Expression Of Polyhedrin-hEGF Fusion Protein In Cultured Cells And Larvae Of Bombyx Mori

Epidermal growth factor (EOF), a single-chain polypeptide of 53-amino acid, which was firstly isolated from the submaxillary gland of mice in 1962, has wide potent applications. For mass production of EGF, Bombyx mori baculovirus expression vector system (BEVS) was adopted in this experiment to express recombinant EGF.hEGF gene with His-tag at the end, which was derived from pET22 -EGF, was in-frame fused to the carboxy-terminal of polyhedrin (Ph) gene, which included the amino-terminal 116aa coding region. The polyhedrin-EGF fusion gene (named Ph-EGF) was then cut out with EcoRV and EcoRI, and was cloned between EcoRV and EcoRI sites of pBacPAK.8 (the result plasmid was named pBacPh-EGF and the Ph-EGF fusing gene was right under the control of Ph promoter). The pBacPh-EGF structure was verified with restriction enzymes digestion and PCR.The pBacPh-EGF plasmid DNA was used to co-transfect BmN cell with the modified Bombyx mori baculovirus Bm-BacPAK DNA, which was first linearized by Aocl. After two rounds of plaque isolation, the recombinant virus (named BmBacPh-EGF) was further verified with PCR and Dot hybridization.The recombinant BmBacPh-EGF virus was used to infect BmN culture cells at a MOI of 10. The cells and the culture supernatant were collected 72 hours post-infection. For detection of recombinant Ph-EGF expression, SDS-PAGE and ELISA analysis were applied. No trace of any newly expressed protein band was noticed in supernant as well as in the cells by SDS-PAGE, except the verification of the substitution of beta-galactosidase gene (the lose of galactosidase protein band), which is a selective marker of the wild-type virus. ELISA test results suggested the expression of EGF in cells, but not in culture supernant. The quantitative calculation suggested the expressed EGF was about 6-7 U g (as EGF standard) per flask (2><106cells) in the cellular extract. The larvae of 5th instar silkworm were inoculated with the recombinant virus and grew a further 5 days. Hemolymph fluid of the infected larvae was collected every day. The ELISA assay showed a maximum expression hi day 4, which was consensus to other genes expressed in the BEVS. SDS-PAGE and Western blotting analysis showed a newly protein band, which can specifically react to EGF antibody, with a molecular weight about 19 kD as expected, was expressed in the larvae hemolymph fluid.Biological activity was determined by EGF dependent Balb/c 3T3 cell line and with MTT colorimetric assay. Extracts of the recombinant virus-infected and mock-infected cells, haemolymph of the recombinant virus infected and mock-infected silkworm larvae could all support the proliferation of Balb/c 3T3 cell. This phenomena implied that there were some EGF-like growth factors in the haemolymph of normal silkworm larvae, which could enhance the proliferation of the cell line.The animal study was designed to determine whether Ph-EGF fusion protein was protective to ethanol induced injury of gastric mucosa in rats. Thirty-two SD rats weighting 200-250g starved for 24hour were divided into four groups which received pupae infected with recombinant BmBacPh-EGF virus, normal pupae, Ranitidine, 0.9% saline respectively for 3days before received absolute ethanol. The results showed that both pupae infected with BmBacPh-EGF virus and normal pupae protect the gastric mucosa against ethanol induced damage.