KR20020020564A - Human VEGF mutant homodimer having a linker, Vector comprising the same, transformants and proteins - Google Patents

Abstract

PURPOSE: Provided are human VEGF(vascular endothelial growth factor) mutant homodimer gene coupled by a linker sequence, an expression vector containing it, and its transformants and proteins. The expression vector and its proteins are used as a therapeutic agent in cardiovascular diseases, Buerger's disease, diabetic foot ulcer and the like. CONSTITUTION: The expression vector, pmut-diVEGF165(KFCC11211), contains Human VEGF mutant homodimer gene is represented by SEQ ID NO:1. Its transformants are characteristically selected from the group consisting of CHO, COS-7(Monkey,African green), NIH/3T3(Mouse fibroblast), BHK-21(Baby hamster kidney,Syrian golden), Hela(Human carcinoma), Vero(Monkey kidney,African green), and C127(Mouse).

Description

Human VEGF mutant homodimer having a linker, Vector comprising the same, transformants and proteins}

The present invention relates to a mutant homodimer gene of human vascular endothelial growth factor (VEGF) linked by a linker sequence, an expression vector comprising the same, a transformant thereof, and a protein thereof.

Chronic ischemic limb disease caused by peripheral vascular disease in the distal lower extremity requires 150,000 patients annually in the United States. As Europe's European Working Group has revealed in the Consensus Document, there are no drug treatments of choice for severe ischemic limb disease, and surgical revascularization of these patients is not yet available. Percutaneous balloon angioplasty or stent implantation can be considered, but unfortunately many patients are not eligible for this treatment. Currently, these patients are undergoing sympathetic blockade to dilate blood vessels, or are expected to have therapeutic effects due to vasodilation and antiplatelet action by drugs such as PgE1.

The chronic ischemic lower limb disease cannot be treated by surgery or interventional procedure, but the treatment that induces neovascularization is expected to be a fundamental treatment for the patient's disease, and Burger's disease, arteriosclerosis, diabetes Sexual foot ulcers.

VEGF is a vascular endothelial growth factor having specificity only for vascular endothelial cells. VEGF was cloned in 1989 and is known to be involved in vasculogenesis at birth and in ischemic diseases, wound healing and female production. In addition, VEGF, which is formed in tumor cells for nutrition and growth of tumor cells in diseases such as tumors, is a 45 kDa basic, heparin-binding, homodimeric glycoprotein. to be. Recombination of mRNA produces four isotopes 121, 165, 189, and 201 (isoform), and VEGF 165 is known to have the strongest effect on vascular endothelial cell growth.

VEFG binds to its receptors Flt-1 (VEGFR-1) and KDR / Flk-1 (VEGFR-2, hereinafter abbreviated as 'KDR') and transmits signals. The receptor has a domain called SH-2, which transmits signals through them, and signals through them are PI-3 kinase, extracellular signal-regulated kinase (ERK), phospholipase C, and FAK ( Much research is being done on these different pathways via focal adhesion kinase. Proliferation and angiogenesis of vascular endothelial cells are mainly caused by KDR, an intracellular receptor for VEGF, but its affinity for KDR is about 50 times lower than that of Flt-1, another receptor for VEGF. Both gene therapy and protein therapy of the disease use a normal VEGF model with low affinity for KDR and thus do not show a high therapeutic effect. Therefore, in order to induce more efficient proliferation of vascular endothelial cells and angiogenesis thereof, there is a growing need for development of expression vectors and recombinant VEGF proteins superior to conventional gene therapy vectors and recombinant proteins.

Accordingly, the present inventors have conducted extensive research and confirmed that the expression vector and the protein thereof containing the mutant homodimeric gene of human VEGF linked by the linker base sequence have a very high affinity for KDR. It became.

An object of the present invention is to provide a mutant homodimeric gene of VEGF that can enhance the affinity of VEGF for the KDR receptor.

Another object of the present invention is to provide an expression vector comprising a mutant homodimeric gene of VEGF that can enhance the affinity of VEGF for the KDR receptor.

Still another object of the present invention is to provide a transformant transformed with the expression vector.

In addition, another object of the present invention to provide a protein produced from the transformant.

1 is a cleavage map of a pmut-diVEGF165 expression vector.

Figure 2 is a gel photograph showing the results of electrophoresis on agarose gels after cleavage of pVEGF165 with restriction enzymes.

Figure 3 is a gel photograph showing the result of electrophoresis on agarose gels after cleavage of pmut-diVEGF165 with restriction enzymes.

Figure 4 is a photograph showing the result of Western blot of VEGF165 protein.

5 is a photograph showing the result of Western blotting of the recombinant mut-diVEGF165 protein.

6 is a graph showing the proliferation effect of CPAE cells by recombinant VEGF165.

Hereinafter, the present invention will be described in more detail.

In the present invention, a mutant homodimer gene of human VEGF linked by a linker base sequence and an expression vector pmut-diVEGF165 including the same are prepared.

Normal human VEGF has a low affinity for the KDR receptor compared to the Flt-1 receptor, and is known to form homodimers in vivo. Thus, even when the mutant VEGF gene and recombinant protein that bind only to the KDR receptor are externally administered, Normal forms and heterodimers can be formed. Therefore, in the present invention, by combining two mutant VEGF genes that bind to the KDR receptor with an artificial linker with higher efficiency, an artificially mutated VEGF homodimer is formed to exclude the possibility of forming a heterodimer, thereby effectively vascular endothelial Cell proliferation and angiogenesis were induced.

The mutant VEGF gene mutVEGF165 (Shen et al., J. Biol. Chem. 1998, 273 (45) 29979-29985) used in the present invention is based on a known VEGF gene (Science 246, 1989, 1306-1309). Although the PCR was performed with each primer including the 5 'end and the 3' end of the gene, and the mutation site to be induced, a variety of known mutation production methods can be used. The two mutVEGF165s were combined with a linker to obtain a mut-diVEGF165 recombinant gene having the nucleotide sequence of SEQ ID NO: 1, and then cloned into a vector to obtain an expression vector pmut-diVEGF165 (Accession No .: KFCC11211; see FIG. 1). The expression vector was introduced into and stored in E. coli for transformation through known techniques, ie, thermal shock, electroporation, and the like. The E. coli / pmut-diVEGF165 E. coli / pmut-diVEGF165 was stored on September 4, 2000 in Korea. (KCCM) was deposited under accession number KFCC11211.

In the present invention, the oligonucleotide having the nucleotide sequence of SEQ ID NO: 7 expressing (Gly-Gly-Gly-Gly-Ser) ₃ was used as a linker. In addition, other linkers that do not modify the structure of the protein may be used. Do.

In the present invention, the mut-diVEGF165 recombinant gene was inserted into the foreign gene insertion site of the pEGFP-N1 vector (Clontech). However, in addition to the pEGFP-N1 vector used in the preparation of the expression vector, other expression vectors may be used depending on the purpose of expression. In addition, the size, nucleotide sequence, etc. of the gene inserted into the foreign gene insertion site of the expression vector can also be variously changed by known techniques.

The expression vector pmut-diVEGF165 prepared above is introduced into an animal cell line to obtain a transformant. In the present invention, the expression vector may be prepared by introducing the expression vector into an animal cell line such as Chinese hamster ovary (CHO). It can be introduced through a known technique, that is, thermal shock, electroporation, etc., in addition to the animal cell line CHO, COS-7 (Monkey, African green), NIH / 3T3 (Mouse fibroblast), BHK-21 (Baby Hamster Kidney, Various transgenic animal cell lines may be used, such as Syrian golden), Hela (Human carcinoma), Vero (Monkey kidney, African green), and C127 (Mouse).

Recombinant mutant VEGF was overexpressed in the animal cell line transformants to obtain recombinant mutant VEGF protein.

The protein has a 371 amino acid sequence as described in SEQ ID NO: 2.

As a result of measuring the physiological activity of the recombinant mutant VEGF protein obtained above, vascular endothelial cells expressed in mutant recombinant VEGF, in particular, mut-diVEGF165 in which two mutant monomers according to the present invention were linked with a linker, were compared to normal VEGF165. The activity against the proliferation of was found to be quite high (see Figure 6).

As a result, it was confirmed that the mut-diVEGF165 expression vector and recombinant mutant homodimer mut-diVEGF165 protein prepared in the present invention can be used more effectively than gene VEGF for gene therapy and protein treatment for various ischemic diseases.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1

Cloning and Expression Vector Production of Normal Human VEGF Gene

Trizol (GIBCO) was used to isolate whole RNA from the human promyelocytic leukemia cell line HL60 (Korea Cell Line Bank) by the protocol of the manufacturer's protocol. CDNA was synthesized using reverse transcriptase (Sambrook et al., Molecular cloning, 1989). The PCR buffer (dNTP 2.5 mM, polymerase 2.5 units, 10) using 250 ng of the cDNA as a template using 20 pmol each of a forward primer (VEGF5Bg) represented by SEQ ID NO: 3 and a reverse primer (VEGF3E) represented by SEQ ID NO: 4, respectively. PCR was performed on polymerase buffer (500 mM KCl, 100 mM TrisCl, 15 mM MgCl _2, 0.1% gelatin). The PCR was performed using a PCR cycler (Perkin Elmer) for 5 minutes at 94 ° C. first, followed by 20 times at 94 ° C., 30 seconds at 60 ° C., 30 seconds at 72 ° C., and finally 30 times. 10 minutes was carried out at 72 ℃ to obtain the result. The resulting product was electrophoresed on 0.9% agarose gel to confirm that a 495 bp DNA fragment was obtained. The amplified fragment was cloned into a pGEM-T (Promega) vector to obtain a pGEM-VEGF ₁₆₅ vector.

In order to determine whether the cloned DNA fragment is the VEGF165 gene, the sequence was analyzed and then compared with the sequence of a known normal VEGF165 gene (Keyt. Et al., J. Biol. Chem. 1996, 271 (10) 5638-5646). It was confirmed that the amplified DNA fragment encodes the VEGF165 gene.

The pGEM-VEGF ₁₆₅ vector VEGF165 gene was cleaved with Bgl II and EcoR I and then inserted into Bgl II and EcoR I sites of the expression vector pEGFP-N1 vector (Clontech), from which EGFP (Enhanced Green Fluorescent protein) was removed, thereby expressing pVEGF165. A vector was obtained (see FIG. 2).

Example 2

Preparation of VEGF Mutant mutVEGF165 by PCR

To prepare a mutant VEGF (Shen, BQ et al., J. Biol. Chem. 273 (45), 1998, 29979-29985) that binds to KDR but not to flt-1, first shown by SEQ ID NO: 5 PCR was performed using a primer (KRK5) and a reverse primer (KRK3) represented by SEQ ID NO: 6.

First, a DNA fragment obtained by the same method as Example 1 using the primers represented by SEQ ID NO: 3 (VEGF5Bg), and 6 (KRK3) and a primer represented by SEQ ID NO: 4 (VEGF3E), and 5 (KRK5) After mixing the DNA fragments obtained in the same manner as in Example 1 using the template and the reverse primer (VEGF3E) shown in SEQ ID NO: 3 and the forward primer represented by SEQ ID NO: 3 (VEGF3E) again PCR was performed in the same manner as 1.

As a result, a 574 bp mutVEGF165 gene was obtained, and the amplified PCR product was sequenced to determine that the 63rd, 64th, and 67th amino acids of normal VEGF were aspartic acid, glutamic acid, and glutamic acid, respectively. (lysine), arginine (arginine), and has an amino acid sequence substituted with lysine. The mutVEGF165 gene was double-cut into Bgl II and EcoR I and inserted into the Bgl II and EcoR I sites of the pEGFP-N1 expression vector (Clontech) to complete the expression vector pmutVEGF165 to express under the CMV (cytomegalovirus) promoter.

Example 3

Induction of binding expression of mutant VEGF via linker

Two genes of mutVEGF165 were digested with Bgl II and EcoR I so that mutant VEGF could form a homodimer in vivo, and the nucleotide sequence of SEQ ID NO: 7 expressing (Gly-Gly-Gly-Gly-Ser) ₃ was The oligonucleotides having been synthesized were then inserted into the linker between two genes of mutVEGF165. Next, the mutVEGF165-linker-mutVEGF165 recombinant gene was inserted into the pEGFP-N1 expression vector in the same manner as in Example 2 to prepare the expression vector pmut-diVEGF165 (see FIGS. 1 and 3).

Example 4

Introduction of Mutant VEGF Expression Vectors into Cells

CHO (Korea Cell Line Bank) cell lines were transformed with the expression vectors pVEGF165, pmutVEGF165, and pmut-diVEGF165, respectively. First, CHO cell lines maintained in RPMI medium containing 10% FBS and 100 μg / ml antibiotics (GIBCO Co., Ltd.) were prepared by inoculating 3 × 10 ⁵ cells per well into 6-well plates, and then the expression vector at 24 hours later. 2 μg and 4 μl of lipofectamine (GIBCO) were mixed with 200 μl of RPMI medium containing no serum and antibiotics and left at room temperature for 45 minutes. The medium is added so that the total volume of the expression vector-containing mixture is 1 ml and then placed in the prepared CHO cell line. Cells were further incubated in a 37 ° C. CO ₂ incubator for 5 hours, then the medium was exchanged, and further cultured for 48 hours before the cells and culture medium were recovered.

Example 5

Isolation of Recombinant Mutant VEGF Protein

Proteins were isolated and quantified from each cell and culture medium obtained in Example 4 (Sambrook J., et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989), and 30 μg of the protein was SDS-PAGE. Fractions and western blotting. In Western blotting, expression levels of VEGF165 and recombinant VEGF165 proteins were measured by the method of Amersham Co., Ltd. using PVDF membrane, antiVEGF antibody, and ECL kit (Amersham). As a result, a band was identified at 42 KDa, indicating that the recombinant VEGF165 protein was successfully expressed (see FIGS. 4 and 5), and the molecular weight was slightly higher than expected, possibly due to glycosylation. I think.

Example 6

Activity Assay of Recombinant Mutant VEGF Protein

CPAE (calf pulmonary artery endothelial cell (ATCC) cell line maintained in RPMI medium containing 20% FBS (Fetal Bovine Serum) (GIBCO) was first inoculated in a 96-well plate at a concentration of 1 × 10 ⁴ . Normal VEGF and mutant recombinant VEGF cultures obtained in Example 4 were each added and further cultured for 5 days. The culture solution contained 80 pg normal VEGF165, 2 pg mutVEGF165, 0.3 pg mut-diVEGF165, respectively.

Next, a cell proliferation assay was performed using a CellTiter 96TM Non-Radioactive Cell Proliferation Assay kit (Promega). Add 15 μl of MTT (3-4,5-dimethylthiaxol-2yl-2,5-diphenyltetrazolium bromide) to each 96-well treated with normal VEGF and mutant recombinant VEGF cultures for 4 hours in a CO ₂ incubator at 37 ° C. After treatment for 100 μl per well of the solution in the kit was added and left for 1 hour at 37 ℃. The 96-well plate was transferred to an ELISA reader, and the OD value was measured at 570 nm to determine the proliferation of vascular endothelial cells.

As a result, it was observed that normal VEGF and recombinant VEGF induced the proliferation of CPAE cells, and compared with the concentration of the recombinant protein treated (80 pg normal VEGF165, 2 pg mutVEGF165, 0.3 pg mut-diVEGF165 treatment), Mutant recombinant VEGF rather than VEGF165, in particular homodimer mut-diVEGF165, in which a mutant monomer was linked by a linker, was found to have significantly higher activity for proliferation of vascular endothelial cells (see FIG. 6).

As seen in the above embodiment, the expression vector and the protein thereof according to the present invention have a high affinity for KDR receptors involved in the proliferation and angiogenesis of vascular endothelial cells, and various cardiovascular diseases such as chronic ischemic leg disease, arteriosclerosis, When used as a therapeutic agent for ischemic diseases such as Burger's disease and diabetic foot ulcers, it can exhibit a high therapeutic effect.

Claims (5)

A mutant homodimeric gene of human VEGF of SEQ ID NO: 1. Expression vector pmut-diVEGF165 comprising a mutant homodimeric gene of human VEGF of SEQ ID NO: 1 (Accession No .: KFCC11211). The transformant transformed with the expression vector pmut-diVEGF165 (Accession No .: KFCC11211) according to claim 2. The method of claim 3, wherein the transformant is CHO, COS-7 (Monkey, African green), NIH / 3T3 (Mouse fibroblast), BHK-21 (Baby Hamster Kidney, Syrian golden), Hela (Human carcinoma), Vero (Monkey kidney, African green), and C127 (Mouse), characterized in that the transformant is selected from the group consisting of. A mut-diVEGF165 protein of SEQ ID NO: 2 expressed from the transformant according to claim 3.