Résumé
The protein production system using a baculovirus Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) as a gene expression vector and its host insect as a natural bioreactor was successful established and its excellent performance in the protein production has been demonstrated. In this paper, the system is used to produce recombinant human epidermal growth factor (rhEGF), which have been widely used in medical and cosmetic treatment. A recombinant AnpehEGF virus has been constructed by replacing the viral polyhedrin gene with the rhEGF gene, and then injected it to Samia cynthia ricini pupae. Amplification and expression of rhEGF gene in the pupae was clearly detected by PCR, Western blot and ELISA analyses. These analyses have also revealed that rhEGF in the pupae was significantly increased at 6 days post-infection, and reached maximum level at the 12th day. The concentrations of rhEGF were 19.77, 24.90, 618.59 and 1 952.46 ng/g pupae at 3, 6, 9 and 12 days post-infection, respectively. However, the rhEGF concentration reduced at later stage (days 15). The rhEGF in the pupae could be purified using ammonium sulfate precipitation and Ni-NTA agrose affinity chromatography. Results demonstrate that Samia cynthia ricini pupae can be used as a bioreactor to produce rhEGF and, if successfully improved, will be a novel method of rhEGF production with lower cost and more efficient.