Cloning And Expression Of Human Epidermal Growth Factor CDNA And Characterization Of Its Product Expressed In Hen Oviduct

Human epidermal growth factor is powerful mitogen for a wide variety of cells in culture and in vivo. It can bind to the extracellular domain of its receptor, which is a kind of tyrosine kinase. leading to itself phosphorylation and cells proliferation.To develop the recombinant hEGF. a pair of primers were synthesized according to previously published hEGF coding sequence with goat p-lactoglobulin signal peptide sequence fused to the 5-end for secretive expression, a cDNA of correct size was amplified from pooled human liver single-stranded cDNAs by high fidelity PCR. Sequence analysis showed that the hEGF coding region was identical to that previously published and the goat p-lactoglobulin signal peptide sequence was introduced correctly. The cDNA was subcloned into prokaryotic expression vector pGEX-6p-l and the resulting recombinant plasmid was transformed to E .coli. BL21 (DE3) for expression. After IPTG induction, an additional band of 32KDa was detected by SOS-PAGE. The cDNA was then subcloned into eukaryotic expression vector pcDNA-3 and the recombinant vector was injected into rabbits. After six times of injection, an antiserum to hEGF was obtained, which was able to recognize GST-hEGF fusion protein expressed in E .coli.The hEGF cDNA was subcloned into hen oviduct-specific expression vector pOV. The recombinant vector was mixed with 25kDa polyethyleneimine and injected into egg-dropping hens via wing vein route. Western Blotting showed that a 6KDa recombinant protein was detectable in egg white from vector-injected hens by the rabbit antiserum. Cell proliferation assay using MTT method showed the recombinant hEGF-containing egg white had promotion or inhibition effect on NIH/3T3 cells, depending on the amounts of egg white used. Based on the cell proliferation assay, the expression of hEGF in vector-injected hen egg white lasted for at least one week. These data indicate the correctness of cloned hEGF cDNA and the feasibility of the construct for production of recombinant hEGF by hen oviduct bioreactor.