CN113354745B - Composition and method for large-scale production of fibroblast growth factor - Google Patents

Abstract

The invention relates to a composition and a method for producing fibroblast growth factor in a large scale. The fibroblast growth factor is fused with the green fluorescent protein and the small ubiquitin-like modifier to realize the soluble large-scale expression of the fibroblast growth factor, and then the small ubiquitin-like modifier protease expressed under the action of the cold shock promoter is promoted to carry out rapid hydrolysis on the fusion protein in cells by utilizing the synergistic effect of the tri (2-carbonyl ethyl) phosphate and the zinc sulfate, so that the production steps are simplified, and the fibroblast growth factor with biological activity is produced on a milligram scale to a kilogram scale.

Description

A kind of composition and method for large-scale production of fibroblast growth factor

technical field

The present invention relates to a composition for fermenting, culturing and purifying fibroblast growth factor, and a fusion protein containing fibroblast growth factor, in particular to a method for large-scale culture and obtaining fibroblast growth factor.

Background technique

Fibroblast growth factor (Fibroblast growth factors, FGFs) superfamily includes 22 members, mainly divided into secretory and intracellular. Secreted fibroblast growth factor (eFGF) includes fibroblast growth factor 1-10 and fibroblast growth factor 16-23, and intracellular fibroblast growth factor (iFGF) includes fibroblast growth factor 11-14. eFGF must activate downstream signaling pathways by binding and activating fibroblast growth factor receptors (FGFRs), members of the cell surface receptor tyrosine kinases (RTKs) family, while iFGF mainly interacts with other proteins to regulate cells Sodium channel switch. In the human body, the fibroblast growth factor superfamily is expressed in almost all tissues, and it plays an extremely important biological function in embryonic development, organ formation and regeneration, tissue repair and the body's metabolism.

Both human and mouse fibroblast growth factor superfamily includes 22 members, fibroblast growth factor-15 and fibroblast growth factor-19 are homologous genes in vertebrates, fibroblast growth factor-15 It has not been found in the human genome, whereas fibroblast growth factor-19, by contrast, has not been found in rats and mice.

Obtaining a large number of active fibroblast growth factors by genetic engineering technology is a necessary condition for the production of fibroblast growth factor-related drugs. Compared with eukaryotic expression systems such as yeast, insect cells and mammalian cells, the use of prokaryotic systems (especially E. coli) to express recombinant proteins is the most simple and economical in terms of time, production costs and site requirements. However, due to the low expression of fibroblasts in prokaryotic systems, the formation of inclusion bodies or the toxicity to the host, it is difficult to produce them on a large scale. In addition, although the expression amount can be increased by the fusion of molecular chaperones, redundant amino acids will appear, resulting in more difficult separation and purification processes and a sharp increase in production costs. All of the above have a serious impact on the druggability of fibroblast growth factor.

SUMMARY OF THE INVENTION

One of the present inventions provides a composition comprising tris(2-carbonylethyl)phosphorus hydrochloride and zinc sulfate.

In a specific embodiment, the mass ratio of the tris(2-carbonylethyl)phosphate hydrochloride to the zinc sulfate is 1:(1 to 50).

The second of the present invention provides the application of the composition according to the one of the present invention in the production of fibroblast growth factor, especially the application of large-scale production of fibroblast growth factor.

The third aspect of the present invention provides a fusion protein containing fibroblast growth factor, which includes a visual reporter protein from N-terminus to C-terminus, Small Ubiquitin-like Modifier (SUMO) and fibroblast growth factor factor.

In a specific embodiment, the fibroblast growth factor is human fibroblast growth factor-1, human fibroblast growth factor-2, human fibroblast growth factor-3, human fibroblast growth factor factor-4, human fibroblast growth factor-5, human fibroblast growth factor-6, human fibroblast growth factor-7, human fibroblast growth factor-8, human fibroblast growth factor-9, human fibroblast growth factor-10, human fibroblast growth factor-11, human fibroblast growth factor-12, human fibroblast growth factor-13, human fibroblast growth factor-14, human fibroblast growth factor-16, human fibroblast growth factor-17, human fibroblast growth factor-18, human fibroblast growth factor-19, human fibroblast growth One of factor-20, human fibroblast growth factor-21, human fibroblast growth factor-22, and human fibroblast growth factor-23.

In a specific embodiment, the amino acid sequence of the human fibroblast growth factor-1 is shown in SEQ ID No. 1, and the amino acid sequence of the human fibroblast growth factor-2 is shown in SEQ ID No. 2 shown, the amino acid sequence of the human fibroblast growth factor-3 is shown in SEQ ID No.3, the amino acid sequence of the human fibroblast growth factor-4 is shown in SEQ ID No.4, the The amino acid sequence of human fibroblast growth factor-5 is shown in SEQ ID No. 5, the amino acid sequence of human fibroblast growth factor-6 is shown in SEQ ID No. 6, and the human fibroblast The amino acid sequence of growth factor-7 is shown in SEQ ID No. 7, the amino acid sequence of the human fibroblast growth factor-8 is shown in SEQ ID No. 8, and the human fibroblast growth factor-9 The amino acid sequence is shown in SEQ ID No.9, the amino acid sequence of the human fibroblast growth factor-10 is shown in SEQ ID No.10, and the amino acid sequence of the human fibroblast growth factor-11 is shown in Shown in SEQ ID No. 11, the amino acid sequence of the human fibroblast growth factor-12 is shown in SEQ ID No. 12, and the amino acid sequence of the human fibroblast growth factor-13 is shown in SEQ ID No. 13, the amino acid sequence of the human fibroblast growth factor-14 is shown in SEQ ID No. 14, and the amino acid sequence of the human fibroblast growth factor-16 is shown in SEQ ID No. 15. The amino acid sequence of the human fibroblast growth factor-17 is shown in SEQ ID No. 16, and the amino acid sequence of the human fibroblast growth factor-18 is shown in SEQ ID No. 17. The amino acid sequence of fibroblast growth factor-19 is shown in SEQ ID No. 18, the amino acid sequence of the human fibroblast growth factor-20 is shown in SEQ ID No. 19, the human fibroblast growth factor The amino acid sequence of -21 is shown in SEQ ID No. 20, the amino acid sequence of the human fibroblast growth factor-22 is shown in SEQ ID No. 21, and the amino acid sequence of the human fibroblast growth factor-23 The sequence is shown in SEQ ID No.22.

In a specific embodiment, the visualization reporter protein is a fluorescent protein.

In a specific embodiment, the visual reporter protein is one of green fluorescent protein, red fluorescent protein, blue fluorescent protein and cyan fluorescent protein; preferably, the amino acid sequence of the green fluorescent protein is as shown in SEQ ID No. 23 shown.

In a specific embodiment, the amino acid sequence of the small ubiquitin-like modifier is shown in SEQ ID No.24.

In a specific embodiment, the visualization reporter protein and the small ubiquitin-like modifier are linked by a polypeptide linker.

In a specific embodiment, the amino acid sequence of the polypeptide linker is shown in SEQ ID No.25.

The fourth aspect of the present invention provides an expression cassette for expressing the fusion protein according to any one of the third aspect of the present invention, comprising a first promoter from the 5' end to the 3' end, an expression cassette encoding the fusion protein Nucleic acid and first terminator. Wherein, RBS is further included downstream of the first promoter and upstream of the nucleic acid encoding the fusion protein, and the nucleotide sequence of the RBS is shown in SEQ ID No.53. An enzyme cleavage site may also be included downstream of the first promoter and upstream of the nucleic acid encoding the fusion protein. Wherein, the enzyme cleavage site is located downstream of the RBS.

In a specific embodiment, the first promoter is a T7 promoter, and the first terminator is a T7 terminator.

In a specific embodiment, the nucleotide sequence encoding the human fibroblast growth factor-1 is shown in SEQ ID No. 26, and the nucleotide sequence encoding the human fibroblast growth factor-2 is shown in SEQ ID No. 26 ID No. 27, the nucleotide sequence encoding the human fibroblast growth factor-3 is shown in SEQ ID No. 28, the nucleotide sequence encoding the human fibroblast growth factor-4 As shown in SEQ ID No. 29, the nucleotide sequence encoding the human fibroblast growth factor-5 is shown in SEQ ID No. 30, and the nucleoside encoding the human fibroblast growth factor-6 The acid sequence is shown in SEQ ID No. 31, the nucleotide sequence encoding the human fibroblast growth factor-7 is shown in SEQ ID No. 32, and the nucleotide sequence encoding the human fibroblast growth factor-8 is shown in SEQ ID No. 32. The nucleotide sequence is shown in SEQ ID No. 33, and the nucleotide sequence encoding the human fibroblast growth factor-9 is shown in SEQ ID No. 34, which encodes the human fibroblast growth factor- The nucleotide sequence of 10 is shown in SEQ ID No. 35, and the nucleotide sequence encoding the human fibroblast growth factor-11 is shown in SEQ ID No. 36, which encodes the growth of human fibroblasts The nucleotide sequence of factor-12 is shown in SEQ ID No. 37, and the nucleotide sequence encoding the human fibroblast growth factor-13 is shown in SEQ ID No. 38, which encodes the human fibroblasts The nucleotide sequence of growth factor-14 is shown in SEQ ID No. 39, and the nucleotide sequence encoding the human fibroblast growth factor-16 is shown in SEQ ID No. 40, which encodes the human fibroblast growth factor-16. The nucleotide sequence of fibroblast growth factor-17 is shown in SEQ ID No. 41, and the nucleotide sequence encoding the human fibroblast growth factor-18 is shown in SEQ ID No. 42, which encodes the human fibroblast growth factor-18. The nucleotide sequence of the source fibroblast growth factor-19 is shown in SEQ ID No. 43, and the nucleotide sequence encoding the human fibroblast growth factor-20 is shown in SEQ ID No. 44, which encodes the The nucleotide sequence of human fibroblast growth factor-21 is shown in SEQ ID No. 45, and the nucleotide sequence encoding the human fibroblast growth factor-22 is shown in SEQ ID No. 46, encoding The nucleotide sequence of the human fibroblast growth factor-23 is shown in SEQ ID No.47.

In a specific embodiment, the nucleotide sequence encoding the visualization reporter protein is shown in SEQ ID No.48.

In a specific embodiment, the nucleotide sequence encoding the small ubiquitin-like modifier is shown in SEQ ID No.49.

In a specific embodiment, the nucleotide sequence encoding the polypeptide linker is shown in SEQ ID No.50.

The fifth invention provides a set for expressing the fusion protein according to any one of the third invention or for expressing the fusion protein according to any one of the third invention and purifying the growth of the fibroblasts The expression cassette of the factor, which includes the expression cassette according to the present invention 4 and the expression cassette for expressing a small ubiquitin-like modifier protease (SUMO protease, such as Ulp1).

In a specific embodiment, the expression cassette for expressing small ubiquitin-like modifier protease comprises a second promoter from the 5' end to the 3' end, a 3'-UTR, encoding a small ubiquitin-like modifier protease Nucleic acids and 5'-UTRs.

In a specific embodiment, the second promoter is a CspA promoter, the 3'-UTR is CspA 3'-UTR, and the 5'-UTR is CspA 5'-UTR.

In a specific embodiment, the amino acid sequence of the small ubiquitin-like modifier protease is shown in SEQ ID No.51.

In a specific embodiment, the nucleotide sequence encoding the small ubiquitin-like modifier protease is shown in SEQ ID No.52.

The sixth aspect of the present invention provides a method for obtaining the fusion protein or the fibroblast growth factor described in any one of the third aspect of the present invention, which comprises the following steps:

1-1) Connect the expression cassette according to the fourth aspect of the present invention or the nucleic acid encoding the fusion protein to a first expression vector to obtain a first recombinant expression vector; wherein, the first expression vector is isopropyl -β-D-thiogalactoside (IPTG) inducible expression vector;

2-1) Linking the expression cassette for expressing small ubiquitin-like modifier protease or the nucleic acid encoding small ubiquitin-like modifier protease in the expression cassette according to the fifth aspect of the present invention to the first recombinant On the expression vector, a second recombinant expression vector is obtained;

3-1) transforming the second recombinant expression vector into the first host to obtain the first recombinant organism;

4-1) culturing the first recombinant organism in a first culture solution until the fusion protein and the small ubiquitin-like modifier protease are expressed to obtain a first culture;

5-1) purifying the first culture to obtain the fibroblast growth factor;

or

1-2) Same as 1-1);

2-2) Linking the expression cassette for expressing small ubiquitin-like modifier protease or the nucleic acid encoding small ubiquitin-like modifier protease in the expression cassette according to the fifth aspect of the present invention to the second expression On the vector, a third recombinant expression vector is obtained;

3-2) transforming the first recombinant expression vector into a second host to obtain a second recombinant organism; transforming the third recombinant expression vector into a third host to obtain a third recombinant organism;

4-2) Culturing the second recombinant organism in the second culture medium to express the fusion protein to obtain a second culture; culturing the third recombinant organism in the third culture medium to express the small Ubiquitin-like modifier protease to obtain a third culture; mixing the second culture and the third culture to obtain a fourth culture;

5-2) purifying the fourth culture to obtain the fibroblast growth factor;

or

1-3) Same as 1-1);

2-3) In the same 3-2), the first recombinant expression vector is transformed into the second host to obtain the second recombinant organism;

3-3) culturing the second recombinant organism in the second culture medium to express the fusion protein to obtain a second culture;

4-3) Purify the second culture to obtain the fusion protein.

In a specific embodiment, the first culture fluid, the second culture fluid and the third culture fluid are independently LB culture fluid or glucose-containing LB culture fluid.

In a specific embodiment, the first expression vector may be one of the expression vectors of the pET series.

In a specific embodiment, the first expression vector is one of pET21a, pET20b and pET28a;

The second expression vector is pCold, which can be purchased from Dalian Bao Bioengineering Co., Ltd. (article number: 3360);

The first host, the second host and the third host are independently Escherichia coli; preferably, the strain of Escherichia coli is BL21 (DE3).

In a specific embodiment, in step 4-2) or step 3-3), the temperature for culturing the second recombinant organism is 28 to 37° C. for 4 to 8 hours;

In step 4-1), the first recombinant organism is cultured under the induction of isopropyl-β-D-thiogalactoside (IPTG) at a temperature of 28 to 37° C. for 4 to 8 hours, Then continue culturing the first recombinant organism for 15 to 60 minutes at a temperature of 10 to 15°C;

In step 4-2), the temperature of culturing the third recombinant organism is 10 to 15° C. and the time is 15 to 60 minutes.

In a specific embodiment, in step 4-2) or step 3-3), the temperature for culturing the second recombinant organism is 32 to 37° C. for 4 to 6 hours;

In step 4-1), the first recombinant organism is cultured under the induction of IPTG at a temperature of 32 to 37°C for 4 to 6 hours, and then continues to be cultured at a temperature of 10 to 15°C. 15 to 30 minutes for recombinant organisms;

In step 4-2), the temperature of culturing the third recombinant organism is 10 to 15° C. and the time is 15 to 30 minutes.

In a specific embodiment, in step 4-1), when the culture temperature drops to the 10 to 15° C., the composition according to one of the present inventions is added to the first culture solution.

In a specific embodiment, the final concentration of the tris(2-carbonylethyl) phosphate hydrochloride in the first culture solution is 0.1 to 1 mmol/L; the zinc sulfate in the first culture solution The final concentration in the liquid is 1 to 5 mmol/L.

In a specific embodiment, in step 4-2), the composition according to one of the present inventions is added to the second culture medium.

In a specific embodiment, the final concentration of the tris(2-carbonylethyl) phosphate hydrochloride in the second culture solution is 0.1 to 1 mmol/L; the zinc sulfate in the second culture solution The final concentration in the liquid is 1 to 5 mmol/L.

In addition, in a specific embodiment, purified fibroblast growth factor-1, fibroblast growth factor-2, fibroblast growth factor-3, fibroblast growth factor-4, fibroblast growth factor- 5. Fibroblast growth factor-6, fibroblast growth factor-7, fibroblast growth factor-10, fibroblast growth factor-11, fibroblast growth factor-12, fibroblast growth factor-13, fibroblast growth factor-14, fibroblast growth factor-16, fibroblast growth factor-18, fibroblast growth factor-19, fibroblast growth factor-20, fibroblast growth factor-21, fibroblasts The activities of cell growth factor-22 and fibroblast growth factor-23 were analyzed accordingly, and the results showed that they all showed their respective activities.

Beneficial effects of the present invention:

Aiming at the problems encountered in prokaryotic expression in the prior art and in order to further improve the quality control accuracy in the process of fibroblast growth factor expression, the present invention provides a composition, and designs a composition containing two Recombinant Escherichia coli expression plasmids with gene expression cassettes of different inducible expression modes, one of which is used to express fusion proteins containing fibroblast growth factor, and fusion of fibroblast growth factor with fluorescent protein and SUMO is beneficial to the large amount of soluble fusion protein expression At the same time, the expression of the protein can be monitored visually by using, for example, green fluorescence produced by green fluorescent protein; the other is used to express the small ubiquitin-like modifier protease. After the induction of the fusion protein is completed, adding the compound combination of the present invention during the low-temperature culture process can realize the hydrolysis effect of the small ubiquitin-like modifier protease on the fusion protein while being induced to express, so as to achieve soluble and natural fibroblasts. For the purpose of large-scale production of fibroblast growth factors with identical amino acid sequences of cell growth factors and corresponding biological functions, the method of the present invention can not only obtain fibroblast growth factors with biological functions, but also greatly simplify the purification steps , significantly reduce the production cost, so it can meet the needs of the production of medicines, daily necessities and scientific research reagents.

The advantages of the present invention are: 1) through visual green fluorescence, the soluble expression and expression level of the recombinant fusion protein can be evaluated or monitored in real time, and in order to achieve the above purpose, the conventional method used requires at least 6 hours; 2) with other Different protease recognition sites, SUMO protease recognizes the three-dimensional structure of SUMO, therefore, there is no random cleavage (hydrolysis); in addition, SUMO protease hydrolysis will not bring any redundant amino acids into the target protein sequence. , so that the fibroblast growth factor consistent with the natural protein sequence can be obtained; 3) the fibroblast growth factor that is difficult-to-expressed in the E. coli host can be soluble by using the unique fusion protein of the present invention Furthermore, the synergistic effect between the compositions of the present invention realizes the rapid hydrolysis of the fusion protein while promoting the expression of SUMO protease in cells for the first time, thus greatly simplifying the production and purification process of fibroblast growth factor. Compared with the time required by the conventional method, the present invention can shorten the purification time by at least 8 to 48 hours; 4) adopt the process of the present invention, without complicated operation technology and special equipment, only need to expand the production scale, such as fermentation scale, The production of fibroblast growth factors ranging from milligrams to kilograms can be achieved by the scale of bacterial fragmentation, centrifugal equipment, and gel chromatography.

Description of drawings

Figure 1 Schematic diagram of the pET-GSFGF9-CspA-Ulp1 recombinant plasmid map

Figure 2 Fluorescence observation after induction of pET-GSFGF17-CspA-Ulp1/BL21 expression

A. The observation results of the fermentation broth under natural light, in which the fermentation broth in the left test tube (pET-GSFGF17-CspA-Ulp1/BL21IPTG induced expression) showed green fluorescence, and the right test tube (pET-GSFGF17-CspA-Ulp1/ The fermentation broth before BL21IPTG induction expression) does not show green fluorescence; B. The observation results of the fermentation broth after pET-GSFGF17-CspA-Ulp1/BL21 IPTG induction expression under UV light.

Figure 3. Expression analysis of fusion protein GFP-SUMO-FGFn

A. Lane 1: total protein of pET-GSFGF8-CspA-Ulp1/BL21 after IPTG induction; Lane 2: total protein of pET-GSFGF9-CspA-Ulp1/BL21 after IPTG induction; Lane 3: pET-GFP after IPTG induction -SUMO/BL21 total protein; Lane M: protein molecular weight marker; Lane 4: pET-GSFGF8-CspA-Ulp1/BL21 total protein before IPTG induction; Lane 5: pET-GSFGF3-CspA-Ulp1/BL21 total protein after IPTG induction protein; lane 6: total protein of pET-GSFGF16-CspA-Ulp1/BL21 after IPTG induction; lane 7: total protein of pET-GSFGF17-CspA-Ulp1/BL21 after IPTG induction; lane 8: pET-GSFGF19 after IPTG induction -CspA-Ulp1/BL21 total protein; Lane 9: pET-GSFGF20-CspA-Ulp1/BL21 total protein after IPTG induction;

B. Lane 1: total protein of pET-GSFGF4-CspA-Ulp1/BL21 after IPTG induction; Lane 2: total protein of pET-GSFGF6-CspA-Ulp1/BL21 after IPTG induction; Lane 3: pET-GSFGF7 after IPTG induction -CspA-Ulp1/BL21 total protein; Lane 4: pET-GFP-SUMO/BL21 total protein after IPTG induction; Lane M: protein molecular weight marker; Lane 5: pET-GSFGF4-CspA-Ulp1/BL21 total protein before IPTG induction protein; lane 6: pET-GSFGF10-CspA-Ulp1/BL21 total protein after IPTG induction; lane 7: pET-GSFGF18-CspA-Ulp1/BL21 total protein after IPTG induction; lane 8: pET-GSFGF21 after IPTG induction -CspA-Ulp1/BL21 total protein; Lane 9: pET-GSFGF22-CspA-Ulp1/BL21 total protein after IPTG induction;

C. Lane 1: total protein of pET-GSFGF11-CspA-Ulp1/BL21 after IPTG induction; Lane 2: total protein of pET-GSFGF23-CspA-Ulp1/BL21 after IPTG induction; Lane 3: pET-GSFGF5 after IPTG induction -CspA-Ulp1/BL21 total protein; Lane 4: pET-GFP-SUMO/BL21 total protein after IPTG induction; Lane M: protein molecular weight marker; Lane 5: pET-GSFGF11-CspA-Ulp1/BL21 total protein before IPTG induction protein; lane 6: total protein of pET-GSFGF12-CspA-Ulp1/BL21 after IPTG induction; lane 7: total protein of pET-GSFGF13-CspA-Ulp1/BL21 after IPTG induction; lane 8: pET-GSFGF14 after IPTG induction -CspA-Ulp1/BL21 total protein.

Fig. 4 Solubilization analysis of fusion protein GFP-SUMO-FGFn after IPTG induction

A: Lane 1: pET-GSFGF8-CspA-Ulp1/BL21 total protein; Lane 2: protein in pET-GSFGF8-CspA-Ulp1/BL21 supernatant; Lane 3: pET-GSFGF8-CspA-Ulp1/BL21 pellet Lane M: protein molecular weight marker; Lane 4: pET-GSFGF5-CspA-Ulp1/BL21 total protein; Lane 5: pET-GSFGF5-CspA-Ulp1/BL21 supernatant protein; Lane 6: pET-GSFGF5 - protein in CspA-Ulp1/BL21 pellet; lane 7: pET-GSFGF23-CspA-Ulp1/BL21 total protein; lane 8: protein in pET-GSFGF23-CspA-Ulp1/BL21 supernatant; lane 9: pET- Protein in GSFGF23-CspA-Ulp1/BL21 precipitates;

B: Lane 1: pET-GSFGF4-CspA-Ulp1/BL21 total protein; Lane 2: protein in pET-GSFGF4-CspA-Ulp1/BL21 supernatant; Lane 3: pET-GSFGF4-CspA-Ulp1/BL21 pellet Lane M: protein molecular weight marker; Lane 4: pET-GSFGF16-CspA-Ulp1/BL21 total protein; Lane 5: pET-GSFGF16-CspA-Ulp1/BL21 supernatant protein; Lane 6: pET-GSFGF16 - protein in CspA-Ulp1/BL21 pellet; lane 7: pET-GSFGF14-CspA-Ulp1/BL21 total protein sample; lane 8: protein in pET-GSFGF14-CspA-Ulp1/BL21 supernatant; lane 9: pET - protein in GSFGF14-CspA-Ulp1/BL21 pellets;

C: Lane 1: pET-GSFGF11-CspA-Ulp1/BL21 total protein; Lane 2: protein in pET-GSFGF11-CspA-Ulp1/BL21 supernatant; Lane 3: pET-GSFGF11-CspA-Ulp1/BL21 in pellet Lane 4: pET-GSFGF12-CspA-Ulp1/BL21 total protein; Lane 5: pET-GSFGF12-CspA-Ulp1/BL21 supernatant protein; Lane 6: pET-GSFGF12-CspA-Ulp1/BL21 precipitate Proteins in ; Lane M: protein molecular weight markers;

D: lane 1: total protein of pET-GSFGF19-CspA-Ulp1/BL21; lane 2: protein in supernatant of pET-GSFGF19-CspA-Ulp1/BL21; lane 3: in pellet of pET-GSFGF19-CspA-Ulp1/BL21 Lane M: protein molecular weight marker; Lane 4: pET-GSFGF17-CspA-Ulp1/BL21 total protein; Lane 5: pET-GSFGF17-CspA-Ulp1/BL21 protein in supernatant; Lane 6: pET-GSFGF17 - Protein in CspA-Ulp1/BL21 pellets.

Figure 5. Functional analysis of tris(2-carbonylethyl)phosphate hydrochloride, zinc sulfate and combinations on proteolytic fusion proteins of small ubiquitin-like modifiers

Black arrows indicate fusion protein GFP-SUMO-FGF12; gray arrows indicate GFP-SUMO hydrolyzed by fusion protein GFP-SUMO-FGF12; hollow arrows indicate fibroblast growth factor-hydrolyzed fusion protein GFP-SUMO-FGF12- 12.

A: Shake flask 1 and flask I, the effect of adding a final concentration of 5 mmol/L zinc sulfate at the beginning of low temperature induction at 15°C on the hydrolysis of fusion protein of small ubiquitin-like modifier protease

Lane 1: pET-GSFGF12-CspA-Ulp1/BL21 induced by low temperature for 30 minutes; Lane 2: induced by low temperature of pET-GSFGF12-CspA-Ulp1/BL21 for 60 minutes; Lane 3: induced by low temperature of pET-GSFGF12-CspA-Ulp1/BL21 for 120 minutes ; Lane 4: pET-GFP-SUMO/BL21 induced at low temperature for 120 minutes;

B: Shake flask 2, the effect of adding tris(2-carbonylethyl) phosphate hydrochloride at a final concentration of 1 mmol/L at the beginning of low temperature induction at 15°C on the hydrolysis of fusion protein of small ubiquitin-like modifier protease

Lane 1: low temperature induction for 0 minutes; lane 2: low temperature induction for 15 minutes; lane 3: low temperature induction for 30 minutes; lane 4: low temperature induction for 60 minutes; lane 5: low temperature induction for 90 minutes; lane 6: low temperature induction for 120 minutes;

C: Shake flask 3, 15°C low temperature induction was initiated by adding a final concentration of 1 mmol/L tris(2-carbonylethyl) phosphate hydrochloride and 1 mmol/L zinc sulfate in combination for small ubiquitin-like modifiers Effects of proteases on hydrolysis of fusion proteins

Lane 1: low temperature induction for 0 minutes; lane 2: low temperature induction for 15 minutes; lane 3: low temperature induction for 30 minutes; lane 4: low temperature induction for 60 minutes; lane 5: low temperature induction for 90 minutes; lane 6: low temperature induction for 120 minutes;

D: Shake flask 4, 15°C low temperature induction was initiated by adding a final concentration of 1 mmol/L tris(2-carbonylethyl) phosphate hydrochloride and 3 mmol/L zinc sulfate in combination to small ubiquitin-like modifiers Effects of proteases on hydrolysis of fusion proteins

Lane 1: low temperature induction for 0 minutes; lane 2: low temperature induction for 15 minutes; lane 3: low temperature induction for 30 minutes; lane 4: low temperature induction for 60 minutes; lane 5: low temperature induction for 90 minutes; lane 6: low temperature induction for 120 minutes;

E: Shake flask 5, 15°C low temperature induction start by adding a final concentration of 0.1 mmol/L tris(2-carbonylethyl) phosphate hydrochloride and 5 mmol/L zinc sulfate in combination to small ubiquitin-like modifiers Effects of protease hydrolysis of fusion proteins

Lane 1: low temperature induction for 0 minutes; lane 2: low temperature induction for 15 minutes; lane 3: low temperature induction for 30 minutes; lane 4: low temperature induction for 60 minutes; lane 5: low temperature induction for 90 minutes; lane 6: low temperature induction for 120 minutes.

Figure 6 Tris(2-carbonylethyl) phosphate hydrochloride and zinc sulfate combination promote the proteolysis of other fusion proteins GFP-SUMO-FGFn by small ubiquitin-like modifiers

During low temperature induction at 15°C, final concentrations of 0.1 mmol/L tris(2-carbonylethyl) phosphate hydrochloride and 5 mmol/L zinc sulfate were added to observe the effect of small ubiquitin-like modifier protease on GFP-SUMO- FGFn fusion proteolytic capacity.

Lane M: protein molecular weight marker; Lane 1: pET-GSFGF9-CspA-Ulp1/BL21 hypothermic induction for 0 min; Lane 2: pET-GSFGF9-CspA-Ulp1/BL21 hypothermic induction for 30 min; Lane 3: pET-GSFGF8-CspA- Ulp1/BL21 induced by low temperature for 30 minutes; Lane 4: pET-GSFGF16-CspA-Ulp1/BL21 induced by low temperature for 30 minutes; Lane 5: induced by low temperature of pET-GSFGF17-CspA-Ulp1/BL21 for 30 minutes; Lane 6: pET-GSFGF19-CspA -Ulp1/BL21 hypothermic induction for 30 minutes.

Figure 7 Hybridization analysis of fusion protein GFP-SUMO-FGFn before and after hydrolysis

Solid arrows indicate fusion protein GFP-SUMO-FGFn; open arrows indicate fibroblast growth factor-n.

A: Lane M: protein molecular weight marker; Lane 1: pET-GSFGF9-CspA-Ulp1/BL21 before IPTG induction; Lane 2: pET-GSFGF9-CspA-Ulp1/BL21 low temperature induction for 30 minutes; Lane 3: pET-GSFGF9-CspA -Ulp1/BL21 low temperature induction starts at 0 minutes;

B: Lane M: protein molecular weight marker; Lane 1: pET-GSFGF18-CspA-Ulp1/BL21 induced by low temperature for 30 minutes; Lane 2: pET-GSFGF18-CspA-Ulp1/BL21 induced by low temperature for 5 minutes; Lane 3: pET-GSFGF18- CspA-Ulp1/BL21 low temperature induction started at 0 minutes.

Figure 8 Analysis of the purification process of fibroblast growth factor-17 (FGF-17)

A: CM ion chromatography gel for separation of FGF-17, wherein, lane 1: buffer 1 containing 0.1M sodium chloride; lane 2: buffer 2 containing 0.3M sodium chloride: lanes 3 and 4: containing 0.6 M NaCl in buffer 3; Lane 4: Buffer 6 containing 1.0 mol/L NaCl; Lane M: protein molecular weight marker;

B: Heparin affinity chromatography gel-purified FGF-17, where lanes 1 and 2: buffer 4 containing 1.2 mol/L sodium chloride.

Figure 9 Activity analysis of fibroblast growth factor promoting Leydig cell proliferation

Detailed ways

The above content of the present invention will be further described in detail below in the form of preferred embodiments, but it does not constitute a limitation to the present invention.

Unless otherwise specified, the reagents, enzymes, etc. in the examples of the present invention can be purchased through commercial channels.

E. coli pET-21a expression vector (Cat. No. 69740) and E. coli BL21(DE3) (Cat. No. 69450) were purchased from Sigma-Aldrich Company.

pCold (Item No. 3360) was purchased from Bao Bioengineering (Dalian) Co., Ltd.

Heparin affinity chromatography gels (Cat. No. 17-0998-03) and ion chromatography gels (CM Sepharose Fast Flow, Cat. No. 17-0719-01; Q Sepharose Fast Flow, Cat. No. 17) used to purify the fusion protein -0510-01) were purchased from GE Corporation of the United States.

The amino acid sequence of human fibroblast growth factor-1 is shown in SEQ ID No. 1, the amino acid sequence of human fibroblast growth factor-2 is shown in SEQ ID No. 2, and the human fibroblast growth factor- The amino acid sequence of 3 is shown in SEQ ID No.3, the amino acid sequence of human fibroblast growth factor-4 is shown in SEQ ID No.4, and the amino acid sequence of human fibroblast growth factor-5 is shown in SEQ ID No. .5, the amino acid sequence of human fibroblast growth factor-6 is shown in SEQ ID No. 6, the amino acid sequence of human fibroblast growth factor-7 is shown in SEQ ID No. 7, and the human The amino acid sequence of fibroblast growth factor-8 is shown in SEQ ID No. 8, the amino acid sequence of human fibroblast growth factor-9 is shown in SEQ ID No. 9, and the amino acid sequence of human fibroblast growth factor-10 The sequence is shown in SEQ ID No.10, the amino acid sequence of human fibroblast growth factor-11 is shown in SEQ ID No.11, and the amino acid sequence of human fibroblast growth factor-12 is shown in SEQ ID No.12. The amino acid sequence of human fibroblast growth factor-13 is shown in SEQ ID No. 13, the amino acid sequence of human fibroblast growth factor-14 is shown in SEQ ID No. 14, and the human fibroblast growth factor The amino acid sequence of -16 is shown in SEQ ID No. 15, the amino acid sequence of human fibroblast growth factor-17 is shown in SEQ ID No. 16, and the amino acid sequence of human fibroblast growth factor-18 is shown in SEQ ID No. 16 As shown in No. 17, the amino acid sequence of human fibroblast growth factor-19 is shown in SEQ ID No. 18, and the amino acid sequence of human fibroblast growth factor-20 is shown in SEQ ID No. 19. The amino acid sequence of fibroblast growth factor-21 is shown in SEQ ID No. 20, the amino acid sequence of human fibroblast growth factor-22 is shown in SEQ ID No. 21, and the amino acid sequence of human fibroblast growth factor-23 is shown in SEQ ID No. 21. The amino acid sequence is shown in SEQ ID No.22.

The amino acid sequence of green fluorescent protein is shown in SEQ ID No. 23; the amino acid sequence of small ubiquitin-like modifier is shown in SEQ ID No. 24; the amino acid of the polypeptide linker between green fluorescent protein and small ubiquitin-like modifier The sequence is shown in SEQ ID No.25.

The nucleotide sequence encoding human fibroblast growth factor-1 is shown in SEQ ID No. 26, and the nucleotide sequence encoding human fibroblast growth factor-2 is shown in SEQ ID No. 27, encoding human The nucleotide sequence of fibroblast growth factor-3 is shown in SEQ ID No. 28, and the nucleotide sequence of encoding human fibroblast growth factor-4 is shown in SEQ ID No. 29, which encodes human fibroblasts The nucleotide sequence of growth factor-5 is shown in SEQ ID No. 30, and the nucleotide sequence of encoding human fibroblast growth factor-6 is shown in SEQ ID No. 31, which encodes human fibroblast growth factor The nucleotide sequence of -7 is shown in SEQ ID No. 32, and the nucleotide sequence encoding human fibroblast growth factor-8 is shown in SEQ ID No. 33, which encodes human fibroblast growth factor-9 The nucleotide sequence is shown in SEQ ID No. 34, the nucleotide sequence encoding human fibroblast growth factor-10 is shown in SEQ ID No. 35, and the nucleotide sequence encoding human fibroblast growth factor-11 is shown in SEQ ID No. 35. The nucleotide sequence is shown in SEQ ID No. 36, the nucleotide sequence encoding human fibroblast growth factor-12 is shown in SEQ ID No. 37, and the nucleotide sequence encoding human fibroblast growth factor-13 The sequence is shown in SEQ ID No. 38, the nucleotide sequence encoding human fibroblast growth factor-14 is shown in SEQ ID No. 39, and the nucleotide sequence encoding human fibroblast growth factor-16 is shown in SEQ ID No. 39 ID No. 40, the nucleotide sequence encoding human fibroblast growth factor-17 is shown in SEQ ID No. 41, and the nucleotide sequence encoding human fibroblast growth factor-18 is shown in SEQ ID No. 42, the nucleotide sequence encoding human fibroblast growth factor-19 is shown in SEQ ID No. 43, and the nucleotide sequence encoding human fibroblast growth factor-20 is shown in SEQ ID No. 44 As shown, the nucleotide sequence encoding human fibroblast growth factor-21 is shown in SEQ ID No.45, and the nucleotide sequence encoding human fibroblast growth factor-22 is shown in SEQ ID No.46 , the nucleotide sequence encoding human fibroblast growth factor-23 is shown in SEQ ID No.47.

The nucleotide sequence encoding green fluorescent protein is shown in SEQ ID No.48; the nucleotide sequence encoding small ubiquitin-like modifier is shown in SEQ ID No.49; the nucleotide sequence encoding green fluorescent protein and small ubiquitin-like modifier is shown in SEQ ID No.49 The nucleotide sequence of the polypeptide linker in between is shown in SEQ ID No.50.

The amino acid sequence of the small ubiquitin-like modifier protease is shown in SEQ ID No.51; the nucleotide sequence of the gene encoding the small ubiquitin-like modifier protease is shown in SEQ ID No.52. The CspA promoter, CspA 3'-UTR and CspA 5'-UTR are located on the pCold vector.

Example 1 Construction of prokaryotic expression vector of visualized fibroblast growth factor fusion protein and acquisition of recombinant strain

Design fusion protein GFP-SUMO-FGFn (where n is 1 to 23, and n is not 15, which is consistent with n in human fibroblast growth factor-n) from N-terminal to C-terminal, green fluorescent protein, polypeptide Linker, small ubiquitin-like modifier and human fibroblast growth factor-n (wherein n is 1 to 23, and n is not 15), the amino acid sequences of each part are as described above; the nucleic acid encoding the fusion protein starts from 5' From the end to the 3' end are green fluorescent protein, polypeptide linker, small ubiquitin-like modifier and human fibroblast growth factor-n (wherein n is 1 to 23, and n is not 15), the nucleosides of each part Acid sequences are as described above.

The nucleic acid encoding the fusion protein GFP-SUMO-FGFn was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and then the synthesized nucleic acid fragments were cloned into the E. coli expression plasmid vector pET-21a by genetic engineering methods to obtain a recombinant plasmid, which was transformed into Escherichia coli. The correctness of the nucleic acid sequence is checked by nucleic acid sequencing. The recombinant plasmid containing the nucleic acid encoding the fusion protein GFP-SUMO-FGFn was named pET-GFP-SUMO-hFGFn (n was 1 to 23, respectively, and n was not 15).

The recombinant plasmid pET-GFP-SUMO-hFGFn was transformed into E. coli host BL21 (DE3), and the positive recombinant bacteria were screened by ampicillin resistance, and named pET-GFP-SUMO-hFGFn/BL21 respectively.

Example 2 Construction of prokaryotic expression vector of small ubiquitin-like modifier protease Ulp1 and acquisition of recombinant strain

The amino acid sequence of the small ubiquitin-like modifier protease Ulp1 and the nucleotide sequence of the nucleic acid encoding the same are described above. The nucleic acid encoding the small ubiquitin-like modifier protease Ulp1 was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and then the synthesized nucleic acid fragment was cloned into the E. coli expression plasmid vector pCold by genetic engineering to obtain a recombinant plasmid, which was transformed into E. coli . The correctness of the nucleic acid sequence was checked by nucleic acid sequencing, and the correct recombinant plasmid was named pCold-Ulp1.

The pCold-Ulp1 plasmid was digested with Pfo I to obtain a DNA fragment containing the CspA promoter, 3'-UTR, nucleic acid encoding small ubiquitin-like modifier protease and 5'-UTR small ubiquitin-like modifier protease expression cassette , and then use T4 polymerase to fill in the end of the restriction site of the DNA fragment of the expression cassette to form a blunt end. The pET-GFP-SUMO-hFGFn was digested with the blunt-end restriction endonuclease EcoICRI to obtain the vector backbone, which was connected with the small ubiquitin-like modifier protease expression cassette and transformed into E. coli. The correctness of the nucleic acid sequence was checked by nucleic acid sequencing, and the correct recombinant plasmid was named pET-GSFGFn-CspA-Ulp1 (n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 16, 17, 18, 19, 20, 21, 22, 23). Take pET-GSFGF9-CspA-Ulp1 as an example to show the plasmid map as shown in Figure 1.

The recombinant plasmid pET-GSFGFn-CspA-Ulp1 was transformed into E. coli host BL21 (DE3), and the positive recombinant bacteria were screened by ampicillin resistance and named pET-GSFGFn-CspA-Ulp1/BL21.

Example 3 Construction of prokaryotic expression vector of fusion protein GFP-SUMO and acquisition of recombinant strain

The designed fusion protein GFP-SUMO is composed of green fluorescent protein, polypeptide linker and small ubiquitin-like modifier from the N-terminal to the C-terminal, and the amino acid sequences of each part are as described above; the nucleic acid encoding the fusion protein is from the 5' end to the 3' end. The sequence is green fluorescent protein, polypeptide linker and small ubiquitin-like modifier, and the nucleotide sequence of each part is as described above.

The nucleic acid encoding the fusion protein GFP-SUMO was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and then the synthesized nucleic acid fragment was cloned into E. coli expression plasmid vector pET-21a by genetic engineering method to obtain a recombinant plasmid and transformed into E. coli. The correctness of the nucleic acid sequence is checked by nucleic acid sequencing. The recombinant plasmid containing the nucleic acid encoding GFP-SUMO was named pET-GFP-SUMO.

The recombinant plasmid pET-GFP-SUMO was transformed into E. coli host BL21 (DE3), and the positive recombinant strains were screened with ampicillin resistance and named pET-GFP-SUMO/BL21 respectively. CspA-Ulp1/BL21 protein-expressing control strain.

Example 4 Induction expression of recombinant strains

Inoculate pET-GSFGFn-CspA-Ulp1/BL21 at 0.1% (v/v) inoculum into 50 mL of fresh liquid LB medium (peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, ampicillin 100 mg/L, pH=7.0), 220 rpm, shaking culture at 37°C for 14 hours. Then, according to the inoculation amount of 5% (v/v), it was inoculated into 1000 ml of fresh liquid LB medium, 220 r/min, shaken at 37 °C and cultured to OD ₆₀₀ = 1.0 (about 2-3 hours), adding the final Isopropyl-beta-D-thiogalactopyranoside (IPTG) at a concentration of 1 mmol/L, 37°C, 160 rpm shaking culture, after 1 hour, the fermentation broth was gradually It exhibits green fluorescence. Figure 2 shows the fluorescence observation of pET-GSFGF17-CspA-Ulp1/BL21 after induction and expression by IPTG. Figure A is the observation result of the fermentation broth under natural light. The fermentation broth in the left test tube It showed obvious green fluorescence (pET-GSFGF17-CspA-Ulp1/BL21 was induced and expressed by IPTG for 1 hour), but the fermentation broth in the right test tube did not show green fluorescence (before pET-GSFGF17-CspA-Ulp1/BL21 was induced and expressed by IPTG); Figure B shows the observation results of pET-GSFGF17-CspA-Ulp1/BL21 fermentation broth under UV light, and the green fluorescence is more obvious. Other fibroblast growth factor recombinant strains have the same results. After being induced by IPTG for 4 hours, centrifuge at 12,000 rpm to collect the precipitate showing green fluorescence, that is, the recombinant bacterial cells after induction of expression. of potassium chloride, 10 mmol/L sodium dihydrogen phosphate, 2 mmol/L potassium dihydrogen phosphate, 0.2% NP-40, pH=7.0) to resuspend the recombinant bacteria to obtain a bacterial suspension. The bacterial suspension was sampled for sodium-dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) detection. For the specific operation method of SDS-PAGE, refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

The pET-GFP-SUMO/BL21 subjected to the same induction operation was used as a control; at the same time, pET-GSFGF8-CspA-Ulp1/BL21 before IPTG induction (Fig. 3A), pET-GSFGF4-CspA-Ulp1/BL21 before IPTG induction ( Figure 3B) and pET-GSFGF11-CspA-Ulp1/BL21 before IPTG induction (Figure 3C) as an additional control, i.e. before adding IPTG, 1 ml of bacterial broth was removed from the fermentation broth for subsequent SDS- PAGE.

The results of SDS-PAGE are shown in FIG. 3 . It can be seen from Figure 3 that the fusion protein GFP-SUMO-FGFn can be expressed in large quantities. The density grayscale scanning of SDS-PAGE results using Image J software showed that the expression of fusion protein GFP-SUMO-FGFn accounted for more than 40% of the total protein.

Example 5 Induction expression of recombinant strains

The temperature of IPTG induction was 28°C, and the induction time was 8 hours, and the others were the same as those in Example 4. The results are comparable to Example 4.

Example 6 Induced solubility analysis of recombinant strains

IPTG-induced expression of fusion protein GFP-SUMO-FGFn was performed by the same operation as in Example 4. After inducible expression, centrifuge at 12,000 rpm to collect the precipitate showing green fluorescence, that is, the recombinant bacterium after inducible expression, and weigh.

The recombinant thalline collected above was added with phosphate buffer (137 mmol/L sodium chloride, 2.7 mmol/L chlorine) at a ratio of 1:10 (mass/volume) to the recombinant bacterium body: phosphate buffer. potassium chloride, 10 mmol/L sodium dihydrogen phosphate, 2 mmol/L potassium dihydrogen phosphate, 0.2% NP-40, pH=7.0) to resuspend the recombinant bacterial cells to obtain a bacterial suspension. The bacterial suspension was placed in a high-pressure homogenizer, and at 4°C, the lysate was obtained by successively homogenizing twice under the pressure conditions of 800 and 1200 bar (bar). The lysate was placed in a sterile centrifuge tube, centrifuged at 12,000 rpm (revolutions per minute) for 1 minute, and the lysate, supernatant and precipitate were taken for SDS-PAGE respectively.

The results of SDS-PAGE of partial pET-GSFGFn-CspA-Ulp1/BL21 total protein, protein in supernatant, and protein in pellet are shown in FIG. 4 . It can be seen from Figure 4 that the fusion proteins GFP-SUMO-FGF4, GFP-SUMO-FGF5, GFP-SUMO-FGF8, GFP-SUMO-FGF11, GFP-SUMO-FGF12, GFP-SUMO-FGF14, GFP-SUMO-FGF16, GFP - The soluble expression levels of SUMO-FGF17, GFP-SUMO-FGF19 and GFP-SUMO-FGF23 accounted for more than 80% of the total fusion protein; and these fusion proteins did not appear to be hydrolyzed, indicating that the process of IPTG induction at 37°C It does not lead to the normal expression of the small ubiquitin-like modifier protease Ulp1, so there is no phenomenon that the fusion protein GFP-SUMO-FGFn is hydrolyzed.

The pET-GSFGFn-CspA-Ulp1/BL21 not shown has the same results after the same induction and expression operation, that is, the expression level of the soluble fusion protein GFP-SUMO-FGFn accounts for more than 80% of the total amount of the fusion protein GFP-SUMO-FGFn; Moreover, these fusion proteins also did not appear to be hydrolyzed.

Based on the above results, it is shown that the fusion expression method can efficiently express the soluble fusion protein GFP-SUMO-FGFn containing fibroblast growth factor-n, and its yield can fully meet the needs of the large-scale production of fibroblast growth factor in the later stage. .

Example 7 Hydrolysis of Fibroblast Growth Factor-Containing Fusion Proteins

According to the method of Example 4, the recombinant strain pET-GSFGFn-CspA-Ulp1/BL21 was fermented at 37°C and rapidly lowered to 15°C after IPTG induction for 4 hours, and a part of the fermentation broth was taken out and dispensed into five shakers with a volume of 1000 ml. In the flasks, the flasks are numbered as flask 1 to flask 5, and 200 ml of fermentation broth is added to each flask.

Taking pET-GFP-SUMO/BL21 subjected to the same induction operation as a control, there is only one control shake flask, numbered as shake flask I.

In shake flask 1 and shake flask 1, zinc sulfate aqueous solution was added respectively, so that the final concentration of zinc sulfate in the fermentation broth was 5 mmol/L.

In shake flask 2I, an aqueous solution of tris(2-carbonylethyl)phosphorus hydrochloride was added so that the final concentration of tris(2-carbonylethyl)phosphorus hydrochloride in the fermentation broth was 1 mmol/L.

In shake flask 3, add tris(2-carbonylethyl)phosphorus hydrochloride aqueous solution to make the final concentration of tris(2-carbonylethyl)phosphorus hydrochloride in the fermentation broth 1 mmol/L; add sulfuric acid Aqueous zinc solution so that the final concentration of zinc sulfate in the fermentation broth is 1 mmol/L.

In shake flask 4, add tris(2-carbonylethyl)phosphorus hydrochloride aqueous solution so that the final concentration of tris(2-carbonylethyl)phosphorus hydrochloride in the fermentation broth is 1 mmol/L; add sulfuric acid Aqueous zinc solution so that the final concentration of zinc sulfate in the fermentation broth is 3 mmol/L.

In shake flask 5, add tris(2-carbonylethyl)phosphorus hydrochloride aqueous solution to make the final concentration of tris(2-carbonylethyl)phosphorus hydrochloride in the fermentation broth 0.1 mmol/L; add sulfuric acid Aqueous zinc solution so that the final concentration of zinc sulfate in the fermentation broth is 5 mmol/L.

Following the addition of the above components, the broth in Shake Flask 1 to Flask 5 and Flask 1 was continued to incubate for 2 hours at 15°C. Among them, and during the fermentation process, samples were taken at 0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes respectively for subsequent SDS-PAGE.

The samples taken from different shake flasks at different time points were centrifuged at 12,000 rpm at high speed, and the recombinant cells were collected respectively, and phosphate buffer was added according to the ratio (mass/volume) of recombinant cells: phosphate buffer to 1:10. Resuspend the recombinant bacteria to obtain a recombinant bacteria suspension. The suspension was placed in a high-pressure homogenizer, and at 4° C., successively homogenized twice under the conditions of 800 and 1200 bar (bar) pressure successively to obtain a lysate. The lysate was placed in a sterile centrifuge tube, centrifuged at 12,000 rpm for 1 minute, and the supernatant was collected for SDS-PAGE analysis. Figure 5 shows the results of pET-GSFGF12-CspA-Ulp1/BL21. According to the electrophoresis results in Figure 5, the addition of zinc sulfate aqueous solution alone could not promote the hydrolysis of the fusion protein GFP-SUMO-FGF12 by SUMO protease even if induced at low temperature for up to 2 hours (Figure 5A); the final concentration of single addition was 1 Although mmol/L tris (2-carbonylethyl) phosphate hydrochloride solution can promote the SUMO protease hydrolysis of fusion protein GFP-SUMO-FGF12 to obtain fibroblast growth factor-12, but the hydrolysis efficiency is low, after 120 minutes of treatment However, when tris(2-carbonylethyl)phosphonium hydrochloride and zinc sulfate were added at the same time, the amount of fusion protein hydrolyzed by SUMO protease was significant It can basically hydrolyze more than 90% of the fusion protein GFP-SUMO-FGFn within 0.5 to 1 hour, indicating that the combination has a synergistic effect and can significantly promote the hydrolysis capacity of the fusion protein by SUMO protease (Figure 5C- E); wherein, in the process of low temperature induction expression, the result of adding a final concentration of 0.1 mmol/L tris(2-carbonylethyl) phosphate hydrochloride and 5 mmol/L zinc sulfate combination shows that the fusion protein is in 15 minutes It was completely hydrolyzed by low temperature-induced SUMO protease (Fig. 5E).

In addition, the test results of other fusion proteins GFP-SUMO-FGFn showed that they were consistent with GFP-SUMO-FGF12. Figure 6 shows a portion of pET-GSFGFn-CspA incubated at 15°C for 30 minutes with a combination of tris(2-carbonylethyl) phosphate hydrochloride and 5 mmol/L zinc sulfate at a final concentration of 0.1 mmol/L. - SDS-PAGE of the supernatant of Ulp1/BL21, the figure shows that the fusion protein GFP-SUMO-FGFn can be completely hydrolyzed by small ubiquitin-like modifier protease within 30 minutes of induction at 15℃.

In order to have a clearer understanding of the progressive hydrolysis of fusion protein GFP-SUMO-FGFn by SUMO protease, anti-human fibroblast growth factor-9 (Thermo Fisher Scientific (China) Co., Ltd., Cat. No.: PA5-111618) and Anti-Human Fibroblast Growth Factor-18 Antibodies (Thermo Fisher Scientific (China) Co., Ltd., Cat. No.: PA5-78261) were directed against pET-GSFGF9-CspA-Ulp1/BL21 and pET-GSFGF18-CspA-Ulp1/ Supernatants of BL21 hypothermic induction for 5 min and 30 min were subjected to western blot analysis. The results are shown in Figure 7. Western blotting confirmed that the fusion protein GFP-SUMO-FGFn could be completely hydrolyzed within 30 minutes.

Example 8 Hydrolysis of Fibroblast Growth Factor-Containing Fusion Proteins

According to the method of Example 4, the recombinant strain pET-GSFGFn-CspA-Ulp1/BL21 was fermented at 32°C and then rapidly reduced to 10°C after 6 hours of IPTG induction, and an aqueous solution of tris(2-carbonylethyl)phosphorus hydrochloride was added. , so that the final concentration of tris(2-carbonylethyl) phosphate hydrochloride in the fermentation broth is 0.1 mmol/L, and an aqueous solution of zinc sulfate is added, so that the final concentration of zinc sulfate in the fermentation broth is 5 mmol/L, Continue to incubate the fermentation broth for 2 hours. Among them, and during the fermentation process, samples were taken at 0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes respectively for subsequent SDS-PAGE. Western blotting results also showed that the fusion protein GFP-SUMO-FGFn could be completely hydrolyzed by SUMO protease within 30 minutes.

Example 9 Large-scale fermentation and separation and purification of fibroblast factor-n

Take out the pET-GSFGF17-CspA-Ulp1/BL21 cryopreserved strain from the -80°C freezer and inoculate 3.7 L of fresh LB medium (10 g/L peptone, yeast extract) at a ratio of 1:100 (v/v) 5 g/L, sodium chloride 10 g/L, ampicillin 100 mg/L, pH=7.0), shaking culture at 37°C, 220 rpm (revolution/min) for 4 hours. The bacterial solution was inoculated into 200L (liter) of LB medium containing 5% (mass/volume) glucose at 10% inoculum amount (volume/volume), and cultured at 37°C for 4-6 hours until the bacterial density reached OD ₆₀₀ ≈ 0.6, all were transferred into 800L (liter) LB medium containing 5% (mass/volume) glucose for culturing for 4 hours, and then inserted into 8000L LB medium containing 5% (mass/volume) glucose for cultivation ( The total volume is about 9000L). When the glucose content in the culture medium drops to 0.1% (mass/volume), start feeding the feed, the feed medium is LB medium containing 20% (mass/volume) glucose, and the fermentation parameters are set: pH=7.0 -7.2; dissolved oxygen >30%; stirring speed 250 to 650 rpm (revolutions per minute). When the wet bacterial weight reaches 35±1 g/L (g/L), add the inducer to IPTG to make the final concentration reach 1 mmol/L, and induce for 4 hours. Then, the temperature of the fermentation broth was rapidly lowered to 15°C, and at the same time, an aqueous solution of tris(2-carbonylethyl)phosphate hydrochloride was added to the fermentation broth, so that tris(2-carbonylethyl)phosphate hydrochloride was fermented in the fermentation broth. The final concentration in the liquid was 0.1 mmol/L, and at the same time, an aqueous solution of zinc sulfate was added to make the final concentration of zinc sulfate in the fermentation broth 5 mmol/L, and the parameters were set as: pH=7.0; dissolved oxygen>30% Stirring speed 400rpm (revolution/min), cultivated for 1 hour, the whole fermentation and induction expression process ended, the total volume of final fermentation broth was about 10000L, and the thalline was collected by centrifugation and weighed to obtain a total of 513 kilograms (KG) of wet weight. Recombinant bacteria, calculated to reach 51.3g/L wet weight of recombinant bacteria.

Since fibroblast growth factors all have heparin binding domains, the fibroblast growth factors hydrolyzed from the fusion protein can be purified by the method of direct adsorption on heparin affinity chromatography gel.

All the collected recombinant cells were crushed by a high pressure homogenizer using the same operation as in Example 6 to obtain a lysate. The lysate was placed in a sterile centrifuge tube, centrifuged at 12,000 rpm (revolution per minute) for 1 minute, the supernatant was collected, and the supernatant was loaded on a CM ion-exchange gel chromatography column. Buffer gradient elution of fibroblast growth factor-17. The buffers used were buffer 1 (50 mmol/L Tris-HCl, 0.1 mol/L sodium chloride, pH=8.0); buffer 2 (50 mmol/L Tris-HCl, 0.3 mol/L) NaCl, pH=8.0); Buffer 3 (50 mmol/L Tris-HCl, 0.6 mol/L NaCl, pH=8.0) and Buffer 6 (50 mmol/L Tris-HCl, 1.0 mol /L sodium chloride, pH=8.0). SDS-PAGE detection, the results are shown in Figure 8A, the analysis shows that fibroblast growth factor-17 mainly exists in the eluate of buffer 3. The eluate containing fibroblast growth factor-17 was loaded onto a heparin affinity chromatography gel column at a flow rate of 8-10 mL/min, followed by buffer 3 (50 mmol/L Tris- HCl, 0.6 mol/l NaCl, pH=8.0), buffer 4 (50 mmol/l Tris-HCl, 1.2 mol/l NaCl, pH=8.0) and buffer 5 (50 mmol/l 1 Tris-HCl, 2.0 mol/L NaCl, pH=8.0) for elution. SDS-PAGE is shown in Figure 8B. The detection results show that fibroblast growth factor-17 is located in the eluate of buffer 4, and the results of electrophoresis band density grayscale scanning (ImageJ software) show that its purity is over 95%.

The purified fibroblast growth factor-17 was quantified by the bicinchoninic acid (BCA) protein quantification method, and converted based on the wet weight of the pET-GSFGF17-CspA-Ulp1/BL21 recombinant bacteria to obtain the fibroblast growth factor The yield of -17, the final purified fibroblast growth factor-17 yield was 5.048 mg/g wet weight.

Similar fermentation results were obtained using the same fermentation method to ferment other pET-GSFGFn-CspA-Ulp1/BL21 and purify fibroblast growth factor-n. The purified yields of these fibroblast growth factors were: fibroblast growth factor-1 6.066 mg/g wet weight, fibroblast growth factor-2 8.62 mg/g wet weight, fibroblast growth factor-3 9.329 mg /g wet weight, fibroblast growth factor-4 7.14 mg/g wet weight, fibroblast growth factor-5 5.224 mg/g wet weight, fibroblast growth factor-6 1.34 mg/g wet weight, fibroblasts Growth Factor-7 2.019 mg/g wet weight, Fibroblast Growth Factor-8 2.46 mg/g wet weight, Fibroblast Growth Factor-9 4.984 mg/g wet weight, Fibroblast Growth Factor-10 6.673 mg/g wet weight, fibroblast growth factor-111.243 mg/g wet weight, fibroblast growth factor-12 1.758 mg/g wet weight, fibroblast growth factor-13 3.153 mg/g wet weight, fibroblast growth factor- 14 1.585 mg/g wet weight, Fibroblast Growth Factor-16 1.892 mg/g wet weight, Fibroblast Growth Factor-18 2.298 mg/g wet weight, Fibroblast Growth Factor-19 1.957 mg/g wet weight, Fibroblast Growth Factor - 201.466 mg/g wet weight, Fibroblast Growth Factor-21 3.428 mg/g wet weight, Fibroblast Growth Factor-22 1.206 mg/g wet weight, Fibroblast Growth Factor-23 2.694 mg /g wet weight.

Example 10 Analysis of proliferative activity of fibroblast growth factor-8, fibroblast growth factor-9 and fibroblast growth factor-17 on Leydig cells

95% of androgens in the human body are synthesized by Leydig cells, and the decrease in the number of Leydig cells will lead to a significant decrease in the production of androgens in the human body.

Human primary Leydig cells (Cat. No. 4510, ScienCell Research Laboratories, Inc, USA) were inoculated into Dulbecco's Modified Eagle Medium (DMEM/F-12, purchased from Thermo Fisher Scientific, USA). , Cat. No.: 11320033), cultured at 37°C to logarithmic growth phase, digested with trypsin solution, then transferred to 96-well cell culture plate (8000 cells/well, 200 μl volume/well), and then separately Add purified fibroblast growth factor-8 (FGF-8), fibroblast growth factor-9 (FGF-9) and fibroblast growth factor-17 (FGF-17) at a final concentration of 100 ng/ml , to add an equal volume of PBS buffer as a negative control. After 24 hours of incubation, 100 µl of the medium was discarded and 100 µl of 5-ethyl-2'-deoxyuridine (EdU, a tracer compound, cell proliferation) containing a final concentration of 10 µmol/l was added. Deoxyribonucleic acid (DNA) is required for replication, during which EdU is inserted into the newly formed nucleotide chain, which fluoresces green when illuminated with light at a wavelength of 450-540 nanometers) freshly modified Duchenne Eagle culture medium, continued to cultivate for 24 hours, and observed with a fluorescence microscope. The results are shown in Figure 9. The results of FIG. 9 show that, compared with the negative control group, FGF-8, FGF-9 and FGF-17 can significantly promote the synthesis of deoxyribonucleic acid (DNA) in Leydig cells.

sequence listing

<110> Wenzhou Medical University

Su Zhijian

<120> A composition and method for large-scale production of fibroblast growth factor

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35 40 45

Val Asn Gly Ser Leu Glu Asn Ser Ala Tyr Ser Ile Leu Glu Ile Thr

50 55 60

Ala Val Glu Val Gly Ile Val Ala Ile Arg Gly Leu Phe Ser Gly Arg

65 70 75 80

Tyr Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Glu His Tyr

85 90 95

Ser Ala Glu Cys Glu Phe Val Glu Arg Ile His Glu Leu Gly Tyr Asn

100 105 110

Thr Tyr Ala Ser Arg Leu Tyr Arg Thr Val Ser Ser Thr Pro Gly Ala

115 120 125

Arg Arg Gln Pro Ser Ala Glu Arg Leu Trp Tyr Val Ser Val Asn Gly

130 135 140

Lys Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gln Lys Ser

145 150 155 160

Ser Leu Phe Leu Pro Arg Val Leu Asp His Arg Asp His Glu Met Val

165 170 175

Arg Gln Leu Gln Ser Gly Leu Pro Arg Pro Pro Gly Lys Gly Val Gln

180 185 190

Pro Arg Arg Arg Arg Gln Lys Gln Ser Pro Asp Asn Leu Glu Pro Ser

195 200 205

His Val Gln Ala Ser Arg Leu Gly Ser Gln Leu Glu Ala Ser Ala His

210 215 220

<210> 4

<211> 183

<212> PRT

<213> Homo sapiens

<400> 4

Met Gly Arg Gly Gly Ala Ala Ala Pro Thr Ala Pro Asn Gly Thr Leu

1 5 10 15

Glu Ala Glu Leu Glu Arg Arg Trp Glu Ser Leu Val Ala Leu Ser Leu

20 25 30

Ala Arg Leu Pro Val Ala Ala Gln Pro Lys Glu Ala Ala Val Gln Ser

35 40 45

Gly Ala Gly Asp Tyr Leu Leu Gly Ile Lys Arg Leu Arg Arg Leu Tyr

50 55 60

Cys Asn Val Gly Ile Gly Phe His Leu Gln Ala Leu Pro Asp Gly Arg

65 70 75 80

Ile Gly Gly Ala His Ala Asp Thr Arg Asp Ser Leu Leu Glu Leu Ser

85 90 95

Pro Val Glu Arg Gly Val Val Ser Ile Phe Gly Val Ala Ser Arg Phe

100 105 110

Phe Val Ala Met Ser Ser Lys Gly Lys Leu Tyr Gly Ser Pro Phe Phe

115 120 125

Thr Asp Glu Cys Thr Phe Lys Glu Ile Leu Leu Pro Asn Asn Tyr Asn

130 135 140

Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly Met Phe Ile Ala Leu Ser Lys

145 150 155 160

Asn Gly Lys Thr Lys Lys Gly Asn Arg Val Ser Pro Thr Met Lys Val

165 170 175

Thr His Phe Leu Pro Arg Leu

180

<210> 5

<211> 252

<212> PRT

<213> Homo sapiens

<400> 5

Met Ala Trp Ala His Gly Glu Lys Arg Leu Ala Pro Lys Gly Gln Pro

1 5 10 15

Gly Pro Ala Ala Thr Asp Arg Asn Pro Ile Gly Ser Ser Ser Arg Gln

20 25 30

Ser Ser Ser Ser Ala Met Ser Ser Ser Ser Ser Ala Ser Ser Ser Pro Ala

35 40 45

Ala Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu Gln Ser Ser Phe Gln

50 55 60

Trp Ser Pro Ser Gly Arg Arg Thr Gly Ser Leu Tyr Cys Arg Val Gly

65 70 75 80

Ile Gly Phe His Leu Gln Ile Tyr Pro Asp Gly Lys Val Asn Gly Ser

85 90 95

His Glu Ala Asn Met Leu Ser Val Leu Glu Ile Phe Ala Val Ser Gln

100 105 110

Gly Ile Val Gly Ile Arg Gly Val Phe Ser Asn Lys Phe Leu Ala Met

115 120 125

Ser Lys Lys Gly Lys Leu His Ala Ser Ala Lys Phe Thr Asp Asp Cys

130 135 140

Lys Phe Arg Glu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser

145 150 155 160

Ala Ile His Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu

165 170 175

Asn Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro

180 185 190

Gln His Ile Ser Thr His Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln

195 200 205

Pro Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro

210 215 220

Ser Pro Ile Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr

225 230 235 240

Asn Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly

245 250

<210> 6

<211> 169

<212> PRT

<213> Homo sapiens

<400> 6

Met Gly Thr Arg Ala Asn Asn Thr Leu Leu Asp Ser Arg Gly Trp Gly

1 5 10 15

Thr Leu Leu Ser Arg Ser Arg Ala Gly Leu Ala Gly Glu Ile Ala Gly

20 25 30

Val Asn Trp Glu Ser Gly Tyr Leu Val Gly Ile Lys Arg Gln Arg Arg

35 40 45

Leu Tyr Cys Asn Val Gly Ile Gly Phe His Leu Gln Val Leu Pro Asp

50 55 60

Gly Arg Ile Ser Gly Thr His Glu Glu Asn Pro Tyr Ser Leu Leu Glu

65 70 75 80

Ile Ser Thr Val Glu Arg Gly Val Val Ser Leu Phe Gly Val Arg Ser

85 90 95

Ala Leu Phe Val Ala Met Asn Ser Lys Gly Arg Leu Tyr Ala Thr Pro

100 105 110

Ser Phe Gln Glu Glu Cys Lys Phe Arg Glu Thr Leu Leu Pro Asn Asn

115 120 125

Tyr Asn Ala Tyr Glu Ser Asp Leu Tyr Gln Gly Thr Tyr Ile Ala Leu

130 135 140

Ser Lys Tyr Gly Arg Val Lys Arg Gly Ser Lys Val Ser Pro Ile Met

145 150 155 160

Thr Val Thr His Phe Leu Pro Arg Ile

165

<210> 7

<211> 141

<212> PRT

<213> Homo sapiens

<400> 7

Met Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile Arg Val Arg Arg Leu

1 5 10 15

Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp Lys Arg Gly Lys Val

20 25 30

Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn Ile Met Glu Ile Arg

35 40 45

Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly Val Glu Ser Glu Phe

50 55 60

Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr Ala Lys Lys Glu Cys

65 70 75 80

Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu Glu Asn His Tyr Asn

85 90 95

Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly Gly Glu Met Phe Val

100 105 110

Ala Leu Asn Gln Lys Gly Ile Pro Val Arg Gly Lys Lys Thr Lys Lys

115 120 125

Glu Gln Lys Thr Ala His Phe Leu Pro Met Ala Ile Thr

130 135 140

<210> 8

<211> 194

<212> PRT

<213> Homo sapiens

<400> 8

Met Gln Val Thr Val Gln Ser Ser Pro Asn Phe Thr Gln His Val Arg

1 5 10 15

Glu Gln Ser Leu Val Thr Asp Gln Leu Ser Arg Arg Leu Ile Arg Thr

20 25 30

Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His Val Gln Val Leu Ala

35 40 45

Asn Lys Arg Ile Asn Ala Met Ala Glu Asp Gly Asp Pro Phe Ala Lys

50 55 60

Leu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg Val Arg Val Arg Gly

65 70 75 80

Ala Glu Thr Gly Leu Tyr Ile Cys Met Asn Lys Lys Gly Lys Leu Ile

85 90 95

Ala Lys Ser Asn Gly Lys Gly Lys Asp Cys Val Phe Thr Glu Ile Val

100 105 110

Leu Glu Asn Asn Tyr Thr Ala Leu Gln Asn Ala Lys Tyr Glu Gly Trp

115 120 125

Tyr Met Ala Phe Thr Arg Lys Gly Arg Pro Arg Lys Gly Ser Lys Thr

130 135 140

Arg Gln His Gln Arg Glu Val His Phe Met Lys Arg Leu Pro Arg Gly

145 150 155 160

His His Thr Thr Glu Gln Ser Leu Arg Phe Glu Phe Leu Asn Tyr Pro

165 170 175

Pro Phe Thr Arg Ser Leu Arg Gly Ser Gln Arg Thr Trp Ala Pro Glu

180 185 190

Pro Arg

<210> 9

<211> 207

<212> PRT

<213> Homo sapiens

<400> 9

Met Pro Leu Gly Glu Val Gly Asn Tyr Phe Gly Val Gln Asp Ala Val

1 5 10 15

Pro Phe Gly Asn Val Pro Val Leu Pro Val Asp Ser Pro Val Leu Leu

20 25 30

Ser Asp His Leu Gly Gln Ser Glu Ala Gly Gly Leu Pro Arg Gly Pro

35 40 45

Ala Val Thr Asp Leu Asp His Leu Lys Gly Ile Leu Arg Arg Arg Gln

50 55 60

Leu Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly Thr

65 70 75 80

Ile Gln Gly Thr Arg Lys Asp His Ser Arg Phe Gly Ile Leu Glu Phe

85 90 95

Ile Ser Ile Ala Val Gly Leu Val Ser Ile Arg Gly Val Asp Ser Gly

100 105 110

Leu Tyr Leu Gly Met Asn Glu Lys Gly Glu Leu Tyr Gly Ser Glu Lys

115 120 125

Leu Thr Gln Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp Tyr

130 135 140

Asn Thr Tyr Ser Ser Asn Leu Tyr Lys His Val Asp Thr Gly Arg Arg

145 150 155 160

Tyr Tyr Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr Arg

165 170 175

Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val Asp

180 185 190

Pro Asp Lys Val Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser

195 200 205

<210> 10

<211> 170

<212> PRT

<213> Homo sapiens

<400> 10

Met Leu Gly Gln Asp Met Val Ser Pro Glu Ala Thr Asn Ser Ser Ser

1 5 10 15

Ser Ser Phe Ser Ser Pro Ser Ser Ala Gly Arg His Val Arg Ser Tyr

20 25 30

Asn His Leu Gln Gly Asp Val Arg Trp Arg Lys Leu Phe Ser Phe Thr

35 40 45

Lys Tyr Phe Leu Lys Ile Glu Lys Asn Gly Lys Val Ser Gly Thr Lys

50 55 60

Lys Glu Asn Cys Pro Tyr Ser Ile Leu Glu Ile Thr Ser Val Glu Ile

65 70 75 80

Gly Val Val Ala Val Lys Ala Ile Asn Ser Asn Tyr Tyr Leu Ala Met

85 90 95

Asn Lys Lys Gly Lys Leu Tyr Gly Ser Lys Glu Phe Asn Asn Asp Cys

100 105 110

Lys Leu Lys Glu Arg Ile Glu Glu Asn Gly Tyr Asn Thr Tyr Ala Ser

115 120 125

Phe Asn Trp Gln His Asn Gly Arg Gln Met Tyr Val Ala Leu Asn Gly

130 135 140

Lys Gly Ala Pro Arg Arg Gly Gln Lys Thr Arg Arg Lys Asn Thr Ser

145 150 155 160

Ala His Phe Leu Pro Met Val Val His Ser

165 170

<210> 11

<211> 225

<212> PRT

<213> Homo sapiens

<400> 11

Met Ala Ala Leu Ala Ser Ser Leu Ile Arg Gln Lys Arg Glu Val Arg

1 5 10 15

Glu Pro Gly Gly Ser Arg Pro Val Ser Ala Gln Arg Arg Val Cys Pro

20 25 30

Arg Gly Thr Lys Ser Leu Cys Gln Lys Gln Leu Leu Ile Leu Leu Ser

35 40 45

Lys Val Arg Leu Cys Gly Gly Arg Pro Ala Arg Pro Asp Arg Gly Pro

50 55 60

Glu Pro Gln Leu Lys Gly Ile Val Thr Lys Leu Phe Cys Arg Gln Gly

65 70 75 80

Phe Tyr Leu Gln Ala Asn Pro Asp Gly Ser Ile Gln Gly Thr Pro Glu

85 90 95

Asp Thr Ser Ser Phe Thr His Phe Asn Leu Ile Pro Val Gly Leu Arg

100 105 110

Val Val Thr Ile Gln Ser Ala Lys Leu Gly His Tyr Met Ala Met Asn

115 120 125

Ala Glu Gly Leu Leu Tyr Ser Ser Pro His Phe Thr Ala Glu Cys Arg

130 135 140

Phe Lys Glu Cys Val Phe Glu Asn Tyr Tyr Val Leu Tyr Ala Ser Ala

145 150 155 160

Leu Tyr Arg Gln Arg Arg Ser Gly Arg Ala Trp Tyr Leu Gly Leu Asp

165 170 175

Lys Glu Gly Gln Val Met Lys Gly Asn Arg Val Lys Lys Thr Lys Ala

180 185 190

Ala Ala His Phe Leu Pro Lys Leu Leu Glu Val Ala Met Tyr Gln Glu

195 200 205

Pro Ser Leu His Ser Val Pro Glu Ala Ser Pro Ser Ser Pro Pro Ala

210 215 220

Pro

225

<210> 12

<211> 243

<212> PRT

<213> Homo sapiens

<400> 12

Met Ala Ala Ala Ile Ala Ser Ser Leu Ile Arg Gln Lys Arg Gln Ala

1 5 10 15

Arg Glu Ser Asn Ser Asp Arg Val Ser Ala Ser Lys Arg Arg Ser Ser

20 25 30

Pro Ser Lys Asp Gly Arg Ser Leu Cys Glu Arg His Val Leu Gly Val

35 40 45

Phe Ser Lys Val Arg Phe Cys Ser Gly Arg Lys Arg Pro Val Arg Arg

50 55 60

Arg Pro Glu Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Phe Ser Gln

65 70 75 80

Gln Gly Tyr Phe Leu Gln Met His Pro Asp Gly Thr Ile Asp Gly Thr

85 90 95

Lys Asp Glu Asn Ser Asp Tyr Thr Leu Phe Asn Leu Ile Pro Val Gly

100 105 110

Leu Arg Val Val Ala Ile Gln Gly Val Lys Ala Ser Leu Tyr Val Ala

115 120 125

Met Asn Gly Glu Gly Tyr Leu Tyr Ser Ser Asp Val Phe Thr Pro Glu

130 135 140

Cys Lys Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser

145 150 155 160

Ser Thr Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe Leu Gly

165 170 175

Leu Asn Lys Glu Gly Gln Ile Met Lys Gly Asn Arg Val Lys Lys Thr

180 185 190

Lys Pro Ser Ser His Phe Val Pro Lys Pro Ile Glu Val Cys Met Tyr

195 200 205

Arg Glu Pro Ser Leu His Glu Ile Gly Glu Lys Gln Gly Arg Ser Arg

210 215 220

Lys Ser Ser Gly Thr Pro Thr Met Asn Gly Gly Lys Val Val Asn Gln

225 230 235 240

Asp Ser Thr

<210> 13

<211> 245

<212> PRT

<213> Homo sapiens

<400> 13

Met Ala Ala Ala Ile Ala Ser Ser Leu Ile Arg Gln Lys Arg Gln Ala

1 5 10 15

Arg Glu Arg Glu Lys Ser Asn Ala Cys Lys Cys Val Ser Ser Pro Ser

20 25 30

Lys Gly Lys Thr Ser Cys Asp Lys Asn Lys Leu Asn Val Phe Ser Arg

35 40 45

Val Lys Leu Phe Gly Ser Lys Lys Lys Arg Arg Arg Arg Arg Pro Glu Pro

50 55 60

Gln Leu Lys Gly Ile Val Thr Lys Leu Tyr Ser Arg Gln Gly Tyr His

65 70 75 80

Leu Gln Leu Gln Ala Asp Gly Thr Ile Asp Gly Thr Lys Asp Glu Asp

85 90 95

Ser Thr Tyr Thr Leu Phe Asn Leu Ile Pro Val Gly Leu Arg Val Val

100 105 110

Ala Ile Gln Gly Val Gln Thr Lys Leu Tyr Leu Ala Met Asn Ser Glu

115 120 125

Gly Tyr Leu Tyr Thr Ser Glu Leu Phe Thr Pro Glu Cys Lys Phe Lys

130 135 140

Glu Ser Val Phe Glu Asn Tyr Tyr Val Thr Tyr Ser Ser Met Ile Tyr

145 150 155 160

Arg Gln Gln Gln Ser Gly Arg Gly Trp Tyr Leu Gly Leu Asn Lys Glu

165 170 175

Gly Glu Ile Met Lys Gly Asn His Val Lys Lys Asn Lys Pro Ala Ala

180 185 190

His Phe Leu Pro Lys Pro Leu Lys Val Ala Met Tyr Lys Glu Pro Ser

195 200 205

Leu His Asp Leu Thr Glu Phe Ser Arg Ser Gly Ser Gly Thr Pro Thr

210 215 220

Lys Ser Arg Ser Val Ser Gly Val Leu Asn Gly Gly Lys Ser Met Ser

225 230 235 240

His Asn Glu Ser Thr

245

<210> 14

<211> 247

<212> PRT

<213> Homo sapiens

<400> 14

Met Ala Ala Ala Ile Ala Ser Gly Leu Ile Arg Gln Lys Arg Gln Ala

1 5 10 15

Arg Glu Gln His Trp Asp Arg Pro Ser Ala Ser Arg Arg Arg Ser Ser

20 25 30

Pro Ser Lys Asn Arg Gly Leu Cys Asn Gly Asn Leu Val Asp Ile Phe

35 40 45

Ser Lys Val Arg Ile Phe Gly Leu Lys Lys Arg Arg Leu Arg Arg Gln

50 55 60

Asp Pro Gln Leu Lys Gly Ile Val Thr Arg Leu Tyr Cys Arg Gln Gly

65 70 75 80

Tyr Tyr Leu Gln Met His Pro Asp Gly Ala Leu Asp Gly Thr Lys Asp

85 90 95

Asp Ser Thr Asn Ser Thr Leu Phe Asn Leu Ile Pro Val Gly Leu Arg

100 105 110

Val Val Ala Ile Gln Gly Val Lys Thr Gly Leu Tyr Ile Ala Met Asn

115 120 125

Gly Glu Gly Tyr Leu Tyr Pro Ser Glu Leu Phe Thr Pro Glu Cys Lys

130 135 140

Phe Lys Glu Ser Val Phe Glu Asn Tyr Tyr Val Ile Tyr Ser Ser Met

145 150 155 160

Leu Tyr Arg Gln Gln Glu Ser Gly Arg Ala Trp Phe Leu Gly Leu Asn

165 170 175

Lys Glu Gly Gln Ala Met Lys Gly Asn Arg Val Lys Lys Thr Lys Pro

180 185 190

Ala Ala His Phe Leu Pro Lys Pro Leu Glu Val Ala Met Tyr Arg Glu

195 200 205

Pro Ser Leu His Asp Val Gly Glu Thr Val Pro Lys Pro Gly Val Thr

210 215 220

Pro Ser Lys Ser Thr Ser Ala Ser Ala Ile Met Asn Gly Gly Lys Pro

225 230 235 240

Val Asn Lys Ser Lys Thr Thr

245

<210> 15

<211> 207

<212> PRT

<213> Homo sapiens

<400> 15

Met Ala Glu Val Gly Gly Val Phe Ala Ser Leu Asp Trp Asp Leu His

1 5 10 15

Gly Phe Ser Ser Ser Leu Gly Asn Val Pro Leu Ala Asp Ser Pro Gly

20 25 30

Phe Leu Asn Glu Arg Leu Gly Gln Ile Glu Gly Lys Leu Gln Arg Gly

35 40 45

Ser Pro Thr Asp Phe Ala His Leu Lys Gly Ile Leu Arg Arg Arg Gln

50 55 60

Leu Tyr Cys Arg Thr Gly Phe His Leu Glu Ile Phe Pro Asn Gly Thr

65 70 75 80

Val His Gly Thr Arg His Asp His Ser Arg Phe Gly Ile Leu Glu Phe

85 90 95

Ile Ser Leu Ala Val Gly Leu Ile Ser Ile Arg Gly Val Asp Ser Gly

100 105 110

Leu Tyr Leu Gly Met Asn Glu Arg Gly Glu Leu Tyr Gly Ser Lys Lys

115 120 125

Leu Thr Arg Glu Cys Val Phe Arg Glu Gln Phe Glu Glu Asn Trp Tyr

130 135 140

Asn Thr Tyr Ala Ser Thr Leu Tyr Lys His Ser Asp Ser Glu Arg Gln

145 150 155 160

Tyr Tyr Val Ala Leu Asn Lys Asp Gly Ser Pro Arg Glu Gly Tyr Arg

165 170 175

Thr Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val Asp

180 185 190

Pro Ser Lys Leu Pro Ser Met Ser Arg Asp Leu Phe His Tyr Arg

195 200 205

<210> 16

<211> 195

<212> PRT

<213> Homo sapiens

<400> 16

Met Thr Gln Gly Glu Asn His Pro Ser Pro Asn Phe Asn Gln Tyr Val

1 5 10 15

Arg Asp Gln Gly Ala Met Thr Asp Gln Leu Ser Arg Arg Gln Ile Arg

20 25 30

Glu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His Val Gln Val Thr

35 40 45

Gly Arg Arg Ile Ser Ala Thr Ala Glu Asp Gly Asn Lys Phe Ala Lys

50 55 60

Leu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg Val Arg Ile Lys Gly

65 70 75 80

Ala Glu Ser Glu Lys Tyr Ile Cys Met Asn Lys Arg Gly Lys Leu Ile

85 90 95

Gly Lys Pro Ser Gly Lys Ser Lys Asp Cys Val Phe Thr Glu Ile Val

100 105 110

Leu Glu Asn Asn Tyr Thr Ala Phe Gln Asn Ala Arg His Glu Gly Trp

115 120 125

Phe Met Ala Phe Thr Arg Gln Gly Arg Pro Arg Gln Ala Ser Arg Ser

130 135 140

Arg Gln Asn Gln Arg Glu Ala His Phe Ile Lys Arg Leu Tyr Gln Gly

145 150 155 160

Gln Leu Pro Phe Pro Asn His Ala Glu Lys Gln Lys Gln Phe Glu Phe

165 170 175

Val Gly Ser Ala Pro Thr Arg Arg Thr Lys Arg Thr Arg Arg Pro Gln

180 185 190

Pro Leu Thr

195

<210> 17

<211> 174

<212> PRT

<213> Homo sapiens

<400> 17

Met Ala Glu Glu Asn Val Asp Phe Arg Ile His Val Glu Asn Gln Thr

1 5 10 15

Arg Ala Arg Asp Asp Val Ser Arg Lys Gln Leu Arg Leu Tyr Gln Leu

20 25 30

Tyr Ser Arg Thr Ser Gly Lys His Ile Gln Val Leu Gly Arg Arg Ile

35 40 45

Ser Ala Arg Gly Glu Asp Gly Asp Lys Tyr Ala Gln Leu Leu Val Glu

50 55 60

Thr Asp Thr Phe Gly Ser Gln Val Arg Ile Lys Gly Lys Glu Thr Glu

65 70 75 80

Phe Tyr Leu Cys Met Asn Arg Lys Gly Lys Leu Val Gly Lys Pro Asp

85 90 95

Gly Thr Ser Lys Glu Cys Val Phe Ile Glu Lys Val Leu Glu Asn Asn

100 105 110

Tyr Thr Ala Leu Met Ser Ala Lys Tyr Ser Gly Trp Tyr Val Gly Phe

115 120 125

Thr Lys Lys Gly Arg Pro Arg Lys Gly Pro Lys Thr Arg Glu Asn Gln

130 135 140

Gln Asp Val His Phe Met Lys Arg Tyr Pro Lys Gly Gln Pro Glu Leu

145 150 155 160

Gln Lys Pro Phe Lys Tyr Thr Thr Val Thr Lys Arg Ser Arg

165 170

<210> 18

<211> 195

<212> PRT

<213> Homo sapiens

<400> 18

Met Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro His Val His Tyr Gly

1 5 10 15

Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr Thr Ser Gly Pro His

20 25 30

Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg Ala Asp Gly Val Val Asp

35 40 45

Cys Ala Arg Gly Gln Ser Ala His Ser Leu Leu Glu Ile Lys Ala Val

50 55 60

Ala Leu Arg Thr Val Ala Ile Lys Gly Val His Ser Val Arg Tyr Leu

65 70 75 80

Cys Met Gly Ala Asp Gly Lys Met Gln Gly Leu Leu Gln Tyr Ser Glu

85 90 95

Glu Asp Cys Ala Phe Glu Glu Glu Glu Ile Arg Pro Asp Gly Tyr Asn Val

100 105 110

Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser Leu Ser Ser Ala Lys

115 120 125

Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu Pro Leu Ser His Phe

130 135 140

Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro Glu Asp Leu Arg Gly

145 150 155 160

His Leu Glu Ser Asp Met Phe Ser Ser Pro Leu Glu Thr Asp Ser Met

165 170 175

Asp Pro Phe Gly Leu Val Thr Gly Leu Glu Ala Val Arg Ser Pro Ser

180 185 190

Phe Glu Lys

195

<210> 19

<211> 210

<212> PRT

<213> Homo sapiens

<400> 19

Met Pro Leu Ala Glu Val Gly Gly Phe Leu Gly Gly Leu Glu Gly Leu

1 5 10 15

Gly Gln Gln Val Gly Ser His Phe Leu Leu Pro Pro Ala Gly Glu Arg

20 25 30

Pro Pro Leu Leu Gly Glu Arg Arg Ser Ala Ala Glu Arg Ser Ala Arg

35 40 45

Gly Gly Pro Gly Ala Ala Gln Leu Ala His Leu His Gly Ile Leu Arg

50 55 60

Arg Arg Gln Leu Tyr Cys Arg Thr Gly Phe His Leu Gln Ile Leu Pro

65 70 75 80

Asp Gly Ser Val Gln Gly Thr Arg Gln Asp His Ser Leu Phe Gly Ile

85 90 95

Leu Glu Phe Ile Ser Val Ala Val Gly Leu Val Ser Ile Arg Gly Val

100 105 110

Asp Ser Gly Leu Tyr Leu Gly Met Asn Asp Lys Gly Glu Leu Tyr Gly

115 120 125

Ser Glu Lys Leu Thr Ser Glu Cys Ile Phe Arg Glu Gln Phe Glu Glu

130 135 140

Asn Trp Tyr Asn Thr Tyr Ser Ser Asn Ile Tyr Lys His Gly Asp Thr

145 150 155 160

Gly Arg Arg Tyr Phe Val Ala Leu Asn Lys Asp Gly Thr Pro Arg Asp

165 170 175

Gly Ala Arg Ser Lys Arg His Gln Lys Phe Thr His Phe Leu Pro Arg

180 185 190

Pro Val Asp Pro Glu Arg Val Pro Glu Leu Tyr Lys Asp Leu Leu Met

195 200 205

Tyr Thr

210

<210> 20

<211> 182

<212> PRT

<213> Homo sapiens

<400> 20

Met His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln

1 5 10 15

Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala

20 25 30

His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln

35 40 45

Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly Val Ile

50 55 60

Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp

65 70 75 80

Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe

85 90 95

Arg Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala

100 105 110

His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His Arg Asp

115 120 125

Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro

130 135 140

Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp

145 150 155 160

Val Gly Ser Ser Asp Pro Leu Ser Met Val Gly Pro Ser Gln Gly Arg

165 170 175

Ser Pro Ser Tyr Ala Ser

180

<210> 21

<211> 150

<212> PRT

<213> Homo sapiens

<400> 21

Met Gly Thr Pro Ser Ala Ser Arg Gly Pro Arg Ser Tyr Pro His Leu

1 5 10 15

Glu Gly Asp Val Arg Trp Arg Arg Leu Phe Ser Ser Thr His Phe Phe

20 25 30

Leu Arg Val Asp Pro Gly Gly Arg Val Gln Gly Thr Arg Trp Arg His

35 40 45

Gly Gln Asp Ser Ile Leu Glu Ile Arg Ser Val His Val Gly Val Val

50 55 60

Val Ile Lys Ala Val Ser Ser Gly Phe Tyr Val Ala Met Asn Arg Arg

65 70 75 80

Gly Arg Leu Tyr Gly Ser Arg Leu Tyr Thr Val Asp Cys Arg Phe Arg

85 90 95

Glu Arg Ile Glu Glu Asn Gly His Asn Thr Tyr Ala Ser Gln Arg Trp

100 105 110

Arg Arg Arg Gly Gln Pro Met Phe Leu Ala Leu Asp Arg Arg Gly Gly

115 120 125

Pro Arg Pro Gly Gly Arg Thr Arg Arg Tyr His Leu Ser Ala His Phe

130 135 140

Leu Pro Val Leu Val Ser

145 150

<210> 22

<211> 228

<212> PRT

<213> Homo sapiens

<400> 22

Met Tyr Pro Asn Ala Ser Pro Leu Leu Gly Ser Ser Trp Gly Gly Leu

1 5 10 15

Ile His Leu Tyr Thr Ala Thr Ala Arg Asn Ser Tyr His Leu Gln Ile

20 25 30

His Lys Asn Gly His Val Asp Gly Ala Pro His Gln Thr Ile Tyr Ser

35 40 45

Ala Leu Met Ile Arg Ser Glu Asp Ala Gly Phe Val Val Ile Thr Gly

50 55 60

Val Met Ser Arg Arg Tyr Leu Cys Met Asp Phe Arg Gly Asn Ile Phe

65 70 75 80

Gly Ser His Tyr Phe Asp Pro Glu Asn Cys Arg Phe Gln His Gln Thr

85 90 95

Leu Glu Asn Gly Tyr Asp Val Tyr His Ser Pro Gln Tyr His Phe Leu

100 105 110

Val Ser Leu Gly Arg Ala Lys Arg Ala Phe Leu Pro Gly Met Asn Pro

115 120 125

Pro Pro Tyr Ser Gln Phe Leu Ser Arg Arg Asn Glu Ile Pro Leu Ile

130 135 140

His Phe Asn Thr Pro Ile Pro Arg Arg His Thr Arg Ser Ala Glu Asp

145 150 155 160

Asp Ser Glu Arg Asp Pro Leu Asn Val Leu Lys Pro Arg Ala Arg Met

165 170 175

Thr Pro Ala Pro Ala Ser Cys Ser Gln Glu Leu Pro Ser Ala Glu Asp

180 185 190

Asn Ser Pro Met Ala Ser Asp Pro Leu Gly Val Val Arg Gly Gly Arg

195 200 205

Val Asn Thr His Ala Gly Gly Thr Gly Pro Glu Gly Cys Arg Pro Phe

210 215 220

Ala Lys Phe Ile

225

<210> 23

<211> 253

<212> PRT

<213> Artificial Sequence

<400> 23

Met Lys Ser Ser His His His His His His Gly Ser Ser Val Ser Lys

1 5 10 15

Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp

20 25 30

Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu Gly

35 40 45

Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly

50 55 60

Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly

65 70 75 80

Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe

85 90 95

Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Ser

100 105 110

Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu

115 120 125

Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys

130 135 140

Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Phe Asn Ser

145 150 155 160

His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala

165 170 175

Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala

180 185 190

Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu

195 200 205

Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu Ser Lys Asp Pro

210 215 220

Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala

225 230 235 240

Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Ile

245 250

<210> 24

<211> 98

<212> PRT

<213> Artificial Sequence

<400> 24

Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro

1 5 10 15

Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser

20 25 30

Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu

35 40 45

Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg

50 55 60

Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp

65 70 75 80

Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile

85 90 95

Gly Gly

<210> 25

<211> 8

<212> PRT

<213> Artificial Sequence

<400> 25

Gly Gly Gly Ser Gly Gly Gly Ser

1 5

<210> 26

<211> 426

<212> DNA

<213> Homo sapiens

<400> 26

atgttcaacc tgccgccggg caactacaag aaaccgaagc tgctgtattg cagcaacggt 60

ggccactttc tgcgtatcct gccggatggt accgtggatg gtacccgtga ccgtagcgat 120

cagcacatcc agctgcaact gagcgcggag agcgtgggcg aagtttacat taaaagcacc 180

gagaccggcc agtatctggc gatggacacc gatggtctgc tgtacggcag ccaaaccccg 240

aacgaggaat gcctgttcct ggagcgtctg gaggaaaacc actacaacac ctatatcagc 300

aagaaacacg cggaaaagaa ctggtttgtg ggtctgaaga aaaacggcag ctgcaagcgt 360

ggtccgcgta cccactatgg tcaaaaagcg attctgttcc tgccgctgcc ggttagcagc 420

gactaa 426

<210> 27

<211> 468

<212> DNA

<213> Homo sapiens

<400> 27

atggcggcgg gtagcatcac caccctgccg gcgctgccgg aagatggtgg cagcggtgcg 60

tttccgccgg gtcacttcaa ggacccgaaa cgtctgtact gcaagaacgg tggcttcttt 120

ctgcgtattc acccggacgg tcgtgtggat ggcgttcgtg aaaagagcga cccgcacatc 180

aaactgcagc tgcaagcgga ggaacgtggt gtggttagca ttaaaggcgt gtgcgcgaac 240

cgttatctgg cgatgaaaga ggacggccgt ctgctggcga gcaaatgcgt taccgatgaa 300

tgcttctttt tcgagcgtct ggaaagcaac aactacaaca cctatcgtag ccgtaagtac 360

accagctggt acgttgcgct gaaacgtacc ggccagtaca agctgggcag caaaaccggt 420

ccgggccaaa aggcgatcct gtttctgccg atgagcgcga aaagctaa 468

<210> 28

<211> 675

<212> DNA

<213> Homo sapiens

<400> 28

atgccggcgg cgggtccggg tgcgcgtctg cgtcgtgatg cgggtggccg tggtggcgtg 60

tatgagcacc tgggtggtgc gccgcgtcgt cgtaagctgt actgcgcgac caaatatcac 120

ctgcagctgc acccgagcgg tcgtgtgaac ggcagcctgg aaaacagcgc gtacagcatc 180

ctggagatta ccgcggtgga agttggtatc gttgcgattc gtggtctgtt cagcggccgt 240

tatctggcga tgaacaagcg tggccgtctg tacgcgagcg agcactatag cgcggagtgc 300

gaatttgtgg aacgtatcca cgaactgggt tacaacacct atgcgagccg tctgtaccgt 360

accgttagca gcaccccggg tgcgcgtcgt cagccgagcg cggagcgtct gtggtatgtg 420

agcgttaacg gtaaaggccg tccgcgtcgt ggtttcaaga cccgtcgtac ccagaagagc 480

agcctgtttc tgccgcgtgt gctggaccac cgtgatcacg aaatggttcg tcagctgcaa 540

agcggtctgc cgcgtccgcc gggcaagggc gtgcaaccgc gtcgtcgtcg tcagaaacaa 600

agcccggata acctggaacc gagccatgtt caggcgagcc gtctgggtag ccaactggaa 660

gcgagcgcgc actaa 675

<210> 29

<211> 552

<212> DNA

<213> Homo sapiens

<400> 29

atgggtcgtg gtggcgcggc ggcgccgacc gcgccgaacg gtaccctgga ggcggaactg 60

gagcgtcgtt gggaaagcct ggtggcgctg agcctggcgc gtctgccggt tgcggcgcag 120

ccgaaagagg cggcggtgca aagcggtgcg ggtgactacc tgctgggtat caaacgtctg 180

cgtcgtctgt attgcaacgt tggtattggt tttcacctgc aggcgctgcc ggatggtcgt 240

atcggtggcg cgcatgcgga cacccgtgat agcctgctgg aactgagccc ggtggagcgt 300

ggtgtggtta gcatttttgg cgtggcgagc cgtttctttg ttgcgatgag cagcaagggt 360

aaactgtacg gcagcccgtt ctttaccgac gaatgcacct tcaaggagat tctgctgccg 420

aacaactaca acgcgtatga aagctacaaa tatccgggca tgtttatcgc gctgagcaag 480

aacggtaaaa ccaagaaagg caaccgtgtg agcccgacca tgaaggttac ccacttcctg 540

ccgcgtctgt aa 552

<210> 30

<211> 759

<212> DNA

<213> Homo sapiens

<400> 30

atggcgtggg cgcatggtga gaagcgtctg gcgccgaagg gtcagccggg tccggcggcg 60

accgaccgta acccgatcgg tagcagcagc cgtcaaagca gcagcagcgc gatgagcagc 120

agcagcgcga gcagcagccc ggcggcgagc ctgggcagcc agggtagcgg tctggaacag 180

agcagctttc agtggagccc gagcggtcgt cgtaccggta gcctgtactg ccgtgttggt 240

atcggctttc acctgcagat ttatccggat ggcaaagtta acggtagcca cgaggcgaac 300

atgctgagcg tgctggaaat tttcgctgtg agccaaggta tcgttggcat tcgtggtgtg 360

ttcagcaaca agtttctggc gatgagcaag aaaggcaagc tgcacgcgag cgcgaaattt 420

accgacgatt gcaagttccg tgagcgtttt caggaaaaca gctacaacac ctatgcgagc 480

gcgatccacc gtaccgagaa aaccggtcgt gaatggtacg tggcgctgaa caagcgtggc 540

aaggcgaaac gtggttgcag cccgcgtgtg aaaccgcaac acattagcac ccacttcctg 600

ccgcgtttta agcagagcga gcaaccggaa ctgagcttca ccgtgaccgt tccggagaag 660

aaaaacccgc cgagcccgat caagagcaaa attccgctga gcgcgccgcg taaaaacacc 720

aacagcgtga agtatcgtct gaaattccgt tttggttaa 759

<210> 31

<211> 510

<212> DNA

<213> Homo sapiens

<400> 31

atgggtaccc gtgcgaacaa caccctgctg gatagccgtg gttggggtac cctgctgagc 60

cgtagccgtg cgggtctggc gggtgaaatt gcgggcgtta actgggaaag cggttacctg 120

gtgggcatca agcgtcagcg tcgtctgtat tgcaacgttg gtattggctt ccacctgcaa 180

gtgctgccgg atggtcgtat cagcggcacc cacgaggaaa acccgtacag cctgctggag 240

atcagcaccg ttgaacgtgg tgtggttagc ctgttcggcg tgcgtagcgc gctgtttgtt 300

gcgatgaaca gcaagggtcg tctgtatgcg accccgagct tccaggaaga gtgcaaattt 360

cgtgagaccc tgctgccgaa caactacaac gcgtatgaaa gcgacctgta ccaaggcacc 420

tatattgcgc tgagcaaata cggtcgtgtg aagcgtggca gcaaagttag cccgatcatg 480

accgtgaccc actttctgcc gcgtatttaa 510

<210> 32

<211> 426

<212> DNA

<213> Homo sapiens

<400> 32

atgagctacg actatatgga gggtggcgat atccgtgtgc gtcgtctgtt ctgccgtacc 60

cagtggtacc tgcgtattga caagcgtggc aaggttaaag gcacccaaga gatgaagaac 120

aactacaaca tcatggaaat ccgtaccgtg gcggttggta tcgtggcgat taagggcgtt 180

gagagcgaat tctacctggc gatgaacaag gaaggtaaac tgtatgcgaa gaaagagtgc 240

aacgaagatt gcaactttaa ggagctgatc ctggaaaacc actacaacac ctatgcgagc 300

gcgaaatgga cccacaacgg tggcgagatg ttcgtggcgc tgaaccagaa gggtatcccg 360

gttcgtggca agaaaaccaa gaaagaacaa aaaaccgcgc actttctgcc gatggcgatt 420

acctaa 426

<210> 33

<211> 585

<212> DNA

<213> Homo sapiens

<400> 33

atgcaggtga ccgttcaaag cagcccgaac ttcacccagc acgtgcgtga gcaaagcctg 60

gttaccgacc agctgagccg tcgtctgatc cgtacctacc aactgtatag ccgtaccagc 120

ggcaagcacg tgcaggttct ggcgaacaaa cgtatcaacg cgatggcgga ggacggtgat 180

ccgttcgcga agctgattgt ggaaaccgac acctttggca gccgtgtgcg tgttcgtggt 240

gcggaaaccg gcctgtacat ctgcatgaac aagaaaggta aactgattgc gaaaagcaac 300

ggcaagggca aagattgcgt gttcaccgag attgttctgg aaaacaacta taccgcgctg 360

caaaacgcga agtacgaggg ctggtatatg gcgttcaccc gtaaaggtcg tccgcgtaag 420

ggcagcaaaa cccgtcagca ccaacgtgaa gttcacttta tgaaacgtct gccgcgtggt 480

caccacacca ccgagcagag cctgcgtttc gaatttctga actacccgcc gtttacccgt 540

agcctgcgtg gcagccaacg tacctgggcg ccggagccgc gttaa 585

<210> 34

<211> 624

<212> DNA

<213> Homo sapiens

<400> 34

atgccgctgg gtgaagtggg caactacttc ggcgtgcagg acgcggttcc gtttggtaac 60

gtgccggttc tgccggtgga cagcccggtt ctgctgagcg atcacctggg ccaaagcgag 120

gcgggtggcc tgccgcgtgg tccggcggtt accgacctgg atcacctgaa gggcatcctg 180

cgtcgtcgtc agctgtattg ccgtaccggt ttccacctgg aaatctttcc gaacggtacc 240

attcaaggca cccgtaaaga ccacagccgt ttcggcatcc tggagtttat cagcattgcg 300

gtgggtctgg ttagcattcg tggtgtggat agcggcctgt acctgggtat gaacgagaag 360

ggcgaactgt atggtagcga gaaactgacc caggaatgcg ttttccgtga gcaatttgag 420

gaaaactggt acaacaccta tagcagcaac ctgtacaagc acgtggacac cggtcgtcgt 480

tactatgttg cgctgaacaa agatggcacc ccgcgtgaag gtacccgtac caagcgtcac 540

cagaaattca cccactttct gccgcgtccg gtggacccgg ataaggttcc ggagctgtat 600

aaagatattc tgagccaaag ctaa 624

<210> 35

<211> 513

<212> DNA

<213> Homo sapiens

<400> 35

atgctgggtc aggacatggt tagcccggag gcgaccaaca gcagcagcag cagctttagc 60

agcccgagca gcgcgggtcg tcatgtgcgt agctacaacc acctgcaagg cgatgttcgt 120

tggcgtaaac tgttcagctt taccaagtat tttctgaaga tcgagaaaaa cggcaaagtt 180

agcggcacca agaaagaaaa ctgcccgtac agcatcctgg agattaccag cgtggaaatc 240

ggtgtggttg cggttaaagc gattaacagc aactactatc tggcgatgaa caagaaaggt 300

aaactgtatg gcagcaagga gttcaacaac gactgcaagc tgaaagaacg tattgaggaa 360

aacggttaca acacctatgc gagctttaac tggcagcaca acggccgtca aatgtatgtg 420

gcgctgaacg gtaaaggtgc gccgcgtcgt ggtcagaaaa cccgtcgtaa gaacaccagc 480

gcgcacttcc tgccgatggt ggttcacagc taa 513

<210> 36

<211> 678

<212> DNA

<213> Homo sapiens

<400> 36

atggcggcgc tggcgagcag cctgatccgt cagaagcgtg aggtgcgcga gccgggtggc 60

agccgtccgg ttagcgcgca acgtcgtgtt tgcccgcgtg gcaccaagag cctgtgccag 120

aaacaactgc tgattctgct gagcaaagtg cgtctgtgcg gtggccgtcc ggcgcgtccg 180

gaccgtggtc cggagccgca gctgaagggt atcgttacca aactgttctg ccgtcagggc 240

ttttacctgc aagcgaaccc ggacggtagc attcaaggca ccccggaaga taccagcagc 300

ttcacccact ttaacctgat cccggttggt ctgcgtgtgg ttaccattca gagcgcgaag 360

ctgggccact acatggcgat gaacgcggag ggtctgctgt atagcagccc gcacttcacc 420

gcggaatgcc gtttcaaaga gtgcgtgttt gaaaactact atgttctgta cgcgagcgcg 480

ctgtatcgtc agcgtcgtag cggtcgtgcg tggtacctgg gtctggataa agagggtcaa 540

gtgatgaaag gtaaccgtgt taagaaaacc aaggcggcgg cgcactttct gccgaaactg 600

ctggaagtgg cgatgtatca agaaccgagc ctgcacagcg ttccggaagc gagcccgagc 660

agcccgccgg cgccgtaa 678

<210> 37

<211> 732

<212> DNA

<213> Homo sapiens

<400> 37

atggcggcgg cgatcgcgag cagcctgatt cgtcagaagc gtcaagcgcg tgagagcaac 60

agcgaccgtg tgagcgcgag caagcgtcgt agcagcccga gcaaagatgg ccgtagcctg 120

tgcgaacgtc acgtgctggg tgttttcagc aaagtgcgtt tttgcagcgg ccgtaaacgt 180

ccggttcgtc gtcgtccgga gccgcagctg aaaggtatcg ttacccgtct gttcagccag 240

caaggctact ttctgcaaat gcacccggac ggtaccattg atggcaccaa ggacgaaaac 300

agcgattata ccctgttcaa cctgatcccg gtgggcctgc gtgtggttgc gattcagggt 360

gtgaaagcga gcctgtacgt tgcgatgaac ggcgagggct acctgtatag cagcgacgtt 420

tttaccccgg aatgcaagtt caaagagagc gtgtttgaaa actactatgt tatctacagc 480

agcaccctgt atcgtcagca agagagcggt cgtgcgtggt tcctgggtct gaacaaggaa 540

ggccaaatta tgaaaggtaa ccgtgtgaag aaaaccaagc cgagcagcca ctttgtgccg 600

aaaccgatcg aggtttgcat gtatcgtgaa ccgagcctgc acgagattgg tgaaaagcag 660

ggccgtagcc gtaaaagcag cggcaccccg accatgaacg gtggcaaggt ggttaaccaa 720

gatagcacct aa 732

<210> 38

<211> 738

<212> DNA

<213> Homo sapiens

<400> 38

atggcggcgg cgatcgcgag cagcctgatt cgtcaaaagc gtcaggcgcg tgagcgtgaa 60

aagagcaacg cgtgcaaatg cgtgagcagc ccgagcaagg gcaaaaccag ctgcgacaag 120

aacaaactga acgtgttcag ccgtgttaaa ctgtttggta gcaagaagcg tcgtcgtcgt 180

cgtccggagc cgcaactgaa gggcatcgtt accaaactgt acagccgtca gggttatcac 240

ctgcaactgc aggcggacgg taccattgat ggcaccaagg acgaagatag cacctacacc 300

ctgttcaacc tgatcccggt gggcctgcgt gtggttgcga ttcaaggtgt tcagaccaaa 360

ctgtatctgg cgatgaacag cgagggctac ctgtatacca gcgagctgtt taccccggaa 420

tgcaagttca aagagagcgt gtttgaaaac tactatgtta cctacagcag catgatctat 480

cgtcagcaac agagcggtcg tggctggtac ctgggtctga acaaagaggg tgaaattatg 540

aaaggtaacc acgtgaagaa aaacaaaccg gcggcgcact tcctgccgaa gccgctgaaa 600

gttgcgatgt ataaagagcc gagcctgcac gatctgaccg aatttagccg tagcggtagc 660

ggcaccccga ccaagagccg tagcgtgagc ggtgttctga acggtggcaa aagcatgagc 720

cacaacgaaa gcacctaa 738

<210> 39

<211> 744

<212> DNA

<213> Homo sapiens

<400> 39

atggcggcgg cgattgcgag cggtctgatt cgtcagaagc gtcaagcgcg tgaacaacac 60

tgggaccgtc cgagcgcgag ccgtcgtcgt agcagcccga gcaagaaccg tggtctgtgc 120

aacggcaacc tggtggacat cttcagcaaa gttcgtattt ttggtctgaa gaaacgtcgt 180

ctgcgtcgtc aggacccgca gctgaaaggc attgtgaccc gtctgtactg ccgtcagggt 240

tactatctgc aaatgcaccc ggacggtgcg ctggatggca ccaaggacga tagcaccaac 300

agcaccctgt tcaacctgat cccggtgggc ctgcgtgtgg ttgcgattca gggtgttaaa 360

accggcctgt atatcgcgat gaacggcgag ggctacctgt atccgagcga gctgtttacc 420

ccggaatgca agttcaaaga gagcgtgttc gaaaactact acgttatcta cagcagcatg 480

ctgtatcgtc agcaagagag cggtcgtgcg tggttcctgg gtctgaacaa ggaaggccaa 540

gcgatgaaag gtaaccgtgt gaagaaaacc aagccggcgg cgcactttct gccgaaaccg 600

ctggaagtgg cgatgtaccg tgaaccgagc ctgcacgatg tgggcgaaac cgttccgaag 660

ccgggtgtga ccccgagcaa aagcaccagc gcgagcgcga tcatgaacgg tggcaagccg 720

gttaacaaga gcaaaaccac ctaa 744

<210> 40

<211> 624

<212> DNA

<213> Homo sapiens

<400> 40

atggcggaag tgggtggtgt gttcgcgagc ctggactggg atctgcacgg ctttagcagc 60

agcctgggta acgtgccgct ggcggacagc ccgggcttcc tgaacgagcg tctgggtcag 120

atcgaaggca agctgcaacg tggcagcccg accgattttg cgcacctgaa aggtatcctg 180

cgtcgtcgtc agctgtactg ccgtaccggt ttccacctgg agatttttcc gaacggtacc 240

gtgcatggta cccgtcatga ccacagccgt ttcggcatcc tggaatttat tagcctggcg 300

gtgggtctga tcagcattcg tggtgttgat agcggcctgt acctgggtat gaacgagcgt 360

ggcgaactgt atggtagcaa gaaactgacc cgtgagtgcg ttttccgtga acaatttgag 420

gaaaactggt acaacaccta tgcgagcacc ctgtacaagc acagcgacag cgagcgtcag 480

tactatgtgg cgctgaacaa agatggcagc ccgcgtgaag gttatcgtac caagcgtcac 540

caaaaattca cccactttct gccgcgtccg gttgacccga gcaagctgcc gagcatgagc 600

cgtgacctgt tccactatcg ttaa 624

<210> 41

<211> 588

<212> DNA

<213> Homo sapiens

<400> 41

atgacccaag gcgagaacca cccgagcccg aacttcaacc agtacgtgcg tgaccaaggt 60

gcgatgaccg atcagctgag ccgtcgtcaa attcgtgaat accagctgta tagccgtacc 120

agcggcaagc acgtgcaggt taccggtcgt cgtattagcg cgaccgcgga ggacggcaac 180

aagttcgcga aactgatcgt ggaaaccgat acctttggca gccgtgttcg tatcaagggt 240

gcggagagcg aaaaatacat ttgcatgaac aagcgtggta aactgatcgg caaaccgagc 300

ggcaagagca aagactgcgt gttcaccgag attgttctgg aaaacaacta taccgcgttt 360

caaaacgcgc gtcacgaggg ctggttcatg gcgtttaccc gtcagggtcg tccgcgtcag 420

gcgagccgta gccgtcagaa ccaacgtgaa gcgcacttca tcaagcgtct gtatcagggc 480

caactgccgt ttccgaacca cgcggagaag cagaaacaat tcgaatttgt tggtagcgcg 540

ccgacccgtc gtaccaaacg tacccgtcgt ccgcagccgc tgacctaa 588

<210> 42

<211> 525

<212> DNA

<213> Homo sapiens

<400> 42

atggcggagg aaaacgtgga cttccgtatc cacgttgaga accagacccg tgcgcgtgac 60

gatgtgagcc gtaagcagct gcgtctgtac caactgtata gccgtaccag cggcaaacac 120

atccaagttc tgggtcgtcg tattagcgcg cgtggtgagg acggcgataa gtacgcgcag 180

ctgctggttg agaccgacac cttcggcagc caagttcgta tcaagggtaa agagaccgaa 240

ttttatctgt gcatgaaccg taagggtaaa ctggtgggca agccggatgg taccagcaaa 300

gaatgcgtgt tcattgagaa ggttctggaa aacaactaca ccgcgctgat gagcgcgaaa 360

tacagcggct ggtacgttgg ttttaccaag aaaggccgtc cgcgtaaggg tccgaaaacc 420

cgtgagaacc agcaagatgt tcacttcatg aagcgttacc cgaaaggcca gccggaactg 480

caaaagccgt ttaaatatac caccgttacc aaacgtagcc gttaa 525

<210> 43

<211> 588

<212> DNA

<213> Homo sapiens

<400> 43

atgcgtccgc tggcgtttag cgatgcgggt ccgcatgtgc actatggttg gggcgatccg 60

atccgtctgc gtcacctgta taccagcggt ccgcatggtc tgagcagctg ctttctgcgt 120

attcgtgcgg acggtgtggt tgattgcgcg cgtggtcaga gcgcgcacag cctgctggaa 180

atcaaggcgg tggcgctgcg taccgttgcg attaaaggtg tgcacagcgt tcgttacctg 240

tgcatgggtg cggacggcaa gatgcagggc ctgctgcaat atagcgagga agactgcgcg 300

ttcgaggaag agatccgtcc ggatggctac aacgtgtatc gtagcgagaa acaccgtctg 360

ccggttagcc tgagcagcgc gaagcagcgt caactgtaca aaaaccgtgg tttcctgccg 420

ctgagccact ttctgccgat gctgccgatg gttccggaag agccggaaga cctgcgtggc 480

cacctggaga gcgatatgtt cagcagcccg ctggaaaccg acagcatgga cccgtttggt 540

ctggtgaccg gcctggaagc ggttcgtagc ccgagctttg agaagtaa 588

<210> 44

<211> 633

<212> DNA

<213> Homo sapiens

<400> 44

atgccgctgg cggaagtggg tggtttcctg ggtggtctgg aaggtctggg ccagcaagtt 60

ggcagccact ttctgctgcc gccggcgggt gaacgtccgc cgctgctggg cgagcgtcgt 120

agcgcggcgg aacgtagcgc gcgtggtggc ccgggtgcgg cgcagctggc gcacctgcac 180

ggtatcctgc gtcgtcgtca gctgtactgc cgtaccggtt tccacctgca aattctgccg 240

gatggtagcg tgcagggtac ccgtcaagat cacagcctgt tcggcatcct ggaatttatt 300

agcgtggcgg ttggtctggt tagcatccgt ggtgttgaca gcggcctgta cctgggtatg 360

aacgataagg gcgagctgta tggtagcgaa aaactgacca gcgagtgcat cttccgtgaa 420

cagtttgagg aaaactggta caacacctat agcagcaaca tttacaagca cggtgacacc 480

ggccgtcgtt attttgttgc gctgaacaaa gatggtaccc cgcgtgatgg tgcgcgtagc 540

aagcgtcacc aaaaattcac ccactttctg ccgcgtccgg tggacccgga gcgtgttccg 600

gaactgtaca aggatctgct gatgtatacc taa 633

<210> 45

<211> 549

<212> DNA

<213> Homo sapiens

<400> 45

atgcatccga ttccggacag cagcccgctg ctgcagtttg gtggccaagt gcgtcaacgt 60

tacctgtata ccgacgatgc gcagcaaacc gaggcgcacc tggagattcg tgaagacggt 120

accgttggtg gcgcggcgga tcagagcccg gaaagcctgc tgcaactgaa ggcgctgaaa 180

ccgggtgtga tccagattct gggcgttaag accagccgtt ttctgtgcca acgtccggat 240

ggtgcgctgt atggcagcct gcacttcgat ccggaggcgt gcagctttcg tgagctgctg 300

ctggaagacg gctacaacgt gtatcaaagc gaagcgcatg gtctgccgct gcacctgccg 360

ggtaacaaga gcccgcaccg tgatccggcg ccgcgtggtc cggcgcgttt cctgccgctg 420

ccgggtctgc cgccggcgct gccggagccg ccgggtatcc tggcgccgca gccgccggac 480

gtgggcagca gcgatccgct gagcatggtt ggtccgagcc aaggccgtag cccgagctat 540

gcgagctaa 549

<210> 46

<211> 453

<212> DNA

<213> Homo sapiens

<400> 46

atgggtaccc cgagcgcgag ccgtggtccg cgtagctacc cgcacctgga gggtgacgtt 60

cgttggcgtc gtctgttcag cagcacccac ttctttctgc gtgttgaccc gggtggccgt 120

gtgcagggta cccgttggcg tcacggccaa gatagcatcc tggaaattcg tagcgttcac 180

gtgggtgtgg ttgtgatcaa ggcggttagc agcggcttct atgtggcgat gaaccgtcgt 240

ggtcgtctgt acggcagccg tctgtatacc gtggactgcc gttttcgtga gcgtattgag 300

gaaaacggtc acaacaccta cgcgagccag cgttggcgtc gtcgtggtca accgatgttc 360

ctggcgctgg atcgtcgtgg tggcccgcgt ccgggtggcc gtacccgtcg ttatcacctg 420

agcgcgcact ttctgccggt tctggttagc taa 453

<210> 47

<211> 687

<212> DNA

<213> Homo sapiens

<400> 47

atgtatccga acgcgagccc gctgctgggt agcagctggg gtggcctgat ccacctgtac 60

accgcgaccg cgcgtaacag ctatcacctg cagattcaca aaaacggtca tgtggatggt 120

gcgccgcacc aaaccatcta tagcgcgctg atgattcgta gcgaggatgc gggttttgtg 180

gttatcaccg gcgttatgag ccgtcgttac ctgtgcatgg acttccgtgg taacattttt 240

ggcagccact atttcgatcc ggagaactgc cgtttccagc accaaaccct ggaaaacggc 300

tacgacgtgt atcacagccc gcagtaccac tttctggtta gcctgggtcg tgcgaagcgt 360

gcgttcctgc cgggtatgaa cccgccgccg tatagccaat ttctgagccg tcgtaacgag 420

atcccgctga ttcacttcaa caccccgatt ccgcgtcgtc acacccgtag cgcggaggac 480

gatagcgaac gtgatccgct gaacgtgctg aaaccgcgtg cgcgtatgac cccggcgccg 540

gcgagctgca gccaggagct gccgagcgcg gaagacaaca gcccgatggc gagcgatccg 600

ctgggtgtgg ttcgtggtgg ccgtgttaac acccatgcgg gtggcaccgg tccggaaggt 660

tgccgtccgt tcgcgaaatt catctaa 687

<210> 48

<211> 762

<212> DNA

<213> Artificial Sequence

<400> 48

atgaagagca gccatcacca ccaccaccat ggcagcagcg tgagcaaagg cgaggaactg 60

ttcaccggcg tggttccgat cctggtggaa ctggacggcg atgttaacgg tcacaagttt 120

agcgttcgtg gtgagggcga aggtgatgcg accaacggca agctgaccct gaaattcatt 180

tgcaccaccg gtaaactgcc ggtgccgtgg ccgaccctgg ttaccaccct gacctacggt 240

gtgcagtgct ttagccgtta tccggaccac atgaaacaac acgatttctt taagagcgcg 300

atgccggaag gttacgttca ggagcgtacc atcagcttca aagacgatgg cacctataag 360

acccgtgcgg aagtgaaatt tgaaggtgat accctggtta accgtatcga actgaaaggc 420

attgacttca aagaggacgg caacatcctg ggtcacaagc tggaatacaa ctttaacagc 480

cacaacgtgt atattaccgc ggacaagcag aaaaacggta tcaaggcgaa ctttaaaatt 540

cgtcacaacg tggaggacgg cagcgttcaa ctggcggatc actaccagca aaacaccccg 600

attggtgatg gtccggttct gctgccggat aaccactatc tgagcaccca gagcaagctg 660

agcaaagacc cgaacgaaaa gcgtgatcac atggtgctgc tggagttcgt taccgcggcg 720

ggcatcaccc tgggtatgga tgaactgtac aaaggtattg ag 762

<210> 49

<211> 294

<212> DNA

<213> Artificial Sequence

<400> 49

atgagcgaca gcgaggtgaa ccaagaagcg aagccggaag tgaaaccgga agttaagccg 60

gagacccaca tcaacctgaa agttagcgac ggcagcagcg agatcttctt taaaattaag 120

aaaaccaccc cgctgcgtcg tctgatggaa gcgttcgcga aacgtcaggg caaggagatg 180

gacagcctgc gttttctgta tgatggcatc cgtattcagg cggaccaaac cccggaagac 240

ctggatatgg aggacaacga tatcattgaa gcgcaccgtg agcaaattgg tggc 294

<210> 50

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 50

ggcggcggct ccggcggcgg ctcc 24

<210> 51

<211> 232

<212> PRT

<213> Artificial Sequence

<400> 51

Met Lys Ser Ser His His His His His His Gly Ser Ser Leu Val Pro

1 5 10 15

Glu Leu Asn Glu Lys Asp Asp Asp Asp Gln Val Gln Lys Ala Leu Ala Ser

20 25 30

Arg Glu Asn Thr Gln Leu Met Asn Arg Asp Asn Ile Glu Ile Thr Val

35 40 45

Arg Asp Phe Lys Thr Leu Ala Pro Arg Arg Trp Leu Asn Asp Thr Ile

50 55 60

Ile Glu Phe Phe Met Lys Tyr Ile Glu Lys Ser Thr Pro Asn Thr Val

65 70 75 80

Ala Phe Asn Ser Phe Phe Tyr Thr Asn Leu Ser Glu Arg Gly Tyr Gln

85 90 95

Gly Val Arg Arg Trp Met Lys Arg Lys Lys Thr Gln Ile Asp Lys Leu

100 105 110

Asp Lys Ile Phe Thr Pro Ile Asn Leu Asn Gln Ser His Trp Ala Leu

115 120 125

Gly Ile Ile Asp Leu Lys Lys Lys Thr Ile Gly Tyr Val Asp Ser Leu

130 135 140

Ser Asn Gly Pro Asn Ala Met Ser Phe Ala Ile Leu Thr Asp Leu Gln

145 150 155 160

Lys Tyr Val Met Glu Glu Ser Lys His Thr Ile Gly Glu Asp Phe Asp

165 170 175

Leu Ile His Leu Asp Cys Pro Gln Gln Pro Asn Gly Tyr Asp Cys Gly

180 185 190

Ile Tyr Val Cys Met Asn Thr Leu Tyr Gly Ser Ala Asp Ala Pro Leu

195 200 205

Asp Phe Asp Tyr Lys Asp Ala Ile Arg Met Arg Arg Phe Ile Ala His

210 215 220

Leu Ile Leu Thr Asp Ala Leu Lys

225 230

<210> 52

<211> 699

<212> DNA

<213> Artificial Sequence

<400> 52

atgaaaagca gccaccacca ccaccaccac ggtagcagcc tggtgccgga gctgaacgaa 60

aaagacgatg accaggttca aaaggcgctg gcgagccgtg agaacaccca gctgatgaac 120

cgtgataaca tcgaaattac cgtgcgtgac ttcaagaccc tggcgccgcg tcgttggctg 180

aacgatacca tcatcgagtt ctttatgaag tacatcgaaa agagcacccc gaacaccgtg 240

gcgtttaaca gcttctttta caccaacctg agcgagcgtg gttatcaggg cgttcgtcgt 300

tggatgaagc gtaagaaaac ccaaatcgat aaactggaca agatcttcac cccgattaac 360

ctgaaccaga gccactgggc gctgggcatc attgatctga agaaaaagac catcggttac 420

gtggacagcc tgagcaacgg cccgaacgcg atgagcttcg cgattctgac cgatctgcaa 480

aaatatgtta tggaggaaag caagcacacc atcggtgaag attttgacct gattcacctg 540

gattgcccgc agcaaccgaa cggttacgac tgcggcatct atgtttgcat gaacaccctg 600

tatggcagcg cggatgcgcc gctggatttc gactataaag acgcgattcg tatgcgtcgt 660

tttatcgcgc acctgattct gaccgacgcg ctgaagtaa 699

<210> 53

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 53

tttgtttaac tttaagaagg aga 23

Claims (26)

1. A composition comprising tris (2-carbonylethyl) phosphate and zinc sulphate in a mass ratio of 1 (1 to 50).

2. A method of obtaining a fibroblast growth factor-containing fusion protein or fibroblast growth factor therein, the fusion protein comprising a visual reporter protein from N-terminus to C-terminus, a small ubiquitin-like modifier and a fibroblast growth factor, the method comprising the steps of:

1-1) connecting an expression cassette of the fusion protein or a nucleic acid for coding the fusion protein to a first expression vector to obtain a first recombinant expression vector; wherein the first expression vector is an isopropyl-beta-D-thiogalactoside inducible expression vector;

2-1) connecting an expression cassette for expressing the small ubiquitin-like modifier protease or nucleic acid for coding the small ubiquitin-like modifier protease to the first recombinant expression vector to obtain a second recombinant expression vector;

3-1) transforming the second recombinant expression vector into a first host to obtain a first recombinant organism;

4-1) culturing the first recombinant organism in a first culture solution until the fusion protein and the small ubiquitin-like modifier protease are expressed to obtain a first culture;

wherein the composition of claim 1 is added to the first culture solution when the culture temperature is lowered to 10 to 15 ℃;

5-1) purifying the first culture to obtain the fibroblast growth factor;

or

1-2) same as 1-1);

2-2) connecting the expression cassette for expressing the small ubiquitin-like modifier protease or the nucleic acid for coding the small ubiquitin-like modifier protease to a second expression vector to obtain a third recombinant expression vector;

3-2) transforming the first recombinant expression vector into a second host to obtain a second recombinant organism; transforming the third recombinant expression vector into a third host to obtain a third recombinant organism;

4-2) culturing the second recombinant organism in a second culture solution until the fusion protein is expressed to obtain a second culture; culturing the third recombinant organism in a third culture solution until the small ubiquitin-like modifier protease is expressed to obtain a third culture; mixing the second culture with the third culture to obtain a fourth culture;

wherein the composition of claim 1 is added to the second culture solution;

5-2) purifying the fourth culture, thereby obtaining the fibroblast growth factor.

3. The method of claim 2, wherein the fibroblast growth factor is one of human fibroblast growth factor-1, human fibroblast growth factor-2, human fibroblast growth factor-3, human fibroblast growth factor-4, human fibroblast growth factor-5, human fibroblast growth factor-6, human fibroblast growth factor-7, human fibroblast growth factor-8, human fibroblast growth factor-9, human fibroblast growth factor-10, human fibroblast growth factor-11, human fibroblast growth factor-12, human fibroblast growth factor-13, human fibroblast growth factor-14, human fibroblast growth factor-16, human fibroblast growth factor-17, human fibroblast growth factor-18, human fibroblast growth factor-19, human fibroblast growth factor-20, human fibroblast growth factor-21, human fibroblast growth factor-23, human fibroblast growth factor.

4. The method according to claim 3, wherein the amino acid sequence of the human fibroblast growth factor-1 is shown as SEQ ID No.1, the amino acid sequence of the human fibroblast growth factor-2 is shown as SEQ ID No.2, the amino acid sequence of the human fibroblast growth factor-3 is shown as SEQ ID No.3, the amino acid sequence of the human fibroblast growth factor-4 is shown as SEQ ID No.4, the amino acid sequence of the human fibroblast growth factor-5 is shown as SEQ ID No.5, the amino acid sequence of the human fibroblast growth factor-6 is shown as SEQ ID No.6, the amino acid sequence of the human fibroblast growth factor-7 is shown as SEQ ID No.7, the amino acid sequence of the human fibroblast growth factor-8 is shown as SEQ ID No.8, the amino acid sequence of the human fibroblast growth factor-9 is shown as SEQ ID No.9, the amino acid sequence of the human fibroblast growth factor-10 is shown as SEQ ID No.10, the amino acid sequence of the human fibroblast growth factor-12 is shown as SEQ ID No.14, the amino acid sequence of the human fibroblast growth factor-12 is shown as SEQ ID No.12, the amino acid sequence of the human fibroblast growth factor-12, the human growth factor-12, the amino acid sequence of the human fibroblast growth factor-12 is shown as SEQ ID No.12, the amino acid sequence of the human fibroblast growth factor-17 is shown as SEQ ID No.16, the amino acid sequence of the human fibroblast growth factor-18 is shown as SEQ ID No.17, the amino acid sequence of the human fibroblast growth factor-19 is shown as SEQ ID No.18, the amino acid sequence of the human fibroblast growth factor-20 is shown as SEQ ID No.19, the amino acid sequence of the human fibroblast growth factor-21 is shown as SEQ ID No.20, the amino acid sequence of the human fibroblast growth factor-22 is shown as SEQ ID No.21, and the amino acid sequence of the human fibroblast growth factor-23 is shown as SEQ ID No. 22.

5. The method according to claim 4, wherein the nucleotide sequence encoding the human fibroblast growth factor-1 is shown in SEQ ID No.26, the nucleotide sequence encoding the human fibroblast growth factor-2 is shown in SEQ ID No.27, the nucleotide sequence encoding the human fibroblast growth factor-3 is shown in SEQ ID No.28, the nucleotide sequence encoding the human fibroblast growth factor-4 is shown in SEQ ID No.29, the nucleotide sequence encoding the human fibroblast growth factor-5 is shown in SEQ ID No.30, the nucleotide sequence encoding the human fibroblast growth factor-6 is shown in SEQ ID No.31, the nucleotide sequence encoding the human fibroblast growth factor-7 is shown in SEQ ID No.32, the nucleotide sequence encoding the human fibroblast growth factor-8 is shown in SEQ ID No.33, the nucleotide sequence encoding the human fibroblast growth factor-9 is shown in SEQ ID No.34, the nucleotide sequence encoding the human fibroblast growth factor-10 is shown in SEQ ID No.35, the nucleotide sequence encoding the human fibroblast growth factor-12 is shown in SEQ ID No.36, the nucleotide sequence encoding the human fibroblast growth factor-13 is shown in SEQ ID No.13, the nucleotide sequence encoding the human fibroblast growth factor-13, the human fibroblast growth factor-13 is shown in SEQ ID No.13, the nucleotide sequence for coding the human fibroblast growth factor-16 is shown as SEQ ID No.40, the nucleotide sequence for coding the human fibroblast growth factor-17 is shown as SEQ ID No.41, the nucleotide sequence for coding the human fibroblast growth factor-18 is shown as SEQ ID No.42, the nucleotide sequence for coding the human fibroblast growth factor-19 is shown as SEQ ID No.43, the nucleotide sequence for coding the human fibroblast growth factor-20 is shown as SEQ ID No.44, the nucleotide sequence for coding the human fibroblast growth factor-21 is shown as SEQ ID No.45, the nucleotide sequence for coding the human fibroblast growth factor-22 is shown as SEQ ID No.46, and the nucleotide sequence for coding the human fibroblast growth factor-23 is shown as SEQ ID No. 47.

6. The method of claim 2, wherein the expression cassette for the fusion protein comprises a first promoter from 5 'end to 3' end, a nucleic acid encoding the fusion protein, and a first terminator.

7. The method of claim 2, wherein the visual reporter protein is a fluorescent protein.

8. The method of claim 2, wherein the visual reporter protein is one of green fluorescent protein, red fluorescent protein, blue fluorescent protein, and cyan fluorescent protein.

9. The method according to claim 8, wherein the amino acid sequence of the green fluorescent protein is shown as SEQ ID No. 23.

10. The method of claim 9, wherein the nucleotide sequence encoding the green fluorescent protein is set forth in SEQ ID No. 48.

11. The method according to claim 2, wherein the amino acid sequence of the small ubiquitin-like modifier is shown in SEQ ID No. 24.

12. The method of claim 11, wherein the nucleotide sequence encoding the small ubiquitin-like modifier is shown in SEQ ID No. 49.

13. The method of claim 2, wherein the visual reporter protein and the small ubiquitin-like modifier are linked by a polypeptide linker.

14. The method of claim 13, wherein the amino acid sequence of the polypeptide linker is as set forth in SEQ ID No. 25.

15. The method of claim 14, wherein the nucleotide sequence encoding the polypeptide linker is set forth in SEQ ID No. 50.

16. The method of claim 6, wherein the first promoter is a T7 promoter and the first terminator is a T7 terminator.

17. The method of claim 2, wherein the expression cassette for expressing a small ubiquitin-like modifier protease comprises a second promoter from 5 'to 3', a 3'-UTR, a nucleic acid encoding a small ubiquitin-like modifier protease, and a5' -UTR.

18. The method of claim 17, wherein the second promoter is a CspA promoter, the 3'-UTR is a CspA 3' -UTR, and the 5'-UTR is a CspA5' -UTR.

19. The method of claim 2 or 17, wherein the amino acid sequence of the SMIF protease is shown in SEQ ID No. 51.

20. The method of claim 19, wherein the nucleotide sequence encoding the small ubiquitin-like modifier protease is set forth in SEQ ID No. 52.

21. The method according to claim 2, wherein the first culture liquid, the second culture liquid and the third culture liquid are independently an LB culture liquid or an LB culture liquid containing glucose.

22. The method according to claim 2, wherein the first expression vector is one of expression vectors of the pET series;

the second expression vector is pCold;

the first host, the second host, and the third host are independently Escherichia coli (Escherichia coli).

23. The method of claim 22, wherein the first expression vector is one of pET21a, pET20b, and pET28 a.

24. The method according to claim 22, wherein the strain of escherichia coli is BL21 (DE 3).

25. The method according to claim 2, wherein in step 4-2) or step 3-3), the second recombinant organism is cultured at a temperature of 28 to 37 ℃ for 4 to 8 hours;

in step 4-1), culturing the first recombinant organism at a temperature of 28 to 37 ℃ for 4 to 8 hours under induction of isopropyl- β -D-thiogalactoside, and then continuing culturing the first recombinant organism at a temperature of 10 to 15 ℃ for 15 to 60 minutes;

in step 4-2), the third recombinant organism is cultured at a temperature of 10 to 15 ℃ for 15 to 60 minutes.

26. The method according to claim 2, wherein in step 4-1), the final concentration of tris (2-carbonylethyl) phosphonium hydrochloride in the first culture liquid is 0.1 to 1 mmol/l; the final concentration of zinc sulfate in the first broth is 1 to 5 mmol/l;

in step 4-2), the final concentration of the tris (2-carbonylethyl) phosphonium hydrochloride in the second culture liquid is 0.1 to 1 mmol/l; the final concentration of zinc sulfate in the second culture solution is 1 to 5 mmol/l.

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