CN1705743A - Porcine uroplakin II promoter and the production method of useful proteins using said promoter - Google Patents

Abstract

The present invention relates to a porcine uroplakin II gene promoter, an expression vector containing the promoter, and a method for producing useful proteins using the vector. The promoter of the present invention promotes the bladder-specific expression of a target protein at high efficiency. An animal, which was transformed using the inventive promoter so as to express the target protein, secretes the target protein in its urine at high concentration, and the protein thus produced shows a superior physiological activity to that of the same kind of the existing protein. As a result, the inventive promoter, the expression vector and transgenic animal using the promoter, can be advantageously used in the production field of useful proteins that are medicinally valuable.

Description

Porcine uroplakin II promoter and method for producing useful proteins using said promoter

technical field

The present invention relates to a porcine uroplakin II gene promoter and a method for producing useful proteins using the promoter.

Background of the invention

In the field of medicine, as a method of maximizing the production of proteins such as EPO having an economic growth value, a large-scale production method using cell culture technology is mainly used. However, in this method, since animal blood is used as a culture medium, production costs increase, and expertise in culture is required. In addition, since it is impossible to completely separate newly produced EPO from animal EPO contained in a culture medium, the resulting EPO has problems of low purity and low activity.

On the other hand, in the method of producing a useful protein using a transgenic animal, the target protein is contained in the body fluid secreted by the animal, and therefore, compared with the existing cell culture technology, the target protein is easily isolated and purified, and retains better active. For this reason, the method is rapidly gaining attention.

In the transgenic animal technology established so far, the mammary gland known to exhibit high protein expression is mainly used as an organ producing the protein of interest. However, the results of animal experiments show that it is basically impossible to produce several important target proteins, such as EPO, through expression in milk, because they are also expressed in other tissues than the mammary gland. In addition, since various proteins such as albumin are also contained in milk in large amounts, the resulting target protein is difficult to purify.

To overcome these problems, it has recently been proposed to use the bladder to produce useful proteins.

Regardless of the sex of the animal, the bladder produces urine throughout the life of the animal, and urine contains only very small amounts of protein and fat components of 5-25 mg/l. Therefore, using bladder makes the isolation and purification of the protein of interest very easy.

However, the efficiency of protein production in animals transformed with bladder-specific promoters has remained low to date.

Therefore, it is urgent to develop a promoter that efficiently promotes the expression of the target protein.

Contents of the invention

Accordingly, an object of the present invention is to isolate a porcine uroplakin II gene promoter that promotes bladder-specific expression of a protein of interest, and to provide a method capable of producing a useful protein in large quantities using the promoter.

In one embodiment, the invention provides the porcine uroplakin II gene promoter.

The porcine uroplakin II promoter preferably has the base sequence of SEQ ID NO:1.

[SEQ ID NO: 1]:

gggctaggagtggaatcagagctggcctatgccacagcaacgcagaatccaaaccacatctccgacctacaccagaccgtcaccataacacaggatccttaacccactgagcaaggtcagggatcaaacccaaatcctcatggatactagt

cgggttcttaacccgctgagccacagtgggcactcctgtttttgtttgtgtcttcgttttttggctgcatctgcagcatacagaa

gttcctgggttaaggattgaacccatgccacagcagcaacccgagccacagcagtgacaacagcctgatccttaactgct

agaccaccagggaacgccccctcaacttttcatgccttggaaaccctgagtcagtacaacctgacaatngntttttttttttttt

ttttttgcttttctagggccacttcccgcggcatgtggagattcgcaggctanaggtctaatcagctgtagccaccggc

ctacaccagagccatagcaacgagggatccgagccgagtctgcaacctacactacagctcatggcaacaccggatcgtt

aacccactgagcaaggccaggggatcgaacccgcaacctcatggttcctagtcagattcgttaaccactgcaccatgaca

ggaactcccaacctgacaattttatcattctgcaccctagttgttgagtaatttgaaaaattcccaagatgtcaaggtcagtgt

gatggttaattttatgtgtcaacctgactaggccatgttgcccffatgtggagtcattgttattctggatgttactgtgaagatat

gttttggatgaaattaacatttaaatcagtgggggaaaaaaagaagttctcgttctggtgcatcagaaacaaatccgacta

ggaaacaagcggttgcaggttcgatccctggcctcacttagtggagtcaggatctggcgttgccgtgagctgtggtacag

gtggcagatgcagctcggatctagcattgctgtggctgtggtgtaggccagcagctgtagctctgattaaaccccaagtct

gggaacctccatatgccgtgggtgtggcccgaaaaagcaaaaaataaataaataaataaatttaaaccaggggattttgag

caaagcagattaccccataatatgggtgggtctcatcaagttcattgtaggccctagtggaacaaagaccgacctccacctt

ctccccatgagaaggaaagaattctgccaaaagaccgccttnggacntaaactgcaactctttcctgagtttccagcatgtt

ggcctcccccatcagactttggacttgccaagcctccgcaattgcatgagccaattccttaaaataaatccgtctatatac

acatcctgttggttctgtttctccagagaaccctgactaacgcagtctgcacccctgaagaccagtggtccccaacactcagc

tgggtgtcacctccaaacactcagccttcctcaaggctctttctagctgtgtcctcctctccccacaacagctgtttcaaactc

tcaccccctcttcagggcgcaatcccttctcctccctgagtttcctacttcccagagaaagcagagaccttcaggagtgtgct

gccttaacttacttccttcatccctcagccttgcaaaagtataagctttcctctgcaccactgccccattcttctctctgcagacag

ggtcattcctaaagccaaacgctaatgcctccacctctgatctgagtcccatcttttccctcctccagaagcttcctcataaatt

ctacccccttttcttccttatctttatctttgaaaacaaaatggaagacagccttcccgttgtggtgcagcggaaacagtggtg

ccttggaagcgctgggacgcaggttcgacccctggcccagcatagtagttaggttaaggatccagtgttgccacagttttggctt

agattgaaactgcagctcagatctgtgtccctggcctgggaacttcatacgccacaggacgcccaaaaagaaaagaaag

aaaaaataaaaaacaaaacagaaaagcctttcctgtacccccaattccctccagttatctctctttcccttcccagccaag

ctctgcaaagagcggtctgcacagttctaactctacctcctcccagttggccctggactttctcagtctggcttctacccccct

cacccgtaggaatctgctctgaaggacacgcacccctcacgatccttggcccagggaacattttttgtaccagcctttcaatc

ctgaccttcatatcatccgacacctcctttggaaaccctccatccactttctcctggttcccctcctaagacccttccgcctt

cttcagccccctccctccatctgtcctttagatgccgcatttcctagtatcctgtcctgcgcggnctcgtccttcccttccacaa

ctctcttcaaggactcttttctccatgtgcgattttgcccatggccccaccttccctctctttaccccagactttcccccggtgctcc

agactcatagactcaattatgaaaacatagttttcatctgatttgcccaagatatttgcattagttaattactgataacagcttatc

ccccaatttagtggcttataaaataaacacttatttctgagaatcagaaacctaggcaggacatagttggggtctcatgaagtt

gcactgaaaatgtccccctgggctaatcatacggaggactgaccagggctggaggatctgttccaagctcattcattcaca

tggccgtaggttggagacagctcttctctggatcttggcaggagcctcaattccttgtcacgtggacctccccttggagggg

gtcccatgtcctccatggtgagtaatccatgagagcaaggtggaaggtgccatgccccattaggacctagcctcaggaggg

acctacgtcacttctgttgtagtctgttggccacacagactaaccctgacacaatgcacccatccatgacctgctgccagtc

cattctccacactgtttccagaatgatatttacataagtaaaactcctcaaaggcttttgagattttttttccccattatagttgatta

taacctcagaggcttttgttttcttcagcataaaaaccaagttccttaacatagcatgtaacccactggccaccctgccagtg

gctagaactctcaccatgtccatccttgaatactgctttctagccaagagctattgtttgcagttcccagaatgtgtcgggata

actcacatctctgagccttttcatgtgctgtccctcactttggaatatccccttccattaggaaggctaatgtccattcattntc

caaaactcagaagcaaatttttttttttttttttttttttttttttttttttttttgctttttagggccgaactctcagcatatggaggttcccaggtta

gccatcaaattggaattgtagctgctggcctacaccacagccatagcaacccagacccaagtcacatctgcaacctacat

cacagatcatggcaatactggatccttaacccactgagtgagcccagggatcaaacacaaattctcatggatactcgccag

gttcattaccactgagccacaacaggaactcctctcctttttatggtcacacctgcagcatatggaagttcctgggccaggg

attgaatctgagtggcagctgtgacaatgccgtatcctttaattcactgtgctgggctgaggggntaaantgcccctcctaa

aaaacctgagctgctgcagttggattcttaatccactgcaccacaagggggaaggtcaagaactgtcttgccatctctgtat

cttatcacctagcatagtacccaccatagagaagttgctcaacaaatgtttactgaatgaataaatgcatgagctggagttcc

cattgcggctcagcagtaacaaacctgactagcattcataagaacttgggttcgatccctagcctcagtgggttaaggatgc

agcattgctgtgagctgtggtgtaggtcgcagacgacactcagatcccacattgctgtcactgtggcgcaggccggcctct

gtagctctgattcgactcctagcctgggaacgtccatatgccacaggtgaggccctaaaaagaaataaataagcaagcaa

gtaagcaagcaggcagtttcttggtgccttgtacccctgtggcctgtgtggtatacaagtaacagctgatccatgtctcagtc

atgtttccccctcagactacctttcctgccccatcttctctccctttgacataattggaaaaacaaattcagaattttgtcccactacc

tttcttgctagctctgtggccttgggaaagctatttattgcctctgagcctctaattttcatctgcaccaaggattaataaaaagg

agaggataagatgaattacttatattaatatttattgaaccagatactgtgctaggcactcttaaataaattagcttgagtgata

gtcatagtatcctggtgagacagattttttttttccttttatggttgcacgtgcaacatatggaagttcctgggctggggtcgaat

tggagctgcaggtgcttgcctatgccacagccatggcaacatcatatacaaaccgcacctgtgacctacaccacagattgc

agcaacgctggatccttcacccaaggagcaaggccaggaatcaaatgtgcatcctcacaaacactatgtccggtttttaac

ccgctgagccacaccaggaactccatggcgagacagattttatactctgtctacagaagaggaaagtgaagctcagaatg

gttaggtaggtaacttggccaagatcaaaaaattcaaagaagatttggggcaagtggtgatatcatggcagcattagaaaa

aataaagaagcatccacttgttttccaacactgaacaactgagattttcttactctcagctttttccagcttcatatccaagga

cagacgctctgccattttcccatcagccaatatttgctgaacactgcacctttacttttaggtccaagtcaccagggggttttcc

cagtttgctcctacagattctgacactatctccacattttttttgcacctttattttaaagcatttttatacctgtcataccttgctaga

taaatgggaaggaatgaatcttcccatttataggtgagaaaattgaggttcaaagtgactcaccaaaagtcatatagcatca

ctcctcaacaggaggacagcagtccccaccagagggtaacatgtccatgagcctagtggacacatttttctaactgactg

ggaagcagcagagtggtattgtgaagggggaatcataggtatcaaacagacttaggttctgatccgagctattctgcttg

caaacaaccatagttcaatttaaaaaaaaaaaaagaaagaaagaaagaaagaaaggagcccccatcctggtgcagtggaa

acaaattcaactaggaactgtgaggttgtgggttcgatcctggccttgctcagtgggttaaggatctggcgttgccatgag

ccgtggtgtaggttgcagactcaactcagatctggcgttgctgtgactgtggctgtgatgtaggctggcagctgtaactccg

gttagaccccagcctgggaacctccatatgcaacctccatatgcggtgggtgtggcctaaaaagaaaaaaaaaaaaaaa

aagaggaattcccttatggctcagcaggttaaggatctggtattgtcactgctgtggctctagttacagccatagtgcaggtt

caatccctggcccaggaacgtctgcatcccacaggtgtgccaaaaaagaaagaaaggaaggagttctgttgtggcaca

ataggattggcaacatcttaggagtactgggacacaggttcaatccctggcccagcacagtgggtaaggagccagtttg

ctggtcaaaaaagaaaagaaaaagtaccatagtttagagtaaatctgttttaggagctattctttggggcagaacagagat

caggagctccttgagagcagaaacttacctttacatccctcgtgcctagcacggttctaggggcatacctggtatttaataaaa

tatagccaactggataggggattggaaggaaagagcagggggagggaacttgagtgagttgaaaaattgagaatccaaa

ggggagacagcctagaaagagtaggtccaagaaagagatcccaggcatttgtggccctggttccctttttccaagccatg

aggaaatcctcagaggaacagagtgctgtggctttaaatgacttcagcgttgtcaatgaatctgctcggctaaaagagttat

cctcttgctccttcgcttgtcctccccctcctctcagctccccaaacccttctcggctgctgtgatgggataattagatgcgag

agctcagcacagatgatgctccagttgcctagcaactaatggtttccatggagaccgcaaagcacagcctccagagcag

ccagtgagcagctcggcagggcagggagaagacgcaactctcagctcctccagaaacctggggagggccaggaggtg

gggaagaagggggggatcggagggcttaaaggcacaggcccctcttatcctcttaaaatctggtcagagctctgccctc

ccctcccctactctgtcccactcataatttcagatggagttgggggcttaggagtggacccaacacaacctacctgcaata

aacccaaccttctttctgcttctggtttgtggctgaaaatggnaaaagaaatctcccaagtgcaagtgtaaacancntcctg

ggttggcaatgggatctgaagagtactaagatccctcagacctggaattccaccattagtctttccctctctccaaagttctc

atgtgcaaaagatcctctttcagtttgcagagcaatgataggatcttctaaaaggagacaaaagccaaggtgcaggaaaa

atagaattcagttcttcacccaaaggcagcctgtcctgggagacaggggtgaaacacttggtcctgatctccatcagagga

tccagagtgtgtgtgtttgttgctggggagggggacacaatatagagcatctggtgactcaaagtatgtgcctcccagagt

agcatcaatcaatgttacctggaagcttgttagaaatgcagaatttcaggcttcacctcagacccactgaatcagaaactgc

atcttaacaagatccctcatgattcatacgcacattaaatttggagaagcgctgacctgagaccctcctcctctctgcttggg

cccatagttctacctttattgtcacctcgtctcacctcgtgctcataccccaggctttgagcctacccttccccccatggggaa

aggacacaaggccaccagcccctcacttccctaccaggacctggccctcctctgggactggagaaggacaaagagga

ccccctctgtggaggtctacgacctctcctgaccaagtagtccactcaccacaagtggctctacctctctgagtctcagtttc

cacatccacaaaaggtggccaatgctatctgccaccccagaatggctgtgagggtggagcaggcaaagcctctgtgccat

cagagaaattgtgtctctttttcattttctcccagtgggtttctttctcgtctttattctttttttttttttttttcctgtctgttgtatttttag

ggccgtgcctgtggcatacggaagttcccagggtaggggtccaatgggagctgtagccccgggcctacgccacagcca

cagcaatgtgggatctgagccacgtctgcaacctacccacagctcacggcaacaccagatccttaacccactgagcaa

ggccagggatcgagccccacgtcctcatggatgctagttgggttcgttaaccgctgagccatgatgataactcctctttctatt

ctttagtcacaaacagtcaacaaaggttgctgaccaaggctgatcgtgcccaccccccagccccccagactgggccagt

gccccacccttgggtctctctggaaatcctgcccagcatcaattggctccactctccaggaggatgggaagccctgtggc

ccctgggactcacaccccctctgcatctcccagagtgcaggacctggtcttcaggagacaccaagaactggctcccccgg

ctctgctgcccccaccccctactaccagtttctctcccattcctgcccagtccaggccccctggggttactctcctctctctgt

acaccagtgcaacctcagaacctgcttccctcctgggaacacccactaccacgtgggagaaggggtcgtctaggggttg

ggccccagatacacttgtaagcaggaacacacgagcccttacatgtgggtgtcccggaagaagggggttttccacccccc

cgctttagtcaccctgcccctctgcagctgcctgagccaccaagacccagccaaggtctcctgccttctggcctgagggc

cagctccccatcctgaaaaacctgtctgggggcctcccctgaggctgtagggcccaaggcctcccctgaggctgtaggg

cccaaggggcaggttgaacaggattcccctctggcccctcctacccccaggacaaaaccagagccccaggacagggc

ctcacttgcctcaggaaaccacagcttgccagcacccagcccagcaccagcccagct

In addition, the porcine uroplakin II promoter of the present invention can be selected from one or more disruption mutations, deletion mutations, insertion mutations, point mutations, replacement mutations, nonsense mutations, Functional equivalents of missense mutations, polymorphic mutations or rearrangement mutations.

In another embodiment, the present invention provides an expression vector comprising all or part of a promoter.

The expression vector of the present invention preferably comprises a promoter and a base sequence encoding the protein of interest at the 3' end of the promoter.

In another embodiment, the present invention provides an animal transformed with a fertilized egg into which an expression vector has been introduced.

In another embodiment, the present invention provides a method for large-scale production of useful proteins, which comprises collecting urine from transgenic animals, isolating and purifying the protein of interest expressed in the urine.

The promoter of the present invention is located at the 5' end of the porcine uroplakin II gene, and regulates the expression of the porcine uroplakin II gene.

The promoter of the present invention can be isolated by screening a porcine genomic library in the following manner.

In order to obtain the partial nucleotide sequence of the porcine uroplakin II gene as a screening probe, the uroplakin II nucleotide sequences of other animals with known nucleotide sequences are compared with each other, and the primer set is constructed with reference to the highly conserved part between species (forward Primers: SEQ ID NO: 2 and reverse primer SEQ ID NO: 3). Then, using the total RNA of porcine bladder as a template, RT-PCR was performed with the primer set.

After obtaining a part of the uroplakin II fragment by RT-PCR reaction, the porcine genome library was screened using the obtained part as a probe. As shown in Figure 2, the probe that is used for the present invention is two probes, by the probe A that contains exon 2-5 part in the uroplakin II gene and the probe that contains exon 1-2 part in the uroplakin II gene B composition.

As shown in Fig. 2, clones containing the uroplakin II gene or promoter were obtained through library screening. By comparing the base sequences among the clones, the base sequence of the promoter was finally determined, so as to obtain the complete base sequence of the porcine uroplakin II promoter.

The promoter thus obtained has a full length of 8847 bp, exhibits high G+C content in the base sequence, features of a housekeeping gene, and contains various Sp1 elements, including AP2 and GATA boxes.

Among various porcine tissues, the promoter of the present invention can only specifically express the target protein in bladder tissue. For the porcine uroplakin II gene, it is expressed in 8-14% of total bladder cells, actively proliferating, especially in the epibasal cells of the bladder epithelium, and exhibits high expression in segmented umbrella cells level.

Therefore, since the promoter of the present invention efficiently induces bladder-specific expression of a protein, the use of the promoter of the present invention allows the generation of an expression vector that expresses an exogenous protein of interest in a bladder-specific manner.

When generating the expression vector of the present invention, the promoter of the present invention is inserted into the existing vector for protein expression as the basic skeleton, and the base sequence encoding the protein of interest is inserted into the 3' end of the promoter, thereby producing the present invention. invention carrier.

A vector that can be used as a basic backbone in the production of the expression vector of the present invention can be a suitable vector selected from commonly used expression vectors, examples of which include pBluescript SK vectors having multiple cloning sites and reverse transcription vectors such as pLNCX.

The expression vector of the present invention can express all proteins used as active ingredients of medical drugs, and examples of these proteins include erythropoietin (EPO), aldosterone, adreno-corticotropin (adreno-corticotropin), blood coagulation factors, gonadotropins, Insulin, prolactin, and vasopressin.

If desired, the expression vector of the present invention may also contain regulators, such as another promoter, enhancer, selectable marker, untranslated region (5'-UTR), 3'-UTR, polyadenylation Signals, ribosome binding sequences, base sequences that can be inserted into specific sites in the genome, and introns at appropriate positions.

The present invention provides the expression vector pUP2/hEPO capable of expressing human EPO under the control of the porcine uroplakin II promoter (Fig. 3). The expression vector pUP2/hEPO is a preferred example of an expression vector containing the uroplakin II promoter.

In the expression vector pUP2/hEPO of the present invention, the pBluescript SK (-) vector is used as the basic backbone, and the gene of encoding human EPO (Lin F.K. et al., Proc. Natl Acad. Sci, USA, Cloning and expression of the human erythropoietin gene , 82:7580-7584, 1985; SEQ ID NO:4) is fused to the 3' end of the uroplakinII promoter of the present invention. The expression vector pUP2/hEPO was deposited on October 17, 2002 at the Korean Type Culture Collection (KCTC) of the Korea Institute of Bioscience and Biotechnology, and the preservation number is KCTC 10352BP.

If necessary, the expression vector pUP2/hEPO of the present invention can also contain a neomycin resistance gene, an insulator or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), so that the establishment of a transgenic cell line is easy to carry out, and the expression level of the protein of interest Maximize and ensure the stability of target protein expression.

The neomycin resistance gene, a gene that exhibits resistance to the G418 reagent used in cell line establishment, can serve as an effective selectable marker in the establishment of animal cell lines expressing proteins under the control of the UPII promoter. The neomycin resistance gene has a sequence of SEQ ID NO: 5 bases.

[SEQ ID NO:5]:

gcggccgcgcgcgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcctgaggcggaaagaaccagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggctt

ttttggaggcctaggcttttgcaaagatcgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgca

cgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccg

ccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaaactgcaag

acgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgg

gaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatcc

atcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcat

cgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcc

agccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttg

ccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggac

atagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccg

ctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgac

caagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttcc

gggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccctagggggaggctaactga

aacacggaaggagacaataccggaaggaacccgcgctatgacggcaataaaaagacagaataaaacgcacggtgttg

ggtcgtttgttcataaacgcggggttcggtcccagggctggcactctgtcgataccccaccgagaccccattggggccaa

tacgcccgcgtttcttccttttccccaccccaccccccaagttcgggtgaaggcccagggctcgcagccaacgtcggggc

ggcaggccctgccatagcctcaggttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtga

agatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtccgatcg

The insulator is a factor that promotes the effect of the regulator adjacent to the promoter and also promotes site-dependent expression, allowing the stable expression of the protein of interest under the control of the UPII promoter. The insulator has a base sequence of SEQ ID NO:6.

[SEQ ID NO:6]:

tcgactctagaggggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgctaggggcagcagcgagccgcccggggctccgctccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacag

cccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagcctgcagacacctgggg

ggatacggggaaaaagctttaggctgaaagagagattaggatgacagaatcatagaacggcctgggttgcaaaggagc

acagtgctcatccagatccaaccccctgctatgtgcagggtcatcaaccagcagcccaggctgcccagagccacatcca

gcctggccttgaatgcctgcagggatggggcatccacagcctccttgggcaacctgttcagtgcgtcaccaccctctggg

ggaaaaactgcctcctcatatccaacccaaacctcccctgtctcagtgtaaagccattcccccttgtcctatcaagggggag

tttgctgtgacattgttggtctggggtgacacatgtttgccaattcagtgcatcacggagaggcagatcttggggataagga

agtgcaggacagcatggacgtgggacatgcaggtgttgagggctctgggacactctccaagtcacagcgttcagaaca

gccttaaggataagaagataggatagaaggacaaagagcaagttaaaacccagcatggagaggagcacaaaaaggcc

acagacactgctggtccctgtgtctgagcctgcatgtttgatggtgtctggatgcaagcagaaggggtggaagagcttgcc

tggagagatacagctgggtcagtaggactgggacaggcagctggagaattgccatgtagatgttcatacaatcgtcaaat

catgaaggctggaaagcctccaagatccccaagaccaaccccaacccaccccaccgtgcccactggccatgtccctcagt

gccacatccccacagttcttcatcacctccagggacggtgaccccccccacctccgtgggcagctgtgccactgcagcac

cgctctttggagaaggtaaatcttgctaaatccagcccgaccctcccctggcacaacgtaaggccattatctctcatccaac

tccaggacggagtcagtgaggatggggctctagaggggacagcccccccccaaagcccccagggatgtaattacgtccc

tcccccgctaggggcagcagcgagccgcccggggctccgctccggtccggcgctccccccgcatccccgagccggc

agcgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagc

ctgcagacacctggggggatacggggaaaaagctttaggctgaaagagagattaggatgacagaatcatagaacggc

ctgggttgcaaaggagcacagtgctcatccagatccaaccccctgctatgtgcagggtcatcaaccagcagcccaggct

gcccagagccacatccagcctggccttgaatgcctgcagggatggggcatccacagcctccttgggcaacctgttcagt

gcgtcaccaccctctgggggaaaaactgcctcctcatatccaacccaaacctcccctgtctcagtgtaaagccattccccct

tgtcctatcaagggggagtttgctgtgacattgttggtctggggtgacacatgtttgccaattcagtgcatcacggagaggc

agatcttggggataaggaagtgcaggacagcatggacgtgggacatgcaggtgttgagggctctgggacactctccaa

gtcacagcgttcagaacagccttaaggataagaagataggatagaaggacaaagagcaagttaaaacccagcatggag

aggagcacaaaaaggccacagacactgctggtccctgtgtctgagcctgcatgtttgatggtgtctggatgcaagcagaa

ggggtccatgtccctcagtgccacatccccacagttcttcatcacctccagggacggtgacccccccacctccgtgggca

gctgtgccactgcagcaccgctctttggagaaggtaaatcttgctaaatccagcccgaccctcccctggcacaacgtaag

gccattatctctcatccaactccaggaacggagtcagtgag

WPRE is a regulator that endows mRNA stability and thus increases the synthesis of the target protein, and enables the massive expression of the target protein under the regulation of the UPII promoter. WPRE has SEQ ID NO: 7 base sequences.

[SEQ ID NO:7]:

accaggttctgttcctgttaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctgtttcgcctcgggctcctcgag

The present invention provides I/pUP2/hEPO vector, pUP2/hEPO(WPRE) vector and I/pUP2/hEPO(WPRE) vector as preferred examples of expression vectors further containing a regulator.

This vector was generated by inserting a neomycin resistance gene into the pUP2/hEPO vector of the present invention, and then inserting WPRE into the 3' end of the EPO gene or inserting an insulator into the 5' end of the UPII promoter.

For example, animals that can be transformed with the expression vector of the present invention include all excreting animals such as pigs, mice, cattle, poultry, sheep and goats.

A method of producing a transgenic animal using the expression vector of the present invention is performed according to a conventional method. That is, fertilized eggs are collected from healthy individuals of the animal to be transformed, and the expression vector of the present invention is introduced into the fertilized eggs. The vasectomized mice were then used to obtain pseudopregnant mice, and fertilized eggs were implanted into the fallopian tubes of pseudopregnant mice serving as surrogate mothers. The offspring obtained from the surrogate mother are then screened for transformed individuals.

Subsequently, urine is collected from the screened individual whose transformation is confirmed, and the protein of interest is isolated and purified from the collected urine, thereby producing a useful protein.

In the method of the present invention for producing useful proteins, urine separation and purification processes can be performed by conventional techniques such as filtration or chromatography.

The transgenic animals of the present invention produced as described above express the protein of interest in a bladder-specific manner, and express the protein of interest in urine at a concentration much higher than that of existing methods.

For example, mice transformed with the expression vector pUP2/hEPO of the present invention exhibit a high EPO expression level of 0.5-1 mg/ml. Although EPO is a difficult protein to express because it causes early death of the embryo, the expression level of EPO in the animals of the present invention is at least 1,000 times higher than that of the protein in urine using the existing uroplakin promoter.

In addition, the protein produced from the transgenic animal of the present invention exhibits better physiological activity than the commercially available protein of the same type.

For example, EPO obtained from mice transformed with the expression vector pUP2/hEPO of the present invention maintained the survival rate of EPO-dependent hepatic cell lines at a higher level than commercially available EPO.

Therefore, the promoter of the present invention and expression vectors and transgenic animals using the same can be advantageously used in the field of production of useful proteins that are difficult to mass-produce.

Description of drawings

Figure 1 illustrates the probe structure used to isolate the porcine uroplakin II promoter of the present invention, and the clones obtained by the probe;

Fig. 2 illustrates the structure of expression vector pUP2/hEPO among the present invention;

Figure 3 illustrates bladder-specific expression of porcine uroplakin II mRNA;

Figure 4 illustrates the bladder epithelium-specific expression of porcine uroplakin II protein;

Figure 5 illustrates the expression levels of porcine uroplakin II protein in bladder cells and the umbrella cell-specific expression of the protein;

Figure 6 illustrates the bladder-specific expression of EPO mRNA in mice transformed with the expression vector pUP2/hEPO of the present invention;

Figure 7 illustrates the expression of EPO protein in mice transformed with the expression vector pUP2/hEPO of the present invention;

Figure 8 illustrates the structure of the expression vector pUP2/hEPO of the present invention;

Figure 9 illustrates the structure of the expression vector pUP2/hEPO of the present invention;

Figure 10 illustrates the structure of the expression vector pUP2/hEPO of the present invention;

Figure 11 illustrates the comparison between the EPO gene expression levels of the expression vectors of the present invention;

Fig. 12 illustrates a comparison between EPO gene expression levels of the expression vectors of the present invention.

best practice

The present invention will be described in more detail below by way of examples. It should be appreciated that the present invention is not limited to or by the Examples.

Example 1: Isolation of the porcine uroplakinII promoter of the present invention

In order to isolate the porcine uroplakinII promoter of the present invention, the following experiments were performed.

1) Preparation of probes by RT-PCR (reverse transcription-polymerase chain reaction)

Since the base sequence of the porcine uroplakin II gene was unknown, mouse and bovine uroplakin II cDNAs whose base sequences were known were compared with each other. A degenerate primer set for amplifying porcine uroplakin II cDNA was generated with reference to highly conserved parts between the two species. The base sequences of the forward and reverse primers are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

Using the primer set, RT reaction was performed on the total RNA of porcine bladder with MuMLV reverse transcriptase, and PCR was performed on the obtained cDNA using Taq polymerase. The reading of the base sequence of the amplified DNA indicated that the amplified DNA was a component of the uroplakin II gene. The amplified DNA was cloned using the pGEM T-easy vector.

To generate probes for isolation of the uroplakin II promoter, 50 μg of cloned DNA was boiled for 3 min and cooled in ice to denature. Add the denatured DNA to the reaction buffer containing primers, dNTP, [α- ^32P ]dCTP (3000Ci/nmol, NEN), then add Klenow fragment to the solution, and react at 37°C for 1 hour. The probes obtained consisted of probe A comprising a part of exons 2-5 of the uroplakin II gene and probe B comprising a part of exons 1-2 of the uroplakin II gene ( FIG. 1 ).

Then, the reaction solution was purified using a Sephadex G-50 column, thereby preparing ³² P-labeled DNA probe A and probe B for porcine uroplakin II promoter detection.

2) Library screening

To isolate the porcine uroplakin II promoter, a porcine genomic library was screened. In this example, a porcine genome library that had been inserted into the λFix II phage vector (Stratagene) was used.

Host bacteria transduced with the library were prepared as follows.

A bacterial colony was inoculated in 5 ml of LB medium containing 0.2% maltose and incubated overnight at 37°C. The 1% culture medium was transferred to 50 ml of fresh LB medium containing 0.2% maltose, and incubated for 2.5 hours. When the absorbance at 600 nm reached about 0.5, the culture solution was centrifuged at 2,500 rpm for 10 minutes. The resulting cell pellet was suspended in 10 ml of sterile magnesium sulfate solution to a final concentration of 1×10 ¹⁰ cells/ml, and stored at 4°C until use.

To determine the titer, the library was serially diluted into SM solution at different concentrations. Plates containing solid LB medium were incubated in a 37°C incubator, the top agar was dissolved, and placed in a water bath maintained at 48°C. 10 μl of each phage solution diluted in different concentrations was mixed with 100 μl of the above-prepared host bacteria, and the host bacteria were infected with the phage at 37°C.

Add the phage-infected host bacteria to the top agar, shake well, and pour onto the LB medium prepared above. After 15 minutes, the plates were incubated face down in a 37°C incubator overnight. Phage plaques were formed on the overnight cultured plate medium for subsequent steps. The plates were cooled at 4° C. for at least 1 hour. The plaques indicated that the phage lysed the host bacteria after the library DNA propagated in the host bacteria.

Provide a serial numbered NC filter and cover the library DNA plate prepared above with the filter in such a way that the middle of the filter touches first. Use a needle to puncture the filter membrane in a direction perpendicular to the filter membrane to mark the position. After 1 minute, carefully separate the filter membrane from the medium.

Each filter membrane was continuously immersed in denaturing solution, neutralizing solution and 2×SSC solution for 1 minute in each solution, and then placed in an oven at 80°C for 2 hours, so that the transferred library DNA was completely immobilized on the filter membrane.

Float each fixed filter membrane in 2×SSC solution to make it wet, then pre-hybridize in a petri dish containing pre-hybridization solution, and shake slowly at 68°C for 1 hour. After pre-hybridization, the probe prepared in part 1) of Example 1 was added to each filter membrane, and hybridization was carried out by shaking slowly at 68° C. for 18 hours. After hybridization, the process of immersing in 2×SSC solution containing 0.1% SDS, and shaking at 65° C. for 10 minutes to wash the filter membrane was repeated twice. After washing, the filters were air-dried and subjected to autoradiography.

Plaques showing positive signs were picked by comparison between autoradiographic results and plates. Plaques were placed in 500 μl of SM solution, and one drop of chloroform was added and mixed well with the solution, and the mixture was stored at 4°C. This screening process was repeated 3 times to finally obtain clones showing positive signs. The DNA contained in each clone was purified using the Qiagenlambda mini kit.

Use the ABI 377 DNA sequencer (Applied Biosystem) to read the DNA base sequence, use the CAP2 sequence assembly (assembly) system to process the sequencing results, use BLAST, SMART, PROSITE, etc. for sequence comparison, and use the Clustal W program for motif analysis .

As a result, when screening was performed with probe A, clones A and B shown in Fig. 1 were obtained. When probe B was used for screening, clones C and D shown in Fig. 1 were obtained. Since each of these clones contains the porcine uroplakin II gene or structural gene at the 3' end, comparison between the clones provides the complete base sequence of the porcine uroplakin II promoter.

The total length of the porcine uroplakin II promoter of the present invention is 8847bp, and its base sequence is shown in SEQ ID NO:1.

3) Verify the expression pattern of the protein expressed under the regulation of the promoter of the present invention

In order to verify the expression pattern of the protein expressed under the control of the promoter of the present invention, the expression of porcine uroplakin II was verified as follows.

3-1) Verify the bladder-specific expression of the protein expressed under the control of the promoter of the present invention

In order to verify whether the proteins expressed under the control of the promoters of the present invention were expressed in a bladder-specific manner, Northern analysis was performed.

The porcine uroplakin II cDNA obtained in Example 1, part 2) was used as a probe, while an actin probe expressed at a constant level in all tissues was provided as a control group. In order to confirm whether the expression of porcine uroplakin II mRNA was present in various porcine body tissues using the probe, total RNA from tissues including bladder, heart, liver, lung, uterus and spleen was subjected to electrophoresis as follows.

Place 0.7g of agarose in a 250ml Erlenmeyer flask, add 58ml of distilled water to dissolve it completely in an electronic rang, and cool it in a water bath kept at 60°C. When the temperature of the agarose gel is adjusted to 60°C, carefully add 7ml of 10× gel running buffer, while shaking, then add 11.9ml of formaldehyde to prepare 1× formaldehyde gel running buffer. Place the solution in a pre-set electrophoresis system and let it stand for 20 minutes to form a gel.

Thoroughly mix 6 μl RNA, 2.5 μl 10× running buffer, 4 μl formaldehyde and 12.5 μl formamide into a centrifuge tube, heat at 65°C for 5 minutes, and then cool on ice. Add 2.5 μl of gel loading buffer, mix well with the sample, and add to the gel that has been pre-electrophoresed at 5V for 5 minutes. The resulting material was electrophoresed at 120 V/cm in 1X running buffer. After electrophoresis, the gel was placed in 0.05N sodium hydroxide solution for about 10 minutes, and the RNA was partially excised to increase the efficiency of the subsequent transfer process.

Place the gel in 0.1M Tris solution (pH 7.5) for 30 minutes, then place it in 20×SSC solution (3M sodium chloride, 0.3M sodium citrate, pH 7.3) for about 30 minutes, then use positively charged The membrane transfers the RNA to the gel. For RNA immobilization, the transferred membrane was left to stand at 80°C for 2 hours.

Place the membrane in a vinyl bag containing the minimum volume of hybridization solution to completely submerge the membrane. Then, store the bag in a shaking incubator at 68° C. for at least 1 hour, then take out the solution, replace it with 15 ml of hybridization solution containing probes, and put it in a shaking incubator at 68° C. overnight.

After hybridization, wash with washing solution 1 (2×SSC, 0.1% SDS) at room temperature for 30 minutes, replace the washing solution, then wash with washing solution 2 (0.2×SSC, 0.1% SDS) at 55°C for 30 minutes, replace the washing Solution 2. After allowing the membrane to dry completely at room temperature, it was autoradiographed to verify the expression of porcine uroplakin II mRNA. The result is shown in Figure 3.

As shown in Fig. 3a, actin mRNA, which served as an internal control group, was expressed consistently in all tissues. On the other hand, as shown in Figure 3b, the uroplakin II mRNA expressed under the control of the promoter of the present invention was specifically expressed only in pig bladder (Figure 3b).

As a result, it can be seen that the promoter of the present invention expresses the protein in a bladder-specific manner.

3-2) Verify the bladder epithelium-specific expression of the protein expressed under the regulation of the promoter of the present invention

Meanwhile, in order to verify whether the protein expressed under the control of the promoter of the present invention is expressed in any cell of the bladder tissue, immunohistochemical staining was performed as described below.

Paraffin fragments of porcine bladder tissue were provided and left in Histoclear solution for 10 minutes to remove paraffin. Sections were dehydrated by dipping in decreasing concentrations of aqueous ethanol solutions, and immersed in methanol containing 3% sodium hydroxide and 0.05N hydrochloric acid containing 0.1% pepsin to prevent non-specific staining of the sections.

Wash twice for 5 minutes with TBS buffer (0.05M Tris, pH 7.4, 0.85% NaCl), and then perform blocking reaction in TBS diluted 1:5 with normal horse serum.

Soak the blocked sections overnight in TBS diluted 1:500 with the primary antibody. At this time, use a polyclonal antibody that can specifically bind to porcine uroplakin II protein as the primary antibody, and use a drop of horse serum from the ABC kit as a negative control group.

The slices that had undergone the primary antibody reaction were washed 5 times with TBS, 5 minutes each time, to remove excess antibody, and then allowed to react with the biotin-conjugated secondary antibody for 30 minutes. Then, the sections were washed 3 times with TBS and reacted with ABC reagent for 30 minutes. The sections were washed again with TBS, washed with PBS containing 1% Triton-X 100 for 30 seconds, and then reacted with 0.05M Tris buffer (pH 7.6) containing 0.5% diaminobenzidine (DAB) and 0.01% hydrogen peroxide color.

After the color reaction, the sections were washed with water, and placed under an optical microscope to observe the colored parts. The result is shown in Figure 4.

As shown in Figure 4a, the control group did not exhibit any positive signal. However, as shown in Figure 4b, the bladder tissue reaction of the antibody and the uroplakin II protein showed that the promoter of the present invention regulates the uroplakin II protein, so that the protein is only specifically expressed in the porcine bladder epithelium, especially in the upper basal cells expressed in the cytoplasm. 3-3) Verify the expression level of the protein expressed under the control of the promoter of the present invention

Because bladder epithelial cells are known to have lower protein synthesis ability than mammary glands, which actively undergo protein synthesis, the identity of proteins expressed under the control of the promoter of the present invention was verified by laser scanning cytometry (hereinafter referred to as "LSC") in the following manner. actual level of expression.

Porcine bladder tissue was carefully dissected and added to DMEM/F12 medium (Gibco) containing 1 mg/ml type I collagenase (Sigma), 0.51 mg/ml hyaluronidase (Sigma) and 50 μg/ml gentamicin , and the digestion reaction was carried out at 37° C. for 1 hour.

After the resulting material was washed with PBS, bulk material was filtered off using a 60 μm nylon membrane (Milipore), and the suspended single cells were bound to Lab-Tek chamber slides (Nunc) coated with 0.1% gelatin. Cells bound to slides were washed with cold PBS, fixed in cold methanol for 15 minutes, and then treated in 0.1% Triton-X 100 solution for 10 minutes.

The fixed cells were blocked in PBS containing 1% BSA for 1 hour, and reacted with the 1:100 solution of the uroplakin II polyclonal antibody prepared in part 3-2) of Example 1 at room temperature for 2 hours. After washing with PBS, cells were reacted with FITC-conjugated anti-mouse IgG secondary antibody (Cappel Laboratories). At this time, a group reacting only with the secondary antibody was prepared as a negative control group.

After the cells were washed three times with PBS containing 0.1% Tween-20, they were stained with 50 μg/ml propidium iodide (PI) to determine the total number of cells. During LSC analysis, a 488 nm argon laser was used to emit fluorescence, and the fluorescence expression was observed using a 530 nm filter for FITC and a 570 nm filter for PI. The result is shown in Figure 5. The results of the analysis of the negative control group are shown in Figure 5a, the results of the analysis of the cells expressing uroplakin II in the bladder cells are shown in Figure 5a, and the results of the analysis of the immunophenotype of the bladder cells expressing uroplakin II are shown in Figure 5c Show.

It can be seen from Figure 5b that about 8-14% of all bladder cells expressed uroplakin II. It can be seen from Figure 5c that most of the cells are actively proliferating and cleaved umbrella cells. Considering that protein levels in urine are usually very low levels of 5-25 mg/l, the above expression levels of uroplakin II are very high. In addition, it is also believed that the use of bladder tissue enables proteins to be isolated and purified with higher efficiency than the use of breast tissue.

The result shows that the promoter of the present invention can make the target protein be highly expressed in the bladder.

Example 2: Generation of the expression vector pUP2/hEPO of the present invention

Using the promoter of the present invention isolated in Example 1, a vector expressing EPO under the control of the promoter was produced by the following method.

Select pBluescript SK(-) as the basic backbone vector, and insert the promoter of the present invention isolated in part 2) of Example 1. Then, the gene (SEQ ID NO: 4) encoding human EPO was inserted into the 3' end of the promoter.

The resulting expression vector has the structure shown in Figure 2, and expresses EPO under the regulation of the uroplakin II promoter of the present invention. The vector is called "pUP2/hEPO", and was deposited at the Korean Type Culture Collection (KCTC) at the Korea Institute of Bioscience and Biotechnology on October 17, 2002, with the accession number KCTC 10352BP.

Example 3: Generation of fertilized egg cells introduced with the expression vector pUP2/hEPO of the present invention

Fertilized egg cells into which the expression vector pUP2/hEPO of the present invention produced in Example 2 were introduced were produced as follows.

1) Collect fertilized eggs

Three days before fertilized eggs were collected, PMSG was administered into the peritoneal cavity of female mice, and two days later, hCG was administered to female mice at 5 o'clock in the afternoon, and then mated with male mice for fertilization. On the morning of the second day of mating and fertilization, observe whether a plug has been formed in the female mouse to verify whether the female mouse is pregnant.

Mice that were confirmed to be pregnant were killed by necking, and the abdominal cavity was cut open with surgical scissors, and the connective tissue part of the uterus was separated. The part between the fallopian tube and the uterus is torn apart with forceps, and the part between the ovary and fallopian tube is cut with scissors. Then, cut off part of the uterine wall with forceps, and separate the fallopian tubes.

Place the isolated oviducts in M2 medium and place them on an insulating plate to prevent their temperature from dropping. Under a microscope, the ampulla of the oviduct was dissected with a 1ml needle, and the embryos were collected. The collected embryos were placed in the hyaluronidase solution that had been exposed to room temperature and allowed to stand until the cumulus cells dispersed.

The resulting solution was washed 2-3 times with M2 medium, centrifuged at 13,000 rpm for 5 min, and then washed 2-3 times with M2 medium. The selected fertilized eggs were washed 2-3 times in M16 medium covered with paraffin oil, then transferred and stored in a 37°C incubator.

2) Microinjection of DNA into fertilized eggs

Using micromanipulation, the expression vector pUP2/hEPO of the present invention was injected into the fertilized eggs collected above.

Example 4: Preparation of transgenic mice producing human EPO under the control of the promoter of the present invention

Using the fertilized eggs produced in Example 3, transgenic mice producing human EPO under the control of the promoter of the present invention were produced in the following manner.

1) Preparation of vasectomized mice

Vasectomized mice used to induce pseudopregnancy in surrogate mothers were generated as follows.

Select and anesthetize a 6-week-old ICR mouse, then use forceps and scissors to cut about 1 cm of epidermis along the pubic bone at a distance of about 1.5 cm above the pubic bone. Placed to the left or right to prevent overlapping incisions, cut the muscularis and move the testes below the scrotum into the abdominal cavity. The testis, epididymis, and vas deferens are separated from each other with forceps, the membrane surrounding the speraduct is separated with forceps, and the vas deferens is cut with heated forceps. After confirming that the vas deferens had been isolated, the muscularis was sutured and the mouse was placed in the greenhouse until awakening.

2) Generation of pseudopregnant mice as surrogate mothers

Before the test day, ICR female mice, which had been confirmed to have estrus, were mated and fertilized with the vasectomized mice produced in Example 3, part 1). On the morning of the test day, observe whether plugs have developed in female mice to verify pseudopregnancy in female mice.

3) Embryo transfer into fallopian tube

The zygotes prepared in Example 2, part 2) were arranged in a micropipette. Slightly cut the epidermis and muscular layer of anesthetized female mice serving as surrogate mothers, and remove the upper part of the ovaries, oviducts, and uterine horns from the body using forceps. The ovary is positioned in such a way that the portion exposed through the ovarian capsule faces upward. Then, a hemostatic device is inserted into the fatty tissue to stabilize the ovary. Under a stereomicroscope, the membranes of the ovarian capsule were removed, and then the oviducts and ovaries were removed to visualize fimbrae. Then, the front tip of the transfer pipette was inserted into the fallopian tube 2-3 mm, and the fertilized eggs were carefully transplanted into the fallopian tube together with the culture medium. Observe that the first of the two air bubbles in the pipette as a marker inserts into the fallopian tube to verify that the fertilized egg has indeed implanted in the fallopian tube.

Offspring were obtained from surrogate dams. In order to screen transgenic mice among them, Northern analysis was performed using exons 1 and 2 of EPO as probes, and the analysis results showed that 12 of 76 mice were transformed.

The expression pattern of EPO protein in transgenic mice was verified, and the results showed that EPO protein was expressed in a bladder-specific manner.

Example 6: Generation of human EPO from transgenic mice of the present invention

1) To verify the expression level of EPO in the urine of the transgenic mice of the present invention, the urine was obtained from the transgenic mice, filtered and analyzed by HPLC. In order to verify the protein composition of each fraction, electrophoresis and western analysis were performed, and the results are shown in Figure 7.

It is evident from the results of electrophoresis in Figure 7a and the results of Western analysis in Figure 7b that the urine obtained from the transgenic mice contained a high concentration of EPO.

The concentration of EPO in urine was calculated at an expression level of 0.5-1 mg/ml, which is significantly higher than the protein expression level in milk from existing transgenic animals.

Therefore, the transgenic animals produced using the promoter of the present invention can efficiently produce the target protein in urine.

2) Verify the physiological activity of the EPO obtained from the transgenic mice of the present invention

In order to verify the physiological activity of the EPO obtained from the transgenic mice of the present invention, the EPO obtained in part 1) of Example 3 was added to EPO-dependent hepatocytes and cultured. At this time, commercially available EPO was added to the control group. The viability of the cells was measured at each time point of 24, 48 and 72 hours after culture, and the results are shown in Table 1.

Table 1: Training time DMEM/F12(%) FBS FBS+Commercial EPO FBS+EPO of the present invention twenty four 38.5±6.8 54.9±4.3 58.2±6.6 72.1±4.7 48 21.6±7.4 39.9±2.9 50.0±2.4 60.4±7.5 72 10.0±4.6 20.8±11.7 39.6±3.8 53.9±4.0

As shown in Table 1, it can be seen that the EPO isolated from the urine of the transgenic mice of the present invention exhibited higher physiological activity than the commercially available EPO in all time periods.

Therefore, the transgenic animals produced using the promoter of the present invention can produce proteins with much higher physiological activity than those obtained by existing methods.

Embodiment 6: Generate the expression vector of the present invention containing regulator and verify its efficiency

1) Construction of expression vectors containing regulators

In order to establish a vector system capable of maximizing EPO production under the control of the UPII promoter of the present invention, a selectable marker and a regulator were introduced into the pUP2/hEPO vector in the following manner to generate a series of improved vectors. 1-1) Construction of pUPII/hEPO-Neo vector

In establishing a cell line capable of expressing a protein under the control of the UPII promoter, in order to insert an efficient selection marker into the vector, a neomycin resistance gene was introduced in the following manner to produce a pUP2/hEPO-Neo vector.

To obtain the neomycin resistance gene, a PCR reaction was carried out using the pEGFP-N1 vector (Clontech) as a template, and forward primer (SEQ ID NO: 8) and reverse primer (SEQ ID NO: 9).

5'-GCGGCCGCGCGCGTCAGGTGGCAC-3'(sEQ ID NO: 8)

5'-CGATCGGACGCTCAGTGGAACGAAAACTC-3' (sEQ ID NO: 9)

The resulting 1.9-kb PCR product was inserted into the pGEM T-easy vector and digested with NotI restriction enzyme to prepare the neomycin resistance gene portion for cloning.

The ampicillin resistance gene site in the pUP2/hEPO vector of the present invention was removed by digestion with NotI and SalI restriction enzymes to prepare a vector for cloning.

The above-prepared neomycin resistance gene was cloned into the vector to produce a pUP2/hEPO-Neo vector in which the neomycin resistance gene was inserted into the existing pUP2/hEPO vector.

1-2) Construction of I/pUP2/hEPO vector

In order to obtain an expression vector capable of stably expressing a protein under the control of the UPII promoter, the insulator gene was introduced into the pUP2/hEPO-Neo vector in the following manner, thereby generating the I/pUP2/hEPO vector.

To obtain the insulator gene, a PCR reaction was performed using the pBC1 vector (Invitrogen) containing the chicken B-globin insulator gene as a template, and forward primer (SEQ ID NO: 10) and reverse primer (SEQ ID NO: 11). To increase PCR efficiency, two copies were amplified.

5'-TCGACTCTAGAGGGACAG-3' (SEQ ID NO: 10)

5'-CTCACTGACTCCGTTCCT-3' (SEQ ID NO: 11)

The resulting 2.4-kb PCR product was inserted into the pGEM T-easy vector and digested with NotI restriction enzyme to prepare the insulator gene for cloning.

The above-prepared insulator gene and the vector of the above section 1-1) were ligated to each other through the NotI site, thereby generating an I/pUP2/hEPO vector (Fig. 8).

1-3) Construction of pUP2/hEPO(WPRE) vector

In order to obtain an expression vector capable of expressing a protein in a large amount under the control of the UPII promoter, the WPRE gene was introduced into the pUP2/hEPO-Neo vector in the following manner to generate a pUP2/hEPO(WPRE) vector.

To clone the WPRE gene, a PCR reaction was carried out using a forward primer (SEQ ID NO: 12) and a reverse primer (SEQ ID NO: 13).

5'-ACCAGGTTCTGTTCCTGTTAATCAACCTC-3' (SEQ ID NO: 12)

5'-CTCGAGGAGCCCGAGGCGAAACAGGCG-3' (SEQ ID NO: 13)

The resulting 0.6-kb PCR product was inserted into the pGEM T-easy vector and inserted into the NcoI restriction site of pUP2/hEPO-Neo generated in part 1-1) of this example. The resulting vector was digested with BspHI restriction enzyme to prepare WPRE gene for cloning.

Meanwhile, the rear portion of the EPO gene in the pUP2/hEPO vector of the present invention was digested with NcoI restriction enzyme to prepare a vector for cloning.

The WPRE gene prepared above was cloned into a vector to generate a pUP2/hEPO(WPRE) vector (Fig. 9).

1-4) Construction of I/pUP2/hEPO(WPRE) vector

In order to generate an expression vector satisfying all maximization of expression level, expression stability and establishment of efficient cell lines under the control of UPII promoter, I/pUP2/hEPO(WPRE) vector was generated in the following manner.

The insulator gene prepared in part 1-2) of this example was connected to the vector in part 1-3) of this example through the NotI site to generate the I/pUP2/hEPO vector ( FIG. 10 ).

2) Verify the efficiency of the expression vector of the present invention

The efficiency of the expression vector produced in Example 6 was verified in the following manner.

2-1) Carry out PCR analysis to the expression vector of the present invention

In order to verify the expression level of the EPO gene by the expression vector of the present invention, real-time PCR was performed as follows.

The four expression vectors of the present invention produced in Example 6 were introduced into the bladder cell line RT4 using a transfection kit (Effectene, Qiagen), and subcultured to establish a stable cell line. Genomic DNA was extracted from each resulting cell line and PCR was performed to verify that the transfection was performed correctly.

To verify the expression level of the EPO gene, total RNA was extracted from the four cell lines, and RT-PCR was performed to amplify the cDNA. PCR was performed using the above cDNA as a template, and forward and reverse primers capable of amplifying the EPO exon region.

To verify the expression level in each cell line, the process was repeated 3 times using the housekeeping gene GAPDH expressed at a constant level in the cells as a control group. The test results are statistically processed with the SAS program, shown in Figure 11 (pUP2=pUP2/hEPO carrier; IUP2=I/pUP2/hEPO carrier; PW=pUP2/hEPO (WPRE) carrier; IW=I/pUP2/hEPO (WPRE) ) carrier).

As shown in Figure 11, the EPO gene expression level of the expression vector of the present invention gradually increases in the order of pUP2/hEPO vector, I/pUP2/hEPO vector, pUP2/hEPO (WPRE) vector and I/pUP2/hEPO (WPRE) vector.

Specifically, the I/pUP2/hEPO (WPRE) vector containing WPRE and an insulator (WPRE) exhibited approximately 50-fold higher expression levels than pUP2/hEPO without an isolating insulator (Fig. 11b).

Thus, vectors of the invention including I/pUP2/hEPO(WPRE) vectors can be advantageously used to generate EPO.

2-2) Western analysis of the expression vector of the present invention

In order to verify the expression level of EPO protein by the expression vector of the present invention, Western analysis was performed as described below.

Cell lines established by introducing each expression vector of the present invention in section 2-1) of Example 6 were placed in lysis buffer containing NP-40 and sonicated to extract proteins from the cell lines.

Each 40 μl protein was electrophoresed on SDS-PAGE gel, transferred to PVDF membrane, and treated with EPO antibody to verify the expression level of IPO protein. To quantify the expression level of EPO protein, this process was repeated twice using an antibody to actin as a control group. The results were processed with the SAS program and are shown in Figure 12 (pUP2=pUP2/hEPO vector; IUP2=I/pUP2/hEPO vector; PW=pUP2/hEPO(WPRE) vector; IW=I/pUP2/hEPO(WPRE) vector) .

As shown in Figure 12, the EPO protein expression level of the expression vector of the present invention gradually increases in the order of pUP2/hEPO vector, I/pUP2/hEPO vector, pUP2/hEPO (WPRE) vector and I/pUP2/hEPO (WPRE) vector.

This result is consistent with the result shown in part 1) of Example 7.

Thus, vectors of the invention including I/pUP2/hEPO(WPRE) vectors can be advantageously used to generate EPO.

industrial application

As described above, the promoter of the present invention induces bladder-specific expression of the protein of interest, and expresses the protein of interest in urine at a much higher concentration than existing methods.

Animals transformed with the expression vector consisting of the promoter of the present invention and the target protein regulated by the promoter can secrete the target protein at a higher efficiency than existing transgenic animals. In addition, proteins obtained from transgenic animals of the present invention exhibit better physiological activity than existing proteins of the same type.

Therefore, the promoter of the present invention, the expression vector using the same, and the transgenic animal can be advantageously used in the field of production of useful proteins of medical value.

Sequence Listing

<110>CHO-A PHARM CO., LTD.

KIM, Jin Hoi

<120> porcine uroplakin II promoter and production using said promoter

A method of producing useful protein

<130>03PP181

<150>KR 10-2002-0067856

<151>2002-11-04

<150>KR 10-2003-0077256

<151>2003-11-03

<160>13

<170>KopatentIn 1.71

<210>1

<211>8847

<212>DNA

<213> pig

<220>

<221> promoter

<222>(1)..(8847)

<223> porcine uroplakin II promoter

<400>1

gggctaggag tggaatcaga gctggcctat gccacagcaa cgcagaatcc aaaccacatc 60

tccgacctac accagaccgt caccataaca caggatcctt aacccactga gcaaggtcag 120

ggatcaaacc caaatcctca tggatactag tcgggttctt aacccgctga gccacagtgg 180

gcactcctgt ttttgtttgt gtcttcgttt tttggctgca tctgcagcat acagaagttc 240

ctgggttaag gattgaaccc atgccacagc agcaacccga gccacagcag tgacaacagc 300

ctgatcctta actgctagac caccagggaa cgccccctca acttttcatg cattggaaac 360

cctgagtcag tacaacctga caatngnttt tttttttttt tttttttgcc ttttctaggg 420

ccacttcccg cggcatgtgg agattcgcag gctanaggtc taatcggagc tgtagccacc 480

ggcctacacc agagccatag caacgaggga tccgagccga gtctgcaacc tacactacag 540

ctcatggcaa caccggatcg ttaacccact gagcaaggcc aggggatcga acccgcaacc 600

tcatggttcc tagtcagatt cgctaaccac tgcaccatga caggaactcc caacctgaca 660

attttatcat ttctgcaccc tagttgttga gtaatttgaa aaattcccaa gatgtcaagg 720

tcagtgtgat ggttaatttt atgtgtcaac ctgactaggc catgttgccc ggatgtggag 780

tcattgttat tctggatgtt actgtgaaga tatgttttgg atgaaattaa catttaaatc 840

agtgggggga aaaaaagaag ttctcgttct ggtgcatcag aaacaaatcc gactaggaaa 900

caagcggttg caggttcgat ccctggcctc acttagtgga gtcaggatct ggcgttgccg 960

tgagctgtgg tacaggtggc agatgcagct cggatctagc attgctgtgg ctgtggtgta 1020

ggccagcagc tgtagctctg attaaaccccc aagtctggga acctccatat gccgtgggtg 1080

tggcccgaaa aagcaaaaaa taaataaata aataaattta aaccaggggga ttttgagcaa 1140

agcagattac cccataatat gggtgggtct catcaagttc attgtaggcc ctagtggaac 1200

aaagaccgac ctccaccttc tccccatgag aaggaaagaa ttctgccaaa agaccgcctt 1260

nggacntaaa ctgcaactct ttcctgagtt tccagcatgt tggcctcccc catcagactt 1320

tggacttgcc aagcctccgc aattgcatga gccaattcct taaaataaat ccgtctatat 1380

atacacatcc tgttggttct gtttctccag agaaccctga ctaacgcagt ctgcacccct 1440

gaagaccagt ggtccccaca ctcagctggg tgtcacctcc aaacactcag ccttcctcaa 1500

ggctctttct agctgtgtcc tcctctcccc acaacagctg tttcaaactc tcacccctct 1560

tcagggcgca atcccttctc ctccctgagt ttcctacttc ccagagaaag cagagacctt 1620

caggagtgtg ctgccttaac ttacttcctt catccctcag ccttgcaaaa gtataagctt 1680

tctctgcacc actgccccat tcttctctct gcagacaggg tcattcctaa agccaaacgc 1740

taatgcctcc acctctgatc tgagtcccat cttttccctc ctccagaagc ttcctcataa 1800

attctacccc cttttcttcc ttatctttat ctttgaaaac aaaatggaag acagccttcc 1860

cgttgtggtg cagcggaaac agtggtgcct tggaagcgct gggacgcagg ttcgacccct 1920

ggcccagcat agtaggttaa ggatccagtg ttgccacagt tttggcttag attgaaactg 1980

cagctcagat ctggtccctg gcctgggaac ttcatacgcc acaggacggc ccaaaaagaa 2040

aagaaagaaa aaataaaaaa caaaacagaa aagcctttcc tgtaccccca attccctcca 2100

gttatctctc tctttccctt cccagccaag ctctgcaaag agcggtctgc acagttctaa 2160

ctctacctcc tcccagttgg ccctggactt tctcagtctg gcttctaccc ccctcacccg 2220

taggaatctg ctctgaagga cacgcacccc tcacgatcct tggcccaggg attttttg 2280

taccagcctt tcaatcctga ccttcatatc atccgacacc tcctttgtga aaccctccat 2340

ccactttctc ctggttcccc tcctaagacc cattccgcct tcttcagccc cctccctcca 2400

tctgtccttt agatgccgca tttcctagta tcctgtcctg cgcggnctcg tccttccctt 2460

ccacaactct cttcaaggac tcttttctcc atgtgcgatt ttgcccatgg cccaccttcc 2520

ctctctttac ccagactttc ccccggtgct ccagactcat agactcaatt atgaaaacat 2580

agttttcatc tgatttgccc aagatatttg cattagttat tactgtataa cagcttatcc 2640

cccaatttag tggcttataa aataaacact tattctgaga atcagaaacc taggcaggac 2700

atagttgggg tctcatgaag ttgcactgaa aatgtccccc tgggctaatc atacggagga 2760

ctgaccaggg ctggaggatc tgttccaagc tcattcattc acatggccgt aggtggggaga 2820

cagctcttct ctggatcttg gcaggagcct caattccttg tcacgtggac ctccccttgg 2880

aggggtccc atgtcctcca tggtgagtaa tccatgagag caaggtggaa ggtgccatgc 2940

catttaggac ctagcctcag gagggaccta cgtcacttct gttgtagtct gttggccaca 3000

cagactaacc ctgacacaat gcacccatcc atgacctgct gccagtccat tctccaacact 3060

gtttccagaa tgatatttac ataagtaaaa ctcctcaaag gcttttgaga ttttttttcc 3120

catttagtt gatttataac ctcagaggct tttgttttct tcagcataaa aaccaagttc 3180

cttaacatag catgtaaccc actggccacc ctgccagtgg ctagaactct caccatgtcc 3240

atccttgaat actgctttct agccaagagc tattgtttgc agttccccaga atgtgtcggg 3300

ataactcaca tctctgagcc ttttcatgtg ctgttccctc actttggaat atccccttcc 3360

atttaggaag gctaatgtcc attcattntc caaaactcag aagcaaattt tttttttttt 3420

tttttttttt tttttttgct ttttagggcc gaactctcag catatggagg ttcccaggtt 3480

agccatcaaa ttggaattgt agctgctggc ctacaccaca gccatagcaa caccagaccc 3540

aagtcacatc tgcaacctac atcacagatc atggcaatac tggatcctta acccactgag 3600

tgagcccagg gatcaaacac aaattctcat ggatactcgc caggttcatt accactgagc 3660

cacaacagga actcctctcc tttttatggt cacacctgca gcatatggaa gttcctgggc 3720

cagggattga atctgagtgg cagctgtgac aatgccgtat cctttaattc actgtgctgg 3780

gctgaggggn taaantgccc ctcctaaaaa acctgagctg ctgcagttgg attcttaatc 3840

cactgcacca caagggggaa ggtcaagaac tgtcttgcca tctctgtatc ttatcaccta 3900

gcatagtacc caccatagag aagttgctca acaaatgttt actgaatgaa taaatgcatg 3960

agctggagtt cccattgcgg ctcagcagta acaaacctga ctagcattca taagaacttg 4020

ggttcgatcc ctagcctcag tgggttaagg atgcagcatt gctgtgagct gtggtgtagg 4080

tcgcagacga cactcagatc ccacattgct gtcactgtgg cgcaggccgg cctctgtagc 4140

tctgattcga ctcctagcct gggaacgtcc atatgccaca ggtgaggccc taaaaagaaa 4200

taaataagca agcaagtaag caagcaggca gtttcttggt gccttgtacc cctgtggcct 4260

gtgtggtata caagtaacag ctgatccatg tctcagtcat gtttccccct cagactacct 4320

ttcctgcccc atctctccct ttgacataat tggaaaaaca aattcagaat tttgtcccac 4380

tacctttctt gctagctctg tggccttggg aaagctattt attgcctctg agcctctaat 4440

tttcatctgc accaaggatt aataaaagg agaggataag atgaattact tatattaata 4500

tttattgaac cagatactgt gctaggcact cttaaataaa ttagcttgag tgatagtcat 4560

agtatcctgg tgagacagat tttttttttc cttttatggt tgcacgtgca acatatggaa 4620

gttcctgggc tggggtcgaa ttggagctgc aggtgcttgc ctatgccaca gccatggcaa 4680

catcatatac aaaccgcacc tgtgacctac accacagatt gcagcaacgc tggatccttc 4740

acccaaggag caaggccagg aatcaaatgt gcatcctcac aaacactatg tccggttttt 4800

aacccgctga gccacaccag gaactccatg gcgagacaga ttttatactc tgtctacaga 4860

agaggaaagt gaagctcaga atggttaggt aggtaacttg gccaagatca aaaaattcaa 4920

agaagatttg gggcaagtgg tgatatcatg gcagcattag aaaaaataaa gaagcatcca 4980

cttgttttcc aacactgaac aactgagatt ttcttactct cacagctttt tccagcttca 5040

tatccaagga cagacgctct gccattttcc catcagacca atatttgctg aacactgcac 5100

ctttactttt aggtccaagt caccagggggt tttcccagtt tgctcctaca gattctgaca 5160

ctatctccac attttttttg cacctttat ttaaagcatt tttatacctg tcataccttg 5220

ctagataaat gggaaggaat gaatcttccc atttataggt gagaaaattg aggttcaaag 5280

tgactcacca aaagtcatat agcatcactc ctcaacagga ggacagcagt ccccaccaga 5340

gggtaacatg tccatggagc ctagtggaca catttttcta actgactggg aagcagcaga 5400

gtggtattgt gaagggggaa tcataggtat atcaaacaga cttaggttct gatccgagct 5460

attctgcttg caaacaacca tagttcaatt taaaaaaaaa aaagaaagaa agaaagaaag 5520

aaaggagccc ccatcctggt gcagtggaaa caaattcaac taggaactgt gaggttgtgg 5580

gttcgatccc tggccttgct cagtgggtta aggatctggc gttgccatga gccgtggtgt 5640

aggttgcaga ctcaactcag atctggcgtt gctgtgactg tggctgtgat gtaggctggc 5700

agctgtaact ccggttagac cccagcctgg gaacctccat atgcaacctc catatgcggt 5760

gggtgtggcc ctaaaaagaa aaaaaaaaaa aaaagaggaa ttcccttatg gctcagcagg 5820

ttaaggatct ggtattgtca ctgctgtggc tctagttaca gccatagtgc aggttcaatc 5880

cctggcccag gaacgtctgc atcccacagg tgtggccaaa aaagaaagaa aggaaggagt 5940

tctgttgtgg cacaatagga ttggcaacat cttaggagta ctgggacaca ggttcaatcc 6000

ctggcccagc acagtgggta aggagccagt gttgctggtc aaaaaagaaa agaaaaagta 6060

ccatagttag agtaaatctg ttttaggagc tattctttgg ggcagaacag agagatcagg 6120

agctccttga gagcagaaac ttacctttac atccctcgtg cctagcacgg ttctaggggc 6180

atacctggta tttaataaat atagccaact ggataggggga ttggaaggaa agagcagggg 6240

agggaacttg agtgagttga aaaattgaga atccaaaggg gagacagcct agaaagagta 6300

ggtccaagaa agagatccca ggcatttgtg gccctggttc cctttttcca agccatgagg 6360

aaatcctcag aggaacagag tgctgtggct ttaaatgact tcagcgttgt caatgaatct 6420

gctcggctaa aagagttatc ctcttgctcc ttcgcttgtc ctccccctcc tctcagctcc 6480

ccaaaccctt ctcggctgct gtgatgggat aattagatgc gagagctcag cacagatgat 6540

gctccagttg cctagcaact aatggtttcc atggagaccg caaagcacag cctccagagc 6600

agccagtgag cagctcggca gggcagggag aagacgcaac tctcagctcc tccagaaacc 6660

tggggagggc caggagtggg gaagaagggg gggatcggag ggcttaaagg cacaggcccc 6720

tcttatcctc ttaaaatctg gtcagagctc tgccctcccc tcccctactc tgtcccactc 6780

ataatttcag atggagttgg gggcttagga gtggacccaa cacaacctac cctgcaataa 6840

acccaacctt ctttctgctt ctggtttgtg gctgaaaatg gnaaaagaaa tctcccaagt 6900

gcaagtgtaa acancntcct gggttggcaa tgggatctga agagtactaa gatccctcag 6960

acctggaatt ccaccatta gtctttccct ctctccaaag ttctcaatgt gcaaaagatc 7020

ctctttcagt ttgcagagca atgataggat cttctaaaag gagacaaaag ccaaggtgca 7080

ggaaaaatag aattcagttc ttcacccaaa ggcagcctgt cctgggagac aggggtgaaa 7140

cacttggtcc tgatctccat cagaggatcc agagtgtgtg tgtttgttgc tggggagggg 7200

gacacaatat agagcatctg gtgactcaaa gtatgtgcct cccagagtag catcaatcaa 7260

tgttacctgg aagcttgtta gaaatgcaga atttcaggct tcacctcaga cccactgaat 7320

cagaaactgc atcttaacaa gatccctcat gattcatacg cacattaaat ttggagaagc 7380

gctgacctga gaccctcctc ctctctgctt gggcccatag ttctaccttt attgtcacct 7440

cgtctcacct cgtgctcata ccccaggctt tgagcctacc cttcccccca tggggaaagg 7500

acacaaggcc accagcccct cacttcccta ccaggaccct ggccctcctc tgggactgga 7560

gaaggacaaa gaggaccccc tctgtggagg tctacgacct ctcctgacca agtagtccac 7620

tcaccacaag tggctctacc tctctgagtc tcagtttcca catccacaaa aggtggccaa 7680

tgctatctgc cacccagaat ggctgtgagg gtggagcagg caaagcctct gtgccatcag 7740

agaaattgtg tctctttttc attttctccc agtgggtttc tttctcgtct ttattctttt 7800

tttttttttt ttttcctgtc tgttgtattt ttagggccgt gcctgtggca tacggaagtt 7860

cccagggtag gggtccaatg ggagctgtag ccccggggcct acgccacagc cacagcaatg 7920

tgggatctga gccacgtctg caacctacac cacagctcac ggcaacacca gatccttaac 7980

ccactgagca aggccaggga tcgagcccac gtcctcatgg atgctagttg ggttcgttaa 8040

ccgctgagcc atgatgataa ctcctctttc tattctttag tcacaaacag tcaacaaagg 8100

ttgctgacca aggctgatcg tgcccacccc ccagcccccc agactgggcc agtgcccacc 8160

ccttgggtct ctctggaaat cctgcccagc atcaattggc tccactctcc aggaggatgg 8220

gaagccctgt ggcccctggg actcacaccc ctctgcatct cccagagtgc aggacctggt 8280

cttcaggaga caccaagaac tggctccccc ggctctgctg cccccacccc ctactaccag 8340

tttctctccc attcctgccc agtccaggcc ccctggggtt actctcctct ctctgtacac 8400

cagtgcaacc tcagaacctg cttccctcct gggaacaccc actaccacgt gggagaaggg 8460

gtcgtctagg ggttgggccc cagatacact tgtaagcagg aacacacgag cccttacatg 8520

tgggtgtccc ggaagaaggg ggttttccac cccccgcttt agtcaccctg cccctctgca 8580

gctgcctgag ccaccaagac ccagccaagg tctcctgcct tctggcctga gggccagctc 8640

cccatcctga aaaacctgtc tgggggcctc ccctgaggct gtagggccca aggcctcccc 8700

tgaggctgta gggcccaagg ggcaggttga acaggattcc cctctggccc ctcctacccc 8760

caggacaaaa ccagagcccc aggacagggc ctcacttgcc tcaggaaacc acagcttgcc 8820

agcacccagc ccagcaccag cccagct 8847

<210>2

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>Forward primer for amplifying porcine uroplakin II gene

<400>2

gatcctgatt ctgctggctb 20

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223>Forward primer for amplifying porcine uroplakin II gene

<400>3

atggtggtca tcacrgtgct 20

<210>4

<211>3602

<212>DNA

<213> Human

<300>

<301>Lin, F.K.

Suggs, S.

Lin, C.H.

Browne, J.K.

Sma11ing, R.

Egrie, J.C.

Chen, K.K.

Fox, G.M.

Martin, F.

Stabinaky, Z.

<302> Cloning and expression of human erythropoietin

<303>Proc.Natl.Acad.Sci.U.S.A.

<304>82

<305>22

<306>7580-7584

<313>1-3602

<400>4

aagcttctgg gcttccagac ccagctactt tgcggaactc agcaacccag gcatctctga 60

gtctccgccc aagaccggga tgccccccag gggaggtgtc cgggagccca gcctttccca 120

gatagcacgc tccgccagtc ccaagggtgc gcaaccggct gcactcccct cccgcgaccc 180

agggcccggg agcagccccc atgacccaca cgcacgtctg cagcagcccc gctcacgccc 240

cggcgagcct caacccaggc gtcctgcccc tgctctgacc ccgggtggcc cctacccctg 300

gcgacccctc acgcacacag cctctccccc accccaccc gcgcacgcac acatgcagat 360

aacagccccg accccccggcc agagccgcag agtccctggg ccaccccggc cgctcgctgc 420

gctgcgccgc accgcgctgt cctcccggag ccggaccggg gccaccgcgc ccgctctgct 480

ccgacaccgc gccccctgga cagccgccct ctcctctagg cccgtggggc tggccctgca 540

ccgccgagct tcccgggatg agggcccccg gtgtggtcac ccggcgcgcc ccaggtcgct 600

gagggacccc ggccaggcgc ggagatgggg gtgcacggtg agtactcgcg ggctgggcgc 660

tcccgccgcc cgggtccctg tttgagcggg gatttagcgc cccggctatt ggccaggagg 720

tggctgggtt caaggaccgg cgacttgtca aggacccccgg aaggggggagg ggggtggggc 780

agcctccacg tgccagcggg gacttggggg agtccttggg gatggcaaaa acctgacctg 840

tgaaggggac acagtttggg ggttgagggg aagaaggttt gggggttctg ctgtgccagt 900

ggagaggaag ctgataagct gataacctgg gcgctggagc caccacttat ctgccagagg 960

ggaagcctct gtcacaccag gattgaagtt tggccggaga agtggatgct ggtagctggg 1020

ggtggggtgt gcacacggca gcaggattga atgaaggcca gggaggcagc acctgagtgc 1080

ttgcatggtt ggggacagga aggacgagct ggggcagaga cgtggggatg aaggaagctg 1140

tccttccaca gccacccttc tccctccccg cctgactctc agcctggcta tctgttctag 1200

aatgtcctgc ctggctgtgg cttctcctgt ccctgctgtc gctccctctg ggcctcccag 1260

tcctgggcgc cccaccacgc ctcatctgtg acagccgagt cctggagagg tacctcttgg 1320

aggccaagga ggccgagaat atcacggtga gaccccttcc ccagcacatt ccacagaact 1380

cacgctcagg gcttcaggga actcctccca gatccaggaa cctggcactt ggtttggggt 1440

ggagttggga agctagacac tgccccccta cataagaata agtctggtgg ccccaaacca 1500

tacctggaaa ctaggcaagg agcaaagcca gcagatccta cggcctgtgg gccagggcca 1560

gagccttcag ggacccttga ctccccgggc tgtgtgcatt tcagacgggc tgtgctgaac 1620

actgcagctt gaatgagaat atcactgtcc cagacaccaa agttaatttc tatgcctgga 1680

agaggatgga ggtgagttcc tttttttttt tttttccttt cttttggaga atctcatttg 1740

cgagcctgat tttggatgaa agggagaatg atcgggggaa aggtaaaatg gagcagcaga 1800

gatgaggctg cctgggcgca gaggctcacg tctataatcc caggctgaga tggccgagat 1860

gggagaattg cttgagccct ggagtttcag accaacctag gcagcatagt gagatccccc 1920

atctctacaa acatttaaaa aaattagtca ggtgaagtgg tgcatggtgg tagtccccaga 1980

tatttggaag gctgaggcgg gaggatcgct tgagcccagg aatttgaggc tgcagtgagc 2040

tgtgatcaca ccactgcact ccagcctcag tgacagagtg aggccctgtc tcaaaaaaga 2100

aaagaaaaaa gaaaaataat gagggctgta tggaatacat tcattattca ttcactcact 2160

cactcactca ttcattcatt cattcattca acaagtctta ttgcatacct tctgtttgct 2220

cagcttggtg cttggggctg ctgaggggca ggagggagag ggtgacatgg gtcagctgac 2280

tcccagagtc cactccctgt aggtcgggca gcaggccgta gaagtctggc agggcctggc 2340

cctgctgtcg gaagctgtcc tgcggggcca ggccctgttg gtcaactctt cccagccgtg 2400

ggagcccctg cagctgcatg tggataaagc cgtcagtggc cttcgcagcc tcaccactct 2460

gcttcgggct ctgggagccc aggtgagtag gagcggacac ttctgcttgc cctttctgta 2520

agaaggggag aagggtcttg ctaaggagta caggaactgt ccgtattcct tccctttctg 2580

tggcactgca gcgacctcct gttttctcct tggcagaagg aagccatctc ccctccagat 2640

gcggcctcag ctgctccact ccgaacaatc actgctgaca ctttccgcaa actcttccga 2700

gtctactcca atttcctccg gggaaagctg aagctgtaca cagggggaggc ctgcaggaca 2760

ggggacagat gaccaggtgt gtccacctgg gcatatccac cacctccctc accaacattg 2820

cttgtgccac accctccccc gccactcctg aacccccgtcg aggggctctc agctcagcgc 2880

cagcctgtcc catggacact ccagtgccag caatgacatc tcaggggcca gaggaactgt 2940

ccagagagca actctgagat ctaaggatgt cacagggcca acttgagggc ccagagcagg 3000

aagcattcag agagcagctt taaactcagg gacagagcca tgctgggaag acgcctgagc 3060

tcactcggca ccctgcaaaa tttgatgcca ggacacgctt tggaggcgat ttacctgttt 3120

tcgcacctac catcagggac aggatgacct ggagaactta ggtggcaagc tgtgacttct 3180

ccaggtctca cgggcatggg cactcccttg gtggcaagag cccccttgac accggggtgg 3240

tgggaaccat gaagacagga tgggggctgg cctctggctc tcatggggtc caagttttgt 3300

gtattcttca acctcattga caagaactga aaccaccaat atgactcttg gcttttctgt 3360

tttctgggaa cctccaaatc ccctggctct gtcccactcc tggcagcagt gcagcaggtc 3420

caggtccggg aaatgagggg tggagggggc tgggccctac gtgctgtctc acagcctg 3480

tctgacctct cgacctaccg gcctaggcca caagctctgc ctacgctggt caataaggtg 3540

tctccattca aggcctcacc gcagtaaggc agctgccaac cctgcccagg gcaaggctgc 3600

ag 3602

<210>5

<211>1916

<212>DNA

<213> Red Jungle Chicken

<220>

<22b>misc_signal

<222>(1)..(1916)

<223> β-globin insulator

<400>5

gcggccgcgc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 60

atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 120

tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt 180

cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 240

ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg 300

caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg 360

cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat ttttttatt 420

tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt 480

tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat 540

gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 600

ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 660

gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 720

agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 780

cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 840

tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 900

gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 960

cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 1020

gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg 1080

cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 1140

ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 1200

agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 1260

cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 1320

cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg 1380

ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 1440

ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 1500

caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct 1560

atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc 1620

ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa 1680

tacgcccgcg tttcttcctt ttccccacccc caccccccaa gttcgggtga aggcccaggg 1740

ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttatactc atatatactt 1800

tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 1860

aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc cgatcg 1916

<210>6

<211>2254

<212>DNA

<213> Artificial sequence

<220>

<223> Cloning vector pEGFP-N1, full sequence, enhanced green fluorescent protein (egfp) and neomycin phosphotransferase genes

<400>6

tcgactctag agggacagcc cccccccaaa gcccccaggg atgtaattac gtccctcccc 60

cgctaggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc cccccgcatc 120

cccgagccgg cagcgtgcgg ggacagcccg ggcacggggga aggtggcacg ggatcgcttt 180

cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat acggggaaaa 240

agctttaggc tgaaagagag atttagaatg acagaatcat agaacggcct gggttgcaaa 300

ggagcacagt gctcatccag atccaaccccc ctgctatgtg cagggtcatc aaccagcagc 360

ccaggctgcc cagagccaca tccagcctgg ccttgaatgc ctgcagggat ggggcatcca 420

cagcctcctt gggcaacctg ttcagtgcgt caccaccctc tgggggaaaa actgcctcct 480

catatccaac ccaaacctcc cctgtctcag tgtaaagcca ttcccccttg tcctatcaag 540

ggggagtttg ctgtgacatt gttggtctgg ggtgacacat gtttgccaat tcagtgcatc 600

acggagaggc agatcttggg gataaggaag tgcaggacag catggacgtg ggacatgcag 660

gtgttgaggg ctctgggaca ctctccaagt cacagcgttc agaacagcct taaggataag 720

aagataggat agaaggacaa agagcaagtt aaaacccagc atggagagga gcacaaaaag 780

gccacagaca ctgctggtcc ctgtgtctga gcctgcatgt ttgatggtgt ctggatgcaa 840

gcagaagggg tggaagagct tgcctggaga gatacagctg ggtcagtagg actgggacag 900

gcagctggag aattgccatg tagatgttca tacaatcgtc aaatcatgaa ggctggaaag 960

cctccaagat ccccaagacc aaccccaacc cacccaccgt gcccactggc catgtccctc 1020

agtgccacat ccccacagtt cttcatcacc tccagggacg gtgacccccc cacctccgtg 1080

ggcagctgtg ccactgcagc accgctcttt ggagaaggta aatcttgcta aatccagccc 1140

gaccctcccc tggcacaacg taaggccatt atctctcatc caactccagg acggagtcag 1200

tgaggatggg gctctagagg gacagccccc ccccaaagcc cccagggatg taattacgtc 126O

cctcccccgc tagggcagc agcgagccgc ccggggctcc gctccggtcc ggcgctcccc 1320

ccgcatcccc gagccggcag cgtgcgggga cagcccgggc acggggaagg tggcacggga 1380

tcgctttcct ctgaacgctt ctcgctgctc tttgagcctg cagacacctg gggggatacg 1440

gggaaaaagc tttaggctga aagagagatt tagaatgaca gaatcataga acggcctggg 1500

ttgcaaagga gcacagtgct catccagatc caaccccctg ctatgtgcag ggtcatcaac 1560

cagcagccca ggctgcccag agccacatcc agcctggcct tgaatgcctg cagggatggg 1620

gcatccacag cctccttggg caacctgttc agtgcgtcac caccctctgg gggaaaaact 1680

gcctcctcat atccaaccca aacctcccct gtctcagtgt aaagccattc ccccttgtcc 1740

tatcaagggg gagtttgctg tgacattgtt ggtctggggt gacacatgtt tgccaattca 1800

gtgcatcacg gagaggcaga tcttggggat aaggaagtgc aggacagcat ggacgtggga 1860

catgcaggtg ttgagggctc tgggacactc tccaagtcac agcgttcaga acagccttaa 1920

ggataagaag ataggataga aggacaaaga gcaagttaaa accccagcatg gagaggagca 1980

caaaaaggcc acagacactg ctggtccctg tgtctgagcc tgcatgtttg atggtgtctg 2040

gatgcaagca gaaggggtcc atgtccctca gtgccacatc cccacagttc ttcatcacct 2100

ccagggacgg tgacccccccc acctccgtgg gcagctgtgc cactgcagca ccgctctttg 2160

gagaaggtaa atcttgctaa atccagcccg accctcccct ggcacaacgt aaggccatta 2220

tctctcatcc aactccagga acggagtcag tgag 2254

<210>7

<211>632

<212>DNA

<213> marmot hepatitis B virus

<220>

<221> misc_signal

<222>(1)..(632)

<223> Marmot hepatitis virus post-transcriptional regulatory element

<400>7

accaggttct gttcctgtta atcaacctct ggattacaaa atttgtgaaa gattgactgg 60

tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta 120

tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct 180

gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt 240

tgctgacgca accccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac 300

tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg 360

ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac 420

gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg 480

ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct 540

gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc 600

ctccccgcct gtttcgcctc gggctcctcg ag 632

<210>8

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223>Forward primer for amplifying the neomycin resistance gene

<400>8

gcggccgcgc gcgtcaggtg gcac 24

<210>9

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> Reverse primer for amplifying the neomycin resistance gene

<400>9

cgatcggacg ctcagtggaa cgaaaactc 29

<210>10

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> Forward primer for amplification of chicken B-globin insulator

<400>10

tcgactctag agggacag 18

<210>11

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> Forward primer for amplification of chicken B-globin insulator

<400>11

ctcactgact ccgttcct 18

<210>12

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223>Forward primer for amplifying post-transcriptional regulatory elements of Marmot Hepatitis Virus

<400>12

accaggttct gttcctgtta atcaacctc 29

<210>13

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> Reverse primer for amplifying post-transcriptional regulatory elements of Marmot Hepatitis Virus

<400>13

ctcgaggagc ccgaggcgaa acaggcg 27

Claims (12)

1. the pig uroplakin II gene promoter that has SEQ ID NO:1 base sequence:

[SEQ?ID?NO：1]

gggctaggagtggaatcagagctggcctatgccacagcaacgcagaatccaaaccacatctccgacctacaccagaccgtcaccataacacaggatccttaacccactgagcaaggtcagggatcaaacccaaatcctcatggatactagtcgggttcttaacccgctgagccacagtgggcactcctgtttttgtttgtgtcttcgttttttggctgcatctgcagcatacagaagttcctgggttaaggattgaacccatgccacagcagcaacccgagccacagcagtgacaacagcctgatccttaactgctagaccaccagggaacgccccctcaacttttcatgccttggaaaccctgagtcagtacaacctgacaatngnttttttttttttttttttttgccttttctagggccacttcccgcggcatgtggagattcgcaggctanaggtctaatcggagctgtagccaccggcctacaccagagccatagcaacgagggatccgagccgagtctgcaacctacactacagctcatggcaacaccggatcgttaacccactgagcaaggccaggggatcgaacccgcaacctcatggttcctagtcagattcgttaaccactgcaccatgacaggaactcccaacctgacaattttatcatttctgcaccctagttgttgagtaatttgaaaaattcccaagatgtcaaggtcagtgtgatggttaattttatgtgtcaacctgactaggccatgttgcccggatgtggagtcattgttattctggatgttactgtgaagatatgttttggatgaaattaacatttaaatcagtggggggaaaaaaagaagttctcgttctggtgcatcagaaacaaatccgactaggaaacaagcggttgcaggttcgatccctggcctcacttagtggagtcaggatctggcgttgccgtgagctgtggtacaggtggcagatgcagctcggatctagcattgctgtggctgtggtgtaggccagcagctgtagctctgattaaaccccaagtctgggaacctccatatgccgtgggtgtggcccgaaaaagcaaaaaataaataaataaataaatttaaaccaggggattttgagcaaagcagattaccccataatatgggtgggtctcatcaagttcattgtaggccctagtggaacaaagaccgacctccaccttctccccatgagaaggaaagaattctgccaaaagaccgccttnggacntaaactgcaactctttcctgagtttccagcatgttggcctcccccatcagactttggacttgccaagcctccgcaattgcatgagccaattccttaaaataaatccgtctatatatacacatcctgttggttctgtttctccagagaaccctgactaacgcagtctgcacccctgaagaccagtggtccccacactcagctgggtgtcacctccaaacactcagccttcctcaaggctctttctagctgtgtcctcctctccccacaacagctgtttcaaactctcacccctcttcagggcgcaatcccttctcctccctgagtttcctacttcccagagaaagcagagaccttcaggagtgtgctgccttaacttacttccttcatccctcagccttgcaaaagtataagctttctctgcaccactgccccattcttctctctgcagacagggtcattcctaaagccaaacgctaatgcctccacctctgatctgagtcccatcttttccctcctccagaagcttcctcataaattctacccccttttcttccttatctttatctttgaaaacaaaatggaagacagccttcccgttgtggtgcagcggaaacaggtggtgccttggaagcgctgggacgcaggttcgacccctggcccagcatagtaggttaaggatccagtgttgccacagttttggcttagattgaaactgcagctcagatctggtccctggcctgggaacttcatacgccacaggacggcccaaaaagaaaagaaagaaaaaataaaaaacaaaacagaaaagcctttcctgtacccccaattccctccagttatctctctctttcccttcccagccaagctctgcaaagagcggtctgcacagttctaactctacctcctcccagttggccctggactttctcagtctggcttctacccccctcacccgtaggaatctgctctgaaggacacgcacccctcacgatccttggcccagggacattttttgtaccagcctttcaatcctgaccttcatatcatccgacacctcctttgtgaaaccctccatccactttctcctggttcccctcctaagacccattccgccttcttcagccccctccctccatctgtcctttagatgccgcatttcctagtatcctgtcctgcgcggnctcgtccttcccttccacaactctcttcaaggactcttttctccatgtgcgattttgcccatggcccaccttccctctctttacccagactttcccccggtgctccagactcatagactcaattatgaaaacatagttttcatctgatttgcccaagatatttgcattagttattactgtataacagcttatcccccaatttagtggcttataaaataaacacttattctgagaatcagaaacctaggcaggacatagttggggtctcatgaagttgcactgaaaatgtccccctgggctaatcatacggaggactgaccagggctggaggatctgttccaagctcattcattcacatggccgtaggttggagacagctcttctctggatcttggcaggagcctcaattccttgttacgtggacctccccttggagggggtcccatgtcctccatggtgagtaatccatgagagcaaggtggaaggtgccatgccatttaggacctagcctcaggagggacctacgtcacttctgttgtagtctgttggccacacagactaaccctgacacaatgcacccatccatgacctgctgccagtccattctccacactgtttccagaatgatatttacataagtaaaactcctcaaaggcttttgagattttttttcccattatagttgatttataacctcagaggcttttgttttcttcagcataaaaaccaagttccttaacatagcatgtaacccactggccaccctgccagtggctagaactctcaccatgtccatccttgaatactgctttctagccaagagctattgtttgcagttcccagaatgtgtcgggataactcacatctctgagccttttcatgtgctgttccctcactttggaatatccccttccatttaggaaggctaatgtccattcattntccaaaactcagaagcaaattttttttttttttttttttttttttttttgctttttagggccgaactctcagcatatggaggttcccaggttagccatcaaattggaattgtagctgctggcctacaccacagccatagcaacaccagacccaagtcacatctgcaacctacatcacagatcatggcaatactggatccttaacccactgagtgagcccagggatcaaacacaaattctcatggatactcgccaggttcattaccactgagccacaacaggaactcctctcctttttatggtcacacctgcagcatatggaagttcctgggccagggattgaatctgagtggcagctgtgacaatgccgtatcctttaattcactgtgctgggctgaggggntaaantgcccctcctaaaaaacctgagctgctgcagttggattcttaatccactgcaccacaagggggaaggtcaagaactgtcttgccatctctgtatcttatcacctagcatagtacccaccatagagaagttgctcaacaaatgtttactgaatgaataaatgcatgagctggagttcccattgcggctcagcagtaacaaacctgactagcattcataagaacttgggttcgatccctagcctcagtgggttaaggatgcagcattgctgtgagctgtggtgtaggtcgcagacgacactcagatcccacattgctgtcactgtggcgcaggccggcctctgtagctctgattcgactcctagcctgggaacgtccatatgccacaggtgaggccctaaaaagaaataaataagcaagcaagtaagcaagcaggcagtttcttggtgccttgtacccctgtggcctgtgtggtatacaagtaacagctgatccatgtctcagtcatgtttccccctcagactacctttcctgccccatctctccctttgacataattggaaaaacaaattcagaattttgtcccactacctttcttgctagctctgtggccttgggaaagctatttattgcctctgagcctctaattttcatctgcaccaaggattaataaaaaggagaggataagatgaattacttatattaatatttattgaaccagatactgtgctaggcactcttaaataaattagcttgagtgatagtcatagtatcctggtgagacagattttttttttccttttatggttgcacgtgcaacatatggaagttcctgggctggggtcgaattggagctgcaggtgcttgcctatgccacagccatggcaacatcatatacaaaccgcacctgtgacctacaccacagattgcagcaacgctggatccttcacccaaggagcaaggccaggaatcaaatgtgcatcctcacaaacactatgtccggtttttaacccgctgagccacaccaggaactccatggcgagacagattttatactctgtctacagaagaggaaagtgaagctcagaatggttaggtaggtaacttggccaagatcaaaaaattcaaagaagatttggggcaagtggtgatatcatggcagcattagaaaaaataaagaagcatccacttgttttccaacactgaacaactgagattttcttactctcacagctttttccagcttcatatccaaggacagacgctctgccattttcccatcagaccaatatttgctgaacactgcacctttacttttaggtccaagtcaccaggggttttcccagtttgctcctacagattctgacactatctccacattttttttgcacctttattttaaagcatttttatacctgtcataccttgctagataaatgggaaggaatgaatcttcccatttataggtgagaaaattgaggttcaaagtgactcaccaaaagtcatatagcatcactcctcaacaggaggacagcagtccccaccagagggtaacatgtccatggagcctagtggacacatttttctaactgactgggaagcagcagagtggtattgtgaagggggaatcataggtatatcaaacagacttaggttctgatccgagctattctgcttgcaaacaaccatagttcaatttaaaaaaaaaaaagaaagaaagaaagaaagaaaggagcccccatcctggtgcagtggaaacaaattcaactaggaactgtgaggttgtgggttcgatccctggccttgctcagtgggttaaggatctggcgttgccatgagccgtggtgtaggttgcagactcaactcagatctggcgttgctgtgactgtggctgtgatgtaggctggcagctgtaactccggttagaccccagcctgggaacctccatatgcaacctccatatgcggtgggtgtggccctaaaaagaaaaaaaaaaaaaaaagaggaattcccttatggctcagcaggttaaggatctggtattgtcactgctgtggctctagttacagccatagtgcaggttcaatccctggcccaggaacgtctgcatcccacaggtgtggccaaaaaagaaagaaaggaaggagttctgttgtggcacaataggattggcaacatcttaggagtactgggacacaggttcaatccctggcccagcacagtgggtaaggagccagtgttgctggtcaaaaaagaaaagaaaaagtaccatagttagagtaaatctgttttaggagctattctttggggcagaacagagagatcaggagctccttgagagcagaaacttacctttacatccctcgtgcctagcacggttctaggggcatacctggtatttaataaatatagccaactggataggggattggaaggaaagagcaggggagggaacttgagtgagttgaaaaattgagaatccaaaggggagacagcctagaaagagtaggtccaagaaagagatcccaggcatttgtggccctggttccctttttccaagccatgaggaaatcctcagaggaacagagtgctgtggctttaaatgacttcagcgttgtcaatgaatctgctcggctaaaagagttatcctcttgctccttcgcttgtcctccccctcctctcagctccccaaacccttctcggctgctgtgatgggataattagatgcgagagctcagcacagatgatgctccagttgcctagcaactaatggtttccatggagaccgcaaagcacagcctccagagcagccagtgagcagctcggcagggcagggagaagacgcaactctcagctcctccagaaacctggggagggccaggagtggggaagaagggggggatcggagggcttaaaggcacaggcccctcttatcctcttaaaatctggtcagagctctgccctcccctcccctactctgtcccactcataatttcagatggagttgggggcttaggagtggacccaacacaacctaccctgcaataaacccaaccttctttctgcttctggtttgtggctgaaaatggnaaaagaaatctcccaagtgcaagtgtaaacancntcctgggttggcaatgggatctgaagagtactaagatccctcagacctggaattccaccatttagtctttccctctctccaaagttctcaatgtgcaaaagatcctctttcagtttgcagagcaatgataggatcttctaaaaggagacaaaagccaaggtgcaggaaaaatagaattcagttcttcacccaaaggcagcctgtcctgggagacaggggtgaaacacttggtcctgatctccatcagaggatccagagtgtgtgtgtttgttgctggggagggggacacaatatagagcatctggtgactcaaagtatgtgcctcccagagtagcatcaatcaatgttacctggaagcttgttagaaatgcagaatttcaggcttcacctcagacccactgaatcagaaactgcatcttaacaagatccctcatgattcatacgcacattaaatttggagaagcgctgacctgagaccctcctcctctctgcttgggcccatagttctacctttattgtcacctcgtctcacctcgtgctcataccccaggctttgagcctacccttccccccatggggaaaggacacaaggccaccagcccctcacttccctaccaggaccctggccctcctctgggactggagaaggacaaagaggaccccctctgtggaggtctacgacctctcctgaccaagtagtccactcaccacaagtggctctacctctctgagtctcagtttccacatccacaaaaggtggccaatgctatctgccacccagaatggctgtgagggtggagcaggcaaagcctctgtgccatcagagaaattgtgtctctttttcattttctcccagtgggtttctttctcgtctttattcttttttttttttttttttcctgtctgttgtatttttagggccgtgcctgtggcatacggaagttcccagggtaggggtccaatgggagctgtagccccgggcctacgccacagccacagcaatgtgggatctgagccacgtctgcaacctacaccacagctcacggcaacaccagatccttaacccactgagcaaggccagggatcgagcccacgtcctcatggatgctagttgggttcgttaaccgctgagccatgatgataactcctctttctattctttagtcacaaacagtcaacaaaggttgctgaccaaggctgatcgtgcccaccccccagccccccagactgggccagtgcccaccccttgggtctctctggaaatcctgcccagcatcaattggctccactctccaggaggatgggaagccctgtggcccctgggactcacacccctctgcatctcccagagtgcaggacctggtcttcaggagacaccaagaactggctcccccggctctgctgcccccaccccctactaccagtttctctcccattcctgcccagtccaggccccctggggttactctcctctctctgtacaccagtgcaacctcagaacctgcttccctcctgggaacacccactaccacgtgggagaaggggtcgtctaggggttgggccccagatacacttgtaagcaggaacacacgagcccttacatgtgggtgtcccggaagaagggggttttccaccccccgctttagtcaccctgcccctctgcagctgcctgagccaccaagacccagccaaggtctcctgccttctggcctgagggccagctccccatcctgaaaaacctgtctgggggcctcccctgaggctgtagggcccaaggcctcccctgaggctgtagggcccaaggggcaggttgaacaggattcccctctggcccctcctacccccaggacaaaaccagagccccaggacagggcctcacttgcctcaggaaaccacagcttgccagcacccagcccagcaccagcccagct

2. the uroplakin II promotor of claim 1, it is selected from a place or many places fracture sudden change, deletion mutantion, insertion sudden change, point mutation, replacement sudden change, nonsense mutation, missense mutation, the polymorphism sudden change that takes place in the SEQ ID NO:1 base sequence or resets the function equivalent of sudden change.

3. contain the expression vector that right requires promotor base sequence in 1 or 2 and is positioned at the base sequence of described promotor 3 ' end coding target protein.

4. the expression vector of claim 3, wherein target protein is erythropoietin (EPO).

5. the expression vector of claim 4, it is into hiding the expression vector pUP2/hEPO that registration number is KCTC 10352BP preservation.

6. the expression vector of claim 4, it is the I/pUP2/hEPO carrier that contains as the SEQ ID NO:6 insulator of the SEQ ID NO:5 neomycin resistance gene of selective marker and UPII promotor 5 ' end.

[SEQ?ID?NO：5]

gcggccgcgcgcgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcctgaggcggaaagaaccagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaagatcgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccctagggggaggctaactgaaacacggaaggagacaataccggaaggaacccgcgctatgacggcaataaaaagacagaataaaacgcacggtgttgggtcgtttgttcataaacgcggggttcggtcccagggctggcactctgtcgataccccaccgagaccccattggggccaatacgcccgcgtttcttccttttccccaccccaccccccaagttcgggtgaaggcccagggctcgcagccaacgtcggggcggcaggccctgccatagcctcaggttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtccgatcg

[SEQ?ID?NO：6]

tcgactctagagggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgctaggggcagcagcgagccgcccggggctccgctccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagcctgcagacacctggggggatacggggaaaaagctttaggctgaaagagagatttagaatgacagaatcatagaacggcctgggttgcaaaggagcacagtgctcatccagatccaaccccctgctatgtgcagggtcatcaaccagcagcccaggctgcccagagccacatccagcctggccttgaatgcctgcagggatggggcatccacagcctccttgggcaacctgttcagtgcgtcaccaccctctgggggaaaaactgcctcctcatatccaacccaaacctcccctgtctcagtgtaaagccattcccccttgtcctatcaagggggagtttgctgtgacattgttggtctggggtgacacatgtttgccaattcagtgcatcacggagaggcagatcttggggataaggaagtgcaggacagcatggacgtgggacatgcaggtgttgagggctctgggacactctccaagtcacagcgttcagaacagccttaaggataagaagataggatagaaggacaaagagcaagttaaaacccagcatggagaggagcacaaaaaggccacagacactgctggtccctgtgtctgagcctgcatgtttgatggtgtctggatgcaagcagaaggggtggaagagcttgcctggagagatacagctgggtcagtaggactgggacaggcagctggagaattgccatgtagatgttcatacaatcgtcaaatcatgaaggctggaaagcctccaagatccccaagaccaaccccaacccacccaccgtgcccactggccatgtccctcagtgccacatccccacagttcttcatcacctccagggacggtgacccccccacctccgtgggcagctgtgccactgcagcaccgctctttggagaaggtaaatcttgctaaatccagcccgaccctcccctggcacaacgtaaggccattatctctcatccaactccaggacggagtcagtgaggatggggctctagagggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgctaggggcagcagcgagccgcccggggctccgctccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagcctgcagacacctggggggatacggggaaaaagctttaggctgaaagagagatttagaatgacagaatcatagaacggcctgggttgcaaaggagcacagtgctcatccagatccaaccccctgctatgtgcagggtcatcaaccagcagcccaggctgcccagagccacatccagcctggccttgaatgcctgcagggatggggcatccacagcctccttgggcaacctgttcagtgcgtcaccaccctctgggggaaaaactgcctcctcatatccaacccaaacctcccctgtctcagtgtaaagccattcccccttgtcctatcaagggggagtttgctgtgacattgttggtctggggtgacacatgtttgccaattcagtgcatcacggagaggcagatcttggggataaggaagtgcaggacagcatggacgtgggacatgcaggtgttgagggctctgggacactctccaagtcacagcgttcagaacagccttaaggataagaagataggatagaaggacaaagagcaagttaaaacccagcatggagaggagcacaaaaaggccacagacactgctggtccctgtgtctgagcctgcatgtttgatggtgtctggatgcaagcagaaggggtccatgtccctcagtgccacatccccacagttcttcatcacctccagggacggtgacccccccacctccgtgggcagctgtgccactgcagcaccgctctttggagaaggtaaatcttgctaaatccagcccgaccctcccctggcacaacgtaaggccattatctctcatccaactccaggaacggagtcagtgag

7. the expression vector of claim 4, it is to contain pUP2/hEPO (WPRE) carrier of otter hepatitis virus post-transcriptional control element (WPRE) early as the SEQ ID NO:7 of the SEQ ID NO:5 neomycin resistance gene of selective marker and EPO gene 3 ' end.

[SEQ?ID?NO：7]

accaggttctgttcctgttaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctgtttcgcctcgggctcctcgag

8. the expression vector of claim 4, it is I/pUP2/hEPO (WPRE) carrier that contains the SEQ ID NO:7 WPRE of the SEQ ID NO:6 insulator of SEQ ID NO:5 neomycin resistance gene as selective marker, UP2 promotor 5 ' end and EPO gene 3 ' end.

9. import the fertilised non-human eggs of expression vector any in the requirement 4 to 8 of having the right.

10. by transplanting the transgenic animal that zygote obtained of claim 9.

11. the transgenic animal of claim 10, it is selected from pig, mouse, ox, poultry, sheep and ram.

12. one kind generates method of useful proteins, it comprises the following steps:

The zygote that has imported expression vector any among the claim 4-8 is implanted in scapegoat's mother animal; And

Obtain transgenic animal from scapegoat's mother animal; And

From the urine of transgenic animal, separate and the useful protein of purifying.

Images