Production, purification and biological activity of recombinan human epidermal growth factor

• Main Authors: Hu Yu Hua, 胡育華
• Other Authors: Liu Yu Tien
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/99504421874000368973

碩士 === 國防醫學院 === 微生物及免疫學研究所 === 94 === Human epidermal growth factor (hEGF), consisting of 53 amino acid residues, is a single chain polypeptide with a molecular weight of about 6.2KD and is widely used in wound healing, corneal transplants, and the treatment of gastric ulcers. In order to obtain hEGF in sufficient quantities, recombinant DNA technology is extensively used. In this study, the hEGF gene was initially synthesized by overlapping PCR technique and then was cloned into the expression and secretion plasmid, pLT105, to create a new recombinant plasmid, pHL401. For improving the expression efficiency of rhEGF, rhEGF gene was subcloned into another expression vector to create recombinant plasmids, pHL502 and pHL503, respectively, which were then introduced into E. coli BL-21 for production of rhEGF. Our results indicated that large amount of expressed rhEGF deposited in cytoplasm fraction of E.coli/pHL502; however, the Caf1 signal peptide could facilitate the secretion of mature rhEGF to the periplasm of E. coli/pHL503. hEGF produced by E. coli/pHL502 is deposited as inclusion body in cytoplasm. The hEGF was isolated from the inclusion bodies by using high concentration of urea and then the dissolved hEGF was renatured by dialysis the lower concentration of urea. Homogeneous hEGF was obtained by using nickel affinity column for purification of hEGF. The biological activity of the purified hEGF was measured with HtTA cell, the result showed that the purified hEGF in this study could promote the growth of the cell under the concentration of 0.3µg/ml.