Expression Of A Human Epidermal Growth Factor In Eschcrichia Coli And Preparation Of Polyclonal Antibody Against It

Human epidermal growth factor is a small single-chain polypeptide of 53 amino acids. It is heat- and acid-stable. The major biological function is to stimulate the growth and proliferation of epidermal and epithelial cell. It is often applied to treat burns, scald, cornea transplant, gastric acid, duodenal ulcered and also also used as cosmetics.Since Cohen firstly discovered epidermal growth factor (EGF) from a mouse in 1960, many growth factors have been identified one after another mainly from human and mammilla such as transforming growth factorα(TGFα), vaccinia growth factor (VGF), shope fibroma growth factor (SFGF), myxoma growth factor (MGF), ampiregulin, arnphireguli (AR), epiregulin(EPl), heparin-binging EGF-like growth factor (HB-EGF), vaccinia virus growth factor (VVGF). They have 50～80 amino acids and homology about 20%～90% in primary structure. In their molecules there have conservatively six cysteines, which form three disulfide bonds, make their bind to EGF receptor inducing similar biological activity. These molecules are defined as an EGF family.In this study using PCR technology, we have manually synthesized the DNA sequence for mature peptide of hEGF. It was cloned into vector pGEM-T and then inserted into the expression vector pGEX-2T resulting the recombination plasmid pGEX-2T-hEGF. This plasmid then was expressed in E.coli, and the 1.0 mM isopropyl-β-D-thiogalactoside (IPTG) was used for inducing protein GST-hEGF expression yielded about 30% of the total protein. By SDS-PAGE the GST-hEGF expressed in prokaryote has been separated. The GST-hEGF bands were grinded to powder with liquid nitrogen. Some 10～20μg GST-hEGF proteins were introduced into mice with Freund adjuvant and the polyclonal antibodies have been produced. This antibody can detect GST-hEGF and EGF-PEA-scFv clearly at 1:10000 dilution.