Expression Of Gp67 Signal Peptide-hEGF Fused Gene In Bombyx Mori Cells And Larvae Of Silkworm And Its Activity Assays

Human epidermal growth factor (hEGF), a single -chain polypeptide of 53-amino acid, has wide potent applications. For mass production of hEGF, Bombyx mori baculovirus expression vector system was adopted in this experiment. Gp67 is the major baculovirus coat protein, its signal peptide can increase target protein productivity in insect baculovirus expression vector system when it's ahead of target gene. In order to increasing hEGF expression in Bombyx moh baculovirus expression vector system, a gp67 signal peptide gene (including 42aa coding region) from BmNPV was cloned by PCR and fused to hEGF gene by Xbal site. The gp67 signal peptide-hEGF fusion gene (named S-hEGF gene. 320bp) was inserted into Bombyx mori baculovirus transfer vector pBacPAK8 and co-transfected with linearized DNA of Bm-BacPAK6 virus into BmN cells. The homologous recombination occurred inside the cells, and the recombinant virus (named BacPAKS-hEGF) was identified by Southern blotting. The recombinant virus BacPAKS-hEGF infected the BmN cells and fifth instars, expression product was run in the Urea-SDS-PAGE, and its immunoreactivity was determined by using ELISA and Western blotting. The bio-activity of the protein product was determined by using Balb/c3T3 cells proliferation test in vitro and by using baby mouse to test whether S-hEGF fusion protein can enhance body weighty and advance eye-opening, tooth-budding date in vivo. Compared with the weighty of standard human epidermal growth factor (6.3kD), the specific protein was about 12kD, indicting the gp67 signal peptide had not been cleaved presumably. The expression activity achieved maximum at the 72h hour in BmN cells(23μg12×106cells) and at 96h hour in larvae (82μ/ml) after infection according to cell activity results. The expression product could enhance the proliferation of the Balb/c3T3 cell line better than standard hEGF. In the baby mouse test, the expression product could enhance body weighty of baby mouse and advance baby mouse's tooth-budding, eye-opening date (8.0± 1.0 and 14.4±0.58 respectively) better than physiological saline (P<0.01) . In this work, normal silkworm could also enhance the proliferation of the cell line and advance baby mouse's tooth-budding x eye-opening date , which indicated that there be some EGF-like growth factors in the silkworm probably.