Overexpression Vector Construction Of RhbFGF And High Cell Density Culture Of Its Recombinant Escherichia Coli

Basic fibroblast growth factor is one of FGFs family and widespread in tissues and cells of human and animal body's. Basic FGF have a few biological roles such as potent mitogen, inducing endothelial cell chemo taxis, neutral nutrition, adjusting cell metabolism and proliferation of vascular endothelial cell. Basic FGF has been showedn curative effect in cure wound and soft tissues lesion, proliferation cardiac muscle vascular formation, cure cardiovascular diseases that can not be cured by surgical operation, keeping neuron survival, cure nerve tissue damage, nerve degeneration, cure central lesion caused by cerebrovascular disease and cerebral trauma by recently 20 years researching. Nevertheless, the concentration of bFGF is tiny in tissues, Quantity of bFGF which separation from tissues can not satisfied its study and clinic application. This study is about utilizing technic of Gene engineering and high cell density culture of recombinant Escherichia coli produce non-fusion recombinant hbFGF. It's results are described as follow:At first, according to the principle of Genetic engineering and knowledge of Biochemistry and Molecular Biology, using techniques of Molecular cloning, pointmutation skill and DNASIS software, modified the 5'-end of hbFGF gene and optimize the secondary structure of TIR of hbFGF mRNA for hbFGF translation. The gene encoding human bFGF, which some base in +1 to +35 rang were substituted, be cloned into pET-3c plasmid vector and expressed in recombinant E.coli JB-2. The product was confirmed as native hbFGF by immunity testing, biological role testing and amino acid sequence analysis. Its expression efficiency was 15% of total cell protein.The results of recombinant E.coli JB-2 culture condition studied showed the follow conditions are favor: temperature as 35℃-37℃; medium pH as 6.8-7.1; dissolve oxygen as over 30% in medium.Acetic ion is inhibitor to the cell growth and hbFGF expression of recombinant E.coli JB-2. The results showed that little acetic ion may decrease the cell growth; the higher acetic ion concentration, the lower hbFGF expression efficiency.