TWI356098B - Promoter of secreted protein acidic and rich in cy - Google Patents

Description

1356098 IX. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a promoter, in particular, to a protein secretory rich in cysteine (secrete (j protein acidic and rich in cysteine, SPARC) The promoter of the gene. [Prior Art]

The transcriptional system of genes in organisms is controlled by a promoter. The promoter is usually located upstream of the gene' with the recognition and binding site of RNA polymerase, allowing RNA polymerase to catalyze transcription. The amount of expression, timing of expression, and regulation of gene expression are closely related to the sequence of the promoter. Many genes that are regulated by a regulator exhibit a promoter region that specifically binds to a regulator, and can regulate gene expression either positively or negatively by binding to a regulator. The cysteine-rich acidic secreted protein, also known as osteonectin or BM40, is a cell-matrix glycoprotein, and the cysteine-rich acidic secreted protein of vertebrate is from about 298 to 304 amine groups. The acid composition consists of a signal sequence containing 17 amino acids that directs secretion of the protein to the outside of the cell. It has 14 highly-retained cysteine to form a disulfide bond (Chun et al., Eur J Oral) 2006; 114: 78-85), and the peptide chain is modified by glycosylation. The cysteine-rich acidic secreted protein can be divided into three functional regions: (丨) Acidic domain: located at the n-terminus of the protein, about 50 amino acids in length, and this region can be combined with 5 to 8 calcium. Ions form a weak affinity binding function, which has the function of inhibiting cell elongation and regulating extracellular matrix production; (2) follicle inhibitory region 133930.doc ί S3 (Follistatin like domain): this region contains about 8 amines behind the acidic region The base acid, which contains a plurality of cysteine and has a function similar to follicle statin, inhibits cell proliferation, and has a copper ion-binding sequence (k) ghk (Iruela-Arispe et al., Mole Biol Cell 1995; 6 : 327-343), has the function of promoting the proliferation of blood vessels; (3) the extracellular ionic binding region (Ec domain): at the C-terminus, having two EF hand positions can form a high affinity binding with the mater ion, Binding to cells or certain collagen also has the effect of inhibiting cell proliferation and elongation (Maurer et al, j Molec Biol 1995; 253: 347-357; Catherine et al, j ceu physiol 2006; 206:211-220) . Although the acidic secreted protein containing cysteine is mainly expressed during embryonic development, it has a great influence on cell differentiation, tissue formation, bone development, morphology and organ development. When it is a biological adult, this protein The performance is reduced 'usually only in the eye, intestines and bones (Bradshaw et al, Proc Natl Acad Sci USA 2003; 100: 6045-6050), and when the skin or tissue is injured and repaired The recombination process has a more pronounced performance (Lane et al., FASEB J 1994; 8: 163-173). The association between the cysteine-rich acidic secretory protein and the extracellular matrix is a regulatory protein (B〇rnstein et al, j Cell Biol 1995; 130: 503-506). In vitro culture of mouse endothelial cells observed that 'cysteine-rich acidic secreted protein inhibits cell type elongation', making cells tend to be round (Murphy-Ullrich et al, J Cell Biol 1991; 115: 1127- 1136), mainly by regulating the expression of extracellular matrix components and extracellular matrix proteins to change the junction between cells and extracellular matrix 133930.doc 1356098

Combine, thereby altering cell type and enhancing migration capacity (Hasselaar et al, J Biol Chem 1991; 266: 13178-13184; Tremble et al, J Cell Biol 1993; 121: 1433-1444); cysteine-rich Acidic secreted proteins can also inhibit cell cycle progression, which may inhibit cell growth by activating the TGF·non-signaling pathway (Schiemann et al., Mol Biol Cell 2003; 14: 3977-3988) to stop the cycle in the mid-G1 phase. (Yan et al., J Histochem Cytochem 1999; 47: 1495-1505); Furthermore, studies on vascular cell culture have also found that cysteine-rich acidic secreted proteins have properties that promote vascular proliferation (Funk et al. j Cell

Physiol 1993; 154: 53-63) °

In addition to the effects on cells, in the in vivo experiment, in the sparc knockout mice (Bradshaw et al., 2003), abnormal changes in skin composition were observed, 'fat layer thickening, in addition, visceral fat and attachment睪 Fat 塾 is significantly thickened' The volume of fat cells has also increased and the number has increased. The concentration of leptin secreted by fat cells in the blood has increased. Therefore, it is speculated that the acidic secreted protein rich in cysteine may be related to regulating fat ( Chavey et al,

Obisity 2006; 14: 1890-1897). In the comparison of the promoter sequences of the cysteine-rich acidic secreted protein gene obtained from mammals such as humans, mice and cattle, there is a reserving repetitive sequence, which is about 7 in front of the gene expression 5 〇 to 25 bp, composed of multiple sets of GGA short fragments, called GGA box (Hafner et al, Matrix Biol 1995; 14: 733-741), can help the rear gene

A large number of performances were performed when necessary. However, the promoter sequence derived from mammals lacked the TATA box required for general developmental gene regulation and the gAAT 133930.doc 1356098 box, while the promoter of the amphibians frog t was selected. The sequence characteristics are exactly the opposite. 'The sequence is unreserved with mammals and does not have a Gga box, but it can find the TATA box on the developmental gene, which is shown in the process of evolution, between amphibians and mammals. Divergence (Sashk〇 et al., Dev Genes Evol 1998; 207: 453-461). The species of the grouper (Epinephehs spp.) is classified into the order of the genus Bonefish (Osteichthyes, the genus n iPercif 〇 rms, the cockroach (Serr£mida illusion, Epie epheHnae, and the genus (Pinepheit 4S)). It is a warm-water fish that is widely distributed in tropical and subtropical waters around the world and is recognized by the aquaculture community as the most important economic fish species in the Asia-Pacific region. Due to the functions regulated by the spare gene, such as appearance development, cell cycle, and immune function (Rempel et al. Genes Immun 2007 ; 8 : 1-13) and fat

^ There are still no studies on the cysteine-rich acidic secretin promoter for grouper. SUMMARY OF THE INVENTION Summary of the present invention The cysteine-rich acidic secreted protein gene can be used to construct a gene expression system. The present invention provides grouper fish

Promoter and functional fragments thereof, the primary object of the invention is to provide 133930.doc 1356098 comprising a promoter selected from the grouper having cysteine rich in the sequence of SEQ ID NO: 1 A promoter consisting of a promoter of an acidic secreted protein gene and a functional fragment of a transcribed polynucleotide molecule operably linked thereto. Another object of the present invention is to provide a carrier comprising the above-described nucleic acid molecule. It is still another object of the present invention to provide a kit comprising the above described carrier.

It is still another object of the present invention to provide a cell comprising the above-described vector. Detailed description of the invention

The main object of the present invention is to provide an isolated nucleic acid molecule comprising a promoter, wherein the promoter is selected from the group consisting of a promoter of a cysteine-rich acidic secreted protein gene and its operably linked A population consisting of a functional fragment of a transcribed polynucleotide molecule, wherein the promoter of the cysteine-rich acidic secreted protein gene has the sequence set forth in SEQ ID NO: 1. The result of analyzing the promoter sequence of the cysteine-rich acidic secreted protein gene promoter according to the present invention is shown in Fig. 1. The sequence analysis of the promoter revealed that the sequence is close to the binding of multiple transcription factors at the starting point of translation. The sequence includes a TATA box, a nuclear factor 1 (NF1) binding site, a GC cassette recognized by Sp 1 , a GC-binding factor (GCF) transcriptional repressor binding site, and a CCAAT/promoter binding protein. (CCAAT/Enhancer-binding protein, C/EBP) binding site and two-10-133930.doc Γ %} 56098 is a transcriptional gene pair regulated by leucine zipper

The MyoD binding site, in particular the C/EBP (leucine zipper)-based protein, plays a very important role in the development and differentiation of liver white adipose tissue and is also involved in anti-mitotic activity (Thurl et al.; Bi〇l) Chem 2001; 276: 29200-29209), and previous studies have indicated that the function of the cysteine-rich acidic secreted protein protein is consistent. The promoter sequence of the cysteine-rich acidic secreted protein gene also has two regulatory positions of the elbow transcription factor, and its main function is to promote the expression of muscle-related genes during embryonic development (M〇hun Et al, EMBO J 1989; 8·1153-1161) 'Explain that during the development of muscle, the cysteine-rich acidic secreted protein gene is likely to be involved. According to the present invention, the cysteine-rich acidic secreted protein promoter has a TATA box but no GGA box, indicating that the retention characteristics of the promoter fragment are close to those of the amphibians, although not wishing to be a theoretical Limitations, but the differences in retention of fish, amphibians, and mammals may be related to embryonic developmental ticks or to the different transcriptional regulators involved in embryonic development of oviparous and viviparous animals (Sashk〇 et al. 1998). According to the present invention, the cysteine-rich acid is confirmed by the still "fish fin cell strain!" The sclerotin promoter can initiate the downstream reporter gene expression, confirming that the SEQ ID sequence of the present invention is The cysteine-rich acid-protein promoter has a promoter function. In a preferred embodiment of the invention, the nucleic acid molecule comprises 133930.doc 1356098 as shown in SEQ ID NO: 1. Preferably, the grouper is a spotted grouper kiss-heart (10). All. This fragment refers to a DNA fragment having the function of a phospholipid-rich acidic secreted protein promoter such as the grouper of the present invention. • In a preferred embodiment of the invention, which further comprises a structural gene, the structural gene is initiated by the semi-proline-rich acidic secretory protein-based promoter or a functional fragment thereof. In the present invention, preferably, the structural gene comprises a reporter gene which is initiated by the grouper's acidic cysteine-rich acid secretion promoter or a functional fragment thereof. The reporter gene can be used to monitor the activity of the cysteine-rich acidic secreted protein promoter and, preferably, can be used to quantify the activity of the semi-proline-rich acidic secreted protein promoter. In a preferred embodiment of the invention, wherein the reporter gene is selected from the group consisting of a fire luciferase gene, a green fluorescent protein, and a galactosidase. In the preferred embodiment of the invention, the nucleic acid molecule can comprise both the reporter gene and the non-reporter structural gene. Since the reporter gene and the non-reporter gene are activated by the same promoter, the amount of expression of the non-reported gene can be known by measuring the amount of the reporter gene, and can be applied to monitor or quantify the non-reported structure. The performance of genes. The invention also provides a vector comprising the nucleic acid molecule described above. The vector can be used for preserving, producing, or for introducing the nucleic acid molecule into an organism or a cell; preferably, the vector comprises an origin that can be replicated in a prokaryote and can be used for a gene The restriction enzyme limit of operation is 133930.doc 12 1356098. Those of ordinary skill in the art to which the present invention pertains can accomplish the construction of such carriers in accordance with the teachings of the present invention. The invention further provides a kit comprising the above described carrier. The kit is easy to handle and can additionally contain the buffer required for the transformation.

The present invention further provides a cell comprising the vector described above, which is obtained by introducing a transformation into a cell, which may be a prokaryotic cell or a eukaryotic cell, preferably a bacterium. The term "transformation" as used herein refers to the alteration of genetic material in a cell via the introduction of a nucleic acid molecule. Such a transformation can be accomplished by a person of ordinary skill in the art having the knowledge of the present invention and general molecular biology, such as by introducing a carrier into a bacterium, using a heat shock mode. The invention is illustrated by the following examples, which are not intended to be construed as limiting the invention. [Embodiment]

Extraction of tissue DNA After placing the grouper on the ice, dissect the muscle tissue and disassemble about 30 mg of the tissue in a lysis buffer (10 mM Tris-cl (pH 8.0), 1 mM EDTA (pH 8.0), 0.1% (w/v). Within SDS), treated with protein hydrolase K at 60 ° C for one hour. Then add the high salt solution and alcohol, mix well, place the supernatant into Genomic DNA Mini Column (VIOGENE) and centrifuge at 800 rpm to precipitate the DNA, pour off the effluent liquid, and centrifuge at 1300 rpm for two minutes to remove the remaining in the column. Alcohol, the DNA was finally dissolved back into 200 pL of pure water and collected by centrifugation and stored at -13-133930.doc _[S] 1356098 20〇C. DNA walking

Experiments were performed with the GenomeWalker Universal Set (BD). The extracted fish DNA was randomly cut into four 2Kb long, end-cut DNA fragments by four restriction enzymes Dra I, EcoR V, Pvu II and Stu I, purified by phenol and gas, and then linked by T4. The enzyme was subjected to a DNA ligation reaction, and the adaptor (5'-GTAAT ACGAC TCACT ATAGG GCACG CGTGG TCGAC GGCCC GGGCT GGT-3', SEQ ID NO: 2) was ligated to DNA and polymerized as four databases. Enzyme chain reaction screening. The method uses a first polymerase chain reaction with a specific set of reverse primers (GSP1-R, 5'-ACTTT GCAGA GCAGG GACAA GAGAA GTC-3', SEQ ID NO: 3) and the forward direction of the adaptor. The primer (5'-GGTAA TACGA CTCAC TATAG GGC-3', SEQ ID NO: 4) was used to amplify the nucleic acid fragment, and the second polymerase chain reaction was followed by another set of specific reverse primers (GSP2-R, 5' -AGCTG TGTGC GGAGC CCGGG ACGAT CTC-3', SEQ ID NO: 5) and the adaptor forward primer (5'-ACTAT AGGGC ACGCG TGGT-3', SEQ ID NO: 6) for more accurate nucleic acid fragment amplification. The resulting product was subjected to electrophoresis analysis, and the amino acid gel at the target nucleic acid position was excised and the nucleic acid sequence was sequenced after the target nucleic acid was ligated into pGEM-T easy vector systems (Promega). The specific primers were designed using the 5-terminal UTR of the full-length cDNA of the known cysteine-rich acidic secreted protein gene and the DNA database formed by cutting the four different restriction enzymes was screened from Dra I data 133930. .doc [S3 1356098

A DNA sequence of about 600 bp in length was obtained from the library, a DNA sequence of about 1000 bp was obtained from the ρνι1 η database, and a 1 bp sequence was obtained from the PVU II database, which contained the entire database from the Dra Ϊ database. A sequence of about 6 〇〇 Qi long was obtained, and the ProteinScan promoter was used to predict the software prediction, and the transcription factor recognition sequence of the acid secreted protein gene known to contain cysteine was obtained, and the binding sites of many transcription factors were obtained. The TATA box, NF1, Sp1, C/EBP and MyoD, it is speculated that the DNA fragment selected from the pvu(R) database is likely to be a promoter fragment of the cysteine-rich acidic secreted protein gene (Fig. 1 ) which has the sequence of SEQ ID NO: 1. Reporting gene expression plastid construction

The promoter sequence of the cysteine-rich acidic secreted protein gene was modified by polymerase chain reaction to cleave the anterior-posterior sequence, so that it was terminated by Ec〇R, and 3, terminated with BamH I identification. The cleavage site carries out a polymerase chain reaction with pGEM_T easy... containing a cysteine-rich acidic secreted protein gene promoter sequence. The amplified nucleic acid fragment was ligated with the restriction enzymes EcoR I and BamH I for 5 hours, and then inserted into the pEGFP-Ι reporter gene expression vector treated with the same restriction enzyme, and the ligation reaction was carried out in the 14_^ water bath using T4 ligase. After 8 hours, the cells were transformed into Escherichia coli (ΗΒ101), and the selected strain was successfully ligated to the expression vector. The EGFP-1 vector was used to attach the green fluorescent protein to the promoter, and the green fluorescent protein was used to judge the expression. The strength and characteristics of the subsequence. Transfection of the cells. The GF1 cells were cultured in a well of 6 wells for about seven minutes. The extracted plastids were 2/zg with 1 〇〇 serum-free cell culture medium and 6 juL PLUSTM 133930.doc. -15- 1356098

Teagent (Invitrogen) was uniformly mixed and allowed to react at room temperature for 15 minutes, and then added to 100/iL of serum-free cell culture medium mixed with 4 and reacted at room temperature for 5 minutes. Add the serum-free cell culture solution to the culture chamber for 3 hours. After the reaction, replace the culture medium with the culture medium with a normal culture concentration of 5% FBS. Observe the cell performance after 48 hours (Liao Zhongjian, 2006) 〇

In order to confirm that the promoter fragment can drive the expression of the downstream gene, a specific primer with a restriction enzyme cleavage site is designed to amplify the fragment, and then cloned into a promoter-free green fluorescent gene gfp expression vector.卯" If the fragment is indeed a functional promoter, the green fluorescent light emitted by the reporter gene can be seen under UV light, and the selected plastid is transfected into GF1 cells to test its intensity (Fig. 2), the control group is to send the empty vector pEGFP-1 without the promoter fragment #, and the green fluorescent expression is not seen completely under the UV illumination, and the acidic secreted protein containing cysteine-rich acid is fed. In the experimental group of the gene promoter plastid pEGFP-1/cysteine-rich acidic secreted protein promoter, green fluorescence was observed in the cells under UV light, which confirmed that the use of cysteine-rich cells The 5-terminal UTR of the full-length cDNA of the acid-secreting protein gene of the amino acid is designed to be the promoter of the downstream gene expression. The above-described embodiments are merely illustrative of the principles and effects of the invention and are not intended to limit the invention. Modifications and variations of the embodiments described above will be apparent to those skilled in the art without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims. [Simplified illustration] 133930.doc 16- 1356098 Figure 1 shows the promoter sequence of the cysteine-rich acidic secreted protein gene.

Fig. 2 is a graph showing the results of functional analysis of the promoter sequence of the acid secreted protein gene rich in cysteine in grouper. The green fluorescent protein produced by the promoter sequence of the cysteine-rich acidic secreted protein gene was observed in GF_丨 cells by transfection. Panel A shows the cell type of pEGFP-Ι vector transfected into the promoter-free sequence in the bright field; Figure B shows the green fluorescence of the field of view under UV light excitation, Εχ: 490 nm, Em : 510 nm, C picture is the cell type of pEGFIM vector transfected into the promoter sequence of the acidic secreted protein gene containing the cysteine-rich acid secreted protein gene in the bright spot; D picture is the C field of view under UV light excitation Green fluorescent performance,

Ex: 490 nm, Em: 5 10 nm, it can be judged that the promoter sequence can indeed drive downstream gene expression. 133930.doc 1356098 Sequence Listing . <110>National Success University <120> Promoter of semi-deaminase-rich acidic secreted protein gene <130> None <160>6 <170> Patentln version 3.3

≪ 210> 1 < 211 > 1050 • < 212> DNA < 213 > grouper < 400> 1 ctgagtgaag tggagcctga ggaggaagca ccggttagcc ccggtttcac tacaggcaga 60 ttcactcgct acaggggcga ggaaataaaa cctgaacagc caatcagagt gatctctctc 120 accgacgagc tccgctgccg attcaacatg ctcaatcggc cgaaaaagtc tcagacgctg 180 acagtgctcc gtcccttcag ctgcaatgtc aaattcaaga cacggggaga gagtggagac 240 agacagagag gacatgtctc atgctgcaga ttgtcctgtt gaatacaata ataaaacaca 300 tcagactcag tgtgtaaagt ttctctgagt tttttccttt cggatgacgg attaaaaaaa 360 caccataaaa aggactcggt gtgcgacggc ctttaatcat gtgactctca gacctttaaa 420 atgacctcag actgagtgaa tcaaatcctg acagtgaaac aaatcatttc tcagggtttt 480 tgtatccact gatttacagg cgtctttttc actgtgggaa gaagtgtttc tgtgccgcat 540 gacgtgaggg acttggaagc tggagtaact gggtctggca attatatcaa actggtttaa 600 gcccagtgct gctcctggac Gcctgatgtc aatgtgtgtg ccaagtttca ggagtttttt 660 133930.doc 1356098 aggtttacca agtgttcaga agcaccatcc aaacatacaa acagatctga atgattataa 720 atagtgactg tagctcatct gtccatttat gggtcgagtc agctccgaac atcaaacaga 780 gaaggaacta ccagaaaaag tttattttct caaactgcag gttcagttca gactcagcgt 840 taggccccgc ccaccagggc aggagggaga gggagggggg gtctgtgtcc aaactgggac 900 gtcccactgg aaatgtcttc tgtgtgagtg gtgaagcagt gagatcgtcc tgggccccgc 960 acacagctga cttctctcct ggtgtccctg ctctgcaaag cttaagactt tgaagatgag 1020 ggtgtggatc gtcttcctcc tgtgcctggc 1050 < 210> 2 < 211 > 48 < 212> DNA <213>Artificialsequence<400> 2 gtaatacgac tcactatagg gcacgcgtgg tcgacggccc gggctggt 48 kmi <210> 3 ® <211> 28 <212> DNA <213>Artificial sequence <400> 3 . actttgcaga gcagggacaa gagaagtc 28 &lt ; 210>4 <211> 23 <212> DNA <213> artificial sequence 133930.doc -2- 1356098 <400> 4 ggtaatacga ctcactatag ggc 23 <210>5 <211> 28 <212> DNA <213> Artificial Sequence <400> 5 agctgtgtgc ggagcccggg acgatctc 28

<210> 6 <211> 19 <212> DNA <213> Artificial sequence <400> 6 actatagggc acgcgtggt 19 133930.doc

Claims (1)

1356098 • Patent Application No. 97145535 _ Announcement Chinese Application ^Lan Scope Replacement (July 1) Monthly Force Correction Replacement Page 10, Patent Application Range: ------- 1. An isolated nucleic acid molecule comprising a promoter, wherein the promoter has a sequence as shown in SEQ ID NO: 1. 6. The spotted fish contains a cysteine-containing acidic secreted protein gene (secretedpr〇teinacidic andrichin cysteine, The promoter of SPARC). 2_According to the request item! The nucleic acid molecule 'where the grouper is a grouper {Epinephelus coioides). 3) A nucleic acid molecule according to claim 2, which comprises the sequence shown as SEQ ID N: i. 4. A nucleic acid molecule according to the claims, further comprising a structural gene which is initiated by the cysteine-rich acidic secretory protein gene promoter. 5 - A nucleic acid molecule according to claim 4, wherein the structural gene comprises a reporter gene. 6. The nucleic acid molecule according to claim 5, wherein the reporter gene is selected from the group consisting of a firefly luciferase gene, a glucose acidase gene, a green fluorescent protein gene, and a β-galactoside A group of enzymes (β-galactosidase) genes. A vector comprising the nucleic acid molecule according to any one of claims 1 to 6. 8. A kit comprising a carrier according to claim 7. A cell comprising the vector according to claim 7. 10. The cell according to claim 9, wherein the cell is a prokaryotic cell. 11. The cell according to claim 9, wherein the cell is an animal cell. 133930-20110707