KR100597316B1 - Novel Promoter Originated from Chlorella Virus and Use Thereof - Google Patents

Abstract

The present invention relates to a promoter derived from Chlorella virus and its use, and more particularly, (a) at least one A box having a nucleotide sequence of SEQ ID NO: 1 and / or (b) a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 It relates to a promoter containing at least one B box having a vector, a vector containing the same, and a method for producing the target protein using the microalgae transformed with the vector.

The promoter according to the present invention has a function of strongly expressing a foreign gene in chlorella, thereby producing a high concentration of recombinant protein in chlorella and plants.

   Chlorella virus, promoter, transgenic plant, transgenic microalgae

Description

Novel Promoter Originated from Chlorella Virus and Use Thereof}             

1 shows the results of SDS-PAGE of chlorella virus isolated from Korean freshwater (lane 1: chlorella virus SS-1; lane 2: chlorella virus SS-2; and lane 3: chlorella virus KH-1).

Figure 2 is a photo of the genomic DNA of Chlorella virus isolated from Korean freshwater treated with a restriction enzyme electrophoresis, a) is a photo of Bam HI, b) is a photo of Hin dIII (lane 1: Chlorella virus SS-1; lane 2: chlorella virus SS-2; and lane 3: chlorella virus KH-1).

 Figure 3 shows a schematic diagram showing the repeating structure of the A box and B box for each virus and the nucleic acid sequence of the A box and B box of each virus.

 4 is a schematic diagram of pCTV, a chlorella transformed recombinant vector.

 5 is a schematic diagram of the recombinant vectors pMGFPble, pTGFPble, pKGFPble and pSGFPble according to the present invention.

Figure 6 is a photograph of fluorescence microscopy expression of GFP expression in transformed Chlorella ellipsodeea (WT: wild type Chlorella; 35S: Chlorella transformed with pMinGFPble; ABABA: Chlorella transformed with pTGFPble; CABA : Chlorella transformed with pKGFPble; and AA: Chlorella transformed with pSGFPble).

The present invention relates to a promoter derived from Chlorella virus and its use, and more particularly, (a) at least one A box having a nucleotide sequence of SEQ ID NO: 1 and / or (b) a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 Eggplant relates to a promoter containing one or more B boxes, a vector containing the same, and a method for producing the protein of interest using the microalgae transformed with the vector.

Producing proteins using transgenic plants is a new field in applied plant biotechnology that can be called molecular agriculture using molecular genetics, plant cell physiology, and plant transformation techniques. Advances in transformation technology have led to the production and purification of foreign proteins from tissues of transgenic plants since the 1980s (Hiatt, A. et al., Nature ., 76:78, 1989; Salmon, V. et al) , Protein Expr Purif ., 127: 135, 1998), the use of transgenic plants to produce foreign proteins can produce industrially expensive proteins that are more available than other protein expression systems (Zhong). , R. et al., Plant Physiol ., 53:64, 1999).

Among them, chlorella, a single-cell eukaryotic green alga, has recently been in the spotlight as a bioreactor. This is because chlorella has a system suitable for the expression of heterologous proteins. It can be inexpensively mass cultivated with a certain amount of light and carbon source, and it grows fast because it splits 2-9 times a day (Sorokin, C. et al., Plant Physiol ., 179: 183, 1958). In addition, eukaryotic chlorella, like other eukaryotes, is transcribed and then modified, thus transforming genes of heterologous eukaryotes, such as producing proteins with biological activity. Transient expression of homologous or heterologous constitutive proteins in chlorella has been reported since the 1990s (Javis, EE et al., Curr Genet ., 317: 321. 1991; Dowson, HN et al., Curr Microbiol ., 356: 362 1997; Hawkins, RL et al., Curr Microbiol ., 335: 341, 1999), and recently, studies have been reported to stably express halibut growth hormone in chlorella (Kim, DH et al., Mar. Biotechnol ., 63:73, 2002).

The number of genes that can be developed is enormous, and in the future, the separation and structural analysis of these genes, as well as the expression of the inserted genes in which organisms at what time or in which tissues, is an important task. However, expression of foreign useful genes cannot be satisfied simply by isolating the gene of interest and selecting a suitable transgenic plant. In order to produce large amounts of high value added proteins at low cost using transgenic plants, one of the first conditions to be considered is the development of promoters with high activity in plants (Norre, F. et al., Plant Mol Biol ., 699: 712, 2002). Indeed, the eukaryotic gene structure differs from prokaryotes such as Escherichia coli and regulatory elements such as promoters, enhancers and silencers are involved. The promoter contains genes that control the timing and amount of transcription, which directly regulates the efficiency of transcription.

Since few promoters have been plant-derived among the promoters developed so far, scientists have been very interested in the promoters of plant viruses and the root-knot bacterium Ti plasmid. One of the first and most important inventions in the field of plant protein expression is the use of plant virus-derived promoters that potently and constitutively express heterologous genes in genetically modified plants. 35S and 19S promoters from Cauliflower mosaic virus (CaMV) have been isolated and widely used (European Patent No. 0131623), and other noparin synthetic genes derived from Agrobacterium T-DNA. (nos), mannopine synthesis genes (mas) and octopin synthesis genes (cos) (European Patent No. 022791, European Patent No. 026546, European Patent No. 0145338), and plant-derived ubiquitin promoters are used (Europe) Patent 342926).

The 35S promoter of CaMV is one of the most widely used promoters for the continuous and efficient expression of genes introduced in plants, and has been mostly used for the production of foreign useful proteins described above. However, this promoter does not always induce strong expression in all host plants, so it is more useful in dicotyledonous plants, and it has been reported that 21bp of the promoter sequence induces intensive tissue expression in the root (Lam, E. et al., PANS, 7890: 7894, 1989).

Thus, attempts have been made to increase the expression levels of these promoters. Examples are the double-strengthened 35S promoter (US Pat. No. 5,164,316) and more recently the super promoter (European Patent 729514) combining portions of the Agrobacterium promoter.

Subsequently, scientists at Fukuoka et al. At the Chugoku National Agricultural Experiment Station in Japan separated NCR promoters from Soybean chlorotic mottle virus (SoyCMV) in Plant Cell Reports. It has been reported to induce potent expression of genes inserted into protoplasts of lobules (Fukuoka, H. et al., Plant Cell Reports, 815: 820, 2000).

In addition, scientists from Rance et al., France, combine the sequences of the Comelia yellow mosaic virus (CoYMV), the Cassava vein mosaic virus (CsVMV), and the active sequences of the CaMV 35S promoter. A new type of promoter has been developed, which has been reported to be used for the production of lactoferrin in the dicotyledonous tobacco and the monocotyledonous corn, with stronger activity than the 35S promoter (Norre, F. et al., Plant Mol Biol., 699) . : 712, 2002).

Recently, promoters that regulate gene expression in response to light, temperature, and stress have been isolated and reported. Typically, gene promoters of peroxidase have been reported in plants (Kwak, SS et al., Plant Mol. Biol. , 831: 838, 2003. Peroxidase is a type of antioxidant that removes free radicals that cause aging and disease, and is particularly strongly expressed by wounds, low temperatures, ultraviolet rays, and ozone. This promoter is used to produce high value-added therapeutic proteins and pharmacologically active substances such as anticancer drug Taxol, and is expected to be used to develop environmentally resistant plants that grow well under environmental stress conditions in the long term.

In many cases, however, these promoters do not meet the criteria of an ideal promoter. All of the promoters described above exhibit a clear form of tissue, or growth specific expression, but frequently the expression forms that appear in this promoter do not exhibit the same expression rates over a wide range, so in each case a specific promoter development is required.

Recently, as efforts are being made to produce useful proteins using transformed microalgae as well as plants, development of suitable promoters must be accompanied at the same time.

Therefore, the present inventors have made intensive efforts to develop a promoter capable of expressing a heterologous gene strongly in a transgenic microalgae, as a result of separating the promoter of the tRNA gene cluster of chlorella virus and producing a vector using the same, the foreign in chlorella It was confirmed that the gene is strongly expressed and the present invention was completed.

After all, the main object of the present invention is to provide a novel promoter derived from chlorella virus.

Another object of the present invention is to provide a method for producing a protein using the vector containing the promoter and the microalgae transformed with the vector.

In order to achieve the above object, the present invention comprises (a) at least one A box having a nucleotide sequence of SEQ ID NO: 1 and / or (b) at least one B box having a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 Provide a promoter.

In the present invention, the promoter may be characterized by having a repeated sequence of the type AA, ABA or ABABA, characterized in that it is derived from chlorella virus. In addition, the promoter may be characterized in that it further comprises a C box of SEQ ID NO: 4, it may be characterized by having a repeated sequence of the CABA type.

The present invention also provides a vector containing the promoter and a recombinant vector containing the gene encoding the promoter and the protein of interest, respectively.

The present invention also provides a transformed microorganism obtained by introducing the recombinant vector into any one host selected from the group consisting of bacteria, fungi and yeasts.

The present invention also provides a method for producing a target protein, characterized in that the microalgae transformed with the recombinant vector and the microalgae transformed.

The present invention also provides a method for producing a target protein, characterized in that the plant or tissue transformed with the recombinant vector and the transformed plant or tissue are cultured.

Chlorella virus encodes 200-300 proteins with linear DNA of more than 330 kb, whereas ordinary viruses synthesize proteins using tRNAs from host cells, while having unique tRNA gene clusters to synthesize their own tRNAs. do. Due to these characteristics, chlorella virus is used as a source of useful genes and transcriptional regulatory elements, and the promoter of the tRNA gene cluster isolated in this experiment has an expression cassette of chlorella virus, which may be advantageous for the expression of heterologous genes using chlorella. It was expected. In addition, since the tRNA gene is expressed as a pre-expressed gene in chlorella, it will be constantly expressed during the synthesis of the protein, so that strong expression can be maintained continuously.

In the present invention, chlorella viruses SS-1, SS-2, and KH-1 were isolated from freshwater in Korea, and promoters of tRNA clusters were isolated from genomic DNA of these chlorella viruses. The promoters had similar repeats A box and B box and consisted of 33 and 84 nucleic acid bases, respectively. The configurations of these A and B boxes consisted of AA for SS-1, CABA for KH-1, and ABABA for SS-2, respectively.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

In particular, the following examples illustrate only GFP as the protein of interest, but instead of GFP, enzymes, inhibitors, hormones, hormone analogs, antibodies, signaling proteins, single chain antibodies, antigens, peptides, polypeptides, binding proteins, binding domains It will be apparent to those skilled in the art to express the target protein using the promoter of the present invention, including adhesion proteins, structural proteins, regulatory proteins, toxin proteins, cytokines, various regulatory factors, and portions thereof.

In addition, in the following examples, the vector of the present invention was transformed into chlorella to confirm the expression of the recombinant protein. However, it will be apparent to those skilled in the art that the recombinant protein can be produced by transforming other plants such as tobacco, Arabesque, tomato, etc. Will do.

Example 1 Isolation of Chlorella Virus

The chlorella virus used in the present invention was isolated by applying fresh water collected in nine cities in Korea to chlorella, which is a host of chlorella virus, and then lysing the infected chlorella cells. First, the collected fresh water was filtered at a pressure of about 20 kPa using a 0.22 μm Al ₂ O ₃ filter (Whatman, USA). In this example, Chlorella NC64A was cultured in MBBM medium (modified Bold's basal medium, Van Etten, JL et al ., Virology , 126: 117, 1983).

5 ml of the filtered freshwater sample was added to 100 ml of 100 ml of Chlorella NC64A culture, and shaken for 5 days at 25 ° C. under light conditions. After chlorella culture was completely dissolved, the supernatant was diluted 10 ^-6 to 10 ^-7 times, and 5 µl was taken. 4 ml of Chlorella (4.0x10 ⁸ cells / ml) and 2 ml of 0.75% (in MBBM medium) agar After mixing, the plate was placed on a culture plate containing MBBM medium to which 1.5% agar was added. A single plaque formed on the culture dish was taken and inoculated into 100 ml fresh chlorella culture. The chlorella lysate was inoculated into a new culture dish and a single plaque was repeated three times to separate pure chlorella virus, thereby obtaining chlorella viruses SS-1, SS-2 and KH-1.

Example 2: Purification of Chlorella Virus

After culturing until the Chlorella NC64A containing the virus isolated in Example 1 was completely dissolved, the lysate was centrifuged at 5000 rpm for 5 minutes. The supernatant was transferred to a new tube, Triton X-100 was added to a final concentration of 0.1%, and mixed at 4 ° C. for 20 minutes. The mixed solution was centrifuged at 20,000 rpm for 60 minutes to precipitate virus particles. The pellet obtained in the above process was suspended in 50 mM Tris-buffer (pH 7.8) and centrifuged for 20 minutes at 20,000 rpm at 4 ° C. in a 10-40% discontinuous sucrose gradient solution. A 30-40% fraction containing virus was obtained, precipitated by centrifugation at 27,000 rpm for 3 hours, suspended in 50 mM Tris-HCl buffer, and then used for SDS-PAGE and genomic DNA separation.

The isolated chlorella virus was electrophoresed using 15% polyacrylamide gel (FIG. 2). As shown in FIG. 2, the chlorella virus of the present invention had several constituent proteins ranging in size from 10 kDa to more than 200 kDa.

Example 3: Isolation of Chlorella Virus Genomic DNA

60 μl of 10 × TEN buffer [100 mM Tris-HCl (pH 7.4), 10 mM EDTA, 1 M NaCl], 60 μl of 1% Na-salcosyl, 0.6 μl dml 60% in 400 μl of purified Chlorella virus w / w) CsCl and a small amount of EtBr were added and reacted at 75 ° C. for 15 minutes, and then the reaction solution was added over a 40 to 60% (w / w) CsCl gradient solution, followed by 18 hours at 25 ° C. and 35,000 rpm. Centrifugation. After obtaining portions containing viral genomic DNA labeled with EtBr, EtBr was extracted and removed using butanol. Viral genomic DNA was precipitated with ethanol and suspended in 1 × TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA]. The genomic DNA of the isolated chlorella virus was treated with restriction enzymes Bam HI and Hin dIII, followed by electrophoresis on 0.7% agarose gel to confirm the cleavage pattern (FIG. 3).

Example 4 Isolation and Analysis of tRNA Gene Cluster Promoters of Chlorella Virus

In order to isolate the tRNA gene cluster promoters of the chlorella viruses SS-1, SS-2 and KH-1 isolated in the present invention, the second tRNA in the tRNA gene cluster is complementary to the known sequence of isoleucine, based on the isoleucine. A primer (5'-TTGGCTCATAAGACCAATGC-3 ': SEQ ID NO: 5) was prepared. In order to confirm the nucleotide sequence of the promoter region in front of the tRNA cluster, direct sequencing of the viral genome digested with restriction enzymes was performed. The purely isolated virus genomic DNA (3-5 ㎍) was digested with Bam HI for at least 15 hours, extracted with phenol / chloroform mixture, precipitated with ethanol and suspended in distilled water. The sequence analysis mixture (1 ~ 1.5㎍ DNA, 1pmol primer, 4μl terminator ready reaction solution, distilled water added to the final 20μl) was added to the PCR instrument and subjected to 25 PCR reactions. Initial denaturation, annealing for 5 seconds at 50 ° C, and extension for 4 minutes at 60 ° C were performed. The PCR result was precipitated with ethanol, dried, dissolved in 25 μl of template suppressing reagent (Perkin Elmer, USA), denaturated at 95 ° C for 2 minutes, and analyzed by sequencing using ABI PRISM 310 analyzer (Perkin Elmer). The nucleotide sequence of the 5 'upper non-transcribed region of the tRNA gene cluster for each virus was obtained. Using the base sequence, primers of SEQ ID NOs: 6 and 7 specific to the promoter region of the tRNA gene cluster were prepared.

SEQ ID NO: 6 (Forward): 5'-CGTAAGCTTGAAAGAATGTAT-3 '

SEQ ID NO: 7 (reverse direction): 5'-GCATACGGATCCGCGCGCGTGTAT-3 '

Using the primers, the promoter regions of the tRNA gene clusters were isolated from the genomic DNA of the virus by PCR, and the sizes of the SS-1, SS-2 and KH-1 viruses were 372bp, 490bp and 373bp, respectively.

As a result of analyzing the nucleotide sequence of each promoter, SS-1, SS-2 and KH-1 chlorella virus had two similarly repeated sequence motifs, as shown in Figure 2, 33 and 84 to 100, respectively It consisted of two nucleotides. The sequence with 33 nucleotides was named A box (SEQ ID NO: 1), and the sequence with about 84 nucleotides was named B box (SEQ ID NOs: 2 and 3) and C box (SEQ ID NO: 4). As shown in FIG. 3, each repeating sequence box was composed of AA for SS-1, CABA for KH-1, and ABABA for SS-2.

SEQ ID NO: 1 ATTAAYTTAAGAAAYATWCTAACACCADAAWCA

Wherein Y is C or T, W is A or T, and D is A or G.

Example 6 Construction of Chlorella Transformation Vectors

In order to confirm whether the promoter derived from chlorella virus obtained in Example 5 can be used in the transformation vector, a transformation vector having the promoter sites of the SS-1, SS-2 and KH-1 was prepared. The transformation vector of this Example was prepared by modifying the pCTV vector (Korean Patent Publication No. 10-2001-0073152) shown in FIG. The pCTV vector is a modified plant transformation vector, has an RBCS2 promoter derived from Chlamydomonas, has a flamycin resistance gene (sh ble) as a selection marker, and the halibut growth hormone gene (fGH) is inserted. First, the pCTV vector was treated with the restriction enzyme Bam HI / XhoI to remove the fGH gene, and the GFP (green fluorescent protein) gene was inserted at the removal position to prepare pMinGFPble. The vector will express the GFP gene under the control of the CaMV35S promoter.

The pMinGFPble was digested with restriction enzyme Hin dIII / Bam HI to remove the CaMV35S promoter site, and the SS-1, SS-2 and KH-1 promoters were inserted at the removal position to prepare pSGFPble, pTGFPble and pKGFPble (FIG. 5). ).

Example 7 Preparation of Recombinant Protein Using Chlorella Transformation Vector

The vector having the tRNA gene cluster promoter of the chlorella virus prepared in Example 6 was transformed into chlorella ellipsoidea (Korea Maritime Microalgae Bank KMCC C-20). The protoplasts of Chlorella ellipsoide (1 × 10 ⁸ ) were centrifuged at 400 × g for 5 minutes to resuspend the precipitate with f / 2 culture medium (medium composition or manufacturer) containing 0.6M sorbitol / mannitol. The tube in which the chlorella was suspended was again centrifuged at 400 × g for 5 minutes, and the precipitate was resuspended in 1 ml of a 0.6M sorbitol / mannitol solution containing 0.05M CaCl ₂ . The 1 × 10 ⁸ cell number of protoplasts was then transferred to four new tubes of 0.4 ml each, each containing 4 μg of pMinGFPble, pTGFPble, pKGFPble, and pSGFPble vectors and a calf thymus cell DNA (Sigma Chemicals) 25 as a carrier. Μg was added. After the tube was reacted at room temperature for 15 minutes, 200 μl of PNC [0.8M NaCl, 0.05M CaCl 2, 40% PEG 4000 (Sigma)] was added, and the mixture was mixed at room temperature for 30 minutes.

Then, 0.6 ml of f / 2 culture medium containing 0.6M sorbitol / mannitol, 1% yeast extract and 1% glucose was added, and cultured for 12 hours under dark conditions at 25 ° C. to re-form the cell wall of chlorella. . Cells were transferred to fresh f / 2 medium containing the selection marker pleomycin (1 μg / ml) and incubated under 25 ° C. dark conditions. From day 5 of culture, the growth of chlorella was observed, and by day 15, cell growth reached stationary phase. In contrast, no growth was observed in the untransformed protoplasts.

The fluorescence of GFP produced in the chlorella ellipsoide transformed with the cultured pMinGFPble, pTGFPble, pKGFPble and pSGFPble was measured to confirm the expression ability of the GFP gene by the tRNA gene cluster promoter of the present invention.

As shown in Figure 6, Chlorella transformed with pTGFPble, pKGFPble and pSGFPble having a tRNA gene cluster promoter derived from the Chlorella virus of the present invention can produce more GFP than chlorella transformed with pMinGFPble having a CaMV35S promoter there was. In addition, as the number of repeating sequences A box and B box increased, the expression was increased. PSGFPble having three A boxes and two B boxes simultaneously showed the highest expression rate.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

 The present invention has the effect of providing a method for producing a target protein using a novel promoter derived from chlorella virus strongly expressing the target gene, a recombinant vector containing the promoter and a microalgae transformed with the recombinant vector.

By using the promoter according to the present invention, it is possible to produce high concentrations of recombinant protein in chlorella and plants, which can be used for molecular farming to produce large amounts of high value proteins at low cost, and to add or improve specific functions to plants. It can also be used in the field of genetic engineering.

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Claims (12)

An oligonucleotide, B box, or C box, wherein B is a nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3, including two or more A boxes having a nucleotide sequence of SEQ ID NO: 1; A box having a nucleotide sequence of SEQ ID NO: 4). The promoter of claim 1 having a repeated sequence of type AA, ABA or ABABA. delete The promoter of claim 1, wherein the promoter has a repeated sequence of type CABA. The promoter according to claim 1, wherein the promoter is derived from chlorella virus. A vector containing the promoter of any one of claims 1 to 5. A recombinant vector containing the promoter of any one of claims 1 to 5 and a gene encoding a protein of interest. A transformed microorganism obtained by introducing the recombinant vector of claim 7 into any one host selected from the group consisting of bacteria, fungi and yeasts. Microalgae transformed with the recombinant vector of claim 7. A method for preparing a protein of claim 9, wherein the microalgae of claim 9 are cultured. Plant or tissue thereof transformed with the recombinant vector of claim 7. A method for producing a protein of interest, comprising culturing the transformed plant of claim 11 or a tissue thereof.

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