JP4544834B2 - Polynucleotides that promote foreign gene expression - Google Patents

Description

【図面の簡単な説明】
【図１】フィブロイン重鎖遺伝子の構造と転写制御領域。フィブロイン重鎖遺伝子は２つのエクソン（太線で示した）が存在する。数字はフィブロイン重鎖遺伝子のヌクレオチド番号を示す（転写開始点を＋１とする）。矢印で示した部分を転写活性測定に用いた。()はin vitro転写系で測定された転写活性（コアプロモーターの活性を1とした場合）(Suzuki et al., 1986, Takiya et al., 1997)である。
【図２】活性測定法の概略である。
【図３】 in vivo測定系における転写制御領域の転写活性。図１で示したフィブロイン重鎖遺伝子領域を用いて転写活性を測定した。フィブロイン重鎖遺伝子コアプロモーターの活性を1とした。白丸は個々の活性値、黒丸はその平均値をあらわす。
【図４】フィブロイン重鎖遺伝子5' 上流領域の転写促進活性。フィブロイン重鎖遺伝子-235から5'上流を遡ってコンストラクトを作製してその転写活性を測定した。
フィブロイン軽鎖遺伝子-600〜+34の活性を1とした。白丸は個々の活性値、黒丸はその平均値をあらわす。
【図５】フィブロイン重鎖遺伝子5' 上流領域部分欠損コンストラクトの構造。フィブロイン重鎖遺伝子-5000〜-235までの転写促進活性の性質を明らかにするために-5000〜-235の間で様々な部分を欠損させたコンストラクトを作製した。
【図６】フィブロイン重鎖遺伝子5'上流領域部分欠損が転写活性に及ぼす影響。図５で示したコンストラクトの転写活性を測定した。フィブロイン軽鎖遺伝子-600〜+34の活性を1とした。白丸は個々の活性値、黒丸はその平均値をあらわす。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a polynucleotide that promotes the expression (transcription activity) of a foreign gene. More particularly, the present invention relates to a polynucleotide useful for mass production of useful recombinant proteins by silkworms, for example.
[0002]
[Prior art and its problems]
Silkworms make cocoons just before they become cocoons. The main components of this cocoon are silk proteins such as fibroin and sericin, which are synthesized in the silkworm silk gland. The silk gland is composed of a posterior silk gland, a middle silk gland, and an anterior silk gland. Fibroin is specifically synthesized and secreted in the posterior silk gland, and sericin is specifically synthesized and secreted in the middle silk gland.
Fibroin is a complex composed of heavy and light chains and occupies about 70% of the total silk protein. Fibroin secreted from the posterior silk gland is gradually sent to the middle silk gland by the peristaltic movement of the silk gland, where it is covered by the secreted sericin, and then sent to the anterior silk gland and spun as silk thread. Is done.
[0003]
The average silk thread to be synthesized reaches about 0.3 to 0.5 g per silkworm. Thus, silkworms are organisms having high protein synthesis ability, and an excellent recombinant protein production system can be constructed by utilizing this ability.
[0004]
As a transient foreign gene expression system in silkworm, a method using silkworm nuclear polyhedrosis virus (BmNPV) as a vector has been established (Patent Document 1). In addition, transgenic silkworms in which foreign genes are permanently incorporated throughout generations have been reported by microinjecting silkworm eggs with a plasmid vector that incorporates piggyBac, a DNA-type transposon derived from the lepidopterous insect Trichoplusia ni. (Non-Patent Document 1).
[0005]
Further, the inventors of this application incorporated a human collagen cDNA linked downstream of a silk protein gene promoter into a silkworm, and produced a transgenic silkworm that produced recombinant human collagen as part of a protein in a silkworm or silk gland. Have been filed for patents (Patent Documents 1 and 2, Japanese Patent Application No. 2002-177536).
[0006]
By the way, in the expression of foreign protein in silkworm, the foreign gene is expressed under the silk protein gene promoter control such as fibroin and sericin in order to express the foreign gene in the silk protein gene expression mechanism.
[0007]
However, as a promoter for gene expression in the silk gland, for example, when the region from -600 to +34 of the fibroin light chain gene is used, the content of the recombinant protein secreted into the silkworm by the transgenic silkworm is 0.8%. (Non-Patent Document 1), productivity is not necessarily high. Therefore, in order to produce a large amount of recombinant protein, it is necessary to develop a promoter / enhancer having higher transcriptional activity and improve the content of the recombinant protein in the straw.
[0008]
On the other hand, for the fibroin heavy chain gene, -30 to +6 (transcription start point is +1) is indispensable for transcription (Non-patent Document 2), so this region is called “core promoter”. ing. In addition, elements that increase promoter activity in the region up to -234 upstream of the core promoter and in the intron (referred to as upstream element and intron element, respectively) have been found (Non-Patent Documents 3 and 4).
[0009]
However, in reality, fibroin heavy chain protein cannot be expressed to the same extent as usual only by these core promoters and elements. For this reason, it is assumed that a region that promotes transcriptional activity is present in the upstream region of the gene. Therefore, in order to express a large amount of foreign protein using the promoter / enhancer of the fibroin heavy chain gene, it is conceivable to use the longest possible region upstream from the upstream element of the fibroin heavy chain gene. However, when an expression cassette for a foreign protein structural gene is constructed using a long promoter / enhancer sequence, there is a problem that handling and production of transgenic silkworms are extremely difficult.
[0010]
The invention of this application has been made in view of the circumstances as described above, and solves the problems of the prior art, and provides a polynucleotide having a high transcription activity and an excellent size. Is an issue.
[0011]
Another object of the invention of this application is to provide a method for expressing a recombinant protein that enhances the transcriptional activity of a gene using the polynucleotide described above.
[0012]
[Patent Document 1]
Japanese Patent Publication No.7-97995
[Patent Document 2]
JP 2001-161214 A
[Patent Document 3]
JP 2002-315580 A
[Non-Patent Document 1]
Tamura et al., Nat. Biotechnol. 18, 81-84, 2000
[Non-Patent Document 2]
Tsuda and Suzuki, Cell, 27, 175-182, 1981
[Non-Patent Document 3]
Suzuki et al., Proc. Natl. Acad. Sci. USA, 83, 9522-9526, 1986
[Non-Patent Document 4]
Takiya et al., EMBO J., 9, 489-496, 1990
[0013]
[Means for Solving the Problems]
This application provides the following inventions (1) to (11) as an invention for solving the above-mentioned problems.
(1) A polynucleotide linked to a foreign protein structural gene in order to promote the expression of a foreign protein in the silkworm silk gland, wherein the following polynucleotides (A) and (B):
(A) a polynucleotide constituting a promoter / enhancer region of a silk protein gene;
(B) a polynucleotide comprising a nucleotide sequence comprising at least the 3141-3874th nucleotide sequence in the 2787-4766th nucleotide sequence of SEQ ID NO: 1,
A polynucleotide to which is linked.
(2) The polynucleotide of the invention (1), wherein a polynucleotide (C) consisting of the nucleotide sequence at positions 1-1153 of SEQ ID NO: 1 is further linked to the 5 ′ side of the polynucleotide (B).
(3) The polynucleotide of the invention (1) or (2), wherein the polynucleotide (A) comprises the 4767-5024th nucleotide sequence of SEQ ID NO: 1.
(4) The invention according to the invention having addition or deletion of one or more bases, or substitution with another base within a range having substantially the same function as the polynucleotide of the inventions (1) to (3) The polynucleotide according to any one of (1) to (3).
(5) One or more complementary sequences of the polynucleotides (A), (B) and (C) in the invention (1) or (2) hybridize under stringent conditions, and the invention (1) To (4) a polynucleotide having substantially the same function.
(6) The polynucleotide of the inventions (1) to (5), wherein the polynucleotide to which the polynucleotides (A), (B) and (C) are linked has a length of 3500 bases or less. Polynucleotide.
(7) An expression cassette in which the polynucleotide according to any one of the inventions (1) to (6) and a foreign protein structural gene are linked.
(8) An expression vector having the expression cassette of the invention (7).
(9) The expression vector according to the invention (8) having an expression cassette sandwiched between a pair of inverted repeats of an insect-derived DNA-type transposon.
(10) A method for expressing a foreign protein, wherein the foreign protein gene contained in the expression cassette of the invention (7) is expressed in a silkworm silk gland.
(11) A transgenic silkworm having the expression cassette of the invention (7) in the genome and expressing a foreign protein in the silk gland.
[0014]
That is, each of the above-mentioned inventions has been completed by paying attention to the fibroin heavy chain gene having high transcription activity and specifying the polynucleotide as the minimum region that promotes the transcription activity of the gene from the upstream region.
[0015]
The sequence of the fibroin heavy chain gene which is the basis of the polynucleotide of the present invention is known (GenBank / AF2266688), and its full length (80009 bp: hereinafter referred to as “known sequence”), the 57714-63450 sequence is shown in the sequence listing. It is shown in SEQ ID NO: 1. Furthermore, position 62414 of the known sequence is the transcription start point (+1), which corresponds to the base of position 5001 of SEQ ID NO: 1. Also, position 57414 of the known sequence is -5000. Corresponds to the first base of SEQ ID NO: 1. In the following description, the 5 ′ side from the transcription start point (+1) is described as a (−) number, and the 3 ′ side is described as a (+) number. For example, the nucleotide sequence at positions 2787-4766 of SEQ ID NO: 1 is described as “-2214 to −235 region”.
[0016]
In the present invention, the term “polynucleotide” refers to a nucleoside phosphate ester (ATP, GTP, CTP, UTP; or dATP, dGTP, dCTP, dTTP) in which purine or pyrimidine is β-N-glycoside-linked to a sugar. It means a molecule that is bound more than one. A polynucleotide and another polynucleotide are “linked” when the 3 ′ terminal nucleotide of one polynucleotide and the 5 ′ terminal nucleotide of the other polynucleotide are linked directly or through another linker sequence. The state that is.
[0017]
Furthermore, in this invention, “protein” means a molecule composed of a plurality of amino acid residues linked to each other by amide bonds (peptide bonds), and “foreign protein” means an animal other than the protein produced by silkworms. It means a protein preferably produced by genetic engineering as a sex protein, particularly a recombinant protein.
[0018]
Furthermore, the “foreign protein structural gene” in the present invention is a polynucleotide containing a region (open reading flame: ORF) encoding a foreign protein, for example, a cDNA of a foreign protein gene.
[0019]
Other terms and concepts in the present invention are defined in detail in the description of the embodiments of the invention and the examples. Various techniques used for carrying out the present invention can be easily and surely implemented by those skilled in the art based on known documents and the like, except for a technique that clearly indicates the source. For example, genetic engineering and molecular biology techniques are described in Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc.
[0020]
Hereinafter, embodiments of the present invention will be described in detail.
[0021]
DETAILED DESCRIPTION OF THE INVENTION
In order to search for sequences having high transcription activity in silk gland cells in addition to the above-mentioned known transcriptional regulatory regions, the inventors of this application first established an experimental system for measuring the transcriptional activity of a promoter in vivo. The firefly luciferase reporter plasmid into which the promoter was inserted was introduced into the silk gland using a gene gun, the gene-introduced silk gland was transplanted to another individual, and luciferase activity was measured 3 days later. Core promoters, upstream elements and intron elements identified in the in vitro transcription system showed the same activity in in vivo systems using gene guns as in in vitro systems. From this result, it was confirmed that the in vivo experimental system was effective in measuring promoter activity.
[0022]
Next, the region that promotes the transcriptional activity of the gene was searched retrospectively upstream of the heavy chain gene. The -5000 to -235 region of the fibroin heavy chain gene was linked to the -234 to +24 region and its transcriptional activity was measured. The -5000 to -235 region was transcribed by the -234 to +24 region. It has been found that there is an action that strongly promotes the activity. Furthermore, when the transcriptional activity was measured by linking to the region -234 to +24 except for the region −3847 to −2215 from the region −5000 to −235, the result was −5000 to −235 to −3847 to −3. It was revealed that the region excluding 2215, that is, only the region of −5000 to −3848 and −2214 to −235 has the same transcriptional promoting activity as −5000 to −235. In addition, by conducting an experiment to measure the transcription promoting effect of a region having various deletion sites, the region from -1860 to -1127 is particularly important for the transcription promotion among the regions from -2214 to -235. It was confirmed that a high transcription promoting effect was exhibited by connecting the region to the region of -5000 to -3848. Each invention of this application has been completed based on the novel findings as described above.
[0023]
Invention (1) of this application is a polynucleotide in which the following polynucleotides (A) and (B) are linked.
(A) A polynucleotide constituting a promoter / enhancer region of a silk protein gene.
(B) at least -1860 to -1127 region (base positions 3141 to 3874 of SEQ ID NO: 1) in the region from -214 to -235 of the fibroin heavy chain gene (in the base sequence of positions 2787-4766 of SEQ ID NO: 1) Sequence).
[0024]
As the polynucleotide (A), for example, a polynucleotide constituting a promoter / enhancer region of a silk protein gene including fibroin heavy chain, fibroin light chain, P25, sericin 1 and sericin 2 genes can be adopted. For example, a region of −234 to +6 can be used as the promoter / enhancer region of the fibroin heavy chain gene. As the promoter / enhancer region of the fibroin light chain, the region of -600 to +34 of its known sequence GenBank / AF541967) can be used. Using the promoter / enhancer region of the fibroin heavy chain, light chain, or P25 gene, the foreign protein can be expressed in the posterior silk gland, and if using the promoter / enhancer of the sericin 1 or sericin 2 gene, The expression of the target foreign protein can be increased in the silk gland. In the present invention, it is particularly preferable to employ a region from -234 to +6 as the promoter / enhancer region of the fibroin heavy chain gene (Invention (3)).
[0025]
As described above, the polynucleotide (B) is characterized by including at least the −1860 to −1127 region in the −2214 to −235 region of the fibroin heavy chain gene. Furthermore, it is preferable that this polynucleotide (B) is linked to a polynucleotide (C) consisting of the -5000 to -3848 region (base sequence 1-153 of SEQ ID NO: 1) of the fibroin heavy chain gene (invention). (2)). The linkage in this case is “5′-polynucleotide (C) -polynucleotide (B) -3 ′”, and a linker sequence consisting of 1 to 30 nucleotide sequences may be interposed between the two, Preferably, the 5 ′ terminal nucleotide of the polynucleotide (C) and the 3 ′ terminal nucleotide of the polynucleotide (B) are directly phosphodiester linked.
[0026]
On the other hand, the polynucleotide (A) constituting the promoter / enhancer region may be linked to the 3 ′ side of the polynucleotide (C) / (B), or may be linked to the 5 ′ side, The former is preferred. Therefore, a preferred configuration of the polynucleotide of the present invention is “5′-polynucleotide (C) -polynucleotide (B) -polynucleotide (A) -3 ′”.
[0027]
Polynucleotides (A), (B), and (C) as described above were screened for genomic libraries using probe DNAs synthesized based on known base sequences, and target genomic gene fragments were obtained. Thereafter, a predetermined region can be obtained by amplifying it by PCR (Polymerase Chain Reaction) using this as a template. Moreover, if it is a short-chain polynucleotide, it can also be acquired by a well-known synthesis method.
[0028]
Furthermore, the polynucleotide of the present invention includes addition or deletion of one or more bases or other bases within the range having substantially the same function as the polynucleotides of the inventions (1) to (3). And those having the substitution (Invention (4)). That is, it is well recognized that a polynucleotide (promoter / enhancer region) that promotes the transcriptional activity of a gene retains its basic activity even if it is modified by various methods. “Addition or deletion of one or more bases or substitution with other bases” means that one or more bases are added or deleted compared to the base nucleotide sequence, or one or more bases This means that the base has a function equivalent to that of the basic non-displacement sequence, although there is displacement such as substitution of the base with another base.
[0029]
The polynucleotide of this invention (5) is hybridized under a condition in which one or more of the complementary sequences of the polynucleotides (A), (B) and (C) in the invention (1) or (2) are stringent. A polynucleotide that is soybean and has substantially the same function as the polynucleotide of any one of the inventions (1) to (4). That is, the single-stranded polynucleotide sequence hybridizes with the partner strand depending on the degree of homology.
At this time, by setting the stringency of the hybridization condition more strictly, a sequence having higher homology can be specified. Stringent conditions are defined by the salt concentration, organic solvent (formamide, etc.) concentration, and temperature conditions in the hybridization and washing steps. Details are described in detail in US Pat. No. 6,100,037 and the like. The polynucleotide of the invention (5) specified by hybridization screening under such stringent conditions has a homology level of 70% or more, preferably 80% or more, more preferably 90% or more.
[0030]
The polynucleotide of the invention (6) is a polynucleotide having the length of the polynucleotide of the inventions (1) to (5) of 3500 bases or less. By removing more than 30% of 5000 bases, it is easy to handle and create transgenic silkworms.
[0031]
Invention (7) of this application is an expression cassette in which any one of the polynucleotides and a foreign protein structural gene are linked. As the foreign protein structural gene, a gene cDNA encoding an arbitrary protein can be used. For cDNA, a cDNA library was prepared using a known method (Mol. Cell Biol. 2, 161-170, 1982; J. Gene 25, 263-269, 1983; Gene, 150, 243-250, 1994). Each can be obtained by a method of isolating each cDNA using a probe DNA prepared based on a known base sequence. The obtained cDNA can be amplified by commonly performed gene amplification methods such as PCR, NASBA (Nucleic acid sequence based amplification), TMA (Transcription-mediated amplification) and SDA (Strand Displacement Amplification). it can. The required amount of each cDNA can also be obtained by RT-PCR using mRNA isolated from cells as a template. The polynucleotide and the foreign protein cDNA can be linked by a known ligation method using DNA ligase.
[0032]
The invention (8) is an expression vector having the expression cassette. As the vector, any insect vector that can be used for transformation of silkworms can be used without particular limitation. For example, silkworm nucleopolyhedrovirus (BmNPV) vectors and plasmid vectors incorporating insect-derived DNA transposons are preferred, with the latter being particularly preferred (Invention (9)). Known insect DNA-type transposons include piggyBac, mariner (Insect Mol. Biol. 9, 145-155, 2000), Minos (Insect Mol. Biol. 9, 277-281, 2000), and the like. Since these transposons exhibit metastatic activity in silkworm cells, silkworms can be transformed with vectors prepared based on these DNA-type transposons. In particular, a plasmid vector prepared based on piggyBac has been successfully transformed by actually injecting it into a silkworm egg (Nat. Biotechnol. 18, 81-84, 2000).
[0033]
By using the above expression vector, a transgenic silkworm (invention (11)) having the expression cassette of the invention (7) in its genome can be produced and expressed in the silk gland of the transgenic silkworm. By expressing the cassette, the target foreign protein can be efficiently produced (Invention (10)).
[0034]
For example, to insert a foreign protein expression cassette into the silkworm genome sequence using the property of piggyBac, for example, the method similar to the method of Tamura et al. (Nat. Biotechnol. 18, 81-84, 2000) Can do. That is, a pair of inverted repeats of piggyBac are incorporated into an appropriate plasmid vector, and a foreign protein expression cassette is inserted so as to be sandwiched by a pair of inverted repeats. This plasmid vector is microinjected into a silkworm egg together with a piggyBac transposase expression vector (helper plasmid). This helper plasmid is a recombinant plasmid vector lacking one or both of piggyBac inverted repeats and substantially incorporating only the transposase gene region of piggyBac. In this helper plasmid, the promoter for expressing transposase may be an endogenous transposase promoter as it is, or a silkworm / actin promoter or a Drosophila HSP70 promoter may be used. In order to facilitate screening of next-generation silkworms, a marker gene can be simultaneously incorporated into a vector incorporating a foreign protein expression cassette. In this case, a promoter sequence such as a silkworm / actin promoter or a Drosophila HSP70 promoter is incorporated upstream of the marker gene, and the marker gene is expressed by its action.
[0035]
Raise larvae (F0 generation) hatched from silkworm eggs microinjected with vector. Some of the F0 generation silkworms have foreign protein expression cassettes incorporated therein. However, in this generation of silkworms, the foreign protein expression cassette is incorporated only in a portion of all the cells in the silkworm body, and in order to obtain a silkworm in which the expression cassette is incorporated in all cells, To obtain transformed silkworms in which the expression cassette is transmitted through germ cells. That is, it is necessary to cross the obtained silkworms of all F0 generations with wild-type silkworms or with F0 silkworms, and to select transformed silkworms having a foreign protein expression cassette from the next-generation (F1 generation) silkworms. Selection of transformed silkworms from F1 generation silkworms is carried out using, for example, PCR or Southern blotting. In addition, when a marker gene is incorporated, it is possible to select using the phenotype. For example, when a fluorescent protein gene such as GFP is used as a marker gene, it can be performed by irradiating an F1 generation silkworm egg or larva with excitation light and detecting the fluorescence emitted by the fluorescent protein. The silkworm thus selected is a transformed silkworm in which a foreign protein expression cassette is incorporated in its chromosome. Therefore, the foreign protein expression cassette is transmitted without loss even in the offspring of these silkworms mated with wild-type silkworms or between transformed silkworms, and the foreign protein or the fusion protein of the foreign protein and silk protein is transmitted throughout the generation. Can be produced.
[0036]
【Example】
Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to the following examples.
[0037]
A reporter vector in which a part of the fibroin heavy chain gene was connected upstream of the luciferase gene was introduced into the silkworm larva silk gland, and the transcriptional activity was examined by measuring the luciferase activity in the posterior silk gland. Firefly luciferase reporter vector pGL3-basic and Renilla luciferase reporter vector pRL-null (Promega, Madison, Wis.) Were used as reporter vectors. pRL-null was cleaved with Pst I and Nhe I, blunt-ended, and self-ligated to remove the human β-globin intron sequence (pRLΔint).
[0038]
The structure of the fibroin heavy chain gene and the transcriptional regulatory region are shown in FIG. The function of the transcriptional regulatory region has already been reported (Suzuki et al., Proc. Natl. Acad. Sci. USA, 83, 9522-9526, 1986, Takiya et al., EMBO J., 9, 489-496, 1990). For use in assay system assay, a reporter vector having the fibroin heavy chain gene transcription regulatory region sequence shown in FIG. 1 was prepared by the following method.
Core promoter ( -37 ~ +6 ) pGL3-basic:
Synthetic oligo DNAs 5′-GATCTTATAAAAAGGTTCAACTTTTTCAAATCAGCATCAGTA-3 ′ (SEQ ID NO: 2) and 5′-AGCTTACTGATGCTGATTTGAAAAAGTTGAACCTTTTTATAA-3 ′ (SEQ ID NO: 3) were hybridized and then inserted into Bgl II / Hind III digested pGL3-basic.
Promoter containing upstream element (core promoter + upstream element, -234 ~ +24 ) pGL3-basic:
DNA fragments corresponding to the fibroin heavy chain gene -234 to +24 region were obtained by PCR using genomic DNA extracted from the adult abdomen as a template. The primers used were 5′-GCTAGCCTTTAAAATATTAAAAGTAAG-3 ′ (SEQ ID NO: 4) and 5′-TGACTTCGCGACTTGAGAGTTGGAACCG-3 ′ (SEQ ID NO: 5). The PCR product was inserted into the restriction enzyme Sma I site at the multiple cloning site of pUC118. Furthermore, fibroin heavy chain gene -234 to +24 was excised by Nru I / Nhe I digestion and subcloned into Sma I / Nhe I digested pGL3-basic.
Whole intron (+68 ~ +1037) Promoter (core promoter + upstream element + intron) containing pGL3-basic:
DNA fragments corresponding to the region of fibroin heavy chain gene +68 to +1037 were obtained by PCR using genomic DNA extracted from the adult abdomen as a template. The primers used were 5′-GCTAGCAAGCGACATACTGAAACAAAATG-3 ′ (SEQ ID NO: 6) and 5′-TCAAGTCGCGAAGTCAAAACCTTTGTGATC-3 ′ (SEQ ID NO: 7). The PCR product was inserted into the restriction enzyme Sma I site at the multiple cloning site of pUC118. Furthermore, fibroin heavy chain gene +66 to +1037 was excised by digestion with Sac I / Nhe I and subcloned into pGL3-basic having a promoter containing the upstream element digested with Sac I / Nhe I.
Intron first half ( +68 ~ +509 ) Containing a promoter (core promoter + upstream element + first half of intron) pGL3-basic:
A DNA fragment corresponding to the region of fibroin heavy chain gene +68 to +509 was obtained by PCR using genomic DNA extracted from the abdomen of the silkworm as a template. The primers used were 5′-GCTAGCATCATAATAAATTAATAATGCGG-3 ′ (SEQ ID NO: 8) and 5′-TCAAGTCGCGAAGTCAAAACCTTTGTGATC-3 ′ (SEQ ID NO: 9). The PCR product was inserted into the restriction enzyme Sma I site at the multiple cloning site of pUC118. Furthermore, the fibroin heavy chain gene +68 to +509 was excised by digestion with Sac I / Nhe I and subcloned into pGL3-basic having a promoter containing an upstream element digested with Sac I / Nhe I.
Intron element ( +156 ~ +454 ) Containing a promoter (core promoter + upstream element + intron element) pGL3-basic:
PGL3 with a promoter containing an upstream element that was blunt-ended after Nhe I digestion of a DNA fragment corresponding to the region of fibroin heavy chain gene +156 to +454 obtained by EcoR V digestion with pGL3-basic having a promoter containing the first half of the intron Obtained by subcloning into -basic.
[0039]
A reporter vector having fibroin light chain and heavy chain gene sequences was prepared by the following method for use in a transcription promoting region search experiment. For the sake of convenience, the numbers [1] to [14] are added to the prepared vectors.
[1] Fibroin light chain gene -600 ~ +34 Have pGL3-basic:
A DNA fragment corresponding to the fibroin light chain gene promoter-600 to +34 region was obtained by PCR as previously reported and cloned into pCR2.1 (Invitrogen, Carlsbad, CA) (pCR-PfL (-600 / + 34), Tomita et al., Nature Biotech., 21, 52-56, 2003). Fibroin light chain genes -600 to +34 were excised from pCR-PfL (-600 / + 34) by Xho I / Hind III digestion and subcloned into Xho I / Hind III digested pGL3-basic.
[0040]
To examine gene transfer efficiency, fibroin light chain gene -600 to +34 was excised from pCR-PfL (-600 / + 34) by Xho I / Hind III digestion, subcloned into Xho I / Hind III digested pRLΔint, and fibroin PRLΔint with the light chain gene -600 to +34 was obtained.
[2] Fibroin heavy chain gene -234 ~ +24 Have pGL3-basic:
Identical to pGL3-basic with a promoter containing upstream elements.
[3] Fibroin heavy chain gene -5000 ~ +24 Have pGL3-basic:
DNA fragments corresponding to the region of the fibroin heavy chain gene 5 ′ upstream region −5000 to +24 were obtained by PCR using genomic DNA extracted from the adult abdomen of silkworm as a template. The primers used were 5′-GCTAGCGCTCAAAGCCTCATCCCAATTTG-3 ′ (SEQ ID NO: 10) and 5′-TGACTTCGCGACTTGAGAGTTGGAACCG-3 ′ (SEQ ID NO: 11). The PCR product was inserted into the restriction enzyme Sma I site at the multiple cloning site of pUC118. Fibroin heavy chain genes -5000 to +24 were excised from this plasmid by Nru I / Nhe I digestion and subcloned into Sma I / Nhe I digested pGL3-basic.
[0041]
PGL3-basic having regions of various lengths between fibroin heavy chain genes -5000 to -235 was prepared as follows.
[Four] Fibroin heavy chain gene -870 ~ +24 Have pGL3-basic:
Fibroin heavy chain gene -870 to +24 was excised from pGL3-basic harboring fibroin heavy chain gene -5000 to +24 by Hind III digestion and subcloned into Hind III digested pGL3-basic.
[Five] Fibroin heavy chain gene -2077 ~ +24 Have pGL3-basic:
PGL3-basic with Kpn I digested fibroin heavy chain gene -5000 to +24 was obtained by self-ligation.
[6] Fibroin heavy chain gene -2885 ~ +24 Have pGL3-basic:
PGL3-basic with the Sac I digested fibroin heavy chain gene -5000 to +24 was obtained by self-ligation.
[7] Fibroin heavy chain gene -3947 ~ +24 Have pGL3-basic:
PGL3-basic having Nhe I / Spe I digested fibroin heavy chain gene -5000 to +24 was obtained by self-ligation.
[0042]
Furthermore, pGL3-basic having various combinations of constructs by deleting a part of the sequence between fibroin heavy chain genes -5000 to -235 was prepared as follows.
[8] Fibroin heavy chain gene -5000 ~ -1127 and -234 ~ +24 Have pGL3-basic:
Excision of fibroin heavy chain gene -234 to +24 and firefly luciferase gene from pGL3-basic having fibroin heavy chain gene -234 to +24 by digestion with Nhe I / Xba I, and Xba I digested fibroin heavy chain gene -5000 to +24 It was obtained by subcloning into pGL3-basic having
[9] Fibroin heavy chain gene -5000 ~ -1861 and -870 ~ +24 Have pGL3-basic:
After digestion with Nde I / Nhe I digested fibroin heavy chain gene -5000 to -1861 from pGL3-basic having fibroin heavy chain gene -5000 to +24, and blunt ended, Sma I digested fibroin heavy chain gene -870 to Obtained by subcloning into pGL3-basic with +24.
[Ten] Fibroin heavy chain gene -5000 ~ -2799 and -2077 ~ +24 Have pGL3-basic:
The heavy chain gene -5000 to +2799 was excised from pGL3-basic having fibroin heavy chain gene -5000 to +24 by digestion with Dra I / Nhe I, blunted, and then blunted after Kpn I digestion. Obtained by subcloning into pGL3-basic with genes -2077 to +24.
[11] Fibroin heavy chain gene -5000 ~ -3718 and -2573 ~ +24 Have pGL3-basic:
Fibrin heavy chain gene -2573 to +24 was excised from pGL3-basic having fibroin heavy chain gene -5000 to +24 by Bgl II digestion and sub-classified into pGL3-basic having Bgl II digested fibroin heavy chain gene -5000 to +24 Obtained by cloning.
[12] Fibroin heavy chain gene -5000 ~ -3848 and -545 ~ +24 Have pGL3-basic:
PGL3-basic having Pst I digested fibroin heavy chain gene -5000 to +24 was obtained by self-ligation.
[13] Fibroin heavy chain gene -5000 ~ -2215 and -545 ~ +24 Have pGL3-basic:
Excision of fibroin heavy chain gene -3847 to -2215 from pGL3-basic having fibroin heavy chain gene -5000 to +24 by Pst I digestion, and Pst I digested fibroin heavy chain genes -5000 to -3848 and -545 to +24 It was obtained by subcloning into pGL3-basic.
[14] Fibroin heavy chain gene -5000 ~ -3848 and -2214 ~ +24 Have pGL3-basic:
Excision of fibroin heavy chain gene -2214--546 from pGL3-basic carrying fibroin heavy chain gene -5000- + 24 by Pst I digestion, and Pst I digested fibroin heavy chain genes -5000--3848 and -545- + 24 It was obtained by subcloning into pGL3-basic.
[0043]
The activity measurement method is shown in FIG. 2 and described below.
1) Preparation of gold particles: Two vectors were mixed 1: 1 to attach 188 ng of DNA per 1 mg of gold particles.
2) Silk gland collection: The silk gland was taken out from the larvae on the 1st or 2nd day of the 5th year, and washed with the Grace medium.
3) Introduction of DNA into the silk gland using a gene gun: Gold particles with DNA attached were implanted into the silk gland using a gene gun (Helios Gene Gun, BioRad, Hercules, CA). 0.02 mg gold particles (3.8 ng DNA) are implanted per silk gland.
4) Transplantation of silk gland: After washing the silk gland with Grace medium, it was transplanted into the dorsal posterior body cavity of larvae of the same age as the individual from which the silk gland was removed. Larvae transplanted with silk glands were raised for 3 days.
5) Collection of silk gland: The transplanted silk gland was removed from the host, washed with Grace medium, and only the posterior silk gland was cut out and collected.
6) Measurement of luciferase activity: Firefly and Renilla luciferase activities in the supernatant were measured using a dual luciferase assay kit (Promega, Madison, Wis.). The posterior silk gland was homogenized in passive lysis buffer and centrifuged. The measured value of firefly luciferase was divided by the measured value of Renilla luciferase and multiplied by the length of the vector to calculate the transcriptional activity. Furthermore, it represented with the relative activity which made the activity of fibroin light chain gene -600- + 34 1.
[0044]
The transcriptional activity of fibroin heavy chain gene core promoter and its surrounding region has been investigated in detail using in vitro transcription system. The promoter activity of these regions was measured using an in vivo activity measurement system to determine the effectiveness of the in vivo activity measurement system. FIG. 3 shows the transcriptional activity of the transcriptional regulatory region in the in vivo measurement system. The activity of the fibroin heavy chain gene core promoter was expressed as a relative activity, where 1. The results are described below.
[0045]
If the activity of the fibroin heavy chain gene core promoter is 1, the activity of core promoter + upstream element, core promoter + upstream element + intron, core promoter + upstream element + first half of intron, core promoter + upstream element + element in intron is 15.9, respectively. It became 20.9, 9.8, 58.9.
[0046]
In in vitro transcription systems, upstream elements and intron elements have been reported to increase core promoter activity by 45 and 5 fold, respectively. In the in vivo activity measurement system, the activity increase effect of upstream elements and intron elements was 15.9 and 3.7 times. Therefore, the in vivo measurement activity system is sufficiently usable although it is inferior in sensitivity to the in vitro transcription system. In this experimental system, long sequences that cannot be handled by in vitro transcription systems can be measured, which is effective for analysis of unknown regions 5 'upstream.
[0047]
FIG. 4 shows the transcription promoting activity of the 5 ′ upstream region of the fibroin heavy chain gene in the posterior silk gland. The results are described below.
[0048]
The previously reported fibroin heavy chain gene -234 to +24 (core promoter + upstream element, [2]) showed only half the activity of the fibroin light chain gene -600 to +34 ([1]) . A sequence ([4]) in which the region of the fibroin heavy chain gene 5 'upstream region -870 to -235 is linked to the fibroin heavy chain gene -234 to +24 ([4]) shows activity corresponding to the fibroin light chain gene -600 to +34. It was. Furthermore, fibroin heavy chain genes -2077 to -235 and -2885 to -235 linked to -234 to +24 ([5], [6]) have activities of fibroin light chain genes -600 to +34, respectively. Three or four times. The activity of the sequence ([7]) connecting the fibroin heavy chain gene-3947 to -235 regions was greatly increased, reaching 15 times that of the fibroin light chain gene -600 to +34. When the fibroin heavy chain gene -5000 to -235 region was linked ([3]), 25 times the activity of the fibroin light chain gene -600 to +34 was observed on average. Some examples exceeded 40 times.
[0049]
The above results indicate that the fibroin heavy chain gene -5000 to -235 region has the effect of strongly promoting the transcriptional activity of the -234 to +24 region. In particular, it was suggested that the region of fibroin heavy chain gene -5000 to -2886 is important.
[0050]
In order to clarify the nature of the transcription promoting activity of the fibroin heavy chain gene -5000 to -235 region, various partially deleted constructs were prepared between the regions of -5000 to -235 (Fig. 5). It was measured. The results are shown in FIG. The results are described below.
[0051]
In the fibroin heavy chain gene -5000 to -235 region, about 1 kb of the deleted construct, the deletion of -1860 to -871 ([9]) showed no effect of promoting transcription. High transcriptional activity was maintained in sequences lacking -1126 to -235 ([8]), -2798 to -2078 ([10]), and -3717 to -2574 ([11]). It can be seen that the region from 1860 to -1127 is important for promoting transcription. However, the region including up to -2077 (Fig. 4, [5]) was about three times more active than the fibroin light chain gene -600 to +34, so the effect of the region -1860 to -1127 alone is not sufficient. it is conceivable that.
[0052]
Of the deficient constructs, those lacking −3847 to −2215 ([14]) showed the highest activity. This sequence contains the fibroin heavy chain genes -5000 to -3848 and -1860 to -1128 regions. A construct having the same length but lacking -22214 to -546 ([13]) does not contain -1860 to -1127 and has low activity.
[0053]
The above results indicate that fibroin heavy chain genes -5000 to -3848 and -1860 to -1127 are particularly preferable for the expression of high transcription promoting activity. The sequence [14] in which the fibroin heavy chain genes -5000 to -3848 and -2214 to -235 are linked to the -234 to +24 region meets this requirement and is shortened to an appropriate length. It is useful for the purpose of making transgenic silkworms.
[0054]
【The invention's effect】
As described above in detail, the invention of this application provides a promoter / enhancer sequence having high transcription activity and a method for expressing a recombinant protein that enhances gene transcription activity using the sequence. By utilizing the present invention, it becomes possible to produce transgenic silkworms that produce a large amount of various recombinant useful proteins in straw, and the produced recombinant proteins can be used in various medical, food, cosmetic, fiber, etc. Can be used in industrial fields.
[0055]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 Fibroin heavy chain gene structure and transcription control region. The fibroin heavy chain gene has two exons (indicated by bold lines). The number indicates the nucleotide number of the fibroin heavy chain gene (the transcription start point is +1). The part indicated by the arrow was used for measuring transcriptional activity. () Is the transcriptional activity measured in an in vitro transcription system (when the activity of the core promoter is 1) (Suzuki et al., 1986, Takiya et al., 1997).
FIG. 2 is an outline of an activity measurement method.
FIG. 3 shows the transcriptional activity of the transcriptional regulatory region in an in vivo measurement system. Transcriptional activity was measured using the fibroin heavy chain gene region shown in FIG. The activity of the fibroin heavy chain gene core promoter was taken as 1. White circles represent individual activity values, and black circles represent average values.
FIG. 4 shows transcription promoting activity in the 5 ′ upstream region of fibroin heavy chain gene. A construct was constructed retrospectively 5 ′ upstream from fibroin heavy chain gene-235 and its transcriptional activity was measured.
The activity of the fibroin light chain gene -600 to +34 was taken as 1. White circles represent individual activity values, and black circles represent average values.
FIG. 5 shows the structure of a fibroin heavy chain gene 5 ′ upstream region partial deletion construct. In order to elucidate the nature of the transcription promoting activity from fibroin heavy chain gene -5000 to -235, constructs in which various portions were deleted between -5000 and -235 were prepared.
FIG. 6 shows the effect of partial deletion of the fibroin heavy chain gene 5 ′ upstream region on transcriptional activity. The transcription activity of the construct shown in FIG. 5 was measured. The activity of the fibroin light chain gene -600 to +34 was taken as 1. White circles represent individual activity values, and black circles represent average values.

Claims (10)

A polynucleotide linked to a foreign protein structural gene to promote the expression of a foreign protein in the silkworm silk gland, comprising the following polynucleotides (A) and (B):
(A) a polynucleotide constituting the promoter / enhancer region of the silkworm fibroin heavy chain gene;
(B) a polynucleotide comprising a base sequence comprising at least the 3141-3874th base sequence in the 2787-4766th base sequence of SEQ ID NO: 1,
A polynucleotide to which is linked. The polynucleotide according to claim 1, further comprising a polynucleotide (C) comprising the nucleotide sequence at positions 1-1153 of SEQ ID NO: 1 linked to the polynucleotide (B). The polynucleotide of claim 1 or 2, wherein the polynucleotide (A) comprises the 4767-5024th nucleotide sequence of SEQ ID NO: 1. A function of adding or deleting one or two or more bases in the base sequence of the polynucleotide according to any one of claims 1 to 3, or substituting with another base, and promoting expression of a foreign protein in a silkworm silk gland A polynucleotide having The polynucleotide according to any one of claims 1 to 4, wherein the polynucleotide is 3500 bases or less in length. An expression cassette in which the polynucleotide of any one of claims 1 to 5 and a foreign protein structural gene are linked. An expression vector carrying the expression cassette of claim 6. The expression vector according to claim 7, comprising an expression cassette sandwiched between a pair of inverted repeats of an insect-derived DNA-type transposon. A foreign protein gene contained in the expression cassette of claim 6 is expressed in a silkworm silk gland. A transgenic silkworm having the expression cassette of claim 6 in its genome and expressing a foreign protein in the silk gland.

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