conservative sequence and is closely related to the viral DNA replication.

Method An ORF72 gene was sub-cloned into the pGEX-4T-1 vector. The protein was over-expressed under different induced conditions and purified by GST-tag affinity chromatography column.

Result It was proved that the recombinant vector was constructed successfully by the restriction map and DNA sequencing. SDS-PAGE gel electrophoresis detection showed that ORF72 protein could be integrated with GST-tag protein on the pGEX-4T-1 and the size of the expressed fusion protein was about 38.2 kDa. The induced conditions in over-expression of the recombinant proteins were optimized. The major recombinant protein was obtained by a feasible condition at 25℃ with 1.0 mmol·L^-1 IPTG for 4 h. Moreover, the recombinant protein was expressed to high levels in the two strains of Escherichia coli BL21 and Rosetta, while the expression in the Rosetta strain was higher than BL21.

Conclusion The recombinant ORF72 was purified using GST-affinity chromatography.