KR100791090B1 - Plant transformation vector comprising tissue-type plasminogen activator gene and plant transformed thereby - Google Patents

Abstract

A plant transformation vector is provided to express an active type plasminogen activator protein by introducing the same into a plant with excellent expression efficiency, thereby being used for developing an effective fibrinolytic enzyme due to very cheap manufacturing cost. A plant transformation vector is characterized in that a TEV(tobacco etch virus) reader sequence having a sequence of SEQ ID : NO. 1, a signal sequence of a glucose-regulated endoplasmic reticular protein derived from Medicago sativa having a sequence of SEQ ID : NO. 2 and a tissue-type plasminogen activator C gene sequence having a sequence of SEQ ID : NO. 3 are linked in sequence. Transgenic bacteria are transformed by the vector, wherein the bacteria are Agrobacterium tumefaciens. A method for preparing a transgenic plant expressing a tissue type plasminogen activator protein or producing the plasminogen activator protein comprises a step of introducing the vector into a plant cell.

Description

Plant Transformation Vector Comprising Tissue-Type Plasminogen Activator Gene and Plant Transformed Thereby}

Figure 1a is a schematic diagram (A) of the parent vector (pBSNB22101) of the plant transformation vector according to the present invention,

1B is a schematic diagram (B) of a plant transformation vector according to the present invention.

(P35S: cauliflower mosaic virus 35S promoter, bar: phosphinothricin acetyl transferase gene, Ti7: terminator sequence of T-DNA gene 7, TEV: tobacco etch virus leader sequence, SS: alfalfa-derived glucose control vesicle protein (glucose signal sequence of regulated endoplasmic reticular protein, T35S: cauliflower mosaic virus 35S terminator)

Figure 2 is the result of restriction enzyme treatment to confirm that the plasminogen activator gene is inserted into the vector.

(Lane 1-12: plant transformation vector, lane M: molecular weight marker)

Figure 3a is a photograph of tobacco transformants with selected herbicide (vasta) resistance genes (bars) (arrows represent wild type tobacco plants),

Figure 3b is the result of confirming the phophinothricin acetyl transferase expressed in the transformant plants using a bar strip (bar strip).

Figure 4a is a result confirming that the recombinant plasminogen activator gene is inserted in the transformant using PCR,

(Lane 1-30: transformant, wt: wild species, p: expression vector)

Figure 4b is the result of confirming the transcription of the recombinant plasminogen activator gene using RT-PCR.

(Lane 1: expression vector PCR, lane 2: transformant PCR, lane 3: wt RT-PCR, lane 4-11: transformant RT-PCR)

Figure 5 shows the results of confirming the recombinant plasminogen activator protein in the transformant via Western blot.

(t-PA: control, wt: wild type, T1, T17, T27, T29: transformant)

Figure 6 shows the result of confirming the amount of recombinant plasminogen activator protein expression in the transformant through ELISA.

(TSP: total soluble protein)

7 shows the fibrin degrading activity of the recombinant plasminogen activator expressed in the transformant through a fibrin-plate method.

(t-PA: control, wt: wild type, rTPA: transformant)

8 is a result of confirming the molecular weight of the recombinant plasminogen activator using Zymography (Zymography).

(t-PA: control, wt: wild type, T17, T29: transformant)

9 shows the results of confirming fibrin specific chain degradation activity using SDS-PAGE.

(t-PA: control, rTPA: transformant)

The present invention relates to a plant transformation vector comprising a plasminogen activator gene in a plant and a plant transformed with the vector, and more particularly, a glucose control vesicle derived from a tobacco etch virus (TEV) leader sequence, alfalfa. The present invention relates to a plant transformation vector and a plant transformed with the vector, wherein a signal sequence of a protein (glucose-regulated endoplasmic reticular protein) and a plasminogen activator gene are sequentially connected.

Tissue-type plasminogen activator (hereinafter, abbreviated as 't-PA') is a serine protease involved in fibrin lysis, and t-PA is a molecular weight of about 68,000 Daltons produced in human tissues such as vascular endothelial cells. It is a glycoprotein of approximately size and is used as a thrombolytic agent. A thrombus is a lump of blood in a blood vessel that causes clogged capillaries, resulting in hand and hand paralysis, cerebral infarction, and myocardial infarction. The main component of blood clots is a glycoprotein called fibrin, which is broken down by plasmin. t-PA not only has high affinity for fibrin, which forms a blood clot, but also has a 100-200-fold increase in the presence of fibrin, which acts locally on the surface of fibrin to convert plasminogen into plasmin. , It is known to have higher specificity than other thrombolytic agents such as Straptocaine or Eurokinase (Bergmann, SR et al., Science 220: 1181-1183. 1981). On the other hand, it is reported that the strap to kinase, euro kinase and the like also have side effects such as severe bleeding phenomenon because it also activates the plasminogen circulating in the blood, but such side effects have not been reported in the plasminogen activator.

Meanwhile, as a method for producing a plasminogen activator, US Pat. Nos. 4,853,330 and 5,869,314 disclose methods for expressing human tissue plasminogen activator proteins in CHO cells, which are ovarian cells of Escherichia coli and Chinese hamsters. US Patent No. 5,106,741 discloses a method for expressing plasminogen activator variants in E. coli and yeast. However, most of microbial expression is expressed in insoluble form, refolding reaction is essential, sugar is not attached, and animal cell expression is inefficient in terms of price competitiveness because of high cost in production cost. There was a disadvantage. Accordingly, there is a need for a method of producing an active form of plasminogen activator protein, which has a significant price competitiveness compared to conventional thrombolytics.

Therefore, the inventors of the present invention, while studying how to express the plasminogen activator protein in plants, TEV (tobacco etch virus) leader sequence, signal sequence of alfalfa-derived glucose-regulated endoplasmic reticular protein and plasmin When the gene-type plasminogen activator gene is introduced into and expressed in plants, the present invention has been completed by confirming that the active form of plasminogen activator protein can be expressed, and its expression efficiency is also excellent.

It is therefore an object of the present invention to provide a plant transformation vector comprising a plasminogen activator gene and a plant transformed by the vector.

Another object of the present invention is to provide a method for producing a transgenic plant expressing plasminogen activator protein using the vector and a method for producing plasminogen activator protein.

In order to achieve the above object, the present invention is a TEV (tobacco etch virus) leader sequence, alfalfa-derived glucose-regulated endoplasmic reticular protein signal sequence and plasminogen activator (tissue-type plasminogen activator) gene Provides a vector for plant transformation, characterized in that the sequentially connected.

The present invention also provides a transformed bacterium transformed with the vector.

The present invention also provides a transgenic plant expressing a plasminogen activator (tissue-type plasminogen activator) protein in which the gene is introduced by the vector.

The present invention also provides a method for producing a transgenic plant expressing a plasminogen activator (tissue-type plasminogen activator) protein, comprising the step of introducing the vector into a plant cell.

The present invention also provides a method for producing a plasminogen activator (tissue-type plasminogen activator) protein comprising the step of introducing the vector into a plant cell.

Hereinafter, the present invention will be described in detail.

In general, the production of plasminogen activator and its variants is produced using CHO cells, Escherichia coli, and expression of plasminogen activator in plant cells has not been reported. However, the inventors have sequentially linked the tobacco etch virus (TEV) leader sequence, the signal sequence of the alfalfa-derived glucose-regulated endoplasmic reticular protein, and the plasminogen activator (tissue-type plasminogen activator) gene. The plasminogen activator protein was first produced in plant cells by introducing the gene into the plant using a plant transformation vector. In addition, the present inventors were able to confirm that the plasminogen activator protein produced in the plant cell is expressed in an active form rather than an insoluble form.

Accordingly, the present invention relates to a sequential connection of a tobacco etch virus (TEV) leader sequence, a signal sequence of alfalfa-derived glucose-regulated endoplasmic reticular protein, and a tissue-type plasminogen activator (tissue-type plasminogen activator) gene. It provides a plant transformation vector characterized by.

The "TEB (tobacco etch virus) leader sequence" is known to bring about an increase in the amount of protein expression in the recombinant translation step. Tobacco etch virus (TEV) leader sequence used in the present invention is GenBank No. It is disclosed in M15239 and has a nucleotide sequence represented by SEQ ID NO: 1.

The 'signal sequence of alfalfa-derived glucose-regulated endoplasmic reticular protein' is GenBank No. It is disclosed in M8023 and has a nucleotide sequence represented by SEQ ID NO: 2.

In the present invention, in order to have a glycosylation and correct folding structure of the plasminogen activator, the signal sequence of the alfalfa-derived glucose-regulated endoplasmic reticular protein Inserted on top of Gen Activator gene

'Tissue-type plasminogen activator gene' used to achieve the object of the present invention preferably has a nucleotide sequence encoding a plasminogen activator protein or a functional equivalent thereof. The functional equivalent of the plasminogen activator protein refers to a polypeptide that exhibits substantially the same physiological activity as the recombinant plasminogen activator protein having the amino acid sequence represented by SEQ ID NO: 3. 'Equivalent physiological activity' refers to fibrin degrading activity. The functional equivalent refers to a polypeptide having sequence homology (ie, identity) of at least 70% or more, preferably 80% or more, and more preferably 90% or more of the amino acid sequence represented by SEQ ID NO: 3. For example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 Sequence homology of%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% It includes a polypeptide having a. Such functional equivalents may result from the addition, substitution or deletion of some of the amino acid sequences of SEQ ID NO: 3. Substitution of amino acids in the above is preferably a conservative substitution. Examples of conservative substitutions of naturally occurring amino acids are: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn) and sulfur-containing amino acids (Cys, Met). The functional equivalent also includes variants in which some of the amino acids are deleted on the recombinant plasminogen activator amino acid sequence of the present invention. Deletion or substitution of these amino acids is preferably located in a region not directly related to the physiological activity of the polypeptides of the invention. Also included are variants in which some amino acids are added to both ends or sequences of the amino acid sequence of the plasminogen activator. The scope of functional equivalents of the present invention also includes polypeptide derivatives in which some chemical structures of the polypeptide have been modified while maintaining the basic backbone of the polypeptide and its physiological activity. For example, this includes structural modifications for altering the stability, shelf life, volatility or solubility of the polypeptide of the present invention.

In one embodiment of the present invention, the plasminogen activator gene represented by SEQ ID NO: 4 was used.

In addition, the present invention can use the gene variants of the plasminogen activator instead of the native plasminogen activator gene, for the variants of the plasminogen activator US Patent Nos. 4,963,357, 5,037,752, 5,087,572, 5,094,953, 5,100,666, 5,106,741, 5,108,901, 5,156,969, 5,185,259, 5,232,847, 5,242,819, 5,244,676, 5,258,180, 5,258,298,198,270,5,262 No. 5,302,390, 5,314,818, 5,338,546, 5,346,824, 5,366,886, 5,376,547, 5,385,732, 5,405,771, 5,411,871, 5,486,471, 5,504,001,5,504,001 5,520,913, 5,556,621, 5,580,559, 5,589,361, 5,614,190, 5,616,486, 5,648,250, 5,714,144, 5,714,145, 5,714,372, 5,728,565, 5,728,566,5,728,566,567 No. 5,736,134, 5,739,012, No. 5 , 553,486, 5,756,093, 5,770,425, 5,770,426, 5,821,105, 5,830,849, 5,840,533, 5,840,564, 5,869,314, 5,908,625, and 6,706,504. Is cited herein.

Tobacco etch virus (TEV) leader sequence of the present invention, the signal sequence of the alfalfa-derived glucose-regulated endoplasmic reticular protein, and for plant transformation comprising a plasminogen activator (tissue-type plasminogen activator) gene The vector may be prepared using a known plant transformation vector as a base vector, and a general binary vector or cointegration vector may be used. A wide variety of binary vectors are widely used in plant transformation, and almost all binary vectors are international centers and university research institutes, such as the Center for the Application of Molecular Biology to International Agriculture, GPO Box 3200, Canberra ACT2601, Australia (CAMBIA). The basic binary vector skeleton is available by using a variety of transformant selection marker genes, promoters, transcriptional termination genes, and the like, in the left and right boundary regions where genes are transferred using Ti plasmids as a parent.

More preferably, the plant transformation vector of the present invention includes the cleavage map disclosed in FIG. 1. That is, the plant transformation vector of the present invention is a cauliflower mosaic virus 35S promoter, a phosphinothricin acetyl transferase gene (herbicide resistance gene) as a selection marker gene, a terminator sequence of T-DNA gene 7, a TEV (tobacco etch). virus leader sequence, a signal sequence of glucose-regulated endoplasmic reticular protein derived from alfalfa, and a cauliflower mosaic virus 35S terminator.

Since the vector according to the present invention is a vector for plant transformation, various plant-functional promoters known in the art may be used, and the cauliflower mosaic virus (CaMV) 35S promoter, the pigwarm mosaic virus 35S promoter, and the sugarcane basilisform virus promoter , Comerina yellow mottle virus promoter, light-induced promoter from subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), rice cytozolic triophosphate isomerase (TPI) promoter, Arabidopsis Adenine phosphoribosyltransferase (APRT) promoter from Arabidopsis, a rice actin 1 gene promoter and a mannopin synthase and octopin synthase promoter, and preferably the Califlower Mosaic Virus (CaMV) 35S promoter use.

In addition, the selection marker genes include, but are not limited to, antibiotic resistance genes, herbicide resistance genes, metabolic genes, luminescent genes, GFP (green fluorescence protein) genes, GUS (β-glucuronidase) genes, and GAL (β-galactosidase) genes. It is not limited to this. Specifically, neomycin phosphotransferase II (NPT II) gene, hygromycin phosphotransferase gene, phosphinothricin acetyltransferase gene or dihydrofolate reductase gene may be used, but phosphinothricin Acetyltransferase is preferred. In an embodiment of the present invention, the t-PA gene (1668 bp), which is linked to a signal sequence of alfalfa-derived glucose-regulating endoplasmic reticulum protein using molecular biological techniques known in the art, is used for the cauliflower mosaic virus 35S promoter, TEV ( cloned into a plant expression vector (p221a) comprising a tobacco etch virus leader sequence and a 35S terminator (p221a), a cauliflower mosaic virus 35S promoter, a phosphinothricin acetyl transferase gene (herbicide resistance gene), T-DNA gene as a selection marker gene Vector pBS221t with a terminator sequence of 7, a tobacco etch virus (TEV) leader sequence, a signal sequence of alfalfa-derived glucose-regulated endoplasmic reticular protein and a T35S califlower mosaic virus 35S terminator -PA was prepared and used (see FIG. 1).

Standard recombinant DNA and molecular cloning techniques, molecular biology techniques known in the art, are described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989; by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1984; and by Ausubel, FM et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience, 1987).

Meanwhile, in one embodiment of the present invention, the recombinant vector of the present invention is transformed into Agrobacterium tumefaciens, and then the gene of the present invention is introduced into plant cells by the transformed Agrobacterium.

Accordingly, the present invention provides a bacterium transformed with the plant transformation vector of the present invention. The bacteria include bacteria of the genus Agrobacterium. Agrobacterium used as a transformation host is Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) or Agrobacterium rhizogenes ( Agrobacterium rhizogenes ) and the like.

The present invention also provides a transgenic plant expressing a tissue-type plasminogen activator protein into which the vector is introduced. The present invention also provides a method for producing a transgenic plant expressing a plasminogen activator (tissue-type plasminogen activator) protein, characterized in that it comprises the step of introducing the plant cell and plasminogen activator (tissue- type plasminogen activator) protein.

The plasminogen activator produced by the present invention can be produced by extraction and purification by known methods from the transgenic plants produced by the method of the present invention as the protein in the active form.

As a method of introducing the recombinant vector according to the present invention into a plant, a method known in the art may be used. For example, but not limited to, transformation with Agrobacterium sp. Microorganisms, particle gun bombardment, silicon carbide whiskers, sonication, electrical Precipitation by electroporation and polyethylene glycol (PEG) can be used. Preferably, Agrobacterium-mediated transformation can be used, and the methods exemplified in the literature can be used (Horsch et al., Science 227: 1229-1231, 1985). For example, Agrobacterium-mediated transformation methods for rice are known in the art and the like (An et al., EMBO J 4: 227-288,1985).

In the present invention, the term "plant" includes all of a whole plant, a part of a plant, callus, plant tissue, plant cells, and plant seeds. Plants to which the method according to the present invention may be applied include monocotyledonous plants or dicotyledonous plants. Examples of the monocotyledonous plant include, but are not limited to, rice, wheat, barley, bamboo shoots, corn, taro, asparagus, onions, garlic, leeks, leeks, soothing, hemp and ginger. Examples of dicotyledonous plants include, but are not limited to, baby pole, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, beetroot, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, mustard, There are watermelons, melons, cucumber pumpkins, gourds, strawberries, soybeans, green beans, kidney beans, buzzfoot trefoils, potatoes, duckweed, perilla, pigeon beans and peas. In one embodiment of the present invention, tobacco was used (see Example 2).

In one embodiment of the present invention, the recombinant vector of the present invention was transformed into Agrobacterium tumefaciens, and then the synthetic gene of the present invention was introduced into plant cells by the transformed Agrobacterium (see Example 2). ).

On the other hand, the transgenic plants introduced the synthetic gene of the present invention by the above method can be re-differentiated into plants through processes such as callus induction, rooting and soil purification using standard techniques known in the art, It can be produced by the planetary breeding method using asexual breeding methods such as grafting and seeds.

In one embodiment of the present invention, in order to confirm that the plasminogen activator gene is stably introduced into the re-differentiated plant as described above, PCR was performed using a plasminogen activator-specific primer in the transformant sample, Confirmed that the plasminogen activator gene was successfully inserted at the DNA level (see FIG. 4).

In another embodiment of the present invention, to confirm whether the plasminogen activator gene is stably expressed as a protein in the redivided plant as described above, Western blot and ELISA using a transformant sample, It was confirmed that the expression is stable (see FIGS. 5 and 6).

In another embodiment of the present invention, the fibrin plate method confirmed whether or not the plasminogen activator protein produced by the method of the present invention is an active form, it was confirmed that there is a degradation activity of fibrin (see Fig. 7), Fibrin specific chain degradation activity using SDS-PAGE was confirmed to show similar activity as that of t-PA of the positive control group (see FIG. 9), indicating that the plasminogen activator protein produced by the method of the present invention was an active form. I could confirm it.

Hereinafter, the present invention will be described in more detail with reference to Examples.

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

<Example 1>

Preparation of plant transformation vector and Agrobacterium transformant

<1-1> Preparation of plant transformation vector

The t-PA gene (1668 bp), which is linked to the signal sequence of the alfalfa-derived glucose control vesicle protein (GenBank accession no. M80235), includes a cauliflower mosaic virus 35S promoter, a tobacco etch virus leader sequence, and a 35S terminator In order to clone into the plant expression vector (p221a) to be expressed as follows.

pCAMBIA 2300 (CAMBIA, Australia) was first cleaved with the restriction enzyme Hind III and dephosphorylated using a calf intestine phosphatase. Thereafter, plasmid pDPG165 (Kausch et al., 2001) was digested with Hind III, and then linked to pCAMBIA 2300 digested with Hind III to prepare plasmid pBS221. In order to remove the restriction enzyme (BamH I, Kpn I, Xba I) sites in the plasmid pBS221, each restriction enzyme was cleaved and polymerized with T7 polymerase.

Meanwhile, in order to insert a polycloning position into pBI221 (Clontech Co., Ltd.), pBI221 was cleaved with Sac I and Xba I, and then synthesized with an oligonucleotide (SEQ ID NO: 5'-GACGCGTGAGCTCGGTACCTCGCGAGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTG-3 '). The polymerase was inserted into the blunt after the polymerization reaction. In pBI221 to which the oligonucleotide was inserted, fragments containing a CaMV 35 promoter, a polycloning position, and a Nos terminator were prepared by primers 1 and 5 shown in SEQ ID NO: 6 and SEQ ID NO: 7 (primer-1; 5'-CGGAATTCAGATTAGCCTTTTCAATTTCAGAAAG- PCR using 3 ', primer-2; 5'-CGGAATTCGATCTAGTAACATAGATGACACC-3' (reaction conditions: 95 degrees 5 minutes, 1 time; 94 degrees 30 seconds, 60 degrees 30 seconds, 72 degrees 2 minutes, 30 times; 72 7 minutes, once), and the vector pBSNB22101 was prepared by cloning with the pBS221 after cleavage using the restriction enzyme EcoR I (see FIG. 1). In order to leave only one selective marker in the vector containing two selective markers (npt II and bar), the vector was cut with Hind III and Xho I to remove one of the selective markers npt II and named pBSB22101. It was. In addition, primer-3 and primer-4 represented by SEQ ID NO: 8 and SEQ ID NO: 9 using pRTL2 (Carrington and Freed et al., J. Virol. 64, 1990) as a template to increase the amount of expression . 5'-CGACGCGTAAATAACAAATCTCAACACAACATATAC-3 ', primer-4: Amplify the TEV leader sequence by PCR using 5'-CGGGATCCAAATAACAAATCTCAACACAACATATA-3' (reaction condition: 95 degrees 5 minutes, 1 time; 94 degrees 30 seconds, 60 degrees 30 Second, 72 degrees 1 minute, 30 times; 72 degrees 7 minutes, once) was cut with Mlu I and Kpn I, and then inserted into the position of Mlu I and Kpn I of pBSB22101 to prepare p221a. The experiment was carried out as follows to attach the signal sequence of the alfalfa-derived glucose-regulating vesicle protein to the 5 'side of t-PA. First, the synthesized oligonucleotide and its complementary oligonucleotide of the signal sequence of the alfalfa-derived glucose-regulating vesicle protein represented by SEQ ID NO: 2 were heated in boiling water for 5 minutes and then annealed at room temperature for 30 minutes. And amplified t-PA PCR using primer 5 represented by SEQ ID NO: 10 and primer 6 represented by SEQ ID NO: 11 (primer 5: 5'-TCTTACCAAGTGATCTGCAGAG-3 ', primer 6: 5'-CCCAAGCTTTCACGGTCGCATGTTGTCACG-3') After mixing with the product was incubated for 30 minutes at 16 degrees using T4 DNA ligase (Takara Company). And PCR using a primer 7 represented by SEQ ID NO: 12 and primer 6 represented by SEQ ID NO: 11 (primer 7: 5'-CGGGGTACCATGAAAATGGAGATGCATCAGATC-3 ') (reaction conditions: 95 degrees 5 minutes, once; 94 degrees 30 Seconds, 60 degrees 30 seconds, 72 degrees 2 minutes, 30 times; 72 degrees 7 minutes, once) to obtain a t-PA PCR product containing the signal sequence of the alfalfa-derived glucose-regulated vesicle protein. The t-PA PCR product was digested with Kpn I and Hind III to clone the 1668 bp t-PA gene into the p221a vector to prepare pBS221t-PA (see FIG. 1B).

Meanwhile, in order to confirm whether the plasminogen activator gene was correctly inserted into the expression vector, 1% agarose gel electrophoresis was performed after cutting with Kpn I and Hind III.

As a result, as shown in Figure 2 it was confirmed the presence or absence of the insertion of the gene of about 2kbp.

<1-2> Agrobacterium transformant preparation

Agrobacterium EHA105 (prodigene) by freeze-thaw method (Glevin et al., Plant molecular biology.Kluwer academic Publishers A3 / 7, 1988) of the pBS221t-PA vector prepared in Example 1-1. .com). Thereafter, the transformed Agrobacterium was incubated in YEP medium (10 g / L bacto yeast extract, 10 g / L bacto peptone, 5 g / L NaCl) for 2 days and resuspended in 1/2 MS medium.

<Example 2>

Preparation of Tobacco Transformants

To express the plasminogen activator gene in tobacco, Agrobacterium EHA105 prepared in Example <1-2> was transformed into tobacco leaves.

Sterilized tobacco seeds were sown and fresh young tobacco leaves were grown aseptically for 2 weeks or longer. Agrobacterium quantified with absorbance 0.4 was co-cultured in cancer medium for two days after infection with the tobacco cotyledons for 30 minutes. Two days later, MS-vitamin-containing medium (Duchefa) was treated with 3.0% sucrose, 1 mg / l BA (benzyladenine), 0.1 mg / l NAA (napthalene acidic acid), 0.3% phytagel, 4 mg / l phosphinothricin and Regeneration was induced in selection medium (pH 5.8) with 500 mg / l carbenicillin added. Callus and leaf tissue were passaged at 14 day intervals. After 4 weeks, the re-differentiated stems induced roots in MS medium and were grown in greenhouses after acclimation. To identify the transformants, resistant transformants were distinguished after two treatments with 0.5% bastard at 4 days intervals.

As a result, as shown in Figure 3, 0.5% (v / v) was able to select a transformant by spraying the Vaster (A of Figure 3), using a bar strip (phosphinothricin acetyl transferase PAT) As a result of confirming that the protein is expressed, it was confirmed that PAT protein was expressed in the transformant to maintain herbicide resistance (FIG. 3B).

Experimental Example 1

Identification of Plasminogen Activator Gene in Transformants Using PCR

Plasminogen in transformed plants using PCR after isolation of genomic DNA from mature leaves of surviving plants after bastard treatment (Edwards, K., et. Al., Nucleic acid Research, 19, 1349, 1991) Insertion of the activator gene was confirmed.

To amplify the plasminogen activator, primer-8 (5'-cggggtactacgagaattatttatgtcttaccaag-3 ') represented by SEQ ID NO: 13 was used as a forward primer, and primer-6 represented by SEQ ID NO: 11 was used as a reverse primer. The PCR conditions were preprocessed at 95 ^o C for 5 minutes and then repeated 30 times at 94 ^o C, 30 seconds at 60 ^o C, 30 minutes at 72 ^o C, and then extended 7 minutes at 72 ^o C ( extension).

As a result, as shown in Figure 4A, the PCR product corresponding to approximately 1.7 kbp was amplified, it was confirmed that the plasminogen activator gene is properly inserted into the transformed plant.

In addition, RT-PCR was performed to determine the transcription of the plasminogen activator gene inserted into the transformant. Total RNA from tobacco leaves (150 mg) was purified using TRIzol reagent (Invitrogen, Breda, Netherlands). For RT-PCR analysis, total RNA samples were contaminated with genomic DNA using DNase (Takara). 1 μg of RNA was removed and buffered with the following composition (1 × One Step RNA PCR Buffer, 0.8 U RNase Inhibitor, Primer 0.4 μM, 0.1 AMV RTase XL, 0.1 U AMV-Optimized Taq DNA Polymerase, 5 The reaction was carried out in mM dNTP and 5MgCl ₂ ) under the following conditions. As a template, the total RNA isolated from the tobacco leaf was used as the forward primer-9 (5'-TCTTACCAAGTGATCTGCAGAG-3 ') represented by SEQ ID NO: 14 and the reverse primer-10 (5'-TCACGGTCGCATGTTGTCAC-3') represented by SEQ ID NO: 15. After 30 minutes at 50 ° C, 2 minutes at 94 ° C, and then repeated 35 times, 30 seconds at 94 ° C, 30 seconds at 60 ° C, 2 minutes at 72 ° C. As a result, it was confirmed that transcription was normally performed in the selected transformants. No bands were seen in the wild species used as a control (FIG. 4B).

Experimental Example 2

Identification of Recombinant Plasminogen Activator Protein in Transformants Using Western Blot

Western blot analysis was performed to determine the molecular weight of the recombinant protein expressed in the transformants.

200 mg of the transformant was homogenized in 1 ml of 1 × PBS buffer. Then, after 30 minutes at 4 ℃ centrifugation at 12,000 rpm for 30 minutes, the supernatant (100) was subjected to Western blotting using the extract of PBS (Phosphate buffer saline) of the transformant. After Western blot, polyclonal antibodies against t-PA (Abcam) were incubated overnight at 4 ^o C, washed five times with PBS-0.05% Tween 20 solution, and then HRP (horseradish peroxidase) After incubation for 4 hours at room temperature with the attached secondary antibody (Pierce) was washed five times, and finally, the color reaction was carried out using 4-chloronaphthol (4-chloronaphthol).

As a result, as shown in FIG. 5, about 68,000 Da of plasminogen activator protein band was confirmed in the transformant, and thus, it was confirmed that the transformant was stably expressed at the protein level.

Experimental Example 3

Expression of Recombinant Plasminogen Activator Protein Expression in Transformants Using ELISA

ELISA was performed to determine antigenicity and expression of the expressed recombinant plasminogen activator protein. The amount of recombinant plasminogen activator protein in the transformant was measured using a soluble t-PA ELISA kit (CalBiochem).

After 1 hour reaction with 50 ㎍ / 100 ㎕ PBS extracted sample prepared in Experimental Example 2 at room temperature and washed three times with PBS-0.05% Tween and after the reaction for 2 hours at room temperature using a secondary antibody HRP attached PBS- After washing three times with 0.05% Tween, 100 μl of TMB (tetramethyl-benzidine) was treated and absorbance was measured at 450 nm.

As a result, as shown in Figure 6, it was confirmed that the protein is expressed at 0.012 ㎍ / mg or more in seven lines, such as transformants 1, 5, 6, 17, 19, 21, 27, 29. On the other hand, there was no change in absorbance in the wild type (wt) used as a control.

Experimental Example 4

Confirmation of Recombinant Plasminogen Activator Enzyme Activity by Fibrin-plate Method

Fibrin degradability of the recombinant enzyme was measured using the fibrin plate method in order to determine the fibrin degrading activity of the recombinant plasminogen activator expressed in the transformant.

Fibrin was formed by mixing 10 ml of 0.8% fibrinogen solution, 0.5 ml of 10 units of thrombin solution, and 0.5 ml of plasminogen, pouring onto a plate, and standing at room temperature for 30 minutes. The fibrin plate was inoculated with 25 μg of PBS extract of t-PA 0, 100 pg, 300 pg, 1000 pg, 3000 pg and transformants T17 and T29, which were positive controls, and left overnight in a high humidity 37 ^° C. incubator.

As a result, as shown in Figure 7, the fibrin degrading activity of the enzyme was confirmed that 25 ㎍ transformant extract sample of the present invention and 3000pg positive control group showed similar activity. No degradation activity was found in the extract of wild-type tobacco used as a negative control.

Experimental Example 5

Confirmation of Molecular Weight of Recombinant Plasminogen Activator Using Zymography

In Experimental Example 4, zymography was performed to measure the molecular weight of the enzyme expressed in the transformant whose fibrin degrading activity was confirmed through the fibrin plate.

5 ml of 0.7% fibrinogen, 5 ml of 2% agarose, and 1 ml of 5 NIH thrombin were mixed and allowed to stand at room temperature for 30 minutes. 100 ㎍ PBS extract sample prepared in Experiment 2, 10% SDS-PAGE, washed three times with 2.5% Triton X-100 solution and placed on agarose fibrin plate and left overnight in a high humidity 37 ^o C incubator It was.

As a result, as shown in Figure 8, it was confirmed that the fibrin is decomposed in the transformants of the two strains (T17, T19), it was confirmed that it has the same molecular weight as t-PA used as a positive control group.

Experimental Example 6

Fibrin-specific Chain Degradation Activity by SDS-PAGE

In order to measure the fibrin specific chain degradation activity of the enzyme expressed in the transformant whose fibrin degradation activity was confirmed through the fibrin plate in Experimental Example 4, SDS after various time incubation in fibrin bound to thrombin and Factor XIIIa (0-24 hours) -PAGE was performed.

0.1 mg of 1 mg / ml fibrinogen dissolved in 5 mM imidazole-salina-5 mM CaCl ₂ , 0.02 ml of 0.25 NIH U thrombin, and 0.02 ml of 0.125 U Factor XIII were mixed and allowed to stand at room temperature for 30 minutes. After washing three times with 5 mM imidazole-salina-5 mM CaCl ₂ buffer, 20 μg of PBS extract sample was incubated for 0 to 24 hours and then subjected to SDS-PAGE.

As a result, as shown in Figure 9, T17 transformant (rTPA) and positive control t-PA (Calbiochem) was found to be similar to the degradation of fibrin γ-γ chain and (B) β chain.

As described above, TEV (tobacco etch virus) leader sequence, the signal sequence of the alfalfa-derived glucose-regulated endoplasmic reticular protein, and the plasminogen activator (tissue-type plasminogen activator) gene are sequentially linked. Plant transformation vector comprising, the plant transformed by the vector and the production method of the plasminogen activator protein using the same because the plasminogen activator protein can be expressed in the active form in the plant cells conventional thrombolytic Compared to the production method, the production cost is very inexpensive, and there is an effect that can produce a thrombolytic without side effects.

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Claims (10)

Tobacco etch virus (TEV) leader sequence, signal sequence of alfalfa-derived glucose-regulated endoplasmic reticular protein, and plant traits characterized in that the plasminogen activator (tissue-type plasminogen activator) gene is sequentially linked Transition vector. According to claim 1, wherein the tobacco etch virus (TEV) leader sequence has a nucleotide sequence of SEQ ID NO: 1, the signal sequence has a nucleotide sequence of SEQ ID NO: 2, the plasminogen activator (tissue- Type plasminogen activator) gene for plant transformation, characterized in that it has a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3. According to claim 1, wherein the plasminogen activator (tissue-type plasminogen activator) gene is a plant transformation vector, characterized in that it has a nucleotide sequence of SEQ ID NO. The vector of claim 1, wherein the plant transformation vector comprises a cleavage map disclosed in FIG. 1B. A bacterium transformed with the vector of any one of claims 1 to 4. 6. The transformed bacterium of claim 5, wherein the bacterium is Agrobacterium tumeficiens . A transgenic plant expressing a plasminogen activator protein into which the vector of claim 1 is introduced. 8. The transgenic plant of claim 7, wherein the plant is a monocotyledonous or dicotyledonous plant. A method for producing a transgenic plant expressing a plasminogen activator (tissue-type plasminogen activator) protein, comprising the step of introducing the vector of claim 1 into a plant cell. A method of producing a plasminogen activator (tissue-type plasminogen activator) protein, comprising the step of introducing the vector of claim 1 into a plant cell.

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