HK1080895B - Hybrid hepatocyte growth factor gene having high expression efficiency of two heterotypes of hepatocyte growth factor - Google Patents

Description

Hybrid hepatocyte growth factor gene for efficiently expressing two different types of hepatocyte growth factors

Technical Field

The present invention relates to highly efficient Hepatocyte Growth Factor (HGF) which simultaneously expresses two different types of HGF.

Background

The present invention relates to the preparation of hybrid HGF gene having higher expression efficiency than HGF cDNA and expressing two different types of HGF and dHGF simultaneously (deletion mutant HGF) by inserting an inherent or exogenous intron between exons 4 and 5 of HGF cDNA.

HGF is a heparin-binding glycoprotein called scatter factor. The gene encoding HGF is located on chromosome 721.1, contains 18 exons and 17 introns, has the amino acid sequence of SEQ ID NO: 1 (Seki T et al, Gene)102: 213-219, 1991). An about 6kb transcript is transcribed from the HGF gene, and from this, an HGF precursor polypeptide consisting of 728 amino acids is synthesized. Meanwhile, dHGF precursor polypeptide consisting of 723 amino acids is synthesized by the other splicing of HGF gene. The biologically inactive precursors are converted by proteases in the serum into the active form of disulfide-linked heterodimers. In the heterodimer, the high molecular weight alpha chain forms an N-terminal hairpin loop of four kringle domain domains and a peptide region resembling the pro-activated plasminogen. The three-loop domain of the three disulfide loop structures consists of about 80 amino acids, which play an important role in protein-protein interactions. The low molecular weight beta chain forms an inactive serine protease-like domain. dHGF, consisting of 723 amino acids, is a polypeptide with 5 amino acids deleted in the first kringle domain of the alpha chain, i.e. F, L, P, S and S.

Recently, both HGF and dHGF have been reported to have some biological functions, such as promoting the growth and morphogenesis of epithelial, melanocyte and endothelial cells. However, they differ in immunological and biological characteristics.

For example, HGF is 20-fold, 10-fold and 2-fold more active than dHGF in promoting DNA synthesis in human umbilical cord endothelial cells, arterial smooth muscle cells and NSF-60 (murine myeloblasts), respectively. The activity of dHGF is 3 times and 2 times higher than that of HGF in promoting the DNA synthesis of LLC-PKI, OK and mouse interstitial cell. HGF was 70 times more soluble in PBS than dHGF. Some monoclonal antibodies against dHGF recognize only dHGF, but not HGF or a reduced form of dHGF,this suggests that the structures of HGF and dHGF are different. Thus, the simultaneous synthesis of HGF and dHGF in vivo indicates that they interact biologically (Shima, N. et al, Biochemical and Biophysical Research Communications)200：808-815，1994)。

HGF secreted by cells from the mesoderm has multiple biological functions, such as 1) inducing epithelial cells to form tubular structures; 2) stimulating endothelial cells to form blood vessels in vivo and in vitro; 3) regenerating the liver and kidney due to their anti-apoptotic activity; 4) kidney, ovary and testis formation; 5) controlling osteogenesis; 6) stimulating the growth and differentiation of hematopoietic precursor red blood cells; and 7) nerve cell sprouting axons (Stella, M.C. and Comoglio, P.M., Journal of International Biochemistry and cell biology (The International Journal of Biochemistry)&CellBiology)31: 1357-1362, 1999)). Based on these different functions, HGF or a gene encoding HGF may be developed as a therapeutic agent for treating ischemic or hepatic diseases. Indeed, HGF may be present in vivo as HGF or dHGF, and thus, co-expression of HGF and dHGF is important to maximize therapeutic effect. Therefore, the present invention aims to develop a hybrid HGF gene capable of simultaneously expressing HGF and dHGF having a high gene therapeutic effect.

Summary of The Invention

Therefore, a first object of the present invention is to provide a hybrid HGF gene that simultaneously expresses two different types of HGF.

According to one aspect of the invention, a hybrid HGF construct comprising human HGF exons 1 to 18, or a degenerate sequence form thereof that does not alter the encoded amino acid sequence, and comprising human HGF intron 4 or a fragment thereof, located between exons 4 and 5, excluding any introns located between other exons.

It is another object of the present invention to provide a recombinant vector comprising the hybrid HGF gene and a microorganism transformed with the above vector.

It is still another object of the present invention to provide a pharmaceutical composition for treating or preventing ischemic or hepatic diseases, which comprises an HGF gene.

Description of the drawings

The above and other objects and features of the present invention will be apparent from the description of the invention when taken in conjunction with the accompanying drawings, which respectively show:

FIG. 1: schematic representation of the HGF-X prototype illustrating the location of the gene fragment,

FIG. 2 is a drawing: the process of cloning gene fragments from HepG2 genomic DNA,

FIG. 3: the process of cloning gene fragments from human placental cDNA,

FIGS. 4A and 4B: the process of preparing expression vector pCK-HGF-X,

FIG. 5: the process of preparing expression vectors pCK-cHGF and pCK-dHGF,

FIG. 6: the process for preparing the expression vector pCP-HGF-X family,

FIG. 7: the process of preparing expression vectors pCP-cHGF and pCP-dHGF,

FIG. 8: the gene expression levels of pCP-cHGF, pCP-dHGF and pCP-HGF-X,

FIG. 9: the gene expression patterns of pCP-cHGF, pCP-dHGF and pCP-HGF-X were observed by electrophoresis on a 12% polyacrylamide gel,

FIG. 10: the gene expression level of pCP-cHGF, pCP-dHGF and pCP-HGF-X7 in vivo,

FIG. 11: cerebral angiogenesis in two groups of rabbits administered pCP and pCP-HGF-X7, respectively.

Detailed Description

The hybrid Hepatocyte Growth Factor (HGF) constructs of the invention comprise human HGF exons 1 to 18, or a denatured form thereof that does not alter the encoded amino acid sequence, and human HGF intron 4 or fragments thereof, located between exons 4 and 5, excluding any introns located between other exons.

The hybrid Hepatocyte Growth Factor (HGF) gene of the present invention comprises cDNA corresponding to exons 1 to 18 and intron cDNA, either intrinsic or exogenous, inserted between exons 4 and 5 thereof. The intron comprises either an inherent intron fragment or a recombination sequence.

One embodiment of the hybrid HGF gene comprising the inherent intron of the present invention is 7112bp long, having the sequence of SEQ ID NO: 2. The hybrid HGF gene expresses both HGF and dHGF simultaneously, and has higher expression efficiency than HGF cDNA. Codon degeneracy allows the coding and/or non-coding regions of the hybrid HGF genes of the invention to be modified or altered without altering the amino acid sequence of the protein and the expression of the gene.

Thus, in comparison to SEQ ID NO: 2 and fragments thereof are within the scope of the invention. "substantially identical" means that the sequence homology is not less than 80%, preferably not less than 90%, and more preferably not less than 95%.

Hybrid HGF genes may contain an intrinsic intron fragment, optionally with a small recombination sequence inserted between exons 4 and 5 of the HGF cDNA. Herein, such hybrid HGF gene comprising the intrinsic intron fragment is designated "HGF-X". Have SEQ ID No: HGFX-6, HGF-X7 and HGF-X8 of 19 to 21 are preferred.

The hybrid HGF gene of the present invention is synthesized and inserted into an expression vector according to known genetic engineering methods. The vector is then introduced into a suitable host cell such as E.coli and yeast. For example, E.coli Top10F was transfected with the HGF-X7 gene of the present invention. The resulting E.coli Top10F 'pCK-HGFX 7 and E.coli Top 10F' pCP-HGFX7 were then deposited at 3.12.2002 under the accession numbers KCCM-10361 and KCCM-10362, respectively.

The gene of the present invention and the protein encoded by it are produced at a high level using the transformed cells.

Depending on the host cell, the vector of the present invention may optionally contain sequences regulating gene expression such as a promoter or terminator, self-replicating sequences and secretion signals.

In addition, the present invention comprises a pharmaceutical composition for treating or preventing ischemic and hepatic diseases, which comprises the hybrid HGF gene as an active ingredient or a vector comprising the gene. Preferably, the composition is formulated for injection.

The compositions of the present invention also comprise a pharmaceutically acceptable carrier. Any of the conventional procedures in the pharmaceutical field are used to prepare oral formulations such as tablets, capsules, pills, granules, suspensions and solutions; injectable formulations such as solutions, suspensions, or dry powders mixed with distilled water prior to injection; topical preparations such as ointments, creams and lotions; and other agents.

Carriers commonly used in the pharmaceutical field may be employed in the compositions of the present invention. For example, formulations for oral administration include binders, emulsifiers, disintegrants, excipients, solvents, dispersants, stabilizers, suspending agents, colorants, and flavorants. Injectable formulations include preservatives, antagonists (unagonizing agents), solubilizing agents or stabilizing agents. Formulations for topical application include bases, excipients, lubricants or preservatives. Any suitable formulation known in the art (Remington pharmaceutical sciences (latest edition), Mack publishing company, Eaton PA)) can be used in the present invention.

The composition of the present invention can be used in clinical applications as a variety of oral and parenteral formulations. Suitable formulations may be prepared using such excipients as additives, enhancers, binders, wetting agents, disintegrants and surfactants or diluents. Solid formulations for oral administration include pills, tablets, dusting powders, granules and capsules. TheseSolid formulations may be prepared by mixing one or more excipients such as starch, calcium carbonate, sucrose, lactose and gelatin with a diphenylbutylated (diphenylbutylated lacton) lignan derivative. Additionally, lubricants such as magnesium stearate and talc are included in the formulations of the present invention. Liquid formulations for oral administration include suspensions, solutions, emulsions and syrups. These preparations may contain wetting agents, sweeteners, aromatics and preservatives in addition to conventional simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, suspensions, emulsions, optionally lyophilized processes and suppositories. Excipients and suspending agents which are not water-miscible include vegetable oils such as propylene glycol, polyethylene glycol and olive oil and injectable esters such as ethyl oleate. Witepsole^®、Macrogol^®、Tween^®61. Cocoa butter, glyceryl laurate and glyceryl gelatin can be used as the basis for suppositories.

The compositions of the invention may be administered orally or by parenteral routes such as intravenous, intramuscular, subcutaneous, intraperitoneal, intraarterial injection or infusion or topically by rectal, intranasal, respiratory or intraocular administration.

It will be understood that the conventional daily dosage of the composition of the present invention will be determined depending on various relevant factors including the disease to be treated, the selected administration route, age, sex and weight of each patient, and the severity of the patient's symptoms, and may be administered once or separately. Therefore, the daily dose should not be construed as limiting the scope of the invention in any way.

The following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: preparation of hybrid Gene construct encoding human HGF

(1) Cloning of HGF Gene fragment derived from genomic DNA

Human HepG2 cells (ATCC accession number: HB-8065) were suspended in TES buffer (10mM Tris-HCl; 1mM EDTA; 0.7% SDS) and treated with 400. mu.g/ml proteinase K at 50 ℃ for 1 h. The genomic DNA was then extracted from the cell suspension by phenol/chloroform extraction and ethanol precipitation as is conventional in the art.

In the PCR amplification, the extracted genomic DNA is used as a template DNA. As a primer pair, synthesized SEQ ID NO: 3 and 4 to obtain a DNA fragment comprising: HGF gene fragment 2(HGF-F2), SEQ ID NO: 3 and 5; HGF-F3, SEQ ID NO: 6 and 7; HGF-F5, SEQ ID NO: 8 and 7; HGF-F7, SEQ ID NO: 9 and 7; HGF-F8, SEQ ID NO: 10 and 7; HGF-F6 (FIG. 1). A PCR amplification mixture was prepared by mixing 1. mu.l of template DNA, 1. mu.l of each primer (10 pmol/1. mu.l), 10. mu.l of dNTP (10mM), 3.5 units of amplification Hi-Fi enzyme (GibcoBRL, USA), and 10. mu.l of enzyme buffer, and adjusting the final volume to 100. mu.l with distilled water. 30 cycles of PCR amplification were performed, each cycle consisting of 94 ℃ for 1min, 55 ℃ for 1min and 72 ℃ for 30 sec. The primers used herein and the amplified gene fragments obtained therefrom are shown in Table 1.

TABLE 1

5' end primer	3' end primer	Amplified fragments
			gHGF3(SEQ ID NO：3)	gHGF4(SEQ ID NO：4)	HGF gene fragment 2(HGF-F2)
gHGF3(SEQ ID NO：3)	gHGF10(SEQ ID NO：5)	HGF gene fragment 3(HGF-F3)
			gHGF5(SEQ ID NO：6)	gHGF7(SEQ ID NO：7)	HGF gene fragment 5(HGF-F5)
gHGF12(SEQ ID NO：8)	gHGF7(SEQ ID NO：7)	HGF gene fragment 7(HGF-F7)
			gHGF13(SEQ ID NO：9)	gHGF7(SEQ ID NO：7)	HGF gene fragment 8(HGF-F8)
gHGF6(SEQ ID NO：10)	GHGF7(SEQ ID NO：7)	HGF gene fragment 6(HGF-F6)

The amplified HGF-F2 comprises a sequence within the range of SEQ ID NO: 2 (HGF-X1; consisting of exons 1 to 4-intron 4-exons 5 to 18) from 392 to 2247; HGF-F3 is a sequence ranging from 392 to 727; HGF-5 is a sequence ranging from 2229 to 5471; HGF-F6 is a sequence ranging from 5117 to 5471; HGF-F7 is a sequence ranging from 3167 to 5471; and HGF-F8 is a sequence ranging from 4167 to 5471.

Each of the amplified HGF gene fragments was inserted into a plurality of cloning sites of pGEM-T easy vector (Promega, Wisconsin, USA) to obtain pGEM-Teasy-HGF 2, pGEM-T easy-HGF-F3, pGEM-T easy-HGF-F5, pGEM-T easy-HGF-F6, pGEM-T easy-HGF-F7 and pGEM-Teasy-HGF 8, respectively (FIG. 2). The nucleotide sequence of the amplified HGF gene fragment was confirmed by sequence analysis.

(2) Cloning of HGF Gene fragment from cDNA

In PCR amplification, human placental cDNA (Clontech, CA, USA) was used as template DNA under the same conditions as described in example 1. As a primer pair, synthesized SEQ id no: 11 and 12, and SEQ ID NO: 13 and 14 were used to obtain the DNA fragments contained in HGF-F1 and HGF-F4, respectively. In addition, cDNAs of HGF gene (cHGF) and deleted HGF gene (dHGF) contain DNA fragments that are expressed using synthetic SEQ ID NO: 15 and 16 as a primer pair. The dHGF gene is an HGF gene in which 5 nucleotide sequences are deleted.

The primers used herein and the amplified gene fragments obtained therefrom are shown in Table 2.

TABLE 2

5' end primer	3' end primer	Amplification product
			gHGF1(SEQ ID NO：11)	gHGF2(SEQ ID NO：12)	HGF gene fragment 1(HGF-F1)
gHGF8(SEQ ID NO：13)	gHGF9(SEQ ID NO：14)	HGF gene fragment 4(HGF-F4)
			cHGF5(SEQ ID NO：15)	cHGF3(SEQ ID NO：16)	HGF Gene cDNA (cHGF) dHGF Gene cDNA (dHGF)

Amplified HGF-F1 and HGF-F4 comprise SEQ ID NOs: 2 ranging in nucleotide sequence from 1 to 402 and from 6533 to 7113. The HGF gene cDNA comprises SEQ ID NO: 1 in the sequence range from 1 to 2184, while the dHGF gene cDNA has the same sequence as the HGF gene cDNA except for deletion of the sequence range from 483 to 495.

Each amplified fragment of HGF gene was inserted into multiple cloning sites of pGEM-T easy vector (Promega, Wisconsin, USA) to obtain pGEM-T easy-HGF-1, pGEM-T easy-HGF-F4, pGEM-T easy-cHGF, and pGEM-T easy-dHGF, respectively (FIG. 3). The nucleotide sequences of the human HGF gene fragment, HGF gene cDNA and dHGF gene cDNA were confirmed by sequence analysis.

(3) Preparation of hybrid HGF Gene construct

A hybrid HGF gene construct of genomic DNA and cDNA was prepared by combining HGF gene fragments cloned in steps (1) and (2) as follows (fig. 4A and 4B).

Plasmid pGEM-T-easy-HGF-F1 was treated with HindIII/BamHI to obtain HGF-F1. Plasmid pCK (see PCT International publication No.: WO/0040737) was treated with HindIII/BamHI, and HGF-F1 was inserted therein to obtain pCK-F1. Plasmids pGEM-T-easy-HGF-F2 and pGEM-T-easy-HGF-F3 were then treated with MluI/BamHI to give HGF-F2 and HGF-F3, respectively. pCK-1 was treated with MluI/BamHI, into which HGF-F2 and HGF-F3 were inserted, to give pCK-F12M and pCKF13M, respectively. The MluI restriction sites of the vectors pCK-F12M and pCKF13M were replaced with the HgaI restriction regions by a region-mediated mutation kit (Stratagene, CA, USA) to obtain pCK-F12M and pCKF13M, respectively.

In addition, plasmid pGEM-T-easy-HGF-F4 was treated with BamHI/XbaI to give HGF-F4. pCK-F12 and pCKF13 were treated with BamHI/XbaI into which HGF-F4 was inserted to give pCK-F124 and pCK-F134, respectively. Then, plasmids pGEM-T-easy-HGF-F5, pGEM-T-easy-HGF-F6, pGEM-T-easy-HGF-F7 and pGEM-T-easy-HGF-F8 were treated with BamHI/XbaI to give HGF-F5, HGF-F6, HGF-F7 and HGF-F8, respectively. pCK-F124 and pCK-F134 were treated with BamHI/XbaI, and then HGF-F5, HGF-F6, HGF-F7 and HGF-F8 were inserted therein to give pCK-F1254, pCK-F1264, pCK-F1274, pCK-F1284, pCK-F1354, pCK-F1364, pCK-F1374 and pCK-F1384, respectively.

Then, pGEM-T easy-cHGF was treated with XhoI to obtain an about 1100bp cDNA fragment of HGF gene (HGF-XhoI). Then, HGF-XhoI was inserted into pCK-F1254, pCK-F1264, pCK-F1274, pCK-F1284, pCK-F1354, pCK-F1364, pCK-F1374 and pCK-F1384 to obtain pCK-HGF-X1, pCK-HGF-X2, pCK-HGF-X3, pCK-HGF-X4, pCK-HGF-X5, pCK-HGF-X6, pCK-HGF-X7 and pCK-HGF-X8, respectively. On the other hand, pGEM-T easy-cHGF and pGEM-T easy-dHGF were treated with BamHI to obtain HGF gene cDNA and dHGF gene cDNA. Then, the HGF gene cDNA and dHGF gene cDNA were inserted into the BamHI restriction site of pCK to give pCK-cHGF and pCK-dHGF, respectively (FIG. 5).

(4) Preparation of expression vectors containing hybrid HGF Gene constructs

Plasmid pCDNA3.1(Invitrogen, USA) was digested with NdeI, treated with Klenow fragment to construct blunt ends, and then digested with NheI to obtain a DNA fragment containing the promoter of human cytomegalovirus.

Plasmids pCK-HGF-X1, pCK-HGF-X2, pCK-HGF-X3, pCK-HGF-X4, pCK-HGF-X5, pCK-HGF-X6, pCK-HGF-X7 and pCK-HGF-X8 were digested with SnaBI, treated with Klenow fragment to generate blunt ends and digested with NheI, and then the above DNA fragments containing the promoter of human cytomegalovirus were inserted therein to obtain pCP-HGF-X1, pCP-HGF-X2, pCP-HGF-X3, pCP-HGF-X4, pCP-HGF-X5, pCP-HGF-X6, pCP-HGF-X7 and pCP-HGF-X8 (FIG. 6), respectively.

Plasmid pcdna3.1(Invitrogen, usa) was digested with NheI, treated with Klenow fragment to generate blunt ends, and digested with NheI to obtain a DNA fragment containing the promoter of human cytomegalovirus. pCK-cHGF and pCK-dHGF were digested with MluI, treated with Klenow fragment to generate blunt ends and digested with NheI, and then the above DNA fragment containing the promoter of human cytomegalovirus was inserted therein to give pCP-cHGF and pCP-dHGF, respectively (FIG. 7).

Example 2: detection of expression efficiency of hybrid HGF Gene constructs and Co-expression of HGF/dHGF

Experiments were performed to test whether the hybrid HGF gene constructs obtained in example 1 (HGF-X1 to HGF-X8) were capable of simultaneously expressing HGF and dHGF, and whether there were any gene expression level differences between the hybrid HGF gene construct and the HGF cDNA.

(1) Efficiency of Gene expression

First, 5. mu.g of pCP-HGF-X2, pCP-HGF-X3, pCP-HGF-X6, pCP-HGF-X7 and pCP-HGF-X8, and 0.5. mu.g of DONAI-LacZ (TAKARA SHUZO, Japan) DNA were transfected to 5X 10 using FuGENE6(Gibco BRL, Maryland, USA) according to the manufacturer's introduction⁶And 293 cells (ATCC CRL 1573). At this time, 5. mu.g of each of pCP-cHGF and pCP-dHGF were used as controls, and DONAI-LacZ DNA was used to calibrate the infection efficiency. 3 hours after transfection, fresh culture solution was added to the cells and the culture was continued for 48 hours. The culture solution thus obtained was divided into two portions. One portion of 293 cell culture was subjected to RNA extraction, and the other portion was used for determination of LacZ activity. LacZ activity was determined using an activity assay kit (Stratagene, CA, USA) according to the manufacturer's instructions.

For comparison of gene expression levels, the content of HGF in cell cultures was determined by enzyme linked immunosorbent assay kit (ELISA, R & D System, MN, usa). After the infection efficiency was calibrated by measuring LacZ activity, the expression level of HGF-X gene was found to be 20-150 times higher than that of HGF cDNA and dHGF cDNA (fig. 8). In particular, HGF-X7 has the highest gene expression level.

(2) Co-expression of HGF and dHGF

To detect co-expression of HGF and dHGF from the hybrid HGF gene construct, total cellular RNAs were extracted from transfected 293 cells by Trizol method (Trizol: Gibco BRL, USA), and RT-PCR was performed to obtain cDNA. The cDNA was then used as a template DNA to synthesize SEQ ID NO: 17 and 18 as primer pairs for PCR amplification. Mu.l of the template DNA, 1. mu.l of each primer (10 pmol/. mu.l), 10. mu.l of dNTP (10mM), 3.5 units of Taq polymerase (TAKARA SHUZO, Japan), and 10. mu.l of enzyme buffer were mixed, and adjusted to a final volume of 100. mu.l with distilled water to prepare a PCR amplification mixture. 30 cycles of PCR amplification were performed, each cycle consisting of 94 ℃ for 1min, 55 ℃ for 1min and 72 ℃ for 90 sec.

The amplified PCR product corresponds to the border region between exons 4 and 5 of the HGF gene; the cDNA of the HGF gene is 142bp, and the cDNA of the dHGF gene is 127 bp. PCR products of at least 1kb in length are amplified without splicing; if alternative splicing occurs, 142bp of HGF gene cDNA and 127bp of dHGF gene cDNA are synthesized and amplified simultaneously. The amplified PCR products were distinguished by electrophoresis on a 12% polyacrylamide gel.

As shown in FIG. 9, bands of 142bp and 127bp were detected in the lanes loaded with the HGF gene cDNA and the dHGF gene cDNA, respectively, and bands of 142bp and 127bp were detected in the lanes loaded with HGF-X. The above results indicate that the hybrid HGF-X gene construct of the present invention expresses both HGF and dHGF.

Example 3: comparison of expression levels of HGF-X7, HGF Gene cDNA and dHGF Gene cDNA (in vivo)

Mu.l of each of pCP-HGF-X7, pCP-cHGF and pCP-dHGF was injected into the tibialis anterior muscle of the hind limb of the mouse with an insulin syringe. After 5 days, mice were sacrificed and muscles around the injection site were removed and triturated in protein extraction buffer (25mM Tris-HCl (pH 7.4), 50mM NaCl, 0.5% sodium deoxycholate, 2% NP-40, 0.2% SDS) to isolate total protein. Total protein content was determined using a DC protein assay kit (Bio-Rad Laboratories, CA, USA) and expressed HGF content was determined using an ELISA kit (R & D System) according to the manufacturer's instructions.

As can be seen from the results shown in FIG. 10, the content of HGF expressed from HGF-X7 was 250 times higher than that expressed from HGF gene cDNA or dHGF gene cDNA.

In combination with the test results of example 2 (in vivo), these results indicate that the expression efficiency of HGF-X gene is much higher than that of HGF gene cDNA or dHGF gene cDNA.

Example 4: gene therapy with HGF-X7 in rabbit ischemic hindlimb model

To examine whether the HGF-X7 gene was effective in treating ischemic hindlimb disease, gene therapy was performed in a rabbit ischemic hindlimb model as follows.

The rabbit ischemic hindlimb model is a standard animal model for ischemic limb disease, and is prepared by Takeshita et al, Journal of Clinical Investigation, 93: 662(1994) by the methods described. One day before surgery (day 0), 30 New Zealand white rabbits (male, weighing 3.8-4.2kg) were anesthetized by intramuscular injection of 5mg/kg xylazine followed by intramuscular injection of 50mg/kg ketamine. The left thigh of the rabbit was then dissected and all the branches of the femoral artery were separated and ligated. A model was prepared by incising the region from the proximal region to the branch point of the saphenous vein and popliteal artery. After surgery, a 15 mg/kg/day intramuscular injection of cefazolin (cefazolin) was performed for 5 days and a 0.3 mg/day injection of morphine was performed for 10 days. 10 days after surgery (day 10), left hind limbs were subjected to angiography, which caused ischemia, and the extent of arteriogenesis was recorded as a baseline level. Rabbits were randomly divided into two groups, and 500. mu.g of plasmid pCPHGF-X7 (test group) or 500. mu.g of plasmid pCP (control group) was injected into four sites of the thigh muscle, respectively. After 40 days of surgery (day 40), the left hind limb was subjected to angiography and the extent of arteriogenesis at the level of the arterioles was determined and compared to day 10.

As can be seen from the results of fig. 11, the degree of arteriogenesis was significantly increased in the test group to which pCPHGF-X7 was administered, compared to the control group to which pCP was administered.

These results indicate that the HGF-X7 gene can be effectively used for gene therapy of ischemic diseases.

While the invention has been described with respect to the specific embodiments set forth above, it will be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

Microorganism international deposit certificate (translation)

Preservation person	Bai Medical Co., Ltd.
		Address	Korea, Hancheng
Date of storage	2002, 3 months and 19 days
		Accession number	KCCM-10361
Microbial classification name	Escherichia coli Top 10F' pCK-HGFX7
		Name of International occlusion Unit	Korea center for culturing microorganisms
Agency seal

Microorganism international deposit certificate (translation)

Preservation person	Bai Medical Co., Ltd.
		Address	Korea, Hancheng
Date of storage	3/12/2002
		Accession number	KCCM-10362
Microbial classification name	Escherichia coli Top 10F' pCK-HGFX7
		Name of International occlusion Unit	Korea center for culturing microorganisms
Agency seal

Sequence listing

<110> Bai therapeutic Co

<120> hybrid hepatocyte growth factor gene for efficiently expressing two different types of hepatocyte growth factors

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<160>21

<170>KopatentIn 1.71

<210>1

<211>2187

<212>DNA

<213> hepatocyte growth factor

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gacttacatc gtcatatctt ctgggaacca gatgcaagta agctgaatga gaattactgc 1320

cgaaatccag atgatgatgc tcatggaccc tggtgctaca cgggaaatcc actcattcct 1380

tgggattatt gccctatttc tcgttgtgaa ggtgatacca cacctacaat agtcaattta 1440

gaccatcccg taatatcttg tgccaaaacg aaacaattgc gagttgtaaa tgggattcca 1500

acacgaacaa acataggatg gatggttagt ttgagataca gaaataaaca tatctgcgga 1560

ggatcattga taaaggagag ttgggttctt actgcacgac agtgtttccc ttctcgagac 1620

ttgaaagatt atgaagcttg gcttggaatt catgatgtcc acggaagagg agatgagaaa 1680

tgcaaacagg ttctcaatgt ttcccagctg gtatatggcc ctgaaggatc agatctggtt 1740

ttaatgaagc ttgccaggcc tgctgtcctg gatgattttg ttagtacgat tgatttacct 1800

aattatggat gcacaattcc tgaaaagacc agttgcagtg tttatggctg gggctacact 1860

ggattgatca actatgatgg cctattacga gtggcacatc tctatataat gggaaatgag 1920

aaatgcagcc agcatcatcg agggaaggtg actctgaatg agtctgaaat atgtgctggg 1980

gctgaaaaga ttggatcagg accatgtgag ggggattatg gtggcccact tgtttgtgag 2040

caacataaaa tgagaatggt tcttggtgtc attgttcctg gtcgtggatg tgccattcca 2100

aatcgtcctg gtatttttgt ccgagtagca tattatgcaa aatggataca caaaattatt 2160

ttaacatata aggtaccaca gtcatag 2187

<210>2

<211>7112

<212>DNA

<213> hepatocyte growth factor heterozygotes

<400>2

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatt ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

aggtaagaac agtatgaaga aaagagatga agcctctgtc ttttttacat gttaacagtc 540

tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600

tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660

tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720

tatttgtgaa ataataacgt aaacttcgtt taaaaggatt cttcttcctg tctttgagaa 780

agtacggcac tgtgcagggg gagaggttga ttgtgaaaaa tcagaggtag atgagaatct 840

tactgagggc tgagggttct ttaaccttgg tggatctcaa cattggttgc acattaaaat 900

cacctgctgc aagcccttga cgaatcttac ttagaagatg acaacacaga acaattaaat 960

cagaatctct ggggagaata gggcaccagt attttttgag ctcccaccat gattccaaag 1020

tgcagccaaa tttgagaacc actgctaaaa gctcaagctt cagattgacc agcttttcca 1080

tctcacctat cgcctaaaga ccaaattgga taaatgtgtt cattacgaca gatgggtact 1140

atttaaagat gagtaaacac aatatactta ggctcgtcag actgagagtt ttaatcatca 1200

ctgaggaaaa acatagatat ctaatactga ctggagtatt agtcaaggct tatttcacac 1260

acaattttat cagaaaccaa agtagtttaa aacagctctc cccttattag taatgcattg 1320

gagggtttac tttaccatgt accttgctga gcactgtacc ttgttaatct catttacttg 1380

taatgagaac cacacagcgg gtagttttat tggttctatt ttacctacat gacaaaactg 1440

aagcataaaa acacttagta agttttcagt gtcatgcaca actaggaagt gacatggcca 1500

gaatataagc ccagtcacca tcactctata acctgcgctt ttaacaactt cagggcatga 1560

cacatttggc cggtcagtag aacccatgct gtgatttgtt tttgcagtgg tggtgatgac 1620

tgccttgttg aatccacttt ttattctatt ccattttggg gacacaattc tgcaagatga 1680

ttcttcatta ggaaacagag atgagttatt gaccaacaca gaaagaaaaa gagtttgttg 1740

ctccacactg ggattaaacc tatgatcttg gcctaattaa cactagctag taagtgtcca 1800

agctgatcat ctctacaaca tttcaataac agaaaacaac aattttcaaa attagttact 1860

tacaattatg tagaaatgcc tctaaaacac agtattttcc ttatattaca aaaacaaaaa 1920

ttataattgg ttttgtcctc ttttgagagt ttgcatggtg ttactccctg catagtgaag 1980

aaaacatttt atttaagtag atggatctaa gtttttcatg aacaaaggaa tgacatttga 2040

aatcaatcct accctagtcc aggagaatgc attagattaa cctagtagag gtcttatttc 2100

accctgagtt ttctatgatc gtgattctct gctggaggag taattgtgaa atagatctct 2160

ctgggaactg gcttcctagt ccaatcagct cttttaccaa tgaacacttc cttgtgatat 2220

agatgtttat ggccgagagg atccagtata ttaataaaat ccctttttgt attcaatgag 2280

ggaaacacat aattttcatc aattagcagc ttattggaat atctgcatga tggtttaaca 2340

cttttaagtg ttgactaaag attaatttta cagaaaatag aaaaagaaat atgtttctgt 2400

ctggaggaat gatttattgt tgacccctaa attgaaatat tttactagtg gcttaatgga 2460

aagatgatga aagatgatga aattaatgta gaagcttaac tagaaaatca ggtgacctga 2520

tatctacatc tgtatccttc attggccacc cagcattcat taatgaatca gatgatggaa 2580

tagatcaagt ttcctaggaa cacagtgaat attaaaagaa aacaaaggga gcctagcacc 2640

tagaagacct agtttatatt tcaaagtata tttggatgta acccaatttt aaacatttcc 2700

tcacttgtct ctcttaaagc cttgccaaca gcaaggacag agaaccaaaa atagtgtata 2760

tatgaataaa tgcttattac agaatctgct gactggcaca tgctttgtgt gtaatgggtt 2820

ctcataaaca cttgttgaat gaacacacat aagtgaaaga gcatggctag gcttcatccc 2880

ttggtcaaat atggggtgct aaagaaaagc aggggaaata cattgggaca ctaacaaaaa 2940

aaaacagtta atttaggtaa aagataaaat acaccacaga atgaagaaaa gagatgaccc 3000

agactgctct ttaaccttca tgtcctagag aggtttttga tatgaattgc attcagaatt 3060

gtggaaagga gcccatcttt tctcttcatt ttgattttat taactccaat gggggaattt 3120

tattcgtgtt ttggccatat ctacttttga tttctacatt attctctctt cctttctacc 3180

tgtatttgtc ctaataaatt gttgacttat taattcacta cttcctcaca gctttttttt 3240

ggctttacaa atccactgga aaggtatatg ggtgtatcac tttgtgtatt tcggtgtgca 3300

tgtgtagagg ggacaaaaat cctctctcaa actataaata ttgagtattt gtgtattgaa 3360

catttgctat aactactagg tttcttaaat aatcttaata tataaaatga tatagaaaaa 3420

gggaaattat agttcgtatt attcatctaa gtgaagagat taaaacccag ggagtaaata 3480

aattgtctaa ggactaaggt tgtatactat ttaggtgata gatatggggc aaccgtatgg 3540

gttttatgat taacaaataa acttctcacc actctaccat atcaactttt ccataaaaga 3600

gagctatagt attctttgct taaataaatt tgattagtgc atgacttctt gaaaacatat 3660

aaagcaaaag tcacatttga ttctatcaga aaagtgagta agccatggcc caaacaaaag 3720

atgcattaaa atattctgga atgatggagc taaaagtaag aaaaatgact ttttaaaaaa 3780

gtttactgtt aggaattgtg aaattatgct gaattttagt tgcattataa tttttgtcag 3840

tcatacggtc tgacaacctg tcttatttct atttccccat atgaggaatg ctagttaagt 3900

atggatatta actattacta cttagatgca ttgaagttgc ataatatgga taatacttca 3960

ctggttccct gaaaatgttt agttagtaat aagtctctta cactatttgt tttgtccaat 4020

aatttatatt ttctgaagac ttaactctag aatacactca tgtcaaaatg aaagaatttc 4080

attgcaaaat attgcttggt acatgacgca tacctgtatt tgttttgtgt cacaacatga 4140

aaaatgatgg tttattagaa gtttcattgg gtaggaaaca catttgaatg gtatttacta 4200

agatactaaa atccttggac ttcactctaa ttttagtgcc atttagaact caaggtctca 4260

gtaaaagtag aaataaagcc tgttaacaaa acacaagctg aatattaaaa atgtaactgg 4320

attttcaaag aaatgtttac tggtattacc tgtagatgta tattctttat tatgatcttt 4380

tgtgtaaagt ctggcagaca aatgcaatat ctaattgttg agtccaatat cacaagcagt 4440

acaaaagtat aaaaaagact tggccttttc taatgtgtta aaatacttta tgctggtaat 4500

aacactaaga gtagggcact agaaatttta agtgaagata atgtgttgca gttactgcac 4560

tcaatggctt actattataa accaaaactg ggatcactaa gctccagtca gtcaaaatga 4620

tcaaaattat tgaagagaat aagcaattct gttctttatt aggacacagt agatacagac 4680

tacaaagtgg agtgtgctta ataagaggta gcatttgtta agtgtcaatt actctattat 4740

cccttggagc ttctcaaaat aaccatataa ggtgtaagat gttaaaggtt atggttacac 4800

tcagtgcaca gtaagctaat aggctgagag aagctaaatt acttactggg gtctcacagt 4860

aagaaagtga gctgaagttt cagcccagat ttaactggat tctgggctct ttattcatgt 4920

tacttcatga atctgtttct caattgtgca gaaaaaaggg ggctatttat aagaaaagca 4980

ataaacaaac aagtaatgat ctcaaataag taatgcaaga aatagtgaga tttcaaaatc 5040

agtggcagcg atttctcagt tctgtcctaa gtggccttgc tcaatcacct gctatctttt 5100

agtggagctt tgaaattatg tttcagacaa cttcgattca gttctagaat gtttgactca 5160

gcaaattcac aggctcatct ttctaacttg atggtgaata tggaaattca gctaaatgga 5220

tgttaataaa attcaaacgt tttaaggaca gatgaaaatg acagaatttt aaggtaaaat 5280

atatgaagga atataagata aaggattttt ctaccttcag caaaaacata cccactaatt 5340

agtaaaatta ataggcaaaa aaaagttgca tgctcttata ctgtaatgat tatcatttta 5400

aaactagctt tttgccttcg agctatcggg gtaaagacct acaggaaaac tactgtcgaa 5460

atcctcgagg ggaagaaggg ggaccctggt gtttcacaag caatccagag gtacgctacg 5520

aagtctgtga cattcctcag tgttcagaag ttgaatgcat gacctgcaat ggggagagtt 5580

atcgaggtct catggatcat acagaatcag gcaagatttg tcagcgctgg gatcatcaga 5640

caccacaccg gcacaaattc ttgcctgaaa gatatcccga caagggcttt gatgataatt 5700

attgccgcaa tcccgatggc cagccgaggc catggtgcta tactcttgac cctcacaccc 5760

gctgggagta ctgtgcaatt aaaacatgcg ctgacaatac tatgaatgac actgatgttc 5820

ctttggaaac aactgaatgc atccaaggtc aaggagaagg ctacaggggc actgtcaata 5880

ccatttggaa tggaattcca tgtcagcgtt gggattctca gtatcctcac gagcatgaca 5940

tgactcctga aaatttcaag tgcaaggacc tacgagaaaa ttactgccga aatccagatg 6000

ggtctgaatc accctggtgt tttaccactg atccaaacat ccgagttggc tactgctccc 6060

aaattccaaa ctgtgatatg tcacatggac aagattgtta tcgtgggaat ggcaaaaatt 6120

atatgggcaa cttatcccaa acaagatctg gactaacatg ttcaatgtgg gacaagaaca 6180

tggaagactt acatcgtcat atcttctggg aaccagatgc aagtaagctg aatgagaatt 6240

actgccgaaa tccagatgat gatgctcatg gaccctggtg ctacacggga aatccactca 6300

ttccttggga ttattgccct atttctcgtt gtgaaggtga taccacacct acaatagtca 6360

atttagacca tcccgtaata tcttgtgcca aaacgaaaca attgcgagtt gtaaatggga 6420

ttccaacacg aacaaacata ggatggatgg ttagtttgag atacagaaat aaacatatct 6480

gcggaggatc attgataaag gagagttggg ttcttactgc acgacagtgt ttcccttctc 6540

gagacttgaa agattatgaa gcttggcttg gaattcatga tgtccacgga agaggagatg 6600

agaaatgcaa acaggttctc aatgtttccc agctggtata tggccctgaa ggatcagatc 6660

tggttttaat gaagcttgcc aggcctgctg tcctggatga ttttgttagt acgattgatt 6720

tacctaatta tggatgcaca attcctgaaa agaccagttg cagtgtttat ggctggggct 6780

acactggatt gatcaactat gatggcctat tacgagtggc acatctctat ataatgggaa 6840

atgagaaatg cagccagcat catcgaggga aggtgactct gaatgagtct gaaatatgtg 6900

ctggggctga aaagattgga tcaggaccat gtgaggggga ttatggtggc ccacttgttt 6960

gtgagcaaca taaaatgaga atggttcttg gtgtcattgt tcctggtcgt ggatgtgcca 7020

ttccaaatcg tcctggtatt tttgtccgag tagcatatta tgcaaaatgg atacacaaaa 7080

ttattttaac atataaggta ccacagtcat ag 7112

<210>3

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> gHGF3 primer

<400>3

gtaaaggacg cgtctacaag ggaacagtat ctat 34

<210>4

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> gHGF4 primer

<400>4

actggatcct ctcggccata aacatct 27

<210>5

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> gHGF10 primer

<400>5

gaagcttagc accatgtggg tgaccaaact cctg 34

<210>6

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> gHGF5 primer

<400>6

tggccgagag gatccagtat attaata 27

<210>7

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> gHGF7 primer

<400>7

cccctcgagg atttcgacag tagtttt 27

<210>8

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> gHGF12 primer

<400>8

gggatccctt cctttctacc tgtatttg 28

<210>9

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> gHGF13 primer

<400>9

gggatcctgg gtaaacacat ttgaa 25

<210>10

<211>28

<212>DNA

<213> Artificial sequence

<220>

<223> gHGF6 primer

<400>10

gggatcctta tgtttcagac aacttcga 28

<210>11

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> gHGF1 primer

<400>11

gaagcttgcc accatgtggg tgaccaaact cctg 34

<210>12

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> gHGF2 primer

<400>12

gggatccaga acgcgtcctt taccgatgat gcag 34

<210>13

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223> gHGF8 primer

<400>13

gggatccctt ctcgagactt gaaagattat gaagc 35

<210>14

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> gHGF9 primer

<400>14

gtctagagcg gccgctatga ctgtggtacc tt 32

<210>15

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> cHGF5 primer

<400>15

ggatccacgc gtagcagcac catgtgggtg accaaa 36

<210>16

<211>36

<212>DNA

<213> Artificial sequence

<220>

<223> cHGF3 primer

<400>16

ggatcctcta gattacttca gctatgactg tggtac 36

<210>17

<211>26

<212>DNA

<213> Artificial sequence

<220>

<223> GHGF 5' primer

<400>17

caaatgtcag ccctggagtt ccatga 26

<210>18

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> GHGF 3' primer

<400>18

ctggattgct tgtgaaacag ggt 23

<210>19

<211>4679

<212>DNA

<213> Artificial sequence

<220>

<223> HGF-X6 Gene

<400>19

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatc ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

aggtaagaac agtatgaaga aaagagatga agcctctgtc ttttttacat gttaacagtc 540

tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600

tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660

tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720

tatttgtgga tcccttcctt tctacctgta tttgtcctaa taaattgttg acttattaat 780

tcactacttc ctcacagctt ttttttggct ttacaaatcc actggaaagg tatatgggtg 840

tatcactttg tgtatttcgg tgtgcatgtg tagaggggac aaaaatcctc tctcaaacta 900

taaatattga gtatttgtgt attgaacatt tgctataact actaggtttc ttaaataatc 960

ttaatatata aaatgatata gaaaaaggga aattatagtt cgtattattc atctaagtga 1020

agagattaaa acccagggag taaataaatt gtctaaggac taaggttgta tactatttag 1080

gtgatagata tggggcaacc gtatgggttt tatgattaac aaataaactt ctcaccactc 1140

taccatatca acttttccat aaaagagagc tatagtattc tttgcttaaa taaatttgat 1200

tagtgcatga cttcttgaaa acatataaag caaaagtcac atttgattct atcagaaaag 1260

tgagtaagcc atggcccaaa caaaagatgc attaaaatat tctggaatga tggagctaaa 1320

agtaagaaaa atgacttttt aaaaaagttt actgttagga attgtgaaat tatgctgaat 1380

tttagttgca ttataatttt tgtcagtcat acggtctgac aacctgtctt atttctattt 1440

ccccatatga ggaatgctag ttaagtatgg atattaacta ttactactta gatgcattga 1500

agttgcataa tatggataat acttcactgg ttccctgaaa atgtttagtt agtaataagt 1560

ctcttacact atttgttttg tccaataatt tatattttct gaagacttaa ctctagaata 1620

cactcatgtc aaaatgaaag aatttcattg caaaatattg cttggtacat gacgcatacc 1680

tgtatttgtt ttgtgtcaca acatgaaaaa tgatggttta ttagaagttt cattgggtag 1740

gaaacacatt tgaatggtat ttactaagat actaaaatcc ttggacttca ctctaatttt 1800

agtgccattt agaactcaag gtctcagtaa aagtagaaat aaagcctgtt aacaaaacac 1860

aagctgaata ttaaaaatgt aactggattt tcaaagaaat gtttactggt attacctgta 1920

gatgtatatt ctttattatg atcttttgtg taaagtctgg cagacaaatg caatatctaa 1980

ttgttgagtc caatatcaca agcagtacaa aagtataaaa aagacttggc cttttctaat 2040

gtgttaaaat actttatgct ggtaataaca ctaagagtag ggcactagaa attttaagtg 2100

aagataatgt gttgcagtta ctgcactcaa tggcttacta ttataaacca aaactgggat 2160

cactaagctc cagtcagtca aaatgatcaa aattattgaa gagaataagc aattctgttc 2220

tttattagga cacagtagat acagactaca aagtggagtg tgcttaataa gaggtagcat 2280

ttgttaagtg tcaattactc tattatccct tggagcttct caaaataacc atataaggtg 2340

taagatgtta aaggttatgg ttacactcag tgcacaggta agctaatagg ctgagagaag 2400

ctaaattact tactggggtc tcacagtaag aaagtgagct gaagtttcag cccagattta 2460

actggattct gggctcttta ttcatgttac ttcatgaatc tgtttctcaa ttgtgcagaa 2520

aaaagggggc tatttataag aaaagcaata aacaaacaag taatgatctc aaataagtaa 2580

tgcaagaaat agtgagattt caaaatcagt ggcagcgatt tctcagttct gtcctaagtg 2640

gccttgctca atcacctgct atcttttagt ggagctttga aattatgttt cagacaactt 2700

cgattcagtt ctagaatgtt tgactcagca aattcacagg ctcatctttc taacttgatg 2760

gtgaatatgg aaattcagct aaatggatgt taataaaatt caaacgtttt aaggacagat 2820

gaaaatgaca gaattttaag gtaaaatata tgaaggaata taagataaag gatttttcta 2880

ccttcagcaa aaacataccc actaattagt aaaattaata ggcaaaaaaa agttgcatgc 2940

tcttatactg taatgattat cattttaaaa ctagcttttt gccttcgagc tatcggggta 3000

aagacctaca ggaaaactac tgtcgaaatc ctcgagggga agaaggggga ccctggtgtt 3060

tcacaagcaa tccagaggta cgctacgaag tctgtgacat tcctcagtgt tcagaagttg 3120

aatgcatgac ctgcaatggg gagagttatc gaggtctcat ggatcataca gaatcaggca 3180

agatttgtca gcgctgggat catcagacac cacaccggca caaattcttg cctgaaagat 3240

atcccgacaa gggctttgat gataattatt gccgcaatcc cgatggccag ccgaggccat 3300

ggtgctatac tcttgaccct cacacccgct gggagtactg tgcaattaaa acatgcgctg 3360

acaatactat gaatgacact gatgttcctt tggaaacaac tgaatgcatc caaggtcaag 3420

gagaaggcta caggggcact gtcaatacca tttggaatgg aattccatgt cagcgttggg 3480

attctcagta tcctcacgag catgacatga ctcctgaaaa tttcaagtgc aaggacctac 3540

gagaaaatta ctgccgaaat ccagatgggt ctgaatcacc ctggtgtttt accactgatc 3600

caaacatccg agttggctac tgctcccaaa ttccaaactg tgatatgtca catggacaag 3660

attgttatcg tgggaatggc aaaaattata tgggcaactt atcccaaaca agatctggac 3720

taacatgttc aatgtgggac aagaacatgg aagacttaca tcgtcatatc ttctgggaac 3780

cagatgcaag taagctgaat gagaattact gccgaaatcc agatgatgat gctcatggac 3840

cctggtgcta cacgggaaat ccactcattc cttgggatta ttgccctatt tctcgttgtg 3900

aaggtgatac cacacctaca atagtcaatt tagaccatcc cgtaatatct tgtgccaaaa 3960

cgaaacaatt gcgagttgta aatgggattc caacacgaac aaacatagga tggatggtta 4020

gtttgagata cagaaataaa catatctgcg gaggatcatt gataaaggag agttgggttc 4080

ttactgcacg acagtgtttc ccttctcgag acttgaaaga ttatgaagct tggcttggaa 4140

ttcatgatgt ccacggaaga ggagatgaga aatgcaaaca ggttctcaat gtttcccagc 4200

tggtatatgg ccctgaagga tcagatctgg ttttaatgaa gcttgccagg cctgctgtcc 4260

tggatgattt tgttagtacg attgatttac ctaattatgg atgcacaatt cctgaaaaga 4320

ccagttgcag tgtttatggc tggggctaca ctggattgat caactatgat ggcctattac 4380

gagtggcaca tctctatata atgggaaatg agaaatgcag ccagcatcat cgagggaagg 4440

tgactctgaa tgagtctgaa atatgtgctg gggctgaaaa gattggatca ggaccatgtg 4500

agggggatta tggtggccca cttgtttgtg agcaacataa aatgagaatg gttcttggtg 4560

tcattgttcc tggtcgtgga tgtgccattc caaatcgtcc tggtattttt gtccgagtag 4620

catattatgc aaaatggata cacaaaatta ttttaacata taaggtacca cagtcatag 4679

<210>20

<211>3679

<212>DNA

<213> Artificial sequence

<220>

<223> HGF-X7 Gene

<400>20

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatc ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

aggtaagaac agtatgaaga aaagagatga agcctctgtc ttttttacat gttaacagtc 540

tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600

tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660

tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720

tatttgtgga tcctgggtag gaaacacatt tgaatggtat ttactaagat actaaaatcc 780

ttggacttca ctctaatttt agtgccattt agaactcaag gtctcagtaa aagtagaaat 840

aaagcctgtt aacaaaacac aagctgaata ttaaaaatgt aactggattt tcaaagaaat 900

gtttactggt attacctgta gatgtatatt ctttattatg atcttttgtg taaagtctgg 960

cagacaaatg caatatctaa ttgttgagtc caatatcaca agcagtacaa aagtataaaa 1020

aagacttggc cttttctaat gtgttaaaat actttatgct ggtaataaca ctaagagtag 1080

ggcactagaa attttaagtg aagataatgt gttgcagtta ctgcactcaa tggcttacta 1140

ttataaacca aaactgggat cactaagctc cagtcagtca aaatgatcaa aattattgaa 1200

gagaataagc aattctgttc tttattagga cacagtagat acagactaca aagtggagtg 1260

tgcttaataa gaggtagcat ttgttaagtg tcaattactc tattatccct tggagcttct 1320

caaaataacc atataaggtg taagatgtta aaggttatgg ttacactcag tgcacaggta 1380

agctaatagg ctgagagaag ctaaattact tactggggtc tcacagtaag aaagtgagct 1440

gaagtttcag cccagattta actggattct gggctcttta ttcatgttac ttcatgaatc 1500

tgtttctcaa ttgtgcagaa aaaagggggc tatttataag aaaagcaata aacaaacaag 1560

taatgatctc aaataagtaa tgcaagaaat agtgagattt caaaatcagt ggcagcgatt 1620

tctcagttct gtcctaagtg gccttgctca atcacctgct atcttttagt ggagctttga 1680

aattatgttt cagacaactt cgattcagtt ctagaatgtt tgactcagca aattcacagg 1740

ctcatctttc taacttgatg gtgaatatgg aaattcagct aaatggatgt taataaaatt 1800

caaacgtttt aaggacagat gaaaatgaca gaattttaag gtaaaatata tgaaggaata 1860

taagataaag gatttttcta ccttcagcaa aaacataccc actaattagt aaaattaata 1920

ggcaaaaaaa agttgcatgc tcttatactg taatgattat cattttaaaa ctagcttttt 1980

gccttcgagc tatcggggta aagacctaca ggaaaactac tgtcgaaatc ctcgagggga 2040

agaaggggga ccctggtgtt tcacaagcaa tccagaggta cgctacgaag tctgtgacat 2100

tcctcagtgt tcagaagttg aatgcatgac ctgcaatggg gagagttatc gaggtctcat 2160

ggatcataca gaatcaggca agatttgtca gcgctgggat catcagacac cacaccggca 2220

caaattcttg cctgaaagat atcccgacaa gggctttgat gataattatt gccgcaatcc 2280

cgatggccag ccgaggccat ggtgctatac tcttgaccct cacacccgct gggagtactg 2340

tgcaattaaa acatgcgctg acaatactat gaatgacact gatgttcctt tggaaacaac 2400

tgaatgcatc caaggtcaag gagaaggcta caggggcact gtcaatacca tttggaatgg 2460

aattccatgt cagcgttggg attctcagta tcctcacgag catgacatga ctcctgaaaa 2520

tttcaagtgc aaggacctac gagaaaatta ctgccgaaat ccagatgggt ctgaatcacc 2580

ctggtgtttt accactgatc caaacatccg agttggctac tgctcccaaa ttccaaactg 2640

tgatatgtca catggacaag attgttatcg tgggaatggc aaaaattata tgggcaactt 2700

atcccaaaca agatctggac taacatgttc aatgtgggac aagaacatgg aagacttaca 2760

tcgtcatatc ttctgggaac cagatgcaag taagctgaat gagaattact gccgaaatcc 2820

agatgatgat gctcatggac cctggtgcta cacgggaaat ccactcattc cttgggatta 2880

ttgccctatt tctcgttgtg aaggtgatac cacacctaca atagtcaatt tagaccatcc 2940

cgtaatatct tgtgccaaaa cgaaacaatt gcgagttgta aatgggattc caacacgaac 3000

aaacatagga tggatggtta gtttgagata cagaaataaa catatctgcg gaggatcatt 3060

gataaaggag agttgggttc ttactgcacg acagtgtttc ccttctcgag acttgaaaga 3120

ttatgaagct tggcttggaa ttcatgatgt ccacggaaga ggagatgaga aatgcaaaca 3180

ggttctcaat gtttcccagc tggtatatgg ccctgaagga tcagatctgg ttttaatgaa 3240

gcttgccagg cctgctgtcc tggatgattt tgttagtacg attgatttac ctaattatgg 3300

atgcacaatt cctgaaaaga ccagttgcag tgtttatggc tggggctaca ctggattgat 3360

caactatgat ggcctattac gagtggcaca tctctatata atgggaaatg agaaatgcag 3420

ccagcatcat cgagggaagg tgactctgaa tgagtctgaa atatgtgctg gggctgaaaa 3480

gattggatca ggaccatgtg agggggatta tggtggccca cttgtttgtg agcaacataa 3540

aatgagaatg gttcttggtg tcattgttcc tggtcgtgga tgtgccattc caaatcgtcc 3600

tggtattttt gtccgagtag catattatgc aaaatggata cacaaaatta ttttaacata 3660

taaggtacca cagtcatag 3679

<210>21

<211>2729

<212>DNA

<213> Artificial sequence

<220>

<223> HGF-X8 Gene

<400>21

atgtgggtga ccaaactcct gccagccctg ctgctgcagc atgtcctcct gcatctcctc 60

ctgctcccca tcgccatccc ctatgcagag ggacaaagga aaagaagaaa tacaattcat 120

gaattcaaaa aatcagcaaa gactacccta atcaaaatag atccagcact gaagataaaa 180

accaaaaaag tgaatactgc agaccaatgt gctaatagat gtactaggaa taaaggactt 240

ccattcactt gcaaggcttt tgtttttgat aaagcaagaa aacaatgcct ctggttcccc 300

ttcaatagca tgtcaagtgg agtgaaaaaa gaatttggcc atgaatttga cctctatgaa 360

aacaaagact acattagaaa ctgcatcatc ggtaaaggac gcagctacaa gggaacagta 420

tctatcacta agagtggcat caaatgtcag ccctggagtt ccatgatacc acacgaacac 480

aggtaagaac agtatgaaga aaagagatga agcctctgtc ttttttacat gttaacagtc 540

tcatattagt ccttcagaat aattctacaa tcctaaaata acttagccaa cttgctgaat 600

tgtattacgg caaggtttat atgaattcat gactgatatt tagcaaatga ttaattaata 660

tgttaataaa atgtagccaa aacaatatct taccttaatg cctcaatttg tagatctcgg 720

tatttgtgga tccttatgtt tcagacaact tcgattcagt tctagaatgt ttgactcagc 780

aaattcacag gctcatcttt ctaacttgat ggtgaatatg gaaattcagc taaatggatg 840

ttaataaaat tcaaacgttt taaggacaga tgaaaatgac agaattttaa ggtaaaatat 900

atgaaggaat ataagataaa ggatttttct accttcagca aaaacatacc cactaattag 960

taaaattaat aggcaaaaaa aagttgcatg ctcttatact gtaatgatta tcattttaaa 1020

actagctttt tgccttcgag ctatcggggt aaagacctac aggaaaacta ctgtcgaaat 1080

cctcgagggg aagaaggggg accctggtgt ttcacaagca atccagaggt acgctacgaa 1140

gtctgtgaca ttcctcagtg ttcagaagtt gaatgcatga cctgcaatgg ggagagttat 1200

cgaggtctca tggatcatac agaatcaggc aagatttgtc agcgctggga tcatcagaca 1260

ccacaccggc acaaattctt gcctgaaaga tatcccgaca agggctttga tgataattat 1320

tgccgcaatc ccgatggcca gccgaggcca tggtgctata ctcttgaccc tcacacccgc 1380

tgggagtact gtgcaattaa aacatgcgct gacaatacta tgaatgacac tgatgttcct 1440

ttggaaacaa ctgaatgcat ccaaggtcaa ggagaaggct acaggggcac tgtcaatacc 1500

atttggaatg gaattccatg tcagcgttgg gattctcagt atcctcacga gcatgacatg 1560

actcctgaaa atttcaagtg caaggaccta cgagaaaatt actgccgaaa tccagatggt 1620

ctgaatcacc ctggtgtttt accactgatc caaacatccg agttggctac tgctcccaaa 1680

ttccaaactg tgatatgtca catggacaag attgttatcg tgggaatggc aaaaattata 1740

tgggcaactt atcccaaaca agatctggac taacatgttc aatgtgggac aagaacatgg 1800

aagacttaca tcgtcatatc ttctgggaac cagatgcaag taagctgaat gagaattact 1860

gccgaaatcc agatgatgat gctcatggac cctggtgcta cacgggaaat ccactcattc 1920

cttgggatta ttgccctatt tctcgttgtg aaggtgatac cacacctaca atagtcaatt 1980

tagaccatcc cgtaatatct tgtgccaaaa cgaaacaatt gcgagttgta aatgggattc 2040

caacacgaac aaacatagga tggatggtta gtttgagata cagaaataaa catatctgcg 2100

gaggatcatt gataaaggag agttgggttc ttactgcacg acagtgtttc ccttctcgag 2160

acttgaaaga ttatgaagct tggcttggaa ttcatgatgt ccacggaaga ggagatgaga 2220

aatgcaaaca ggttctcaat gtttcccagc tggtatatgg ccctgaagga tcagatctgg 2280

ttttaatgaa gcttgccagg cctgctgtcc tggatgattt tgttagtacg attgatttac 2340

ctaattatgg atgcacaatt cctgaaaaga ccagttgcag tgtttatggc tggggctaca 2400

ctggattgat caactatgat ggcctattac gagtggcaca tctctatata atgggaaatg 2460

agaaatgcag ccagcatcat cgagggaagg tgactctgaa tgagtctgaa atatgtgctg 2520

gggctgaaaa gattggatca ggaccatgtg agggggatta tggtggccca cttgtttgtg 2580

agcaacataa aatgagaatg gttcttggtg tcattgttcc tggtcgtgga tgtgccattc 2640

caaatcgtcc tggtattttt gtccgagtag catattatgc aaaatggata cacaaaatta 2700

ttttaacata taaggtacca cagtcatag 2729

Claims (17)

1. A hybrid HGF gene comprising human HGF exons 1 to 18, or a degenerate sequence form thereof which does not change the encoded amino acid sequence, and human HGF intron 4 located between exons 4 and 5 or a fragment of intron 4 comprising nucleotides 1 to 246 and 4635 to 4941 of intron 4, excluding any introns located between other exons.

2. Hybrid HGF gene according to claim 1 having the amino acid sequence of SEQ ID NO: 2.

3. Hybrid HGF gene according to claim 1 having an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

4. A vector comprising the hybrid HGF gene of claim 1.

5. The vector according to claim 4, further comprising one or more sequences regulating gene expression, self-replicating sequences or secretion signals.

6. The vector of claim 5 selected from pCK-HGF-X2, pCK-HGF-X3, pCK-HGF-X6, pCK-HGF-X7, pCK-HGF-X8, pCP-HGF-X2, pCP-HGF-X3, pCP-HGF-X6, pCP-HGF-X7, or pCP-HGF-X8.

7. A cell comprising the vector of claim 4.

8. The cell according to claim 7, which is a yeast cell or an E.coli cell.

9. The cell of claim 8, which is escherichia coli Top 10F' pCK-HGF-X7, accession number: KCCM-10361, depository: korean microbial culture center, preservation time: 19/3/2002, or escherichia coli Top 10F' pCP-HGF-X7, accession number: KCCM-10362, depository: korean microbial culture center, preservation time: 3/19/2002.

10. A pharmaceutical composition comprising the hybrid HGF gene of claim 1 and a pharmaceutically acceptable carrier.

11. Use of the hybrid HGF gene of claim 1 for the preparation of a medicament for the prevention or treatment of ischemic hind limb disease.

12. Use according to claim 11, wherein the hybrid HGF gene is located on a vector.

13. The use of claim 12, wherein the vector is selected from the group consisting of pCK-HGF-X2, pCK-HGF-X3, pCK-HGF-X6, pCK-HGF-X7, pCK-HGF-X8, pCP-HGF-X2, pCP-HGF-X3, pCP-HGF-X6, pCP-HGF-X7, and pCP-HGF-X8.

14. Use according to claim 11, wherein the hybrid HGF gene is configured in the form of a pharmaceutical composition suitable for direct injection.

15. A method of simultaneously producing two isoforms of HGF and dHGF protein comprising the steps of culturing the cell of any one of claims 7 to 9 in a suitable culture medium and collecting HGF and dHGF protein.

16. The hybrid HGF gene of claim 1, wherein the fragment of intron 4 further comprises nucleotides 3686 to 4634 of intron 4.

17. The hybrid HGF gene of claim 1, wherein the fragment of intron 4 further comprises nucleotides 2686 to 4634 of intron 4.