JPH0937786A - Promoter and vector containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new promoter that is capable of controlling protein manifestation and makes large transgenic animals produce milk that contains useful substances by cloning a β-casein-manifesting promoter domain of swine. SOLUTION: This new promoter is obtained by cloning a β-casein-manifesting promoter domain, and has a base sequence of the formula. This promoter can control manifestation of protein so that useful substances such as proteins can be produced in milk of large transgenic animals such as swine transducted with a vector incorporated with this promoter. Specifically, this promoter can be obtained by labeling, as a probe, DNA fragments prepared by cloning by the PCR method a swine chromosome DNA by using a primer consisting of a part of a base sequence of bovine β-casein, screening the swine chromosome DNA library by using the foregoing probe, and cloning the positive clone.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a promoter for expressing a human type useful protein in the milk of a large transgenic animal such as pig, and an expression vector containing the promoter.

[0002]

2. Description of the Related Art In recent years, biotechnology has been developed, and studies have been conducted to secrete useful substances such as proteins in milk using transgenic animals such as mice, rats, goats, sheep, pigs and cows. There is. There are many types of substances produced in milk, and their amounts are large. Moreover, it is known that the gene expression of milk protein is not only regulated by steroid hormone peptide sex hormone but also regulated by the interaction between cells and the pressure of milk accumulated in the vesicles that the producing cell receives. When a useful substance is produced by a transgenic animal, a promoter capable of efficiently expressing a structural gene encoding the useful substance and controlling the gene expression in a tissue-specific manner is not threatening the life of the recombinant. .

Currently used as this promoter is the mouse WAP (whey acidic protein) promoter which is specifically expressed in the mammary gland secreting milk,
There are mouse, rat and bovine β-casein promoter, sheep β-lactoglobin promoter and the like. It has already been practiced to let goats, sheep, pigs, cows and the like secrete the active substance in milk using the above existing promoters. However, the mouse WAP promoter is an exogenous promoter except for small animals such as mice and rats and is not suitable for mass production of useful substances.

Further, mouse, rat, bovine β-casein promoter, and sheep β-lactoglobin promoter have been used as promoters containing full length, but there are cases in which the expression level varies among individuals and tissue specificity is not shown. However, there is a problem that it is not practical.

[0005]

As described above, the promoter for producing a useful substance in the milk of transgenic animals has a problem that production amount and tissue specificity are low and individual differences occur. Therefore, it is an object of the present invention to provide a promoter that controls the expression of a useful substance such as a human protein into the milk of a large transgenic animal such as pig, and an expression vector containing the promoter.

[0006]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies in view of the above-mentioned problems, and as a result, unlike the promoters of small animals such as mice and rats, the β-casein promoter was derived from a porcine chromosomal library which is a large animal. The region was cloned, the nucleotide sequence was determined, and the following invention was completed. The present invention is a promoter having the nucleotide sequence of SEQ ID NO: 1 (the transcription start point is +1) that controls protein expression. The present invention is also an expression vector containing the above promoter.

The present invention selects a large animal such as a pig, not a small animal such as a mouse or a rat, as an animal to be used as a production system, and presents a gene sequence for controlling the β-casein protein specifically produced in pig mammary gland. Revealed. That is, the ratio of β-casein protein in the total protein contained in milk is high, and the activity of the promoter that controls the expression is also strong. In addition, β-casein protein is not expressed in other tissues and can be expressed tissue-specifically in the mammary gland, and can be stably produced in large amounts in milk.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The promoter of the present invention and an expression vector containing the promoter include a replication origin for transient expression and a selectable marker gene. The type is not limited as long as it is a combination with a gene sequence having a dot, an integration region, and a selection marker.

In the present invention, in order to clarify the promoter of the present invention, the homology with the known bovine β-casein promoter nucleotide sequence was utilized. Generally, in many organisms, the gene sequences of the more closely related species are conserved.
That is, PCR using a porcine chromosomal gene as a template with a primer corresponding to the bovine β-casein promoter region
A probe for the porcine β-casein promoter was prepared by the (polymerase chain reaction) method. Then, a porcine chromosomal library was screened using the above probe, the porcine β-casein promoter region was cloned, the nucleotide sequence was determined, and the promoter of the present invention was obtained.

When the 5'upstream region 200 bp of the promoter of the present invention shown in SEQ ID NO: 1 (transcription start point is +1) is compared with rat and bovine β-casein promoter regions, rat and bovine β-casein promoters are shown. The same region as the sequence found in the region is -158 to -146,
It existed at -101 to -88 and -65 to -44. This sequence is unique to the casein promoter not found in the promoters of other milk proteins such as WAP.alpha.-lactalbumin. Furthermore, TATA BOX (TATAAA) required for transcription initiation at -33 to -27.
A sequence) was also confirmed. Therefore, the promoter of the present invention is a promoter that expresses β-casein protein in a mammary gland tissue-specific manner.

The promoter of the present invention and the expression vector containing the promoter express the protein in the milk of transgenic animals by incorporating genes for proteins such as albumin and other serum proteins and collagen and other proteins that are abundant in living tissues. Can be made. In particular, by obtaining a human-type protein, it can be used as a drug or a substitute for biological tissues such as artificial skin, artificial blood vessels and artificial bones. The transgenic animal using the promoter of the present invention and the expression vector containing the promoter as a production system is not limited as long as it is a mammal, but pigs are particularly preferable.

[0012]

【Example】

(1) Screening method Primers A and B shown in FIG. 1 were synthesized (DNA / RNA Synthesizer, manufactured by ABI) from the bovine β-casein base sequence. Primer A corresponds to the 5'upstream region of the bovine β-casein chromosomal gene (-156 to -145 of SEQ ID NO: 1), and primer B is the first exon (33 to SEQ ID NO: 1 to 33).
It corresponds to 15). Using pig chromosomal DNA as a template and using the above primers A and B, a denaturation reaction at 94 ° C. for 1 minute, an annealing reaction at 50 ° C. for 2 minutes, and a primer extension reaction at 72 ° C. for 3 minutes are 30 cycles of PCR ( A DNA thermal cycler (manufactured by Perkin Elmer Cetus) was performed to amplify a fragment of about 200 bp. Digest this fragment with Digoxigenin-11-dUT
Pigmented with P (DIG Labeling kit, BMY)
-Used as a probe for the casein gene. Pig chromosome DNA library / EMBL3 SP6 / T7 by plaque hybridization using the above probe
1.0 x 10 ⁶ clones (made by Clontech) were DIG De
Primary screening was performed using a tection kit (manufactured by BMY).

The primary screen yielded 4 positive clones. When this was further subjected to secondary screening by plaque hybridization, three positive clones were obtained. DNA was extracted from the obtained clones by ultracentrifugation, and BamHI, Xho
After digestion with I, SacI, and SfiI (all manufactured by Toyobo Co., Ltd.), electrophoresis was carried out on a 0.8% agarose gel for Southern hybridization. As a result, it was determined that there were two types of three positive clones.

(2) Subcloning of SacI fragment The DNA of two clones determined to be positive by Southern hybridization was digested with SacI. The plasmid pUC119 was digested with SacI and then treated with alkaline phosphatase (manufactured by BMY), and a part thereof and the SacI digest of the above phage DNA were ligated (ligation kit, manufactured by Takara Shuzo) to transform E. coli DH5α. The plasmid DNA was extracted from some of the transformants by the boiling method, and after digestion with SacI, the DNA was converted to 0.
Southern hybridization was performed by electrophoresis on an 8% agarose gel. From this result, about 6.4 kb () and about 12.3 kb containing the porcine β-casein promoter region were obtained.
A subclone having the insert of () was obtained. Create a restriction map for these two subclones,
Also, about 300 bp sequence (DN
A sequencer, manufactured by Pharmacia),
, Both were determined to contain a porcine β-casein promoter region of about 3.2 kb.

(3) Nucleotide sequence determination The clone was treated with a kilosequencing dilation kit (Takara Shuzo) to determine the nucleotide sequence of SEQ ID NO: 1 of the 3.2 kb porcine β-casein promoter region. Further, the region 200 bp upstream from the transcription initiation point was compared as shown in FIG. 2 and confirmed to be the casein promoter.

(4) Construction of expression vector -940 to -91 of porcine β-casein promoter region
1 with the addition of a KpnI recognition site at the 5'end corresponding to 9
The primer C shown in 1 and the primer D shown in FIG. 1 corresponding to 67 to 46 of the first exon and having a SacI recognition site added to the 5 ′ end were synthesized. Using the subclones of these primers C and D as templates, the 1.0 kb porcine β-casein promoter region was amplified by the same PCR as above and ligated with the pCRII vector (Invitrogen) to transform DH5α. DNA was extracted from the obtained 6 clones (to) by the boiling method to obtain KpnI.
Digested with SacI. Digest Pikagene Basic Vector (Toyo Ink Co., Ltd.) with KpnI SacI,
A part thereof and the KpnI SacI digested products of the above 1 to were ligated to transform E. coli DH5α (FIG. 3). 6 clones obtained individually (BCAP
The DNA was extracted from 1.0 / luc-1 to 6) by the ultracentrifugation method.

(5) Activity measurement in cells COS1 cells were added to a 35 mm cell culture dish at 1.0 × 1.
It was cultured in DMEM medium containing 0 ⁵ seeds 10% FCS. After 24 hours, each BCAP1.0 / luc-1 to 6 was introduced into COS1 cells by the lipofection method using lipofectin (manufactured by BMY). 24 hours later,
Luciferase assay system (Toyo Ink Co., Ltd.)
Was used to measure the luminescence amount of luciferase using a luminometer (Integral time 30 seconds) to confirm the transcriptional activity of the porcine β-casein promoter. The results are shown in Table 1.

[0018]

[Table 1]

[0019]

INDUSTRIAL APPLICABILITY The promoter of the present invention and the vector containing it have low individual production and tissue specificity of the promoter for producing useful substances such as proteins in the milk of conventional transgenic animals, resulting in individual differences. It is possible to control the expression of useful substances such as proteins in the milk of large transgenic animals such as pigs by overcoming the above problems.

[0020]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 3218 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence -3158 CCCACTAT TTCCTGATTC TTGATTAACT TTATAAATTA -3121 ATCTCAATAG CCATATTTAT CATAAATCAG ATGTTATTTT ATGTGGCTAA TCTTATGGAA -3061 AGAATTAAAA TATTCCATCT ATATAAATAA TGCTATGCAA TCTCATGGAA AGAATTAAAA -3001 TATTACATCT ATATAAATAA TGCTATGCAA TCTCATGGAA AGAATTAAAA TATTACATCT -2941 ATATAAATAA TGCTATGCTA CCTATTCTTA ACTAATAATT TGGTGGAATT AACAAATTTT -2881 GTTTTACATT GTTGCCATAT CTGAATCATT ATATAAACTT CACTCTTTTT TTATCCTATT -2821 TCCACCCCCG CCCATGTTTT GGAAGTTATT AAACAGAGTC TCAAATATTA TTTAGAAAAA -2761 ATGAAGTTTT TAGTAACAGC TCTATTTCTA AAGAGCTGCA GTCTCCTGAA AAAAAACAAT - 2701 GCAAATTCAC AGGCCCTCTG TTTCTTTGCC TCTAAAATAT ATTTAAAACA TCATATGGAT -2641 GATATCTAGG GTCTCTTCTA GTCCTTATTT TCTGAACAAT TTTTGGTCCT TTTATTGTAC -2581 TTGTAAATTCCC AGCTTTTCTT-25 TTGATCCATCTT-25 TAGCCCCCATT-25 GCTG CTGACCTACA -2461 CCACAGCCAC AGCCACGCAG GATCCTATCT GCATGGGTGC TGGTCAGATT TGCTAACCAC -2401 TGAGTCACGA CAGGAACTCC CTTATCAGCT TTTCTATTAC TCTATTTCTT GAGGGTGTTA -2341 TTCACATGGT TTCAGCTATT ACCTTTTTGC TTTACCTATA TCTCATGCCT GCATATTTCA -2281 GATTCATCAT CCAATATTGG AAAAAGCTTT GACTGAACAA ATTCTGCTTA TAACAAATAG -2221 TACTAGAACA CTTTGCATAG ATCATATATT ACTCCTCACC CAGGGTTGTC TTATAAAATT -2161 TCTGTGGATG AAATTCTCAC TTTGATATAT AAAAAATGGC AACGCTTGGG TTATTCATTG -2101 CAAAAGCTTG GTTTGCCCAT ATGGCACTTT GCTCCTTACT GGTCATTGTG CTCTGAGGCT -2041 TACCTGGCCA GTGGCACCTG ATGTTATCTT AAATCTCTGG CTTTTCAACT CTCCCTTGGG -1981 CAAGTCTTTT TCTCTGGTGT ATTGTTAGTG ATTYCCTTGA TCAAGGTTTT ACCTACTTTT -1921 CTGGCCCAAT CAAGTCCATA ACTGTATCTA TTCATATAAT TCAAAATTGG TGAGCGATAG -1861 TCATAAGGGA ATGTTGTATT TATTGCACAA TAGGTAAAGC ATCTTGCTGA GAAAACACAA -1801 AGGAAGTATC AAATGGTTAG GGCACACTTG CTTTTGTATT CCTTTTCTTA GAAATTATAC -1741 TCTTTTGCCT ATGGCCTTGG CAACCAAGAG CTAACACATA AAGGCACAGT GAACAGCCAA -1681 GACCTTTTCT AGTTATACCT ATGACACTGG GGTCATTTC A TCATTTCTTT TCATGGCTTC -1621 CTCCTGGCTC CAATATGAAC ACTCTTAGAA TTAATATTAG CTGAATAATT TGAATACACA -1561 GTAGACACTG AGTTGGGTTT CTGAGGAATA CAAAACCTAT ATGACAGTCA GGAATATTCT -1501 CCTCCCACAT ACATCTAGCA TGATATGAAT GCAAGGTATC TTATGAAGAA AAATCATTAA -1441 TTATGCCGAT ACTCTACTTG CTTACTTCAT AACAGCATCT ATCTATCTAT ACTTAAGTAC -1381 TGAATATATC TTTACAGGCA ATGAATAGAA GAAATAAAGA TAAAAATGAG TAGTCTAATA -1321 CAATCAGCCA ATTACATTTA TGAACATCTA TCACTAAAGA GGCAAAGAAA CTTGAAGACA -1261 ACTTTTATAC GTGGGCAACT AAGAAATACT TTTGAGGCCT GGCTCAGACT CTATTATAGC -1201 ACGTTAGGTG AGACCCTCCT CCTGTCYGGG CTTCATCTTC TTCTTCCTTC CATCATTTGG -1141 CCTTCATGAA TATTAGCTGA CATACATTGA TTCACTATAG AAGATATGAG ACCAAACTTG -1081 AGAGGATCGA TTTGTTTTTC TTTTCTTTCT TTCTTCTTCT TCTTCTTCTT TTTTTTTTTT -1021 TTTTTTTTTT TTTTTTAAGG CTGCACCCAA AGCACATGGA AGTTTCCAGG CCAGGGGTCA -961 AATCAGAGTT GCAGCTGTGG TCTATACCAC AGCCATAGCA ACACCAGATC TGAGCTGCAC - 901 CTGTGACCAA GGTCTCTTGT GGCGATGCGG ATTCTTAACC CACTGAGCAA GGCCAGGGAT -841 TGAACCTGCA TCCTCATGGA CACCATGTTT TGTT CTTGAC CCGCTGAGCC ACAAAGGGAA -781 TTCCTAGAAG CACCACTTTA ATAAATTTGT ACCAGTATCA TTTTTTTCTC TAGAAATATT -721 CTCTAAATGT TACTTTCTGT CTTAAAACCC TTCAAAAAGT CCTCACTATC TAGAGAATAA -661 AATTTGCACT CCTTAGAATT CTTTGCCCAC TGTTATGTCA CCCCTTTGTC TTTCTTCTCA -601 TCTCCATCTA CTCGCTACTC CTTGCACTTC ATAACCTAAA TTCACTATTT GATTTAGTTG -541 TAGTGACTTT GTTATCATGT TCAAGACCAT ATTCTTCCTT TCGTTTTAGC CTACATCTCT -481 CTTTCTTCCT TAGGTCTGAG CTGTCATCAC AGGCTTGTCA TGTCATTTCT CCATTATGGC -421 ATAAAATGGT AACATCTATT TAGTTAGCAT GAAATAGTGA CCTTCGTGTG GCCTGTGTAT -361 CTCCAATACC TATTAGAATT TCCCCACAAG AAAGCTCTTG AAAACCTACC AAGTGCTAAA -301 GAGACCTTAT TGTGGCTACC ATAACTTGGG GACTGGGCCA GAATGTCACT GTCCCCCAGC -241 CAACGTCTGT ACTTATTGAA CAGTTTCATT TCCTGGATGG ATTTTCTTTA TCAGATGGTA -181 AATTATCCAC TTGTTAAAAT GCTCCTCAGA ATTTCTGGGG ATAGATAATA GGAAGAAATC -121 ATTTTCTAAT CATGCAGATT TCTTGGAATT CAAACTCACT ATTGATTTTA TTTTCCAACC - 61 ACACAATTAG CATGTCATTA AATACTGTAT AAAAATAGCC ACAGAAGTGG ATGATTATCC -1 ATTCACCTCC TCCTTCACTT CTTGTCCTCC ACTTTGGAGA AA AGGTAAGA ATTTCAGATT 60

[Brief description of drawings]

FIG. 1 shows each primer used in Examples.

FIG. 2 shows a region 200 bp upstream from the transcription start site of the porcine, bovine, and mouse β-casein promoter regions.

FIG. 3 shows the vector of the present invention.

Claims (2)

[Claims] 1. A promoter whose nucleotide sequence is represented by SEQ ID NO: 1 which controls protein expression. 2. An expression vector containing the promoter according to claim 1.