Transforming and secreting expression of human egf in mutant strain WYBS2001 of Bacillus and its functions.
Mutant strain WYBS2001 of B. subtilis with strong anti-pathogenic activity was obtained by mutagenic ultraviolet rays. The gene fragment of Human Epidermal Growth Factor(hegf) of 175 bp was synthesized by PCR and the restriction sites Pst I and Hind III, original code and the signal sequence CTTAGA of secreting vector pUS186 were induced in the fragment. The DNA sequencing result revealed that the synthesized fragment was identical with that of human egf. Then the biological engineering strain WYBS2001T with human egf was obtained by transforming pUSE which was constructed by cloning egf into the secreting plasmid pUS186, into mutant strain WYBS2001. The result of RIA showed that hEGF can be found in the supernatant of the cultures and its content was 7.6 ng/ml. And the content can be increased if the proteinase inhibitor was added into the medium. After several generations' culturing, WYBS2001T positive engineering strain can still secrete and express hEGF steadily. The result of experiment showed hEGF had biological activity of proliferation and growth of human cell K562 in vitro. WYBS2001T engineering strain had obvious effect on healing the burned animals' models. This research showed microecological gene-engineering bacteria has good applying foreground.
Wang
Guan-Lin
GL
College of Life Science, Liaoning Normal University, Dalian 116029, China. guanlinwang@163.com
Yi
Qing
Q
Fang
Hong-Jun
HJ
D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002056:Burns / Q000382:microbiology; D002455:Cell Division / Q000187:drug effects; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D014945:Wound Healing
D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D004247:DNA