KR20070055647A - Promoters originated from chlorella virus hs-1 and use thereof - Google Patents

Abstract

The present invention relates to a promoter derived from chlorella virus strain HS-1 isolated from Korea and its use, and more particularly, to encode a protein necessary for the replication of the virus and to express it before the virus is replicated by the host transcriptional organ. The present invention relates to a promoter isolated from an initially expressed gene, a vector containing the same, and a method for producing a target protein using the microalgae transformed with the vector.

The early expression gene 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, early expression gene promoter, transgenic plant, transgenic microalgae

Description

Promoters Originated from Chlorella Virus HS-1 and Use Thereof}

1 is a DNA polymerase gene promoter (A) and DNA ligase by PCR amplification of genomic DNA of eight chlorella viruses isolated from freshwater in Korea in order to amplify the early expression gene promoter from chlorella virus. Gene promoter (B), chitinase gene promoter (C) is a picture of the electrophoresis, lane 7 represents Chlorella virus HS-1.

Figure 2 is a schematic diagram analyzing the nucleic acid base sequence and each base sequence of the DNA polymerase gene promoter isolated from the early expression gene of Chlorella virus HS-1.

Figure 3 is a schematic diagram of the analysis of the nucleic acid base sequence and each base sequence of the DNA ligase gene promoter isolated from the early expression gene of Chlorella virus HS-1.

Figure 4 is a schematic diagram of the analysis of the nucleic acid base sequence and each base sequence of the chitinase gene promoter isolated from the early expression gene of Chlorella virus HS-1.

5 is a schematic diagram of pCTV, a chlorella transformation vector.

6 is a schematic diagram of recombinant vectors pMinGFP, pPGFP, pLGFP and pCGFP.

7 is a photograph of fluorescence microscopy of expression of green fluorescent protein (GFP) in wild type chlorella and transformed chlorella.

FIG. 8 is a schematic diagram comparing the expression intensity of green fluorescent protein (GFP) in a transformed chlorella using a fluorescence spectrometer and expressing the values in relative values.

The present invention relates to a promoter from the Chlorella virus HS-1 strain and its use. More specifically, the DNA polymerase gene promoter, DNA ligase, which encodes a protein necessary for the replication of the virus and is isolated from the initial expression gene expressed before the virus is replicated by the host's transcriptional organs. The present invention relates to a gene promoter, a chitinase gene promoter, a vector containing the same, and a method for preparing a 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), using a transgenic plant to produce foreign proteins can produce industrially expensive proteins that are more available than other protein expression systems. (See Zhong, R. et al., Plant Physiol., 53-64, 1999).

Among them, chlorella, a single-cell eukaryotic green alga, has recently been used as a bioreactor because chlorella has a system suitable for the expression of heterologous proteins. With a certain amount of light and a carbon source, it can be inexpensively mass cultivated and grow fast because it splits 2-9 times a day (see 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). Recently, studies have been reported on the stable expression of 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 quantities of high value added proteins at low cost using transgenic plants, one of several conditions that should be considered first is the development of high activity promoters in plants (Norre, F. et al. , Plant Mol Biol., 699-712, 2002).

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 genes derived from agrobacterium T-DNA are noparin synthetic genes. (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 double mosaic virus (CaMV) is one of the most widely used promoters for the continuous and efficient expression of genes introduced in plants and has been used mostly 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 is reported that 21bp of promoter sequence induces intensive tissue expression in the roots (see Lam, E. et al., PANS , 7890-7894, 1989). Thus, attempts have been made to increase the expression levels of these promoters, examples of which include a double promoter 35S promoter (US Pat. No. 5,164,316) and a part of the Agrobacterium promoter (European Patent No. 729514). There is.

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.

However, all of the promoters described above do not meet the criteria of ideal promoters because they exhibit clear tissue or growth-specific expression, but do not exhibit the same expression rates over a wide range, and in each case require specific promoter development. . Therefore, at the time when efforts are being made to produce useful proteins using not only plants but also transformed microalgae, the development of suitable promoters for this should be accompanied.

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 initial expression gene of Chlorella virus strain HS-1 isolated in Korea, and using the vector It was confirmed that the strong expression of foreign genes in Chlorella to complete the present invention.

After all, the main object of the present invention is to provide a novel promoter from chlorella virus strain HS-1.

It is another object of the present invention to provide a method comprising preparing a target protein using a vector comprising the initial expression gene promoter and the microalgae transformed with the initial expression gene vector, and the recombinant vector comprises a group consisting of bacteria, fungi and yeast. It is to provide a method for producing a target protein, characterized in that the transformed microorganisms and plants obtained by introducing into any one of the host selected from the culturing and cultivating them.

The present inventors provide promoters of early expression genes, particularly DNA polymerase, ATP-dependent DNAligase and chitinase genes, which are known to be essential for the replication of DNA from the Chlorella virus strain HS-1, and through analysis of these promoters, TATA boxes, CAAT boxes We have identified cis-acting elements that appear to affect transcription, including the NTBBF1 box and the CCAAT box.

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

Chlorella virus encodes 200-300 proteins with more than 330kb of linear DNA. Genes encoded by PBCV -1, a representative virus belonging to the Phycodnaviridae , can be divided into early and late genes according to their expression start time. Initially expressed genes encode proteins necessary for the replication of the virus and are expressed before the virus is replicated by the host's transcriptional organs, which is why the promoters of these genes can be used to construct chlorella's transformation vectors. To provide. PBCV-1 does not encode its own DNA dependent RNA polymerase and is known to utilize enzymes from the host. Therefore, it is expected that the promoters of DNA polymerase, ATP-dependent DNA ligase and chitinase genes, which are known as essential genes for DNA replication among early expression genes of the virus, will enable the expression of heterologous genes using chlorella.

In the present invention, eight chlorella viruses were isolated from freshwater in Korea, and the initial expression gene promoter was isolated based on the genomic DNA of chlorella virus PBCV-1 from the genomic DNA of these chlorella viruses. Among them, all of the initial expression gene promoters were isolated, and the initial expression gene promoter of HS-1 having the highest homology with PBCV-1 was selected. The analysis of the initial expression gene promoter revealed that there were two TATA boxes in the DNA polymerase gene promoter, three in the DNA ligase gene promoter and four in the chitinase gene promoter, as well as the CAAT box, NTBBF1 box, GATA box and CCAAT box. It was confirmed that there is a site to which many transcription elements can bind, including.

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 in detail, it will be apparent to those of ordinary skill in the art and the art that the scope of the present invention is not limited by these examples. .

In particular, the following examples illustrate only green fluorescence protein (GFP) as the protein of interest, but instead of GFP, enzymes, inhibitors, hormones, hormone analogs, antibodies, signaling proteins, single chain antibodies, antigens, peptides It is known to those skilled in the art to express a target protein using a promoter of the present invention, including a polypeptide, a binding protein, a binding domain, an adhesion protein, a structural protein, a regulatory protein, a toxin protein, a cytokine, various regulatory factors, and a part thereof. It will be self explanatory.

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, the 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 an Al ₂ O ₃ filter (Whatman, USA). In this embodiment, Chlorella (Chlorella) medium to NC64A MBBM (modified Bold's basal medium ) ( See: Van Etten, JL et al ., Virology , 126: 117, 1983).

5 ml of the filtered freshwater sample was added to 100 ml of 100 ml Chlorella NC64A culture solution, followed by shaking culture at 25 ° C. for 5 days under light conditions. After the chlorella culture was completely dissolved, the supernatant was diluted 10 ^-6 to 10 ^-7 times, and then 5 µl was taken. 4 ml of chlorella (4.0x10 ⁸ cells / ml), 2 ml of 0.75% (in MBBM medium) After mixing with agar (agar) was placed on a culture dish containing MBBM medium added 1.5% agar. 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 isolate pure chlorella virus, thereby obtaining eight chlorella viruses.

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 precipitate obtained in the above procedure was suspended in a 50 mM Tris-buffer (pH 7.8), and centrifuged at 4 ° C. and 20,000 rpm for 20 minutes in a 10-40% discontinuous sucrose gradient solution. Fractions containing 30-40% of the virus were obtained, precipitated by centrifugation at 27,000 rpm for 3 hours, and then suspended in 50 mM Tris-HCl buffer for use in genomic DNA separation.

Example 3: Isolation of Chlorella Virus Genomic DNA

60 μl of 10 × TEN buffer [100 mM Tris-HCl (pH7.4), 10 mM EDTA, 1 M NaCl], 60 μl of 1% Na-salcosyl, 0.6 μl in 400 μl of purified Chlorella virus 60% (w / w) of 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 40-60% (w / w) CsCl gradient solution, followed by 25 ° C., 35,000. Centrifugation for 18 hours at rpm. 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].

Example 4 Isolation and Analysis of Initial Expression Gene Promoters of Chlorella Virus

In order to isolate the early expression gene promoter of the chlorella virus HS-1 isolated in the present invention, the DNA sequence including about 420 bp upstream of the DNA polymerase, DNA ligase and chitinase genes can be amplified. Polymerase chain reaction (PCR) was carried out with the primers prepared as a basis. DNA fragments were PCR amplified mixture (50-100 ng DNA, 10 × buffer (100 mM Tris-Cl (pH8.3), 500 mM KCl, 15 mM MgCl ₂ ), 2.5 mM dNTP, 4 μl Taq polymerase (5 u / 35 μl) and 100 pmol forward primer and reverse primer) were placed in a PCR device and subjected to 35 PCR reactions.The PCR reactions were predenaturation at 94 ° C. for 10 minutes, denaturation at 94 ° C. for 1 minute, and annealing at 50 ° C. for 1 minute. Extension was carried out for 1 minute at ℃ and postextension for 7 minutes at 72 ℃. PCR results were confirmed by electrophoresis on 1.2% agarose gel. PCR results were confirmed to the expected size in each promoter portion, but not in all eight chlorella viruses used to perform PCR (see Figure 1). Of the eight chlorella viruses, HS-1 was amplified by all three early expression gene promoters and showed the highest homology with the nucleotide sequence of PBCV-1 on the nucleotide sequence. . Initially expressed gene promoters isolated from HS-1 were analyzed using a database, plant cis-acting regulatory DNA element (PLACE). In the DNA polymerase gene promoter, two TATA boxes, three CAAT boxes and five GATA boxes were identified (see FIG. 2). In the DNA ligase gene promoter, one CCAAT box, three TATA boxes, five CAAT boxes, and four GATA boxes were identified (see FIG. 3). In the Chitinase gene promoter, one CCAAT box, an NTBBF1 box, four TATA boxes, five CAAT boxes, and four GATA boxes were identified (see FIG. 4). No homology was found between the three early expressed gene promoters isolated from HS-1.

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 region of the initial expression gene of HS-1 was prepared. The transformation vector of this Example was prepared by modifying the pCTV vector (see: Kim, D.-H. et al., Mar. Biotechnol. 4 (1): 63-73) shown in FIG. The pCTV vector is a modified plant transformation vector, which has a RBCS2 promoter derived from Chlamydomonas, and has a pleomycin resistance gene (sh ble) inserted into the flounder growth hormone gene (fGH) and a selection marker. First, the pCTV vector was treated with the restriction enzyme Bam HI / Xho I to remove the fGH gene, and the GFP gene was inserted into the removal position to prepare pMinGFPble. The vector expresses the GFP gene under the control of the CaMV35S promoter, which is cleaved with the restriction enzyme Hin dIII / Bam HI to remove the CaMV35S promoter site, and inserts the initial expression gene of HS-1 into the removal site. pPGFPble, pLGFPble and pCGFPble were constructed (see Figure 6).

Example 7 Preparation of Recombinant Protein Using Chlorella Transformation Vector

A vector having a gene promoter for initial expression of chlorella virus prepared in Example 6 was transformed into chlorella vulgaris (Korea Maritime Microalgae Bank KMCC FC-001). Protoplasts of Chlorella vulgaris (1 × 10 ⁸ ) were centrifuged at 400 × g for 5 minutes to resuspend the precipitate in MBBM culture (medium composition or manufacturer) containing 0.6 M sorbitol / mannitol. The tube in which the chlorella was suspended was again centrifuged at 400 xg for 5 minutes and the precipitate was resuspended in 1 ml of 0.6 M sorbitol / mannitol solution containing 0.05 M CaCl ₂ . Then, 1 × 10 ⁸ cell number of protoplasts were transferred to four new tubes, each 0.4 ml, and 4 µg of chlorella transformation vector and 25 µg of calf thymic cell DNA (Sigma Chemicals) were added as carriers. . After the tube was reacted at room temperature for 15 minutes, 200 μl of PNC (0.8 M NaCl, 0.05 M CaCl ₂ , 40% PEG 4000 (Sigma)) was added, and mixed at room temperature for 30 minutes.

Thereafter, 0.6 ml of MBBM culture solution containing 0.6 M 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 MBBM medium containing pleomycin (1 μg / ml), the selection marker, 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 vulgaris transformed with the cultured pMinGFPble, pPGFPble, pLGFPble and pCGFPble was measured to confirm the expression ability of the GFP gene by the early expression gene promoter of the present invention (see Fig. 7). It was confirmed that chlorella transformed with pPGFPble, pLGFPble and pCGFPble having an early expression gene promoter derived from chlorella virus produced GFP like chlorella transformed with pMinGFPble having a CaMV35S promoter and expression of GFP using a fluorescence spectrometer. When the intensity was measured and compared with the relative values, pCGFPble showed the highest expression rate (see FIG. 8).

As described and demonstrated in detail above, the present invention provides a novel initial expression gene promoter derived from Chlorella virus that strongly expresses a gene of interest, a recombinant vector comprising the promoter, and a target protein using a microalgae transformed with the recombinant vector. It provides a method for producing. In addition, by using the promoter of the present invention, it is possible to produce high concentrations of recombinant protein in chlorella and plants, so that the genetics to add or improve specific functions to the molecular agriculture and plants that produce a large amount of high value-added protein at low cost It can also be used in engineering.

Claims (7)

 Promoter selected from DNA polymerase gene, DNA ligase gene, chitinase gene group isolated from early expression gene of Chlorella virus HS-1. A recombinant vector comprising the promoter of claim 1. The transformed microorganism obtained by introducing the recombinant vector of claim 2 into any one host selected from the group consisting of bacteria, fungi and yeasts. Microalgae transformed with the recombinant vector of claim 2. Method for producing a target protein, characterized in that the culture of the transformed microalgae of claim 4. Plant transformed with the recombinant vector of claim 2. Method of producing a target protein, characterized in that the culture of the transformed plant of claim 6.

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