KR100506582B1 - The apolipoprotein aⅰ-transgenic mud loach over-producing hdl-cho - Google Patents

Abstract

The present invention relates to a transgenic loach which has been transplanted with loach apolipoprotein AI gene, and comprises a step of injecting and incubating an expression vector containing the cDNA of loach apolipoprotein A1 described in SEQ ID NO: 1 into loach embryos. The transformed loach of the present invention produced by the present invention produces high-density lipoprotein-cholesterol (HDL-CHO) because it mass-produces high-density lipoprotein, which overexpresses Apo AI and thus plays a key regulatory function in cholesterol metabolism. It can be developed as a health food containing.

Description

Transformed loach grafted with loamy apolipoprotein A1 gene overproducing high density lipoprotein-cholesterol {THE APOLIPOPROTEIN AI-TRANSGENIC MUD LOACH OVER-PRODUCING HDL-CHO}

The present invention relates to a transgenic loach in which loach apolipoprotein AI gene is transplanted, and the loach has a trait useful for cardiovascular diseases such as hypercholesterolemia and arteriosclerosis by overproducing high density lipoprotein-cholesterol (HDL-CHO). It is to provide.

Transgenic technology through gene transfer is a technology that allows an individual to have the desired new genotype and phenotype by injecting a new genetic material into a cell and transplanting it on a chromosome of a host. In particular, fish is a taxon with the widest genetic diversity among vertebrates, and has been reported for transgenic material. In addition, recent studies have shown that genes whose evolution has been lost in higher vertebrates, including humans, have been preserved with high activity in fish (Hew, CL and GL Fletcher, 2001). . The role of aquatic biotechnology in aquaculture Aquaculture, 197:. 191-204; Nam, YK, YS Cho, SE Douglas, JW Gallant, ME Reith and DS Kim, 2002. Isolation and transient expression of a cDNA encoding L-gulono- g-lactone oxidase, a key enzyme for L-ascorbic acid biosynthesis, from the tiger shark Scyliorhinus torazame Aquaculture, 209:. 271-284). Therefore, transformation of fish is not only economically useful production of aquaculture system (Nam, YK, JK Noh, YS Cho, HJ Cho, KN Cho, CG Kim and DS Kim, 2001. Dramatically accelerated growth and extraordinary . gigantism of transgenic mud loach (Misgurnus mizolepis) transgenic Res, 10:. 353-362), useful vertebrate model, and start getting the limelight recently in terms of development (Powers, DA, 1989. Fish as model systems Science, 246. (352-358) Iyengar, A., F. Muller and N. Maclean, 1996. Regulation and expression of transgenes in fish-a review.Transgenic Res., 5: 147-166) It is becoming very important in terms of the development of functional animal materials, and it is emerging as a sharp competitive field all over the world.

During development of such a transgenic animal system, traits related to lipid metabolism are considered to be one of the most important molecular therapeutic traits in the world because they have a direct and close relationship with the nervous, heart and vascular circulatory systems. In particular, a great deal of research is being conducted on the discovery of genes related to cholesterol metabolism, collection of gene expression information, and interpretation of biological function and action information. At the same time, new model systems are being actively developed to perform these studies (Ruben, EM, RM Krauss, EA Spangler, JG Verstuyft and SM Clift, 1991. Inhibition of early atherosclerosis in transgenic mice by human apolipoprotein A I. Nature (Lond.) 353: 265-267; Jiang, XC, OL Francone, C. Bruce, R. Milne, J Mar, A. Walsh, JL Breslow and AR Tall, 1996. Increased preβ-high density lipoprotein, apolipoprotein AI, and phospholipid in mice expressing the human phospholipid transfer protein and human apolipoprotein AI transgenes.J. Clin. Invest. , 98: 2373-2380;.. Cohen, RD, LW Castellani, J. -H Qiao, BJV Lenten, AJ Lusis and K. Reue, 1997. Reduced aortic lesions and elevated high density lipoprotein levels in transgenic mice overexpressing mouse apolipoprotein A-ⅳ J Clin Invest. , 99: 1906-1916.

Among them, apolipoproteins are the major constituents of lipoproteins and play an important role in the in vivo fat transfer and metabolism of all vertebrates (Gotto, AM Jr., HJ Pownall and RJ Havel, 1986. Introduction to the plasma lipoproteins.Methods Enzymol. , 128A: 3-41). Many Apo proteins have a bipolar helix that can react with and interact with hydrophobic lipids as well as hydrophilic cellular components, thereby inducing lipid transport and metabolism (Segrest, JP, MJ Jones, H. De). Loof, CG Brouillette, YV Venkatachalapathi and GM Anantharamaiah, 1992. The amphipathic helix in the exchangeable apolipoproteins: A review of secondary structure and function.J. Lipid Res. , 33: 141-166). In particular, apolipoprotein AI (hereinafter abbreviated as Apo AI) is a major protein component of high density lipoprotein (HDL) and is closely involved in cholesterol metabolism of living tissues and organs including the arteries. HDL is known to function as an extracellular receptor that binds cholesterol released from tissues and organs to prevent excessive cholesterol accumulation in tissues, and plays a key role in the reverse cholesterol transport (RCT), a biological response to excess cholesterol. (Plump, AS, SK Erickson, W. Weng, JS Partin, JL Breslow and DL Williams, 1996. Apolipoprotein AI is required for cholesteryl ester accumulation in steroidogenic cells and for normal adrenal steroid production.J. Clin. Invest. , 97 : 2660-2671). Apo AI, a major component of HDL, is a protein that determines the activity of the biological defense pathways, and a decrease in serum Apo AI-HDL concentration directly contributes to atherosclerosis and many cardiac diseases. Thus, Apo A I is very important as a biomarker for predicting circulatory disease risk (Francis, MC and JJ Frohlich, 2001. Coronary artery disease in patients at low risk-apolipoprotein A as as independent risk factor.Atherosclerosis , 155: 165-170). Furthermore, Apo A I is found to be involved not only in the extracellular RCT response but also in the extracellular release of intracellular cholesterol as well as its antioxidant and anti-inflammatory effects (Wang, XL, RB Badenhop, AS Sim and DEL Wilcken, 1998.The effect on transcription efficiency of the apolipoprotien A I gene of DNA variants at the 5 'untranslated region.Int . J. Clin.Lab.Res . , 28: 235-241; Sviridov, D., N. Fidge , G. Beaumier-Gallon and C. Fielding, 2001. Apolipoprotein AI stimulates the transport of intracellular cholesterol to cell-surface cholesterol-rich domains (caveolae) .Biochem . J. , 358: 79-86).

In view of the above, the present inventors attempted to perform the discovery and function and usefulness of the gene by setting the loach Apo AI gene as a target gene as part of the development of functional fish gene resource utilization technology. Unlike other fish, loach is not found in the abdominal fat at all, even in the case of aquaculture, and the liver (liver) is unlikely to contain little fat, unlike other fish is believed to have a very high activity of apolipoprotein in the body. . Therefore, using the loach Apo AI gene, it is expected to obtain transgenic fish which can play a role in preventing excessive cholesterol accumulation in tissues and organs, as well as lipid metabolism functions useful for atherosclerosis and cardiovascular diseases. The large amount of high density lipoprotein (HDL) accumulated in the body through the mass expression of Apo A I is expected to produce a much higher health supplement effect than intake loach.

SUMMARY OF THE INVENTION An object of the present invention is to provide a useful renal functional transgenic fish that contains a high content of components useful for atherosclerosis and cardiovascular disease as well as preventing cholesterol accumulation by participating in cholesterol metabolism of living tissues and organs including arteries. I would like to.

In order to achieve the above object, in the present invention, cDNA of loach apolipoprotein AI (mApo AI) described in SEQ ID NO: 1, an expression vector comprising the same, and a high-density lipoprotein-cholesterol transformed with the expression vector (HDL-CHO) Provides overgrown loach ( Misgurnus mizolepis ).

Here, the transgenic loach which overproduces the HDL-CHO of the present invention is produced by a method comprising microinjecting an expression vector containing a cDNA of mApo AI into a loach fertilized egg and incubating it.

In the present invention, a useful transgenic fish system for searching and cloning loach Apo AI gene to analyze its structure, constructing loach Apo AI expression vector using recombinant DNA technology, and overexpressing Apo AI through gene transfer. Developed.

Subsequently, transgenic loach that has been engineered with lipid metabolism was developed as a model for studying lipid metabolism and molecular therapy, and evaluated as a new value-added generation model containing a large amount of useful human components.

Specifically, functional transgenic loach, which induces excessive cholesterol stress artificially using various experimental feeds including cholesterol, and expresses Apo AI produced according to the present invention, is rapidly and rapidly increased in the body. It was evaluated whether it could react sensitively. As a result, these transformants showed a significant increase in HDL-CHO, which rapidly regulates excess cholesterol transport (RCT) reactions, which can cause the transformants to accumulate excessively in tissues and blood vessels. This is because the RCT ability is much superior to the general loach.

These results indicate that fish cholesterol metabolism can be manipulated by regulating the expression of the Apo AI gene, as well as the disease model caused by the accumulation of hypercholesterolemia, as well as hypercholesteramia and atherosclerosis through improved RCT ability. It is also possible to develop a molecular therapy model that resists atherogenesis.

In addition, fenofibrate administration, a compound that affects the expression rate of Apo AI genes of different species, was determined that loach Apo AI gene is subjected to the human type Apo AI gene regulation mechanism.

That is, the loach Apo AI gene according to the present invention has a very similar protein structure and function despite the homology of the human Apo AI gene with a low DNA sequence, and the transgenic loach expressing the gene is a key regulator of cholesterol metabolism. It can produce large quantities of HDL, which is responsible for its function, thereby acquiring new capacity to cope with the accumulation of excess cholesterol strongly in the body, thereby preventing diseases caused by excess cholesterol such as atherosclerosis.

As a result, the transgenic loach, in which the loach Apo AI gene was transplanted according to the present invention, overexpresses Apo AI and thus produces a large amount of HDL, which plays a key regulatory function in cholesterol metabolism, and thus contains a high content of HDL-CHO. It can be developed as a health food.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only illustrative of the present invention, and the scope of the present invention is not limited to these.

Example 1 Exploration, Discovery and Cloning of Loach Apolipoprotein Gene

1-1. cDNA Library Fabrication

To prepare the cDNA library, the whole tissue was crushed using 1 ml TriPure ^™ Isolation Reagent (Roche Molecular Biochemicals, Germany) per 100 mg of the tissues of the Misgurnus mizolepis liver tissue, and then at room temperature for 10 minutes. The mixture was left for a minute, followed by chloroform extraction and centrifugation (12,500 rpm, 15 minutes). Centrifugation was performed to take supernatant containing RNA, and then the same amount of isopropanol was added to induce precipitation of total RNA. Only mRNA was purified purely from 250 μg total RNA using mRNA Isolation Kit (Roche, Germany). Lysis buffer (lysis buffer; 0.1 M Tris, 0.3 M LiCl, 10 mM EDTA, 1% lithium dodecyl sulfate, 5 mM dithiothreitol, pH 7.5) was added to the RNA sample and left at 65 ° C. for 2 minutes, followed by 100 2 mol of pmol / μl and biotin-labeled oligo d (T) ₂₀ were mixed thoroughly to induce a hybrid reaction between poly (A) and oligo d (T). Thereafter, 1.5 mg of streptavidin magnetic particles were added and left at 37 ° C. for 5 minutes to induce a binding reaction between biotin and streptavidin. When the reaction was completed, using a magnetic particle separater (Roche Co., Germany) washed three times with 250 μl of washing buffer (10 mM Tris, 0.2 M LiCl, 1 mM EDTA, pH 7.5) , Poly (A) mRNA was separated from the magnetic particles by warming at 65 ℃ with DEPC treated sterile water for 2 minutes. CDNA library was prepared using the isolated poly (A) tail mRNA.

Experimental procedures for cDNA library fabrication were performed using Stratagene's ZAP-cDNA Synthesis Kit and ZAP-cDNA GigapackIII Gold Cloning Kit. Based on the manufacturer's protocol, cDNA was synthesized using reverse transcriptase from mRNA, and both ends of XhoI and EcoRI were manipulated by manipulating the ends of the synthesized cDNA to facilitate cloning. A size fraction was performed using a Sepharose column to select only cDNA strands of 0.6 kb or more among the synthesized cDNA fragments. The column was prepared using a Falcon 1 ml pipette and 10 ml sterile syringe according to Stratagene's cDNA Synthesis Kit manual, and the media of the column was Sepharose CL2. After loading cDNA on the prepared drip column, 12 fractions of 100 μl each were obtained. After confirming the length of the cDNA in each fraction by electrophoresis, the cDNA was selected to be 0.6 kb or more to perform the vector and ligation. In order to package the ligated cDNA / vector into bacteriophage, 4 μl of 1.0 μg ligated DNA was immediately mixed with the prepared packaging extract (Stratagene Co., USA), followed by induction for 2 hours at room temperature (23 ° C.). It was. When the reaction was completed, 500 μl of SM buffer (100 mM NaCl, 8 mM MgSO ₄ , 50 mM Tris-HCl; pH 7.5, 0.01% gelatin) and 20 μl of chloroform were added, mixed slowly, and stored at 4 ° C. and used for the experiment. Titers were examined to analyze packaging efficiency.

In order to prepare host cells, bacterial glycerol stock (XL1-Blue MRF 'and SOLR cells) were plated on agar plates and incubated at 37 ° C for 16 hours, and one colony was taken to obtain a liquid medium (10 in 1X LB). Inoculated with mM MgSO ₄ , 0.2% maltose) and incubated at 37 ° C. until no more than OD ₆₀₀ = 1.0, and then adjusted to OD ₆₀₀ = 0.5 with 10 mM MgSO ₄ to be used in the experiment. For primary library titering, add 200 μl of host cells diluted to OD ₆₀₀ = 0.5 and 10 μl of packaged lambda phage to 10 mM MgSO ₄ , followed by 15 minutes at 37 ° C. Reacted. Upon completion of the reaction, 3 ml of NZY top agar (melted and cooled to ˜48 ° C.), 15 μl of 0.5 M isopropyl-1-thio-β-D-galactopyranoside (IPTG) and 250 mg / ml X-gal 50 μl of (in N, N-dimethylformamide) was immediately added to the NZY agar plate, left at room temperature for 10 minutes, and incubated at 37 ° C. for 16 hours. Mass in vivo excision was performed to sub-clones cDNA clones contained in lambda phage into plasmid vectors. For the cleavage reaction, the host cells of the phages, XL1-Blue MRF 'and SOLR cell strains were grown to OD ₆₀₀ = 1.0 (8 × 10 ⁸ cells) in LB medium supplemented with 10 mM MgSO ₄ and 0.2% maltose. 10 mM MgSO ₄ was adjusted to a concentration of OD ₆₀₀ = 1.0 and used in the experiment. First, phage stock: XL1-Blue MRF 'cell: ExAssist helper phage was mixed at 1: 10: 100 and reacted at 37 ° C. for 15 minutes to allow phage to attach to all host cells. After the reaction was completed, 20 ml of LB medium was added and shaking culture was performed at 37 ° C. for 3 hours. After incubation, the cells were inactivated at 67 ° C., and the cell residues were centrifuged (1,000 × g, 10 minutes). )). To confirm that the cleavage was effective, 1 μl of the cut phage was infected with 200 μl of the prepared SOLR cell at 37 ° C. for 15 minutes. The cleaved phagemid was engineered to contain ampicillin resistance genes and thus plated in LB-ampicillin agar medium (50 μg / ml) to selectively obtain only infected SOLR cells (cells containing phagemid).

1-2. Screening and Structural Analysis of Loach Apolipoprotein AⅠ Gene

To prepare the template for PCR for sequencing, bacterial colonies containing cDNA were inoculated in LB-ampicillin liquid medium and incubated at 37 ° C. for 16 hours. After the incubation was completed, the cells were recovered by centrifugation, resuspended in proteinase K (100 µg / ml in DW) solution, left at room temperature for 2 hours, and reacted at 50 ° C for 30 minutes. After the reaction was completed, the proteinase K was inactivated at 94 ° C. for 15 minutes, centrifuged (12,000 rpm, 5 minutes) to remove the cell residues, and the supernatant containing the plasmid was taken and stored at -20 ° C. Used.

Each bacterial colonies obtained were incubated overnight in 2 ml LB-ampicillin liquid medium. Cells were recovered by centrifugation, suspended in a solution of proteinase K (100 μg / ml in DW), and reacted at room temperature for 2 hours at 50 ° C for 30 minutes, followed by proteinase K at 94 ° C for 15 minutes. The supernatant containing the plasmid was obtained by inactivation and centrifugation again (12,000 rpm, 5 minutes). To extend the primer, add 11 μl of lysate, 2 μl of SK primer (5 'CGCTCTAGA ACTAGTGGATC 3'), 2 μl of BigDye terminator (Perkin Elmer Co., USA) containing fluorescently labeled ddNTP, and 6 μl of 2.5X dilution buffer. The mixture was prepared and PCR cycling was performed. The PCR reaction was performed at 94 ° C. for 3 minutes, followed by 10 seconds at 96 ° C., 5 seconds at 50 ° C., and 4 minutes at 60 ° C. After the PCR reaction was completed, the PCR product was subjected to ethanol precipitation and 70% ethanol washing was performed. Finally resuspended in 3.5 μl loading buffer (5: 1 deion formamide to 25 mM EDTA, 50 mg / ml blue dextran) and used for analysis. Sequencing gel was prepared according to the manufacturer's protocol (PE Co., USA), and the nucleotide sequence data was obtained using an ABI 377 sequencer (PE Co., USA).

The data obtained from the sequencing gel was analyzed using Sequencher 3.1.1 (Gene Codes Co., USA), the sequencing software, and then unnecessary genome data including the sequence of the vector portion was removed and Genbank search was performed. Similarity comparisons at base and amino acid sequence levels with other fish species were performed simultaneously with the NIH BLAST programs blastx and blastn (www.ncbi.nlm.nih.gov). In addition, the obtained base sequence and the amino acid sequence inferred therefrom were compared and analyzed for homology by multiplexing using a complex sequence alignment program such as MultAlin (www.toulouse.inra.fr) and Clustal W (.ad.jp).

1-3. result

To ensure complete cloning of the apolipoprotein gene, the entire sequence of the cloned insert was determined using the forward and reverse primers for the ML003 clone, which has a relatively high homology with the carp close to the flexible relationship. . The identified loach apoprotein gene had 762 bp encoding 254 amino acids of 1090 bp in length, followed by a poly (A) signal sequence followed by 274 bp from the 5 'UTR region of 32 bp and the TAA stop codon. Two of them appeared, and 893 bp or poly (A) sequence was identified, resulting in a 393 UTR region of 293 bp up to the transcriptional termination point. Figure 1 shows the nucleotide sequence and the corresponding amino acid sequence of the loach Apo AI cDNA gene.

The apolipoprotein consisting of 254 amino acid sequences inferred from these nucleotide sequences had a molecular weight of about 29.3 kDa and a pH of 6.12. The average hydrophobicity was -0.500787, particularly at the N-terminal region. Sequence multi-array analysis of the apolipoprotein amino acid sequence of loach with those of currently known fish, mammalian mice and humans revealed 34-43% homology with other fish, including carp and 60% with close relationships, In addition, they showed only 16% low sequence homology with humans, and the gene relationship based on these sequence homology was well represented.

Example 2 Development of Vector for Transplantation of Loach Apoprotein AI and Preparation of Transgenic Fish

2-1. Development of Vector for Transplanting Loach Apoprotein AⅠ

For the preparation of apo-protein gene expression vectors pBluscript II SK (+) (pBS ; Stratagene, USA) ML003 pFV4a plasmid and the plasmid is cloned into the vector (Dr. PB Hackett, University of Minnesota , USA) 2 ㎍ the restriction enzyme Not After reacting with I and Apa I 10 U for 2 hours and cutting, it was separated by size by electrophoresis. Gel fragments containing the bands were recovered using a Geneclean kit (Bio 101, USA) and ligated using T4 DNA ligase. Subsequently, the E. coli host cell XL-1 blue MRF 'strain treated with 50 mM CaCl ₂ was transformed, and white colonies which appeared after incubation were randomly selected and incubated in 2 ml LB culture medium at 37 ° C. overnight. 1.5 ml of the culture solution was extracted with plasmid DNA according to the alkali dissolution method, and a part thereof was cut with a restriction enzyme to confirm whether the correct insert was included.

2-2. Production of Transgenic Loach with Transplanted Loach Apolipoprotein AI

Mature female and male loach (4-5 years old) were intraperitoneally injected with HCG at a concentration of 10 IU / g of fish, and fertilized with sperm. While the fertilized eggs were stored at 15 ° C., the expression vector pFV4mApoA I was microinjected in the center of the blast cell of the fertilized egg which reached the 1-cell stage. Micromicroscopic injection was performed according to the method of Nam et al . (2001), and the circular and linear DNA was 1: 1 by using a 5 μm diameter capillary at a DNA concentration of 100 μg / ml. Mixed in proportions and transplanted into 3 fertilized loach fertilized eggs from each male and female.

After 3 months of incubation, caudal fins were excised to search for transgenic fish and DNA was extracted by SDS / proteinase K method (Nam et al ., 2001). For Southern blots, 10 μg of DNA was cut with restriction enzyme DraII, electrophoresed on a 0.7% agarose gel and transferred to a nylon membrane using a non-isotopic labeling and detection kit (Roche, Germany). Individuals transplanted with the Apo AI gene were selected. At this time, the loach Apo AI cDNA was labeled with digoxigenin (digoxygenin) and used.

2-3. result

The apolipoprotein gene for preparation of the expression vector was obtained from the ML003 clone including the entire nucleotide sequence of the EST results, and the pFV4a vector including the regulatory region of the carp β-actin gene was used as a promoter region of the expression vector. Figure 2 is a schematic diagram showing the configuration of the expression vector containing loach apolipoprotein AI cDNA gene.

Table 1 below shows the gene transplantation rates in hatched larvae generated from fertilized eggs microscopically injected with the expression vector pFV4mApoAⅠ. Here, it can be seen that the microtransplantation resulted in a gene transplantation rate of 22 to 41%, an average of 33.3%.

Gene Transfer Rate (%) Average(%) Trial I Trial II Trial Ⅲ 22 41 37 33.3 ± 10.01

Figure 3 shows the results of Southern blot analysis of transgenic loach, which is lodged with loach apolipoprotein AI gene. That is, 27 out of 81 analyzed animals showed Southern positive patterns. As a result of sorting their gene insertion patterns, all of the selected transgenic fishes had bands in the high molecular weight region, and generally 1 to 2 copies (copy) ) Was inserted into DNA in the nucleus.

Example 3 Functional Analysis of Apolipoprotein AI Gene in Transgenic Loach

3-1. Induction Analysis of T-CHO / HDL-CHO Concentration by Apolipoprotein AI Expression in Transgenic Fish Lines

The Southern blot showed positive signal and high molecular weight DNA band, and selected the individuals whose integration was confirmed, and extracted the blood and measured T-CHO and HDL-CHO to determine whether the Apo AI gene was expressed in the transformed fish. It was investigated directly. F ₁ TG fish were produced by mating the control females with artificial insemination in two selected transgenic males. The expression rate and pattern of F ₁ fish were examined by measuring T-CHO and HDL-CHO after collecting 20 transformants from each produced F1 line.

The blood collection for measuring T-CHO was collected from 200 to 300 μl using a 1.0 ml / dL (26 1 / 2G) syringe, and the collected blood was centrifuged at 5,000 rpm at 4 ° C. for 5 minutes, followed by a new tube of serum only. Stored in. Serum from each individual stored was analyzed using an enzyme-linked immunosorbent assay (ELISA) plate reader (Bio-Rad, USA). As analytical reagent, a reagent for measuring total cholesterol (Asan Tech, Korea) was used, and the assay method was 300 μl enzyme solution (cholesterol esterase + cholesterol oxidase; Asantech T-CHO test solution, Korea) in 2 μl of fish serum. ) And then mixed with a microwell plate of serum and enzyme solution at room temperature for 10 minutes. At this time, PBS was used for the blank test and standard solution (Cholesterol 300 mg / dl) was used for the standard test, and the absorbance was measured at 490 nm (500 nm). Analytical reagent for measuring HDL-CHO is a reagent for measuring HDL-CHO (Asan Tech, Korea). Selective dissolution of only HDL-CHO using reagent A (surfactant + ASOD: surfactant having high affinity with HDL) And reagent B (cholesterol esterase + cholesterol oxidase: a reagent for quantifying HDL-CHO dissolved by enzymatic reaction). In the analytical method, 300 μl Reagent A and 100 μl Reagent B were mixed with 3 μl of fish serum, and the microwell plate mixed with serum and measurement reagent was reacted at room temperature for 5 to 10 minutes. At this time, PBS was used for the blank test, and standard solution (HDL-CHO 50 mg / dL) was used for the standard test, and the absorbance was measured at 655 nm (600 nm; sub-700 nm) using an ELISA plate reader.

3-2. Results-Expression of first generation transgenic fish

T-CHO analysis of 13 first-generation Fo fish showed a T-CHO reading of 190 in the control group and 128 to 445 in the transformed Fo group, indicating diversity among individuals. Individuals with the highest T-CHO levels had an expression rate of about 2.3 times that of the control fish. Table 2 below shows serum concentrations (total readings at 490 nm) of total CHO values in transformed and control loach. Where each value is the average of three experiments.

Test group Incubation time 5 minutes 10 minutes Control group-1 control-2 control-3 control-3-4 control-5 control-6 234233197195175120 221218210197182138 Mean ± SD 192 ± 43 194 ± 31 Transformation Group-1 Transformation Group-2 * Transformation Group-3 * Transformation Group-4 Transformation Group-5 * Transformation Group-6 Transformation Group-7 * Transformation Group-8 Transformation Group-9 Transformation Transition group-10 * transformation group-11 * transformation group-12 * transformation group-13 * 120262268224272199259183223345349299392 128277271230296208266174221368370326445

* Tables indicate that overexpression of the transplanted gene may result in individuals showing significantly higher concentrations of total CHO.

In the results of the analysis of HDL-CHO, the control group showed a value of 120 when incubated for 10 minutes, but in the transforming group, 94 ~ 261 showed that the HDL concentration increased up to 2.2 times depending on the individual. Table 3 below shows the serum concentrations of HDL-CHO (any reading at 655 nm) in the transgenic and control loach. Where each value is the average of three experiments.

Test group Incubation time 5 minutes 10 minutes Control group-1 control-2 control-3 control-3-4 control-5 control-6 10810212111593118 11912413712598126 Mean ± SD 110 ± 11 121 ± 13 Transformation Group-1 Transformation Group-2 * Transformation Group-3 * Transformation Group-4 * Transformation Group-5 * Transformation Group-6 * Transformation Group-7 * Transformation Group-8 * Transformation Group -9 * transformation group -10 * transformation group -11 * transformation group -12 * transformation group -13 * 97209141174131184191174209162210207218 94213166180175195188181234189239240261

* Tables indicate that individuals may show significantly higher concentrations of HDL-CHO by overexpression of the transplanted gene.

Table 4 below shows the expression levels of Apo AI genes between the individuals according to the analysis of the T-CHO and HDL-CHO amounts between the individuals of transgenic Fo fish including pFV4mAPO cDNA. As shown here, individuals with both levels lower than the control (TG 1), individuals with both levels slightly higher than the control (TG 2, 3, 5 & 7), and individuals with only one higher level (TG 4, 6, 8, 9, 10 & 12), and both levels were divided into higher individuals (TG 11, 13).

system % Relative to control in 10 min incubation summary T-CHO HDL-CHO Transformation Group-1 66 77 TG-containing, TG mAPO AI cDNA not expressed Transformation Group-2 143 176 Moderate increase in both, total CHO ↑ <HDL-CHO ↑ Transformation Group-3 140 137 Moderate increase in both, total CHO ↑ = HDL-CHO ↑ Transformation Group-4 118 148 Increasing only in HDL-CHO Transformation Group-5 153 144 Moderate increase in both, total CHO ↑ = HDL-CHO ↑ Transformation Group-6 107 161 Increasing only in HDL-CHO Transformation Group-7 137 155 Moderate increase in both, total CHO ↑ = HDL-CHO ↑ Transformation Group-8 90 149 Increasing only in HDL-CHO Transformation Group-9 114 193 Significant increase only in HDL-CHO Transformation Group-10 190 156 Significant increase in total CHO, increase in HDL-CHO, total CHO ↑> HDL-CHO ↑ Transformation Group-11 191 197 Significant increase in both, total CHO ↑ = HDL-CHO ↑ Transformation Group-12 168 198 Moderate increase in total CHO, significant increase in HDL-CHO, total CHO ↑ <HDL-CHO ↑ Transformation group-13 229 215 Significant increase in both, total CHO ↑ = HDL-CHO ↑

3-3. Results-Expression of second generation transgenic fish

After fertilization of sperm from both strains with high expression levels of T-CHO and HDL-CHO in transgenic Fo fish, especially HDL-CHO, the hatching rate and initial survival rate are shown in Table 5. Here, the difference in hatching rate according to the egg quality of the female was observed, but there was no difference in the hatching rate and the initial survival rate according to the hatching population, the transgenic fo fish.

Female number Male number (TG) Incubation rate (%) Initial survival rate (%) One TG 006TG 009 83.581.4 85.988.8 2 TG 006TG 009 75.677.4 87.586.4

The results of analysis of their expression patterns with blood T-CHO and HDL-CHO concentrations of 20 F1 fish obtained from both strains are shown in Table 6 below. For TG # 006 F1 fish, a significant increase in the concentrations of T-CHO and HDL-CHO was observed in 14 of 20 animals analyzed, ranging from 354-532 mg / dl serum in T-CHO. HDL-CHO had an arbitrary value of 279-508, which was 1.4-2.2 times higher than that of the control group. In the case of TG # 009 F ₁ fish, 17 animals showed an increase in all assays, indicating that 85% of the TG fish were present, and that TG # 006 F was increased in both T-CHO and HDL-CHO. Slightly higher than ₁ fish level, the expression of Apo AI gene was higher in F ₁ fish of TG # 009.

TG Fish Number TG Fish Frequency (%) T-CHO range (mg / dl) HDL-CHO range (arbitrary unit) One TG 14/20 (70) NTG 6/20 (30) 354 ~ 532292 ~ 324 279 to 508 203 to 246 2 TG 17/20 (85) NTG 3/20 (15) 407-518 240-361 340 ~ 682195 ~ 279

Example 4 Evaluation of the Utility of Apolipoprotein AI Gene in Transgenic Loach

4-1. Changes in Cholesterol Concentrations of Transgenic Fishes by Feeding Porcine Scaffold, Cholesterol and Fenofibrate

A pig grinder, already well known for its high LDL-CHO, was crushed with a feed grinder and fed to control loach at 0, 5 and 10% in loach for 4 days and then measured for changes in T-CHO and HDL-CHO concentrations. Thus, the control loach was analyzed whether it is possible to induce changes in blood concentrations when feeding a high concentration of cholesterol feed.

In order to investigate the concentration change of T-CHO and HDL-CHO during the feeding of cholesterol feed from the transgenic loach, the feed containing 4% cholesterol in the transgenic loach of the two F ₁ strains was used. Serum levels of T-CHO and HDL-CHO were examined at two-day intervals after daily feeding. At this time, the control group was divided into the group fed with no cholesterol and the general loach fed with 4% cholesterol-added feed, and the cholesterol accumulation and HDL-CHO conversion efficiency of the control and transformed loach were analyzed.

On the other hand, whether the loach Apo AI gene developed in the present invention can exhibit a function similar to that of human Apo A I by using fenofibrate, which is known as an inducing compound that can significantly increase blood levels of T-CHO and HDL-CHO in humans. Was investigated. Fenofibrate is a compound that affects the expression rate of Apo AI genes of different species. It is known that fenofibrate reduces the expression of Apo AI mRNA in rats but increases the expression of Apo AI in humans.

By mixing fenofibrate with feed and investigating changes in the concentrations of T-CHO and HDL-CHO in fish bodies according to fenofibrate treatment period, the function of loach Apo AI gene is affected by fenofibrate. This experiment was conducted to find out what type it belongs to. For this experiment, feed containing 1% fenofibrate was fed to the control group and the loach of TG # 009 group, and blood was collected after 6 and 10 days to observe the T-CHO and HDL-CHO concentration changes. .

4-2. Results-Increasing T-CHO Serum Levels by Feeding Scaffolds

The blood concentration of T-CHO was 236.5 in those fed the control diet for 4 days. However, the concentration of T-CHO in the experimental group fed 5% pig scaffold for 4 days was increased by 78% to 301.0, showing a significant increase. This means that even in the case of loach, feeding CHO with increased cholesterol levels increases blood CHO. However, in the case of feeding 10% pig scaffolding, the feeding intake was very low, and the measurement of blood T-CHO concentration was not significantly different from the control group. Figure 4 is a graph showing the change in blood of total cholesterol (T-CHO) when feed the loaf scaffold supplemented loach.

4-3. Results-Increased blood levels of T-CHO / HDL-CHO by cholesterol-added diets

FIG. 5 is a graph showing changes in blood T-CHO and HDL-CHO when cholesterol-supplemented feed is supplied to loach, and FIGS. 5A, 5B, 5C, and 5D show cholesterol levels 0%, 1%, 2%, and 4, respectively. The case where% is added is shown. In this case, when the feed was fed to the control group for 6 days with various concentrations of cholesterol, there was a significant increase in both T-CHO and HDL-CHO according to the treatment period. As a result of T-CHO analysis, no significant increase was observed in the control group as the treatment period increased, but the 1 ~ 4% CHO supply group showed a significant increase according to the treatment period, especially in the 4% treatment group. There was a significant difference from day 2 of feeding, and on day 6 of feeding, T-CHO concentration of 433 mg / dl was nearly 1.6 times higher than that of the control group. In the case of blood HDL-CHO also showed a similar tendency to T-CHO. That is, the control group showed the same HDL-CHO blood concentration value during the experiment, but in the other treatment groups, the apparent HDL-CHO amount increased with the concentration. In particular, as in the case of T-CHO, the 4% and 6-day treatment groups showed a blood concentration of 251 mg / dL, which is 2.39 times higher than that of the control group. It can be seen that both amounts are increased.

As a result of the preliminary experiment, it was found that 4% of CHO treatment increased T-CHO and HDL-CHO in the control loach, and thus the concentrations of blood T-CHO and HDL-CHO in transgenic (TG) fish. Was analyzed. 6 is a graph showing the changes in blood T-CHO and HDL-CHO of the control loach and transformed loach when fed the cholesterol-added feed, Figure 6a is cholesterol 0% in the control group, Figure 6b is cholesterol 4% in the control group, FIG. 6C shows a case where 4% cholesterol is added to transform group-I and FIG. 6D shows 4% cholesterol added to transform group-II. From here, the CHO-free feed group did not change at all during the experimental period, as in the previous results. In addition, the results of the 4% feed control also showed that the concentration of both T-CHO and HDL-CHO increased with the treatment period, similar to the above results. However, the transgenic fish group showed a tendency to increase T-CHO slightly compared to the control group, but the HDL-CHO showed a tendency to increase 1.3-1.7 times compared to the control group at 4 days after treatment and 6 days after treatment. The 1.8-2.3 fold increase suggests that transgenic fish are more potent in cholesterol response than controls.

4-4. Results-Changes in Cholesterol Concentration of Transgenic Fish by Feeding Fenofibrate

In experiments to determine the changes in T-CHO and HDL-CHO concentrations in fish body with fenofibrate treatment period, 1% of fenofibrate was supplied, and in the control group, both T-CHO and HDL-CHO increased slightly with treatment period. Was observed, but there was no significant difference according to the treatment period. However, when fenofibrate was fed to transgenic fish, T-CHO increased 1.1-fold and 1.8-fold for HDL-CHO after 6 days of treatment. After 10 days, T-CHO increased 1.3 times and HDL-CHO increased 1.8 times. This shows that loach Apo A I gene can have similar function to human Apo A I. 7 is a graph showing changes in blood T-CHO and HDL-CHO of control and transformed loach when fed fenofibrate (1%) feed, Figure 7a is T-CHO, and Figure 7b is HDL-CHO Indicates.

The results obtained from the above experiments are summarized as follows:

First, in a basic experiment using scaffold-added feed, T-CHO increased 78% when fed 4 days or more with a pig scaffold of 5% concentration. It can be seen that it can artificially induce an increase in the concentration of T-CHO. Second, the conditions were reconfirmed by experiments inducing T-CHO and HDL-CHO increase using purified cholesterol.

Based on the results of these T-CHO and HDL-CHO induction experiments using general loach, functional transgenic loach that expresses Apo AI produced according to the present invention can respond quickly and sensitively to increased cholesterol levels in the body. Was evaluated. According to this study, 4% cholesterol-containing feeds that could cause up to two-fold changes in HDL-CHO in common loach were administered to transgenic loach in the same environment for the same period of time, resulting in increased T-CHO. There was no significant difference, but HDL-CHO showed a significant increase of 180-230% of the increase in the control group at 6 days of treatment. This is because the transformants rapidly regulate excess cholesterol, which may be excessively accumulated in tissues and blood vessels, through a reverse cholesterol transport (RCT) reaction. Ruben, et al ., 1991; Plump et al ., 1996).

According to the results of the present invention, fish cholesterol metabolism can be manipulated by regulating the expression of the Apo A I gene, hypercholesteramia and the disease model caused by excessive accumulation of cholesterol, as well as improving RCT ability. It is possible to develop molecular therapeutic models that resist atherosclerosis.

In addition, fenofibrate administration experiments showed that loach Apo AI is similar in human gene expression regulation to Apo AI. When fenofibrate was administered to the control and transgenic loach at a concentration of 1% in the feed, a relatively small increase in T-CHO / HDL-CHO was observed in the control, but a maximum of 180% was increased in the transgenic loach. The AI gene was judged to be subject to the human type Apo AI gene regulation mechanism (Berthou, L., N. Duverger, F. Emmanuel, S. Langouёt, J. Auwerx, A. Guillouzo, J. -C. Fruchart, E Rubin, P. Denefle, B. Staels and D. Branellec, 1996, Opposite regulation of human versus mouse apolipoprotein A I by fibrates in human apolipoprotein A I transgenic mice.J. Clin. Invest. , 97: 2408-2416).

That is, the loach Apo AI gene according to the present invention has a very similar protein structure and function despite the homology of the human Apo AI gene with a low DNA sequence, and the transgenic loach expressing the gene is a key regulator of cholesterol metabolism. It can produce large quantities of HDL, which is responsible for its function, thereby acquiring new capacity to cope with the accumulation of excess cholesterol strongly in the body, thereby preventing diseases caused by excess cholesterol such as atherosclerosis.

New functional transgenic loach produced according to the present invention can be developed as a functional food material containing a large amount of human useful substances through development as a culture material. In other words, the loach transformed loach according to the present invention Apo AI gene is overexpressed its own Apo AⅠ and thus contains a large amount of high-density liporotin (HDL), a useful component of the human body responsible for the regulation of cholesterol It is clear that much better health benefits can be created than with regular loach as a food.

Moreover, the transplantation of fish genes that function similar to human genes as in the present invention can generate useful information and added value for human disease and molecular therapy research (Walter, RB, 2001. Introduction: Aquaria fish models of human disease.Mar. Biotechnol. , 3: S1-S2). In other words, when discovering fish-like genes that are closely related to human diseases or metabolism and exerting their functions in experimental fish through transformation, useful information for functional genomics and disease treatment of human genes can be obtained. do. Specifically, in the present invention, it was possible to develop a new functional fish that acquired a specific phenotype of overexpression of loach Apo A I gene, which has a similar function to a human, which is a function study of a corresponding human gene (ie, human Apo A I), development of a therapeutic agent. In addition, it may provide useful experimental models for the development of diagnostic reagents.

Figure 1 shows the nucleotide sequence of the loach Apolipoprotein AI cDNA gene and the corresponding amino acid sequence.

Figure 2 is a schematic diagram showing the configuration of the expression vector containing loach apolipoprotein AI cDNA gene.

Figure 3 shows the results of Southern blot analysis of the transformed loach loach transplanted loach Apolipoprotein AI gene.

Figure 4 is a graph showing the change in blood concentrations of total cholesterol (T-CHO) when feed the pig scaffold added loach.

FIG. 5 is a graph showing changes in blood T-CHO and HDL-CHO when cholesterol-supplemented feed is fed to loach, and FIGS. 5A, 5B, 5C, and 5D show cholesterol 0%, 1%, 2%, and 4%, respectively. The case of addition is shown.

6 is a graph showing the changes in blood T-CHO and HDL-CHO of the control loach and transformed loach when fed the cholesterol-added feed, Figure 6a is 0% cholesterol in the control group, Figure 6b is 1% cholesterol in the control group, FIG. 6C shows a case where 4% cholesterol is added to transform group-I and FIG. 6D shows 4% cholesterol added to transform group-II.

7 is a graph showing changes in blood T-CHO and HDL-CHO of control and transformed loach when fed fenofibrate 1% feed, Figure 7a shows T-CHO, and Figure 7b shows HDL-CHO.

<110> PUKYONG NATIONAL UNIVERSITY OF KOREA <120> THE APOLIPOPROTEIN AI-TRANSGENIC MUD LOACH OVER-PRODUCING HDL-CHO <130> 1-300 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1110 <212> DNA <213> Misgurnus mizolepis <220> <221> CDS (222) (33) .. (794) <400> 1 ggcacgagac cagctacatc aacagatcca gg atg agg ttt ata gct 47 Met Arg Phe Ile Ala 1 5 ctt gca gtc aca gtt ctg ctg gca ggt tgc cag gca cgt ttc atg cag 95 Leu Ala Val Thr Val Leu Leu Ala Gly Cys Gln Ala Arg Phe Met Gln 10 15 20 gat gca cct cca tcg cag ctg gaa cat gtg aag tct gtt ctg cag gtg 143 Asp Ala Pro Pro Ser Gln Leu Glu His Val Lys Ser Val Leu Gln Val 25 30 35 tat gca gat caa ctg aaa caa tct gca cac aaa gcc ctc aat cac ctc 191 Tyr Ala Asp Gln Leu Lys Gln Ser Ala His Lys Ala Leu Asn His Leu 40 45 50 gat gac aca gag ttc aaa gac tac aaa ggg ttc ctg ggc cag tcc gtg 239 Asp Asp Thr Glu Phe Lys Asp Tyr Lys Gly Phe Leu Gly Gln Ser Val 55 60 65 gac aac ctc cat ggc tac ttt cag cat gcc ttc caa ggc att gcc cca 287 Asp Asn Leu His Gly Tyr Phe Gln His Ala Phe Gln Gly Ile Ala Pro 70 75 80 85 ttt gcc ggc cag ttc gct gaa gtc ttg gct cca aag atc gag cag ttc 335 Phe Ala Gly Gln Phe Ala Glu Val Leu Ala Pro Lys Ile Glu Gln Phe 90 95 100 aag aag gat atg gag gac atg cgc aag cag ctc gaa ccc aag cga gag 383 Lys Lys Asp Met Glu Asp Met Arg Lys Gln Leu Glu Pro Lys Arg Glu 105 110 115 gag ctg agg gct gtg ata gag aag cac ttt cag gag tac agc act gag 431 Glu Leu Arg Ala Val Ile Glu Lys His Phe Gln Glu Tyr Ser Thr Glu 120 125 130 ctc aag ccc atc gtc gat gag tac ttg gcc aaa cac gac aag gaa atg 479 Leu Lys Pro Ile Val Asp Glu Tyr Leu Ala Lys His Asp Lys Glu Met 135 140 145 gcg gag ctg aag gtc aag ctg gag cct gtg gta gag agc ttg aag cag 527 Ala Glu Leu Lys Val Lys Leu Glu Pro Val Val Glu Ser Leu Lys Gln 150 155 160 165 aaa att cct gtt aac tgg gag gag acc aag tcc aag ctg ctg ccc atc 575 Lys Ile Pro Val Asn Trp Glu Glu Thr Lys Ser Lys Leu Leu Pro Ile 170 175 180 ttg gag att gtg cgt aac aag att act gct cag gtc cag gat ctg aag 623 Leu Glu Ile Val Arg Asn Lys Ile Thr Ala Gln Val Gln Asp Leu Lys 185 190 195 gcc cag cta gag ccc tac atc cag gac tat aaa gat tca gtg gag aaa 671 Ala Gln Leu Glu Pro Tyr Ile Gln Asp Tyr Lys Asp Ser Val Glu Lys 200 205 210 ggc gcc ctg gaa ttc cgt gag aaa gtc cga tcc gga gaa ctg agg aaa 719 Gly Ala Leu Glu Phe Arg Glu Lys Val Arg Ser Gly Glu Leu Arg Lys 215 220 225 aag atg gac gaa ctg gga gcg gag gtc aag ccc cac ttt gag gct att 767 Lys Met Asp Glu Leu Gly Ala Glu Val Lys Pro His Phe Glu Ala Ile 230 235 240 245 ttt gca gct ctc caa aag tcc ttt gag taaagc aatcgctttt taacattccc 820 Phe Ala Ala Leu Gln Lys Ser Phe Glu 250 acttctttct tcctttccac ttatacccaa tacgaaaacc tcagcctttc tcagagaata 880 caccagtctg tctttcttct agagtacact tcatatgcac ttttcaccaa accactaaac 940 ttatggactt taatcttatg atcaaacaga gatataaaca tgagaacaca attcggttta 1000 acgttcacag aaattcttag caaaatcctt tgttaaatgc tttttttggt aaaaattgat 1060 ttgcaatgtt aaataaaata aaaactgacc aaaaaaaaaa aaaaaaaaaa 1110 <210> 2 <211> 254 <212> PRT <213> Misgurnus mizolepis <400> 2 Met Arg Phe Ile Ala Leu Ala Val Thr Val Leu Leu Ala Gly Cys Gln 1 5 10 15 Ala Arg Phe Met Gln Asp Ala Pro Pro Ser Gln Leu Glu His Val Lys 20 25 30 Ser Val Leu Gln Val Tyr Ala Asp Gln Leu Lys Gln Ser Ala His Lys 35 40 45 Ala Leu Asn His Leu Asp Asp Thr Glu Phe Lys Asp Tyr Lys Gly Phe 50 55 60 Leu Gly Gln Ser Val Asp Asn Leu His Gly Tyr Phe Gln His Ala Phe 65 70 75 80 Gln Gly Ile Ala Pro Phe Ala Gly Gln Phe Ala Glu Val Leu Ala Pro 85 90 95 Lys Ile Glu Gln Phe Lys Lys Asp Met Glu Asp Met Arg Lys Gln Leu 100 105 110 Glu Pro Lys Arg Glu Glu Leu Arg Ala Val Ile Glu Lys His Phe Gln 115 120 125 Glu Tyr Ser Thr Glu Leu Lys Pro Ile Val Asp Glu Tyr Leu Ala Lys 130 135 140 His Asp Lys Glu Met Ala Glu Leu Lys Val Lys Leu Glu Pro Val Val 145 150 155 160 Glu Ser Leu Lys Gln Lys Ile Pro Val Asn Trp Glu Glu Thr Lys Ser 165 170 175 Lys Leu Leu Pro Ile Leu Glu Ile Val Arg Asn Lys Ile Thr Ala Gln 180 185 190 Val Gln Asp Leu Lys Ala Gln Leu Glu Pro Tyr Ile Gln Asp Tyr Lys 195 200 205 Asp Ser Val Glu Lys Gly Ala Leu Glu Phe Arg Glu Lys Val Arg Ser 210 215 220 Gly Glu Leu Arg Lys Lys Met Asp Glu Leu Gly Ala Glu Val Lys Pro 225 230 235 240 His Phe Glu Ala Ile Phe Ala Ala Leu Gln Lys Ser Phe Glu 245 250

Claims (6)

CDNA of the Misgurnus mizolepis apolipoprotein AI described in SEQ ID NO: 1. DNA encoding the amino acid sequence set forth in SEQ ID NO: 2. Expression vector containing cDNA of Misgurnus mizolepis apolipoprotein AI. delete Misgurnus mizolepis overproducing high density lipoprotein-cholesterol, transformed with the expression vector of claim 3. A method of producing a transformed loach ( Misgurnus mizolepis ), which overproduces high-density lipoprotein-cholesterol, comprising the step of incubating the locus of the expression vector of claim 3 in a loach embryo.