KR100509957B1 - Mutant basic fibroblast growth factor having increased secretion efficiency - Google Patents

Abstract

The present invention relates to a mutant bFGF protein with increased secretion efficiency, a gene encoding the protein, a recombinant vector comprising the gene, and a transformed cell transduced with the recombinant vector. It relates to a variant bFGF protein described, a variant bFGF gene encoding the protein, a recombinant vector for animal cell expression comprising the gene and a transformed cell transduced with the recombinant vector. Since the variant bFGF protein of the present invention is excellent in extracellular secretion efficiency, it can be usefully used for mass production of bFGF protein that can be used for ischemic cardiovascular disease due to trauma treatment, angiogenesis, arteriosclerosis or vascular restenosis.

Description

Mutant basic fibroblast growth factor having increased secretion efficiency

The present invention relates to a mutant bFGF protein with increased secretion efficiency, more specifically, a mutant bFGF protein as set forth in SEQ ID NO: 6, a mutant bFGF gene encoding the protein, and for expression of an animal cell comprising the gene. It relates to a recombinant vector and transformed cells transduced with the recombinant vector.

Fibroblast growth factor (hereinafter abbreviated as "FGF") is a protein that was first isolated from the pituitary gland of cattle in the 1970s and has biological activity to promote the growth of fibroblasts and endothelial cells. So far, more than 20 kinds of FGFs are known to exist in humans. Among them, basic (pI 9.6) FGFs, named FGF-2 or bFGF, have a molecular weight of about 18 kDa consisting of 155 amino acids. As a protein, it is secreted mainly in the pituitary gland and is known to promote the growth of various cells, including vascular endothelial cells (Bikfalvi et al ., 1997, Endocr Rev. , 18, 26-45).

Human bFGF gene was cloned in the 1980s and characterized by expression in E. coli (Abraham et al ., 1986, EMBO J. , 5, 2523-2523; Squires et al ., 1988, J. Biol. Chem) , 263, 16297-1630). In particular, bFGF promotes growth of vascular endothelial cells and smooth muscle cells, and thus exhibits excellent efficacy in trauma and vasculature. Developed. Recently, bFGF is known to be a potent inducer of neovascularization in ischemic cardiovascular disease due to atherosclerosis or vascular restenosis, and many studies have been conducted to develop protein or gene therapy using it (Sosnowski et al . , 1999, Curr. Opin. Mol. Ther ., 1, 573-579; Post et al ., 2001, Cardiovasc. Res ., 49, 522-531).

Most growth factors and cytokines that secrete extracellularly and affect other cells and tissues contain secretion signal peptides at the ends of each protein to be secreted out of the cell. Doing. These growth factors and cytokine proteins are synthesized as immature proteins in the cytoplasm, then transferred to the cell membrane by secretory signal peptides through post-translational modification and secreted outside the cell in the form of mature proteins (Heijne, 1986, Nucleic). Acids Res ., 14, 4683-4690).

As discussed above, most of the FGF proteins contain secretory peptides, but specifically, no secretory peptides are found in the amino acid sequence of the bFGF protein. Although bFGF plays a role in the formation of neovascularization and cellular protection against ischemic stimulation in vivo, it is known that the amount of bFGF is secreted to the outside of the cell is very small, because it is not present in the secretion signal peptide. Thus, many studies have been carried out to attach secretory signal peptides at the ends of other proteins such as FGF-4 to the ends of bFGF proteins to promote extracellular secretion of bFGF (Ueno et al ., 1997, Arteriosclerosis, Thrombosis, and Vascular Biology , 17, 2453-2460). However, side effects such as bFGF gene or protein binding to secretory signal peptides have been reported when cells or animal tissues are treated (Rogelj et al ., 1988, Nature , 331, 173-175). Rogelj et al ., 1989, J Cellular Biochem , 39, 13-23).

Therefore, the present inventors prepared a variant bFGF modified with an amino acid sequence without additionally binding a new amino acid sequence such as a secretion signal peptide to bFGF and introduced it into cells, resulting in an increase in the extracellular secretion efficiency of bFGF. By confirming that the present invention was completed.

An object of the present invention is to provide a mutant bFGF protein, a mutant bFGF gene encoding the protein, a recombinant vector for animal cell expression comprising the gene, and a transformed cell transduced with the recombinant vector.

In order to achieve the above object, the present invention provides a variant bFGF protein set forth in SEQ ID NO: 6.

The present invention also provides a variant bFGF gene encoding the protein.

The present invention also provides a recombinant vector for animal cell expression comprising the gene.

The present invention also provides a transformed cell in which the recombinant vector is transduced into a host cell.

In addition, the present invention provides a recombinant adenovirus for gene therapy wherein the variant gene is introduced.

In addition, the present invention provides a method for producing a large amount of variant bFGF using the variant gene.

Hereinafter, the present invention will be described in detail.

The present invention provides variant bFGF protein set forth in SEQ ID NO: 6.

The protein of the present invention is a variant bFGF protein prepared by replacing the 17th amino acid glycine of the wild type bFGF protein of SEQ ID NO: 5 with isoleucine.

Wild type bFGF is widely used as a therapeutic agent for trauma and vasculature due to its property of promoting growth of vascular endothelial cells and smooth muscle cells. It can also be used to treat ischemic cardiovascular disease due to atherosclerosis or vascular restenosis because it is a potent factor that can induce neovascularization. However, since wild-type bFGF does not contain a secretion signal peptide, the amount secreted to the outside of the cell is so small that there are many difficulties in mass production for use as a therapeutic agent.

However, the mutant protein of the present invention has about 1.3 times higher secretion efficiency to the extracellular cell than the wild type bFGF protein by replacing the amino acid sequence of the wild type bFGF protein (see FIG. 5A). Therefore, the variant bFGF protein of the present invention can be usefully used for mass production of bFGF or for treatment of trauma, angiogenesis, arteriosclerosis or cardiovascular disease using bFGF.

The present invention also provides a variant bFGF gene encoding the protein.

The variant bFGF gene of the present invention may be any nucleotide sequence capable of encoding the protein, and preferably has a nucleotide sequence set forth in SEQ ID NO: 4. The gene set forth in SEQ ID NO: 4 is a variant prepared by replacing the base encoding Glycine (GGC), which is the 49th to 51st sequence of the wild type bFGF gene, set forth in SEQ ID NO: 3 with a base that encodes isoleucine (ATC) bFGF gene.

The present invention also provides a recombinant vector for animal cell expression comprising the gene.

Recombinant vector for animal cell expression of the present invention can be prepared by inserting the mutant bFGF gene described in SEQ ID NO: 4 in a general animal cell expression vector. In the preferred embodiment of the present invention, the pcDNA3.1 vector is used as the animal cell expression vector. However, the present invention is not limited thereto, and all animal cell expression vectors that can be generally used may be used. In a preferred embodiment of the present invention, a vector in which the mutant bFGF gene described in SEQ ID NO: 4 is inserted into a pcDNA3.1 vector, which is an animal cell expression vector, was prepared and named "pcDNA / neobFGF" (see FIG. 4). .

The present invention also provides a transformed cell in which the recombinant vector is transduced into a host cell.

In preparing the transformant, animal cells or animal cell lines are preferably used as host cells, and more preferably cells or cell lines derived from humans are used, but such host cells are not particularly limited. In the preferred embodiment of the present invention, a case of using a COS cell line as a host cell is illustrated.

In addition, the present invention provides a recombinant adenovirus for gene therapy wherein the variant gene is introduced.

The recombinant adenovirus of the present invention is to prepare a shuttle vector inserting the mutant bFGF gene described in SEQ ID NO: 4, and to induce homologous recombination with the adenovirus it was prepared to include a variant bFGF gene.

The shuttle vector may be any shuttle vector that can be used generally, pCA13 (Microbix, Canada) was used as the shuttle vector in the present invention. In addition, the adenovirus may also use all adenoviruses that can be generally used, and in the present invention, adenoviruses remove E1 / E3 and deleted from human adenovirus type 5 (deleted). replication defective) Adenovirus pHRLd1324 was used.

In a preferred embodiment of the present invention, a shuttle vector pCA13 / neobFGF into which the mutant bFGF gene described in SEQ ID NO: 4 is inserted is prepared (see FIG. 7), and the homologous recombination with adenovirus pHRLd13424Bst is induced to produce recombinant adenovirus Ad / CMV / neobFGF was prepared (see FIG. 8). In addition, as a result of performing PCR by infecting the prepared Ad / CMV / neobFGF recombinant adenovirus to animal cells and recovering the virus, it was confirmed that the recombinant adenovirus contained the mutant bFGF gene described in SEQ ID NO: 4. It was.

The present invention also provides a method for producing variant bFGF by culturing the transformed cells after transducing a recombinant expression vector comprising a variant bFGF gene described in SEQ ID NO: 4 into a host cell.

In the above, the host cell is not limited to a specific cell, and in a preferred embodiment of the present invention, a transformant having pcDNA / neobFGF introduced into a COS cell line was used. In the present invention, by culturing the transformed cells and measuring the amount of mutant bFGF secreted therefrom, it was confirmed that it produces about 1.3 times as much amount as the COS cell line without the transduction of pcDNA / neobFGF (Fig. 5a). Thus, the method of the present invention can be usefully used for the mass production of bFGF proteins that can be used to treat ischemic cardiovascular disease due to trauma, angiogenesis, atherosclerosis or vascular restenosis.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 cDNA Cloning of Natural Type bFGF

Human epidermal-derived fibroblasts (foreskin fibroblasts) were cultured to clone cDNA of native bFGF. After 2 × 10 ⁹ cells were obtained, total RNA was isolated using a Trizol reagent (Life Technologies, USA). PCR kit containing 10 μg of total RNA isolated as a template and containing oligo-dT primers and AMV reverse transcriptase (RNA PCR Kit Ver.2.1, Takara, Japan) ) Was used to synthesize a single strand of cDNA complementary to the total mRNA.

Taq DNA polymerization was carried out using 10 μg of the synthesized cDNA as a template and a primer described in SEQ ID NO: 1 corresponding to the upstream bFGF gene and a primer described in SEQ ID NO: 2 corresponding to the downstream bFGF gene. A polymerase chain reaction (hereinafter, abbreviated as "PCR") by an enzyme (Taq DNA polymerase) was performed. PCR reaction was performed for 1 minute at 94 ° C for denaturation of the template, 1 minute at 55 ° C for annealing the template and primer, and 1 minute at 72 ° C for extension. 30 cycles were performed using a PCR reactor (PTC-100 thermal controller, MJ Research, USA). As a result of performing a 1% agarose gel electrophoresis on the amplified product amplified from the reaction, it was confirmed that the DNA fragment of about 480 base pairs (hereinafter, abbreviated as "bp") was amplified. (FIG. 1).

The amplified DNA fragments were separated and purified and inserted into the pGEM-Teasy vector (3.0 kbp, Promega, USA), which is a plasmid vector capable of directly cloning the PCR product, using T4 DNA ligase. pGEMT / bFGF ". The prepared pGEMT / bFGF vector was transformed into E. coli strain XL1-Blue (Stratagene, USA). The transformed E. coli strain was plated and cultured on an LB (Luria Botani) plate medium containing 150 µg / ml of antibiotic ampicillin, and transformed with a pGEMT / bFGF vector to form white colonies. It was possible to obtain a strain. Plasmid DNA was isolated from the transformed white colonies and subjected to electrophoresis by restriction enzyme Bam HI, and it was confirmed that there was a DNA band having a size of about 3.45 kbp equal to the DNA size of pGEMT / bFGF (FIG. 2). After separating the DNA from the identified band and DNA sequencing using restriction enzyme map analysis and DNA sequencing (ABI system, USA), the DNA fragment of 480 bp amplified by the PCR reaction is sequence number It was confirmed that the human bFGF gene described as 3.

Example 2 Construction of Mutant bFGF Gene

In order to increase the extracellular secretion efficiency by modifying the amino acid sequence of the bFGF protein, a mutant bFGF gene in which glycine, which is the 17th amino acid at the amino terminus of the native bFGF protein, is replaced with isoleucine To prepare. Specifically, the bFGF gene described in SEQ ID NO: 7 in which the codon corresponding to the 17th amino acid is changed from GGC (glycine) to ATC (isoleucine) so that the 17th amino acid glycine from the amino terminus of the bFGF protein can be replaced with isoleucine. Primers corresponding to the upstream primer and downstream of the bFGF gene described by SEQ ID NO: 8 were designed. The primers were designed to include the amino acid start codon (ATG) and the end codon (TGA), followed by the site GATATC that the restriction enzyme Eco RV can recognize.

PGEMT / bFGF, which is a vector containing the native type bFGF gene cloned in Example 1, was used as a template, and the primers described in SEQ ID NO: 7 and SEQ ID NO: 8 were used for 1 minute at 94 ° C. 30 cycles of PCR reactions were carried out with one cycle of heat denaturation, primer binding for 1 minute at 55 ° C., and extended reaction at 72 ° C. for 1 minute. As a result of agarose electrophoresis, the DNA fragment amplified above was a DNA fragment having a size of about 465 bp, which was a vector containing a mutant bFGF gene described in SEQ ID NO: 4.

The amplified DNA fragments were isolated, cut with restriction enzyme Eco RV and inserted into the Eco RV restriction enzyme locus of pBluescript KS (+) vector (Stratagene, USA) by reaction with T4 DNA ligation, followed by mutation described in SEQ ID NO: 4. A new plasmid containing type bFGF (neobFGF) was prepared and named "pBlue / neobFGF" (FIG. 3).

The pBlue / neobFGF vector was transformed into E. coli XL1-Blue strain, and then transformed into E. coli LB plate medium containing antibiotics ampicillin, X-gal, IPTG, and transformed E. coli to form white colonies. PBlue / neobFGF was isolated from the selected E. coli colonies and subjected to restriction enzyme mapping and DNA sequencing.Based on the 17th glycine amino acid sequence of human bFGF protein, the base codon GGC was encoded as isoleucine. It was confirmed that the mutant bFGF gene described in SEQ ID NO: 4 is substituted.

Example 3 Construction of Mutant bFGF Vector for Animal Cell Expression

In order to analyze the expression and secretion efficiency of the mutant bFGF gene prepared in Example 2, an animal cell expression vector was prepared. To this end, first, the pBlue / neobFGF vector prepared in Example 2 was digested with restriction enzymes Eco RI and Xho I to obtain a variant bFGF gene fragment as set forth in SEQ ID NO: 4. The expression vector for animal cells, pcDNA3.1 (Invitrogen, USA) was digested with the same restriction enzymes as above, and the mutant bFGF gene fragment was inserted using T4 DNA ligase. An expression vector of 5.9 kbp into which the variant bFGF gene fragment was inserted was named "pcDNA / neobFGF" (FIG. 4), and the vector was transformed into E. coli strains to confirm the transduction of the expression vector. In addition, as a control for comparing the mutant bFGF gene and the extracellular secretion efficiency prepared above, the wild type bFGF gene (wild type bFGF, hereinafter referred to as "wtbFGF") described in SEQ ID NO: 3 vector pcDNA3.1 vector And transformed into a transform vector, and named it "pcDNA / wtbFGF".

Example 4 Extracellular Secretion Efficiency Analysis of pcDNA / neobFGF in Animal Cell Lines

Animal cell lines were infected with the vector pcDNA / nebFGF, which is a vector containing the mutant bFGF prepared in Example 3, and the vector pcDNA / wtbFGF, which comprises the native type bFGF, described in SEQ ID NO: 3. After transfection, the secretion efficiency of bFGF was compared. Specifically, 1 μg of the pcDNA / neobFGF prepared in Example 3 and the natural type pcDNA / wtbFGF prepared in Example 3 were treated in a COS cell line (ATCC, USA) grown at about 70-80% in a cell culture vessel containing DMEM medium. After transfection, lipofectamine (lipofectamine, Gibco BRL, USA) was used. At this time, in order to correct the transduction efficiency, beta-galactosidase (beta-galactosidase, LacZ) was expressed and transduced together with the vector pcDNA / LacZ (Invitrogen, USA) which can convert the infection efficiency. The transduced cells were incubated at room temperature for about 3 hours and then replaced with DMEM medium, and cells and cell cultures were obtained 48 hours after culture. The obtained cell culture was quantified by proportionally converting the amount of bFGF secreted to the outside of the cell by performing an Enzyme linked immunosorbent assay (Quantikine bFGF ELISA kit, R & D Systems, USA). In addition, the obtained cells were used to correct infection efficiency using pcDNA / LacZ as a control. Infection efficiency was measured by transducing pcDNA / LacZ, which expresses beta-galactosidase, and measuring the amount of beta-galactosidase expressed in cells when pcDNA / neobFGF was transduced. Correction was made by calculating proportionally.

As a result, the cells transduced with pcDNA / neobFGF in the COS cell line increased the extracellular secretion of bFGF by about 1.3 times or more compared with the cells transduced with pcDNA / wtbFGF (FIG. 5A). In addition, the same results were obtained in the experiments performed by transducing the 293 cell line, which is another animal cell line (FIG. 5B).

Example 5 Analysis of bFGF Proteins Secreted in Animal Cell Lines Transduced with pcDNA / neobFGF

Western blots were performed to qualitatively analyze neobFGF proteins secreted from COS cell lines transduced with pcDNA / neobFGF vectors. Specifically, cell culture was isolated 48 hours after transducing the pcDNA / neobFGF vector into the COS cell line. The culture solution was administered to a Heparin Sepharose column (Amersham Pharmacia, Sweden), washed several times with sodium phosphate buffer (pH 7.4), and the buffer solution containing 1 M sodium chloride (NaCl) was concentrated in concentration gradient ( gradient was added to purify the bFGF protein secreted out of the COS cells. The purified bFGF protein was electrophoresed on 15% SDS-polyacrylamide gel (SDS-polyacrylamide gel electrophoresis), and the protein contained in the electrophoretic gel was polyvinylidene fluoride (PVDF) paper (Gelman, USA) and Western blot was performed in a conventional manner using an antibody against bFGF (SantaCruz, USA).

As a result, it was confirmed that the mutant bFGF described in SEQ ID NO: 6 having a size of 18 kDa was secreted from the COS cell line transduced with pcDNA / neobFGF (FIG. 6). The present inventors deposited the transformant E. coli pcDNA / neobFGF containing the variant bFGF with the transduction of pcDNA / neobFGF to the Korea Microbiological Conservation Center on October 9, 2003 (Accession No .: KCCM 10515).

Example 6 Construction of Recombinant Adenovirus Containing NeobFGF

Using a neobFGF to prepare a shuttle vector containing the neobFGF gene for use in gene therapy, and then induce homologous recombination of the shuttle vector and adenoviruses recombinant adenovirus containing neobFGF Was prepared. Specifically, "pBlue / neobFGF" prepared in Example 2 was cut with restriction enzymes Eco RI and Xho I to obtain neobFGF gene fragments, and shuttle vector pCA13 (6.95 kbp, Microbix, Canada) was also used as the restriction enzyme as described above. After cleavage, a recombinant shuttle vector having a size of 7.4 kDa in which the neobFGF gene was inserted into the shuttle vector was prepared by reaction with T4 DNA ligase, which was named "pCA13 / neobFGF" (FIG. 7). In addition, a recombinant shuttle vector into which a natural bFGF gene was inserted was also prepared in the same manner as above and named "pCA13 / wtbFGF".

In addition, to prepare a recombinant adenovirus for gene therapy containing neobFGF, pHRLdl324Bst (Qbiogene, E1 / E3 removal and deleted-replication defective adenovirus derived from human adenovirus type 5). USA). Recombinant adenovirus for gene therapy comprising neobFGF was prepared by inducing homologous recombination reaction with the pCA13 / neobFGF shuttle vector and adenovirus pHRLdl324Bst prepared above. First, the virus was linearized by treatment of pHRLdl324Bst with restriction enzyme Bst BI, and co-transformed with pCA13 / neobFGF in E. coli BJ5183 (Qbiogene, USA). The newly recombined adenovirus was named "Ad / CMV / neobFGF" through the reaction (Fig. 8). In addition, adenovirus Ad / CMV / wtbFGF containing natural bFGF and Ad / CMV / LacZ virus capable of expressing beta-galactosidase for infection efficiency control were also prepared in the same manner.

In order to confirm that the neobFGF gene was properly inserted into the prepared adenovirus, the prepared Ad / CMV / neobFGF recombinant adenovirus was infected with 293 cell lines and then grown, and the virus was purified to perform a PCR reaction. Specifically, 293 cell lines were cultured at about 90% in a culture vessel, and the recombinant adenovirus Ad / CMV / neobFGF prepared above was infected with 10 moi, and after 2-3 hours, medium was exchanged, followed by 2-3 days at 37 ° C. Incubated for After the incubation, the cells and the cell culture solution were obtained and centrifuged. The virus was recovered by centrifugation after cooling / thawing three times. The virus recovered above was used as a template and analyzed by PCR using primers described in SEQ ID NO: 1 and SEQ ID NO: 2.

As a result, it was confirmed that the mutant bFGF gene described in SEQ ID NO: 4 exists in the recombinant adenovirus Ad / CMV / neobFGF (FIG. 9).

As described above, the mutant bFGF protein of the present invention is highly secreted when introduced into a cell, and thus may be useful for mass production of bFGF and gene therapy using the same.

Figure 1 is a DNA fragment photograph of the bFGF gene obtained by PCR amplification using a primer having a nucleotide sequence described in SEQ ID NO: 1 and SEQ ID NO: 2.

Lane 1: φX174 DNA / Hae III Marker (Fermentas, Lithuania)

Lane 2: bFGF gene amplification products

Figure 2 is a photo of the electrophoresis of pGEMT / bFGF isolated from the strain transformed with pGEMT / bFGF and then treated with restriction enzyme Bam HI.

Lane 1: Lamda DNA / Hind III Marker (Fermentas, Lithuania)

Lane 2: sample treated with pGEMT / bFGF with restriction enzyme Bam HI

Figure 3 is a schematic diagram showing the process of cloning the human bFGF gene described in SEQ ID NO: 3.

Figure 4 is a schematic diagram showing a process for preparing a recombinant vector for expressing animal cells (pcDNA / neobFGF) comprising the mutant bFGF gene described in SEQ ID NO: 4.

Figure 5a is a graph quantifying the amount of bFGF secreted extracellularly by ELISA by expressing a recombinant vector (pcDNA / neobFGF) containing a mutant bFGF gene in COS cell line, an animal cell line.

Figure 5b is a graph quantifying the amount of bFGF secreted extracellularly by ELISA by expressing a recombinant vector (pcDNA / neobFGF) comprising a variant bFGF gene in animal cell line 293 cell line.

Figure 6 is a photograph of analysis of the bFGF secreted after expression of the recombinant vector (pcDNA / neobFGF) containing a variant bFGF gene in an animal cell line.

Lane 1: 18 kDa human bFGF recombinant protein from Sigma

Lane 2: Concentration of Cultures from COS Cells Transduced with pcDNA / LacZ

Lane 3: bFGF isolated from culture obtained from COS cells transduced with pcDNA / neobFGF

7 is a schematic diagram illustrating a process of preparing a shuttle vector (pCA13 / neobFGF) containing a mutant bFGF gene.

8 is a schematic diagram illustrating a process for preparing a recombinant adenovirus (Ad / CMV / neobFGF) by homologous recombination of a shuttle vector (pCA13 / neobFGF) and adenovirus (pHRLdl324Bst) comprising a mutant bFGF gene.

9 is a PCR amplification photograph confirming that the mutant bFGF gene is inserted into the recombinant adenovirus (Ad / CMV / neobFGF) into which the mutant bFGF gene is inserted.

Lane 1: PCR amplification product based on pcDNA / neobFGF

Lane 2: PCR amplification products based on Ad / CMV / LacZ

Lane 3: PCR amplification product based on Ad / CMV / wtbFGF

Lane 4: PCR amplification product based on Ad / CMV / neobFGF

<110> JANG, Yangsoo CHUNG, Ji Hyung <120> Mutant basic fibroblast growth factor having increased secretion efficiency <130> <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> upstream primer for bFGF <400> 1 aggaccatgg cagccgggag c 21 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> downstream primer for bFGF <400> 2 gtaaaatcag ctcttagcag acat 24 <210> 3 <211> 468 <212> DNA <213> Homo sapiens <400> 3 atggcagccg ggagcatcac cacgctgccc gccttgcccg aggatggcgg cagcggcgcc 60 ttcccgcccg gccacttcaa ggaccccaag cggctgtact gcaaaaacgg gggcttcttc 120 ctgcgcatcc accccgacgg ccgagttgac ggggtccggg agaagagcga ccctcacatc 180 aagctacaac ttcaagcaga agagagagga gttgtgtcta tcaaaggagt gtgtgctaac 240 cgttacctgg ctatgaagga agatggaaga ttactggctt ctaaatgtgt tacggatgag 300 tgtttctttt ttgaacgatt ggaatctaat aactacaata cttaccggtc aaggaaatac 360 accagttggt atgtggcact gaaacgaact gggcagtata aacttggatc caaaacagga 420 cctgggcaga aagctatact ttttcttcca atgtctgcta agagctga 468 <210> 4 <211> 468 <212> DNA <213> Homo sapiens <400> 4 atggcagccg ggagcatcac cacgctgccc gccttgcccg aggatggcat cagcggcgcc 60 ttcccgcccg gccacttcaa ggaccccaag cggctgtact gcaaaaacgg gggcttcttc 120 ctgcgcatcc accccgacgg ccgagttgac ggggtccggg agaagagcga ccctcacatc 180 aagctacaac ttcaagcaga agagagagga gttgtgtcta tcaaaggagt gtgtgctaac 240 cgttacctgg ctatgaagga agatggaaga ttactggctt ctaaatgtgt tacggatgag 300 tgtttctttt ttgaacgatt ggaatctaat aactacaata cttaccggtc aaggaaatac 360 accagttggt atgtggcact gaaacgaact gggcagtata aacttggatc caaaacagga 420 cctgggcaga aagctatact ttttcttcca atgtctgcta agagctga 468 <210> 5 <211> 155 <212> PRT <213> Homo sapiens <400> 5 Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg 35 40 45 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu 50 55 60 Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 65 70 75 80 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys 130 135 140 Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155 <210> 6 <211> 155 <212> PRT <213> Homo sapiens <400> 6 Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15 Ile Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg 35 40 45 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu 50 55 60 Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 65 70 75 80 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys 130 135 140 Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155 <210> 7 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> upstream primer for mutant bFGF <400> 7 agcaccgata tcatggcagc cgggagcatc accacgctgc ccgccttgcc cgaggatggc 60 atcagc 66 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> downstream primer for mutant bFGF <400> 8 gtaaaagata tctcagctct tagcagacat 30

Claims (9)

Variant bFGF protein set forth in SEQ ID NO: 6. A variant bFGF gene encoding the protein of claim 1. 3. The variant bFGF gene of claim 2, wherein the variant bFGF gene is set forth in SEQ ID NO: 4. Recombinant vector (pcDNA / neobFGF) for animal cell expression comprising the gene of claim 2. A transformed cell in which the recombinant expression vector of claim 4 is introduced into a host cell. The transformed cell of claim 5, wherein the host cell is a COS cell line. Recombinant adenovirus (Ad / CMV / neobFGF) for gene therapy wherein the variant bFGF gene of claim 2 is introduced. A method for producing the variant bFGF of claim 1 by culturing the transformed cell after transducing a recombinant expression vector comprising the variant bFGF gene of claim 2 into a host cell. The method of claim 8, wherein the host cell is a COS cell line.