..I II GST GST

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2 ..I II GST GST

3 FGF FGF-7 GST- GST- 25 IPTG 0.3mmol/L OD h GST- 4.2% 1L 25mg GST- GSH, GSH-Sepharose 4B Heparin-Sepharose CL-6B GST- Heparin -Sepharose CL 6B N- GST 17KD 1L 5.2mg 52% Western Blot MTT 0.1ng/mL 10.0ng/mL 100.0ng/mL 24.5± ±1.2

4 Expression of Keratinocyte Growth Factor in Escherichia.coli and its Purification and Bioactivity ABSTRACT Keratinocyte Growth Factor () is a member of Fibroblast Growth Factor(FGF-7). By acting predominantly in a paracrine manner, can stimulate the proliferation, differentiation and migration of epithelial cells. A serious studies have provided insight into the function of in repair processes of various tissues and organs, including skin, cornea, bowel. In addition, it is a protective factor for gastrointestinal injury induced by radiation and chemotherapy. Interest in producing large amo unt and bioactive through genetic engineering method has been heightened due to the limited amount of in the body. Based on previous work, this paper focus on the expression, purification and bioactive test of for the sake of providing groundwork for future study. By changing the inducing temperature, inducer IPTG concentration, the density of bacteria and inducing time, the satisfying condition for expression of soluble fusion protein GST- could be obtained at mmol/L IPTG, OD 600 equals to 0.97, 5h inducing time. Under the improved condition, the soluble GST- comprised about 4.2% of total cellular protein and 25mg GST- could be harvested in 1L fermentation. A method which combined two-step of affinity chromatography and trypsin incubation was used to achieve purification since the fusion protein GST- could bind to GSH-Sepharose 4B and Heparin-Sepharose CL-6B and Heparin-Sepharose CL-6B protects the active core of from trypsin proteolysis. the 17KD derived from GST- was purified to homogeneity. 5.2mg purified, a total recovery of 52%, was harvested. Western-Blot analysis showed that the purified could be detected by rabbit against human. MTT assay was used to investigate the effect of recombined on the

5 proliferation of mouse corneal epithelial cells, which were cultured through tissue culture method. Results showed that enhanced cellular proliferation in a dose dependent manner: when the concentration of was 0.1ng/mL, it began to show the ability to stimulate the proliferation of culture epithelial cells(p<0.05), when the concentration increased to 10ng/mL, it showed the most intensive proliferative effect on cultured cells. However, this kind of effect decreased when the concentration of increased to 100ng/mL. The effects of were evaluated on the model of scalded wounds in mice. Results showed that the topical application of could accelerate wound healing: the wound area of treated group was smaller than the control one at the same time after scalded; the average healing time was (24.5±1.7) days for treated groups while (21.2±1.2) days for control one. Key Words keratinocyte growth factor expression purification bioactivity

6 [1], [2] PDGF bfgf EGF - GM-CSF TGF-â IGF-I [3] PDGF-ââ FDA [4] 8 bfgf

7 2000 EGF(, ) bfgf EGF EGF [5], 1 mrna bfgf 5 bfgf keratinocyte growth factor 1989 Rubin [6]

8 FGF afgf bfgf FGF-7 [7] 2/3 30%-45% [8] 15 q cdna M H K W I L T W I L P T L L Y R S C F H ATG CAC AAA TGG ATA CTG ACA TCC ATC CTC CCA ACT TTC CTC TAC AGA TCA TGC TTT CAC I I C L V G T I S L A C N D M T P E Q M ATT ATC TCT CTA CTG GGA ACT ATA TCT TTA GCT TGC AAT GAC ATG ACT CCA GAG CAA ATG A T N V N C S S P E R H T R S Y D Y M E GCT ACA AAT GTG AAC TGT TCC AGC CCT GAG CGA CAC ACA AGA AGT TAT GAT TAC ATG GAA G G D I R V R R L F C R T Q W Y L R I D GGA GGG GAT ATA AGA GTG AGA AGA CTC TTC TGT CGA ACA CAG TGG TAC CTG AGG ATC GAT K R G K V K G T Q E M K N N Y N I M E I AAA AGG GAC CCA AGA GAT GAA GAA TAA TTA CAA TAT CAT GGA AAT CAG GAC AGT GGC AGT R T V A V G I V A I K G V E S E F Y L A AGG ACA GTG GCA GTT GGA ATT GTG GCA ATC AAA GGG GTG GAA AGT GAA TTC TAT CTT GCA M N K E G K L Y A K K E C N E D C N F K ATG AAC AAG GAA GGA AAA CTC TAT GCA AAG AAA GAA TGC AAT GAA GAT TGT AAC TTC AAA E L I L E N H Y N T Y A S A K W T H N G GAA CTA ATT CTG GAA AAC CAT TAC AAC ACA TAT GCA TCA GCT AAA TGG ACA CAC AAC GGA G E M F V A L N Q K G I P V R G K K T K GGG GAA ATG TTT GTT GCC TTA AAT CAA AAG GGG ATT CCT GTA AGA GGA AAA AAA ACG AAG 190 K E Q K T A H F L P M A I T AAA GAA CAA AAA ACA GCC CAC TTT CTT CCT ATG GCA ATA ACT TAA [9] 1. cdna Fig. 1 cdna sequence and deduced amino acid sequence of human human, h [10] FGF [11] R FGF FGF 4 FGFR flg FGFR-2(bek) FGFR-3 FGFR-4 R ( K-sam ) FGFR-2 bek [12] R,3 R FGFR-2 IG

9 47% [12] I- 2. R FGFR-2 Fig 2. Structure of human R and FGFR-2 R afgf bfgf FGFR-2 afgf bfgf [13] R IG3 K 25 R R IG3 R [14] K-sam K-sam- KATO- K-sam- FGF [15] Liu [16] R 125 I- afgf bfgf Wordinger [17] IL-1 IL-6 TGF- [18] PDGF TNF IL-1 IL-1 Western Blot mrna Mizel [19] IL-1 Chedid [18] IL-1 IL-1 mrna [20] ; 17- [21] mrna PTHrP PTHrP

10 mrna PTHrP PTHrP PTHrP [22] Zhou [23] -39~ -46bp TGAGGTCAG AFT ATF1 ATF2 Brauchle [24] Chedid [25] mrna IL mrna R mrna [26] [27] camp IL-6 Simonet [28] Alarid [29] Sugimura [30] Lovicu [31]

11 [32] MatsubaraY [33] [34] Parrott [35] mrna / [36] Werner [37] FGF mrna afgf bfgf FGF-5 mrna mrna Staiano-cocio [38] IL-1 IL-1 Werner [39] 60%-80% Sugahara [40]

12 [41] p53 Bax Bc1-x AEC DNA AEC DNA [42] Waters [43] calu-3 H 2 O 2 H 2 O 2 C PKC [44] [45] [46] [47] Johnson [48] 85% DNA Farrell [49] [50] [51] [52] mrna

13 [53 54] D- TNF Senaldi [55] [56] Zheng [57] HPV16 DNA upa tpa Ropiquet [58] PNT1A mrna Zhang [59] mrna mrna Otte [60] mrna R mrna Visco [61] 18 R Ishiwata [62] mrna R mrna ROS (GSH) GSH -S- [63] (GST) mrna Estivariz [64] 3 R mrna

14 mrna [65] [66] [67] [68] 2 cdk2 [69] 4 cdk4 Frank [70] CHL1 Na + - Na - [71] [72] CAD Gassmann [73] mrna Castafnino [74] Balb/MK (NDF) NDF Balb/MK DNA [73]

15 Zsengeller. [75] Bosch. [76] E.coli GVHD GVHD Hill [77] Krijanovski [78] -3~7 LPS TNF2 T GVHD GVHD [79] [80] Gillis [81] R mrna [82] Parrott [83] 1.3

16 Ron [84] pet8c Nco² BamH ² BL21(DE3) 4h 1~2% 21KD 163 Hsu [85] -a -b -a 25~29KD 1.4 h DH5á

17 Degree papers are in the Xiamen University Electronic Theses and Dissertations Database. Full texts are available in the following ways: 1. If your library is a CALIS member libraries, please log on and submit requests online, or consult the interlibrary loan department in your library. 2. For users of non-calis member libraries, please mail to etd@xmu.edu.cn for delivery details.

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