KR20000000576A - Gene expression vector containing regulatory region of misgurnus mizolepis beta-actin gene - Google Patents

Abstract

PURPOSE: A fish-specific gene expression vector containing regulatory region of Mistunes mizolepis beta-actin gene is provided which can express usable exogenotes effectively in host cells variously originated from fish or mammal therefore, can be useful in developing novel fish. CONSTITUTION: A recombinant plasmid expression vector (pmlbetaa41:KCTC 88889P) is produced by: preparing genomic DNA library of Mistunes mizolepis; isolating a beta-actin encoding gene and sequencing it; cloning of beta-actin encoding gene and its regulatory region, followed by binding the regulatory region fragment to the green fluorescence protein (GFP) encoding gene.

Description

Gene expression vector comprising loach beta-actin gene regulatory region region

The present invention relates to a gene expression vector comprising a loach beta-actin gene regulatory region region, and more particularly, to analyze the nucleotide sequence of the beta-actin gene and gene expression control region of Korean loach, and from the loach beta produced -Fish specific expression vector comprising an actin gene regulatory site region for efficient expression of the desired gene of interest in the fish.

In the taxonomy, Misgurnus mizolepis, belonging to the carp and the family, is inhabited in Northeast Asia including China, Taiwan, Japan, and Russia. It is a representative freshwater fish species of our country which is widely used (Yongbobogam, Medicinal Combination). Although only a few years ago, loach was seen anywhere in the country, such as rice fields and rivers, and was also responsible for obtaining foreign currency by exporting it to Japan.However, the amount of natural harvest is decreasing every year due to the pollution of rivers, agricultural waterways, and the abuse of pesticides. to be. Considering that these phenomena are not limited to loach, there is a need for the development of new aquaculture techniques and the improvement of economic and useful varieties for the conservation and efficient use of domestic fish resources. As part of this, various studies should be conducted to secure various useful genetic resources of fish, and to manufacture transgenic fish that manipulates these genes and directly implants them using biotechnology techniques.

Research into the manufacture of transgenic fish began in the late 1970s, using genes from mammals and their regulatory sites. For the expression of genes artificially introduced in these transgenic fish, (1) when no expression of the gene occurs, (2) the expression of the gene is detected but does not have a physiological effect, and (3) Three results of physiological changes were expressed. If the expression of the gene does not occur, it is interpreted that the gene regulatory region of the mammal or mammalian virus is not recognized in fish. In addition, the physiological effect of certain heterogeneous genes expressed in fish is not because the substrate-specific protein-protein interaction does not occur smoothly in the cell. In other words, the expression product of the gene to be transplanted must have structural similarity with that of the fish to be effective. These results suggest that homologous fishes are optimal as genes to be used for transgenic fishes and their regulatory sites, and that successive species are higher in the species of closest species with the closest possible flexibility.

In the early stages of research on fish transformation, most mammalian-derived vectors were used. It has been reported that several promoters of higher vertebrates and viruses induce foreign gene transcription with high efficiency in fish cells (Foster, R., PE). Olson and L. Gedamu, 1989. Mol. Cell. Biol. 9: 4105-4108; Gedamu, L., PE Olson and M. Zaffarullah, 1990. Regulation of rainbow trout metallothioneine genes.In: Transgenic models in Medicine and Agriculture. Church, R. (ed.) New York: Willy-Liss, pp. 101-108; Inoue, K., S. Yamashita, J. Hata, S. Kabeno, S. Asada, E. Nagahisa and T. Fujita, Cell Differ. Dev., 29: 123-128; Liu, Z., B. Moav, AJ Faras, KS Guise, AR Kapuscinski and PB Hackett, 1990. Bio / Technol 8: 1268-1272; Friedenreich, H and M. Schartl, 1990. Nucl.Acids Res., 18: 3299-3305; Bearzotti, M., E. Perrot, C. Michard-Vanhee, G. Jolivet, J. Attal, MC Theron, C. Puissant, M. Drano, JJ Kopchick, R. Po well, F. Gannon, L. M. Houdebine and D. Chourrot, 1992. J. Biotechnol. 26: 315-325).

Zhu et al. (Zhu, J., K. Xu, G. Li, Y. Xie, and L. He, 1986. Tongbao 31: 988-990) are the human promoters for the metallothioneine gene in mice. By combining the growth hormone genes and transplanting them into loach, goldfish and koi carp, they succeeded in making transgenic fish three times larger than the control group. However, the application of vertebrate and viral promoters to fish may limit the transcriptional factors necessary for their expression in fish cells, and commercially, consumers may feel resentful of such produced fish. (Du, SJ, Z. Gong, CL Hew, CH Tan and GL Fletcher, 1992. Mol. Mar. Biol. Biotechnol. 1 (4/5): 290-300).

In view of this, it is urgently necessary to secure a specific regulatory site and structural gene derived from the fish itself, and to develop an expression vector using the same in order to manufacture a transgenic fish that expresses a foreign gene more stably. It is becoming. Accordingly, the protamine gene of salmon (Jankowski, JM and GH Dixon, 1987. Biosci. Rep. 7: 955-963), the metallothioneine B (MT) gene of rainbow trout (Zafarullah, M , K. Bonham and L. Gedamu, 1988. Mol. Cell. Biol., 8: 4469-4476), the beta-actin gene of carp (Liu, Z., B. Moav, AJ Faras, K. Guise, AR Kapuscinski and PB Hackett, 1990. Mol. Cell. Biol., 10: 3432-3440), and the antifreeze protein (AFP) gene (Gong, Z., CL Hew and JR Biekind, 1991. Mol. Regulatory sites such as Mar. Biol. Biotechnol. 1: 64-72). In particular, the MT gene regulatory site shows strong expression in various tissues by treatment of toxic substances, heavy metals, glucocorticoids, and the like. However, derivatives treated for induction of expression may be toxic or cause tumors during metabolism in the body, and heavy metal derivatives may accumulate in the body of fish and be delivered to the end consumer. (Liu, Z., B. Moav, AJ Faras, KS Guise, AR Kapuscinski and PB Hackett, 1990. Bio / Technol. 8: 1268-1272).

Actin genes are genes of cytoskeletal proteins present in eukaryotes and are often used as regulatory site regions for stable and continuous expression of introduced foreign genes. In vertebrates, there are at least six isoforms with slight differences in amino acid sequences, which vary in expression during development. Unlike alpha-actin, which has muscle-specific expression, the promoter of the beta-actin gene is also expressed in all non-muscular cells, including muscle, and therefore, when a foreign gene is coupled to the promoter and introduced into an individual, expression of a desired foreign gene can be efficiently expressed. (Quitschke, WW, ZY Lin, LDP-Zilli and BM Paterson, 1989. J. Biol. Chem. 264 (16): 9539-9546).

That is, when the beta-actin gene regulatory site is used for the production of expression vectors that can be expressed in fish, (1) the promoter is potent to induce expression in all tissues, and (2) the complexity of the promoter structure It is possible to induce and regulate expression at the stage, and (3) the beta-actin gene regulatory site has a high homology between species, and thus has the advantage that it can fully function in all fish species (Liu, Z., B. Moav, AJ Faras, K. Guise, AR Kapuscinski and PB Hackett, 1990. Mol. Cell. Biol., 10: 3432-3440).

In view of the above, the inventors of the present invention, in order to prepare a fish-specific gene expression vector capable of delivering useful genes in vivo and inducing strong expression in fish, genes encoding beta-actin in loach and its regulation After cloning the site, a recombinant plasmid expression vector was prepared by combining the beta-actin gene regulatory site fragment and the fragment encoding the green fluorescence protein (GFP) gene derived from jellyfish. Was found to have a high expression rate in various cultured cells, leading to the completion of the present invention.

That is, an object of the present invention is to provide a gene control site of fish origin suitable for expressing a useful gene in all fish species, and to prepare an expression vector comprising such a gene control site, thereby efficiently expressing a desired foreign gene in the fish. I want to induce.

1 shows a restriction map of bacteriophage DNA comprising loach beta-actin gene,

Figure 2 is a schematic diagram showing the cloning process of the plasmid pmlβa41 containing loach beta-actin gene regulatory region region,

Figure 3 is a schematic diagram showing the manufacturing process of the pmlβaGFP plasmid expression vector recombinant the locus beta-actin gene regulatory region and the gene encoding the fluorescent protein of jellyfish origin,

Figure 4 is a photograph comparing the expression control pattern of GFP by the plasmid pmlβa41 comprising the CMV promoter and loach beta-actin gene regulatory site region in the E25B2 cell line, Figure 4a is the expression result by the CMV promoter, Figure 4b is pmlβa41 Photo showing the results of expression by.

In order to achieve the above object, the present invention provides a DNA comprising a beta-actin gene and a beta-actin gene regulatory site of loach represented by the nucleotide sequence of SEQ ID NO: 1.

In addition, the present invention provides an expression vector comprising a beta-actin gene regulatory site of loach and a transformant comprising the expression vector.

In the present invention, a genome library containing all genes is prepared from Korean loach, and the beta-actin gene is cloned therefrom to reveal its nucleotide sequence and structural properties, and the homology of the other species of the known base sequence is analyzed. It was. In addition, by preparing an expression vector comprising a beta-actin gene regulatory site, the locus beta-actin gene is analyzed by recombining the green fluorescent protein gene of jellyfish origin and analyzing the expression pattern in culture cells and in vivo of various origins. Expression vectors comprising regulatory sites have proved to be functional expression vectors suitable for expressing useful genes in fish.

1. Preparation of Loach Genomic DNA Library

First, genomic DNA was extracted from the blood of Korean loach, partially digested with restriction enzyme Sau3A I, ultracentrifuged to separate 15-23 kb DNA fragments, and these genomic DNA fragments were ligated to the λGEM-11 vector. Packaged phages are mixed with E. coli phage extracts to infect E. coli host cells and cultured to produce loach genomic DNA libraries.

2. Searching and Sequencing Beta-Actin Genes

As a beta-actin gene probe, a 1.5 kb gamma-actin gene fragment cloned from a mouse embryonic cell line cDNA library is used. After labeling with [α ³² P] dCTP, only labeled probes are isolated purely through a spin column.

After culturing Escherichia coli host cell KW251 transformed with a loach genomic DNA library, the phage clone was repeated by transferring phage to a filter, hybridizing the DNA attached to the filter with the gamma-actin probe, and then recovering the positive plaques. Pure separation.

To isolate single positive phage DNA, E. coli host cell KW251 is infected with phage and incubated until lysis, followed by chloroform and centrifugation to obtain upper phage lysate. Phage lysates were reacted with RNase A and DNase I, precipitated with NaCl and PEG, protein shells were removed from the precipitated phage, and centrifuged to recover DNA, precipitated and dried, and then loach present in λGEM-11 phage vector. A restriction enzyme map of the beta-actin gene is created. That is, by cutting the DNA by single or proper combination of restriction enzymes such as Bam HI, EcoR I, Hind III, Pst I, Sac I, Xba I, Xho I, etc., and comparing the size of the fragments shown by electrophoresis, Map the locations of restriction enzymes.

To subcloce the loach beta-actin gene clone into the plasmid vector pBluscript II KS (-), the restriction enzymes Xho I and EcoR I were reacted with λ phage DNA and pBS vector and electrophoresed to insert the correct sized insert DNA and vector. After recovery it is ligated using T4 DNA ligase. This transformed the E. coli host cell XL-1 blue MRF 'strain, incubated in an agar plate containing ampicillin and X-gal, extracted the plasmid DNA from the colonies, digested some with restriction enzymes and contained the correct insert. It was confirmed as pmlβa34.

Restriction enzymes, such as BamH I, Kpn I, Sac I, Xba I, to a 3.4 kb clone (pmlβa34) containing the loach beta-actin gene subcloned into the pBS vector to map the position of each restriction enzyme Based on this, a sequence of deletion mutants is obtained according to the Erase-a-base method using Exonuclease III to analyze the base sequence.

3. Cloning of Beta-Actin Gene Regulatory Sites

Sty I at 3702 bp of pmlβa34 and Not I at 670 bp located in the pBS vector were cut and blunt ligated to obtain a pmlβa34S / N plasmid from which the beta-actin structural gene region was removed. This was then completely cleaved with Xho I followed by partial cleavage with BssH II to obtain a 444 bp regulatory site fragment.

In addition, for cloning the beta-actin gene regulatory region, 3.9 kb fragments of Xba I, Xho I located 5 ′ above the 3.4 kb clone were also cloned into the Xba I, Xho I positions of the pBS plasmid vector to obtain the pmlβa39 plasmid. The base sequence is analyzed. Next, in order to facilitate the recombination of the useful genes, the restriction enzyme Sac I cleavage site located 182 bp away from Xba I was cut with restriction enzyme Sac I and blunt ligation removed. This was completely cleaved with Xho I and then partially cleaved with BssH II to obtain a fragment of 6576 bp.

A fragment of Xho I, BssH II 444 bp of pmlβa34S / N was combined to prepare a plasmid pmlβa41 having a complete beta-actin regulatory site of 4146 bp of the translation start codon.

4. Expression of Fluorescent Proteins by Modulation of Beta-Actin Gene Expression

In order to confirm the expression control ability of the beta-actin gene regulatory region, a green fluorescence protein (GFP) gene derived from Aequorea victoria, which is very useful as a marker gene, is used. The green fluorescent protein expression vector phGFP-S65T was cleaved with Mlu I and Sac I to remove the cytomegalovirus (CMV) promoter portion, which is a 586 bp expression control site, and the plasmid pmlβa41 was cleaved with restriction enzymes BssH II and Sac I. The fragment of 4194 bp is ligated to prepare plasmid pmlβaGFP which is regulated by the green fluorescent protein expression by the beta-actin gene regulatory site.

In order to confirm the expression control ability of the beta-actin gene regulatory site, the plasmid pmlβaGFP vector is transduced into the cell line in culture to measure the expression level of GFP. PhGFP-S65T with cytomegalovirus (CMV) promoter was transduced and compared, and as cultured cell lines, salmon embryonic cell line (CHSE-214) and human erythropoietic cell line (Human erythropoietic cell line) were compared. K562), African Green Monkey kidney cell line CV1 drived E25B2, and Mouse erythroid leukemia cell line (MEL).

The cells into which the genes were introduced were collected 60-72 hours after introduction, and the expression rate was analyzed by counting the number of GFP-expressing cells under fluorescence microscopy, and some were counted by LacZ staining to express cells expressing the E. coli LacZ gene [GFP]. Obtain the value of / [LacZ] and quantify the expression of GFP.

As a result, expression vectors comprising loach beta-actin gene regulatory sites have been shown to be able to operate normally in cultured cell lines of various origins, including mammals and fish, and to efficiently express the genes of interest. It has been confirmed that it can be usefully used.

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

Example 1 Preparation of Loach Genomic DNA Library

Blood was collected from the tail vein of the loach and the genomic DNA was extracted according to the method of Blin and Stanford (Blin & Stafford, 1976, Nucleic Acid Res., 3: 2303). The extracted genomic DNA (200 μg) was partially digested with restriction enzyme Sau3A I (0.04 U / μg DNA, 30 min), followed by ultracentrifugation (22,000 rpm, 22 hours, 20 ° C.) in a 10-40% sucrose density gradient solution. After receiving 30 fractions, the size of each fragment contained in each fraction was confirmed by electrophoresis. Among them, 15-23 kb fragments of suitable size for phage vector were separated.

These 15-23 kb genomic DNA fragments were ligated to the BamH I cleaved λGEM-11 vector (Promega, USA) according to the manufacturer's experimental method (Genomic cloning manual. Promega, USA) and E. coli phage extract (Promega, USA) 50 μl and 5 μl of Regate were mixed and packaged by standing at room temperature for 3 hours, followed by addition of 445 μl of phage buffer solution (20 mM Tris-HCl; pH 7.4, 100 mM NaCl, 10 mM MgSO ₄ ) and 25 μl of chloroform. It was. Packaged phages were infected with E. coli host cell KW251 (F-, supE44, galK2, galT22, metB1, hsdR2, mcrB1, mcrA, [argA81: Tn10], recD1014, Promega, USA) and plated overnight. As a result of counting the plaques shown, a loach genomic DNA library with a titer of 1.5 × 10 ⁶ pfu / μg DNA was prepared and amplified to prepare an amplification library of 1.26 × 10 ⁹ pfu / μg DNA.

Example 2: Search and Sequence Analysis of Beta-Actin Genes

① Construction of beta-actin gene probe

As a beta-actin gene probe, a 1.5 kb gamma-actin gene fragment cloned from the mouse embryonic cell line cDNA library was used, and the preparation of the probe was carried out using a random primed labeling kit (BM, Germany). According to the method, after labeling with [α ³² P] dCTP for 1 hour at 37 ° C., fragments that were too small for use as probes and unsynthesized dNTPs were removed through a spin column, and only the labeled probe was purely separated. .

② Plaque hybridization

The loach genomic DNA library was mixed with 0.3 ml of E. coli host cell KW251 by adjusting the titer to form about 50,000 plaques on 10 150 mm plates, and incubated at 37 ° C. for 20 minutes to infect the phage. . E. coli host cell KW251, transformed according to Benton & Davis' method (Benton & Davis, 1977, Science, 196: 180-182) and mixed with 7 ml of 0.7% top agarose, 150 mm plate Incubated for 16 hours at 37 ℃. The nylon filter was then placed on a petri dish to transfer phage to the filter and after 1 minute the filter was removed. The filter was sequentially filtered for 2 minutes in denaturing solution (0.5 M NaOH, 1.5 M NaCl), 5 minutes in neutral solution (1 M Tris; pH 7.4, 1.5 M NaCl), 2X SSC (0.3 M NaCl, 30 mM sodium citrate) After soaking in for 1 minute, the DNA was UV-crosslinked and air dried to firmly adhere to the nylon filter.

Pre-hybrid hybrid at 65 ° C. for 1 hour using DNA firmly attached to the filter and the previously prepared gamma-actin probe using Southern method (Southern EM, 1975, J. Mol. Biol., 98: 503-517) And hybridize at 42 ° C. for 16 hours, then rinse off excess probes with primary wash solution (2X SSC, 0.1% SDS) and secondary wash solution (0.1X SSC, 0.1% SDS), and covered with an intensifying screen) and exposed to an X-ray film at −70 ° C. for 24 hours and then developed with an automatic film developer. Plaques on the plate that matched the positive signal shown on the film were recovered with a Pasteur pipette, placed in 1 ml of SM buffer solution, then 2 drops of chloroform were added and allowed to stand for 4 hours at room temperature to allow phages to come into solution. For the isolation of complete positive single plaques, the plaques obtained in the first screen were incubated with titer adjusted to form about 50 plaques, and then re-scanned twice in the same manner as above. Positive phage clones from dogs were purely isolated.

③ Beta-actin gene phage DNA extraction and restriction enzyme mapping

In order to isolate single positive phage DNA, 20 μl of phage was inoculated at 500 μl of KW251 cultured overnight for 20 minutes at 37 ° C., and cultured until dissolution in 100 ml of LB containing MgSO ₄ at a final concentration of 10 mM. Then, 500 μl of chloroform was added and centrifuged (8,000 g, 10 minutes) to obtain an upper phage lysate. Phage lysate was transferred to a 250 ml centrifuge tube and RNase A and DNase I were added at a final concentration of 1 μg / ml and reacted at 37 ° C. for 30 minutes, followed by 5.8 g of NaCl and 9.3 g of PEG 8000. And centrifuged (10,000 g, 20 min, 4 ° C.) to precipitate phage. The precipitated phage was dissolved in 10 ml of phage buffer (20 mM Tris-HCl; pH 7.4, 100 mM NaCl, 10 mM MgSO ₄ ), centrifuged, and then 100 µl of 10% SDS and 0.5 M EDTA (pH) in the supernatant. 8.0) 100 μl each was added and reacted at 68 ° C. for 15 minutes. Then, the same amount of phenol was added to remove the protein shell of the phage, followed by centrifugation for 5 minutes to recover the supernatant containing DNA. In order to remove the remaining phenol, the same amount of chloroform was treated, and only the supernatant was taken. Then, 0.1 times of 5 M NaCl and 1 times of isopropanol were added to precipitate the DNA, and the inorganic salts were removed with 70% ethanol and dried. The obtained DNA was dissolved in 1 ml of TE.

Single or proper combination of restriction enzymes such as Bam HI, EcoR I, Hind III, Pst I, Sac I, Xba I, Xho I to create restriction enzyme maps of loach beta-actin genes present in the λ Gem-11 phage vector After cutting each DNA, the size of each restriction enzyme was mapped by comparing the size of the fragments shown by electrophoresis.

1 shows a restriction map of bacteriophage DNA comprising loach beta-actin gene.

Southern blots were performed using the same probes used for phage search to identify fragments containing beta-actin genes in phage DNA.The beta-, 3.4 kb of Xho I and EcoR I, showed positive signals. Fragments containing the actin gene were identified.

④ subcloning of beta-actin gene into plasmid vector

Xho I and EcoR, restriction enzymes cleaved to include beta-actin genes identified by Southern blot, for subcloning loach beta-actin gene clones into the plasmid vector pBluscript II KS (-) (Stratagene, USA; pBS) Each 10 U of I was reacted with 2 μg λ phage DNA and pBS vector for 2 hours and electrophoresed to recover the correct size of the inserted DNA and the vector, followed by ligation using T4 DNA ligase.

Transformation was modified in one method (Hanahan O., 1983. J. Mol. Biol., 166: 557-580), and 2 μl of regate was treated with 0.5 M CaCl ₂ E. coli host cell XL-1 blue The mixture was mixed with 100 μl of MRF 'strain and reacted at 4 ° C. for 30 minutes, then heat-treated at 42 ° C. for 90 seconds, and 1 ml of LB culture was added thereto, followed by incubation at 37 ° C. for 45 minutes. The transformant was centrifuged (13,000 rpm, 20 seconds) and resuspended in 200 µl, then plated in agar plate containing ampicillin (50 µg / ml) and X-gal (50 µg / ml) and 37 ° C. Incubated overnight at, then randomly selected white colonies were incubated overnight at 37 ° C. in 2 ml LB broth. 1.5 ml of the plasmid DNA was extracted according to the alkali dissolution method, and a part thereof was cut with a restriction enzyme to confirm whether the correct insert was contained, and then named as pmlβa34.

⑤ restriction enzyme mapping and sequencing of beta-actin gene

Restriction enzymes, such as BamH I, Kpn I, Sac I, Xba I, to a 3.4 kb clone (pmlβa34) containing the loach beta-actin gene subcloned into the pBS vector to map the position of each restriction enzyme Based on the Erase-a-base method using Exonuclease III (Henikoff, S., 1984. Gene 28: 357), to obtain subclones in which one direction is sequentially deleted. The base sequence was analyzed.

First, 13 µg of the plasmid pmlβa34 was cleaved into Sac I, wherein the restriction enzyme cleavage strand forms a 3 'end, which is not degraded by Exo III, and BamH I which provides a 5' end, which is degraded by Exo III, and phenolchloroform DNA digested by extraction and ethanol precipitation was recovered and dissolved in 56.3 μl of distilled water. Here, 6.2 μl of 10-fold Exo III reaction buffer solution and 3.4 μl of Exo III (175 U / μl) were added and 2.5 μl were dispensed at intervals of 30 sec while reacting at 37 ° C., and 7.5 μl of SI nuclease mixed solution was prepared in advance. Was put in a test tube put on ice. After dispensing with a total of 25 test tubes, the mixture was reacted with S I nuclease at room temperature for 30 minutes, and then, 1 μl of the S I reaction stopping solution was added thereto, followed by heat treatment at 68 ° C. for 10 minutes to stop the reaction. 2 μl of the 10 μl reaction solution per each tube was electrophoresed to confirm continuous defects, and the remainder was added with 1 μl of the Klenow mixed solution and 1 μl of the dNTP mixed solution, followed by reaction at 37 ° C. for 5 minutes to form a single strand. Fill the remaining ends with. Subsequently, 40 μl of the ligation mixture solution was added and reacted at room temperature for 2 hours to transform the E. coli host cell XL-1 blue MRF ′ in the same manner as described above.

The following shows the composition of each reaction solution.

Restriction enzyme reaction

10 μl pmlβa34 (1.3 μg / μl)

1 μl BamH I (10 U / μl)

1 μl Sac I (10 U / μl)

5 μl 10-fold buffer solution

33 μl of distilled water

S I nuclease mixed solution

18.8 μl 10 × S I buffer solution

S I nuclease (50 U / μl) 0.4 μl

168.3 μl of distilled water

S I Reaction Stop Solution

0.3 M Tris (pH 8.0)

0.05 M EDTA

Clenau Mixed Solution

50 μl Klenow reaction buffer solution

Klenow (5 U / μl) 1.5 μl

Clenau Reaction Buffer Solution

20 mM Tris (pH 8.0)

10 mM MgCl ₂

Ligation Mixed Solution

80 μl of 10-fold ligation reaction buffer solution

100 μl 40% PEG

0.1 M DTT 80 μl

5 μl T4 DNA ligase (1 U / μl)

535 μl of distilled water

The colonies were randomly selected from 25 plates cultured overnight by incubating the transformants, incubated overnight at 2 ml of LB, and plasmid DNA was extracted by alkaline lysis and digested with restriction enzymes to confirm the size of the defect. . Sequencing was performed by the method of diedeoxy chain termination of Sanger et al. (Sanger, F., S. Nicklen and AR Coulson, 1977, Proc. Natl. Acad. Sci., USA, 74: 5463-5467). By using a sequencing kit (Sequenase version 2.0, USB, USA), the base sequence of each clone is read through the base sequence of each of the clones, and the base sequence of each clone through the overlapping base sequence, genes using a BLAST server The amino acid sequence and gene structure were analyzed through GenBank search.

SEQ ID NO: 1 shows the base sequence of the cloned loach beta-actin gene and its corresponding amino acid sequence, and the base sequence of the beta-actin gene regulatory site 5 'high.

As a result of analyzing the nucleotide sequence of the beta-actin gene, the beta-actin gene is composed of six exons following the GT-AT exon-intron boundary law, including exon 1 that is transcribed into RNA but does not encode amino acids. The actin protein could be assumed to consist of 375 amino acids starting from the second exon translation start codon (ATG). Therefore, the cloned 7336 bp DNA contains not only the beta-actin gene but also the upper end of the 5 'gene of 4339 bp and the lower end of the 3' gene of 1170 bp.

The nucleotide sequence of the loach beta-actin gene shows homology of 89.4% with common carp, 87.1% with mouse and 86.4% with human, and with homology of 98.4%, 97.9% and 98.1%, respectively. Have In 1990, Liu et al. Found that the nucleotide sequence of the carp beta-actin gene was 91% homologous with chicken, 88.5% with human, 87.9% with rat, and 99.5%, 99%, 99% homologous with amino acid sequence, respectively. The proximal promoter region up to 100 bp from the transcription initiation site and 340 bp on the 3 'side of intron 1 were involved in the expression, indicating that the nucleotide sequence of this region showed high homology between species. In this proximal promoter region, the nucleotide sequences of the CAAT box (CCAAT), the CArG motif (CCTTATATGG), and the TATA box (TATAAAA) were also identified in the proximal promoter region. It has been shown that the useful gene expression vector using loach beta-actin gene regulatory sites obtained through the present invention can be functionally operated in several species.

Example 3: Cloning of Beta-Actin Gene Regulatory Sites

As a result of sequencing analysis, there was an ATG nucleotide sequence of translation initiation codon 394 bp in front of Xho I at 4093 bp of plasmid pmlβa34, so that the Sty I site at 3700 bp and Not I at 670 bp existed in the pBS vector. The site was cleaved and blunt ligated to obtain the pmlβa34S / N plasmid from which the beta-actin structural gene site was removed. In blunt ligation, 1 µg of plasmid pmlβa34 was added to the reaction buffer solution and restriction enzymes Sty I 10 U and Not I 10 U, followed by reaction at 20 µl of the reaction volume for 2 hours, followed by electrophoresis on 10 µl of agarose gel for complete cleavage. 10 μl of the remaining reaction solution and 60 μl of the SI reaction solution (15 U / 0.3 μl of SI nuclease, 6 μl of 10-fold reaction buffer solution, 53.7 μl of distilled water) were treated at room temperature for 30 minutes to form a single strand at the cut end. This was done by removing. 10 μl of the 70 μl reaction solution was then mixed with 40 μl of ligation mixed solution (4 μl of 10-fold reaction buffer solution, 4.9 μl of 40% PEG, 0.4 μl of 0.1 M DTT, 0.28 μl of T4 DNA ligase, 30.4 μl of distilled water) The mixture was reacted overnight at room temperature, and 10 µl of these were transformed into E. coli host cell XL-1 blue MRF 'in the same manner as described above. The colonies shown were arbitrarily selected and cultured to extract plasmids, and the clones from which the beta-actin structural gene region was removed through restriction enzyme reactions were named pmlβa34S / N. This was then completely cleaved with Xho I and then partially cleaved with BssH II to obtain a 444 bp regulatory site fragment.

In addition, for cloning the beta-actin gene regulatory region, the 3.9 kb fragments of Xba I and Xho I on the 5 'top of the 3.4 kb clone were also cloned into the Xba I and Xho I positions of the pBS plasmid vector to obtain the pmlβa39 plasmid. For analysis, Sph was cut into Kpn I in the outer pBS vector outside the Xho I and Xba I above the regulatory site, transferred to the same restriction enzyme site of the pUC19 vector (NEB, USA), and formed 3'-end, which was not degraded by Exo III. The nucleotide sequence was analyzed by Erase-a-base method by cutting into Xba I which was digested by Exo III by providing I and 5 'ends.

Next, in order to facilitate the recombination of useful genes, sequencing of the pmlβa39 plasmid revealed that the restriction enzyme Sac I cleavage site, located 186 bp away from Xba I, was cleaved with restriction enzyme Sac I and blunt ligation. As before, it was completely cleaved with Xho I and then partially cleaved with BssH II to obtain a fragment of 6584 bp. The plasmid pmlβa41 having the full beta-actin regulatory site of the translation initiation codon top 4151 bp was prepared by combining the fragments of Xho I, BssH II 444 bp of pmlβa34S / N obtained above.

Figure 2 is a schematic diagram showing the cloning process of plasmid pmlβa41 including loach beta-actin gene regulatory region region.

The plasmid pmlβa41 prepared as described above was transformed into Escherichia coli XL-1 blue MRF 'and deposited in the Biotechnology Research Institute as Accession No. KCTC 8889P dated May 12, 1998.

Example 4 Expression of Fluorescent Proteins by Modulation of Beta-Actin Gene Expression

① Preparation of Fluorescent Protein Expression Vector

Green fluorescence protein (GFP) gene derived from Aequorea victoria, which is very useful as a marker gene, was used to confirm the expression control ability of loach beta-actin gene regulation site. The green fluorescent protein expression vector (phGFP-S65T; Clonetech, USA) was cleaved with Mlu I and Sac I to remove the cytomegalovirus (CMV) promoter portion, which is a 586 bp expression control site, and the plasmid pmlβa41 with the restriction enzyme BssH II. A 4202 bp fragment obtained by cleavage with Sac I was ligated to prepare plasmid pmlβaGFP which is controlled to express green fluorescent protein by beta-actin gene regulatory site.

Figure 3 is a schematic diagram showing the manufacturing process of the pmlβaGFP plasmid expression vector recombinant the locus beta-actin gene regulatory site and the gene encoding the fluorescent protein of jellyfish origin.

② Expression of fluorescent protein by regulation of beta-actin gene expression

In order to confirm the expression control ability of the beta-actin gene, the plasmid pmlβaGFP vector prepared above was transduced into the cell line in culture to measure the expression level of GFP. As a control, phGFP-S65T carrying a cytomegalovirus (CMV) promoter was transduced and compared, and the transformation efficiency into cultured cells was quantitatively analyzed by introducing a pCMV-LacZ vector together. Cultured cell lines include salmon salmon embryonic cell line (CHSE-214), human erythropoietic cell line (K562), monkey green monkey kidney cell line CV1 derived E25B2, and rat blood cells. A cancer cell line (Mouse erythroid leukemia cell line; MEL) was used. The CHSE-214, K562, and MEL cell lines were transduced by electroporation, and the E25B2 cell line by calcium phosphate precipitation.

The CHSE-214 cell line was cultured in DMEM medium containing 10% fetal bovine serum (Sigma, USA; FBS), treated with trypsin-EDTA, and detached from the culture vessel, followed by PBS and HBS (21 mM HEPES; pH 7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na ₂ HPO ₄ , 6 mM glucose) and resuspended in HBS to a final density of 5 × 10 ⁶ cells / ml. Among them, 800 μl of cells and 10 μg / 200 μl of plasmid DNA were mixed in a cuvette, mixed for 3 minutes on ice, mounted on an electroporator, and subjected to electric shock under conditions of 500 μs and 330 V. Next, after standing in ice for 10 minutes, the cells were transferred to the culture vessel, the culture solution was added, and cultured at 18 ° C. for 72 hours.

K562 cells in RPMI 1640 medium containing 10% calf serum (Hyclone, USA; BCS), MEL cells in 37 ° C., 5% CO ₂ , 100 in DMEM medium containing 10% BCS (Hyclone, USA) Cells were recovered by centrifugation after suspension culture with% humidity, and plasmid DNA was transduced by the electroporation method as described above. Electroporation conditions were electroshock at 50 kPa and 500 V twice for K562 cells and 50 kPa and 1000 V for MEL cells.

E25B2 cells were cultured at 37 ° C., 5% CO ₂ , 100% humidity in DMEM medium containing 10% BCS (Hyclone, USA), and transduction was performed by Graham, FL, van der Eb, AJ, 1973. Virology 52: 456-467) calcium phosphate precipitation method was used. Cells were harvested by treatment with trypsin-EDTA the day before transduction and incubated overnight with 1 × 10 ⁶ cells per 100 mm plate and replaced with fresh culture 2 hours before transduction. Plasmid DNA was dissolved in 20 μl of TE by ethanol precipitation of 10 μg per plate, and 50 μl of 2.5 M CaCl ₂ and 430 μl of 0.1X TE were mixed well. Next, 500 μl of 2 × HBS (42 mM HEPES; pH 7.05, 274 mM NaCl, 10 mM KCl, 1.4 mM Na ₂ HPO ₄ , 12 mM glucose) was added to a polypropylene test tube, and the mixture was gently vortexed. DNA / CaCl ₂ mixture was added dropwise. The DNA / CaCl ₂ / phos- phate mixture was allowed to stand at room temperature for 30 minutes, and when white precipitate was confirmed, it was gently crushed, added dropwise to the cell culture medium, shaken 2-3 times, and incubated in the incubator for 60 hours.

The cells into which the genes were introduced were collected 60-72 hours after introduction, and the expression rate was analyzed by counting the number of GFP-expressing cells under fluorescence microscopy, and some were counted by LacZ staining to express the cells expressing E. coli LacZ genes [GFP]. The expression level of GFP was quantitatively analyzed by determining the value of / [LacZ].

Figure 4 is a photograph comparing the expression control pattern of GFP by the plasmid pmlβa41 comprising the CMV promoter and loach beta-actin gene regulatory site region in the E25B2 cell line, Figure 4a is the expression result by the CMV promoter, Figure 4b is pmlβa41 It is a photograph showing the expression result by.

In the CHSE-214, K562, and MEL cell lines, the expression regulation by CMV promoter induced GFP expression in more cells, but the expression level per cell was more strongly regulated by loach beta-actin regulatory sites in light of GFP brightness. Showed an aspect. In addition, GFP expression by the beta-actin regulatory site in E25B2 cells showed about half the expression control capacity compared to the CMV promoter used as a strong expression control site as a whole.

Table 1 compares the expression regulation patterns of GFP by the CMV promoter and plasmid pmlβa41 including loach beta-actin gene regulatory site region in E25B2 cell line.

As shown in Table 1 and FIG. 4 above, due to homology between the loach and other known species in the beta-actin gene regulation site, the expression vector comprising the locus beta-actin gene expression control site is a salmon embryonic cell line (CHSE). -214), human hematopoietic cancer cell line (K562), monkey kidney cell line (E25B2), mouse hematopoietic cancer cell line (MEL), it was confirmed that the gene of interest can be efficiently expressed in a variety of cell lines.

As described above, it can be seen that the expression vector comprising the loach beta-actin gene regulatory site of the present invention can be efficiently expressed in the target gene by operating normally in cultured cell lines of various origins including mammals and fish. have.

That is, the expression vector including the loach beta-actin gene regulatory site of the present invention can be usefully used for the expression of useful genes in fish, and thus useful genes such as growth hormone genes, pathogenic genes and other bioactive substance producing genes. Expression in fish could be used to develop new breeds of good fish.

(Sequence list)

SEQ ID NO: 1

Length of sequence: 7314

Type of sequence: nucleic acid

Number of chains: two prints

Topology: Linear

Type of molecule: genomic DNA

origin:

Creature: Loach (Misgurnus mizolepis)

Claims (5)

DNA comprising a beta-actin gene and a beta-actin gene regulatory site of loach represented by the nucleotide sequence of SEQ ID NO: 1. An expression vector comprising a beta-actin gene regulatory site of loach. The expression vector pmlβa41 (KCTC 8889P) according to claim 2 The transformant transformed with the expression vector of claim 2. The fish of claim 4 transformed with the expression vector of claim 2.