CN1705743B - Porcine uroplakin II promoter and the production method of useful proteins using said promoter - Google Patents
Porcine uroplakin II promoter and the production method of useful proteins using said promoter Download PDFInfo
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- CN1705743B CN1705743B CN200380101482XA CN200380101482A CN1705743B CN 1705743 B CN1705743 B CN 1705743B CN 200380101482X A CN200380101482X A CN 200380101482XA CN 200380101482 A CN200380101482 A CN 200380101482A CN 1705743 B CN1705743 B CN 1705743B
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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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
Technical field
The present invention relates to pig uroplakin II gene promoter and use this promotor to produce method of useful proteins.
Background of invention
At field of medicaments, produce the method for protein as EPO, the main large scale production method that utilizes cell culture technology that adopts with economic growth value as maximization.But in the method, owing to use animal blood as substratum, production cost increases, and need have expertise to cultivation.In addition, owing to newly-generated EPO can not be separated fully with animal EPO in being contained in substratum, so the EPO that finally makes has low-purity and SA problem.
On the other hand, using transgenic animal to produce in the method for useful proteins, target protein is included in the secreted body fluid of animal, therefore, compares with existing cell culture technology, and target protein is easy to separate and purifying, and keeps better active.Owing to this reason, this method causes people's attention just rapidly.
In the transgenic animal technology of setting up so far, mainly use the known mammary gland of high protein expression that shows as the organ of producing target protein.But animal test results shows: can not produce several important target proteins substantially by the expression in milk, as EPO, this is because also express in their its hetero-organizations outside mammary gland.In addition, owing to also be contained in a large number in the milk such as albuminous range protein, the target protein of gained is blamed with purifying.
In order to overcome these problems, proposed the use bladder recently and produced method of useful proteins.
No matter the sex of animal how, bladder generates urine in whole animal life in the cycle, and urine only contains 5-25mg/l protein and lipid fraction very in a small amount.Therefore, use bladder that the separation of target protein and purifying are very easy to.
But development still is in low-level with the protein production efficient of the animal of bladder specificity promoter conversion so far.
Therefore, press for a kind of efficient promotion target protein expression promoter of exploitation.
Summary of the invention
Therefore, an object of the present invention is the specific expressed pig uroplakin II gene promoter of promotes target protein bladder, and provide and to use this promotor mass production method of useful proteins.
In one embodiment, the invention provides pig uroplakin II gene promoter.
Pig uroplakin II promotor preferably has the base sequence of SEQ ID NO:1.
[SEQ?ID?NO：1]：
gggctaggagtggaatcagagctggcctatgccacagcaacgcagaatccaaaccacatctccgacctaca
ccagaccgtcaccataacacaggatccttaacccactgagcaaggtcagggatcaaacccaaatcctcatggatactagt
cgggttcttaacccgctgagccacagtgggcactcctgtttttgtttgtgtcttcgttttttggctgcatctgcagcatacagaa
gttcctgggttaaggattgaacccatgccacagcagcaacccgagccacagcagtgacaacagcctgatccttaactgct
agaccaccagggaacgccccctcaacttttcatgccttggaaaccctgagtcagtacaacctgacaatngntttttttttttttt
ttttttgccttttctagggccacttcccgcggcatgtggagattcgcaggctanaggtctaatcggagctgtagccaccggc
ctacaccagagccatagcaacgagggatccgagccgagtctgcaacctacactacagctcatggcaacaccggatcgtt
aacccactgagcaaggccaggggatcgaacccgcaacctcatggttcctagtcagattcgttaaccactgcaccatgaca
ggaactcccaacctgacaattttatcatttctgcaccctagttgttgagtaatttgaaaaattcccaagatgtcaaggtcagtgt
gatggttaattttatgtgtcaacctgactaggccatgttgcccggatgtggagtcattgttattctggatgttactgtgaagatat
gttttggatgaaattaacatttaaatcagtggggggaaaaaaagaagttctcgttctggtgcatcagaaacaaatccgacta
ggaaacaagcggttgcaggttcgatccctggcctcacttagtggagtcaggatctggcgttgccgtgagctgtggtacag
gtggcagatgcagctcggatctagcattgctgtggctgtggtgtaggccagcagctgtagctctgattaaaccccaagtct
gggaacctccatatgccgtgggtgtggcccgaaaaagcaaaaaataaataaataaataaatttaaaccaggggattttgag
caaagcagattaccccataatatgggtgggtctcatcaagttcattgtaggccctagtggaacaaagaccgacctccacctt
ctccccatgagaaggaaagaattctgccaaaagaccgccttnggacntaaactgcaactctttcctgagtttccagcatgtt
ggcctcccccatcagactttggacttgccaagcctccgcaattgcatgagccaattccttaaaataaatccgtctatatatac
acatcctgttggttctgtttctccagagaaccctgactaacgcagtctgcacccctgaagaccagtggtccccacactcagc
tgggtgtcacctccaaacactcagccttcctcaaggctctttctagctgtgtcctcctctccccacaacagctgtttcaaactc
tcacccctcttcagggcgcaatcccttctcctccctgagtttcctacttcccagagaaagcagagaccttcaggagtgtgct
gccttaacttacttccttcatccctcagccttgcaaaagtataagctttctctgcaccactgccccattcttctctctgcagacag
ggtcattcctaaagccaaacgctaatgcctccacctctgatctgagtcccatcttttccctcctccagaagcttcctcataaatt
ctacccccttttcttccttatctttatctttgaaaacaaaatggaagacagccttcccgttgtggtgcagcggaaacagtggtg
ccttggaagcgctgggacgcaggttcgacccctggcccagcatagtaggttaaggatccagtgttgccacagttttggctt
agattgaaactgcagctcagatctggtccctggcctgggaacttcatacgccacaggacggcccaaaaagaaaagaaag
aaaaaataaaaaacaaaacagaaaagcctttcctgtacccccaattccctccagttatctctctctttcccttcccagccaag
ctctgcaaagagcggtctgcacagttctaactctacctcctcccagttggccctggactttctcagtctggcttctacccccct
cacccgtaggaatctgctctgaaggacacgcacccctcacgatccttggcccagggacattttttgtaccagcctttcaatc
ctgaccttcatatcatccgacacctcctttgtgaaaccctccatccactttctcctggttcccctcctaagacccattccgcctt
cttcagccccctccctccacttgtcctttagatgccgcatttcctagtatcctgtcctgcgcggnctcgtccttcccttccacaa
ctctcttcaaggactcttttctccatgtgcgattttgcccatggcccaccttccctctctttacccagactttcccccggtgctcc
agactcatagactcaattatgaaaacatagttttcatctgtgatttgcccaagatatttgcattagttattactgtataacagcttatc
ccccaatttagtggcttataaaataaacacttattctgagaatcagaaacctaggcaggacatagttggggtctcatgaagtt
gcactgaaaatgtccccctgggctaatcatacggaggactgaccagggctggaggatctgttccaagctcattcattcaca
tggccgtaggttggagacagctcttctctggatcttggcaggagcctcaattccttgtcacgtggacctccccttggagggg
gtcccatgtcctccatggtgagtaatccatgagagcaaggtggaaggtgccatgccatttaggacctagcctcaggaggg
acctacgtcacttctgttgtagtctgttggccacacagactaaccctgacacaatgcacccatccatgacctgctgccagtc
cattctccacactgtttccagaatgatatttacataagtaaaactcctcaaaggcttttgagattttttttcccattatagttgattta
taacctcagaggcttttgttttcttcagcataaaaaccaagttccttaacatagcatgtaacccactggccaccctgccagtg
gctagaactctcaccatgtccatccttgaatactgctttctagccaagagctattgtttgcagttcccagaatgtgtcgggata
actcacatctctgagccttttcatgtgctgttccctcactttggaatatccccttccatttaggaaggctaatgtccattcattntc
caaaactcagaagcaaatttttttttttttttttttttttttttttgctttttagggccgaactctcagcatatggaggttcccaggtta
gccatcaaattggaattgtagctgctggcctacaccacagccatagcaacaccagacccaagtcacatctgcaacctacat
cacagatcatggcaatactggatccttaacccactgagtgagcccagggatcaaacacaaattctcatggatactcgccag
gttcattaccactgagccacaacaggaactcctctcctttttatggtcacacctgcagcatatggaagttcctgggccaggg
attgaatctgagtggcagctgtgacaatgccgtatcctttaattcactgtgctgggctgaggggntaaantgcccctcctaa
aaaacctgagctgctgcagttggattcttaatccactgcaccacaagggggaaggtcaagaactgtcttgccatctctgtat
cttatcacctagcatagtacccaccatagagaagttgctcaacaaatgtttactgaatgaataaatgcatgagctggagttcc
cattgcggctcagcagtaacaaacctgactagcattcataagaacttgggttcgatccctagcctcagtgggttaaggatgc
agcattgctgtgagctgtggtgtaggtcgcagacgacactcagatcccacattgctgtcactgtggcgcaggccggcctct
gtagctctgattcgactcctagcctgggaacgtccatatgccacaggtgaggccctaaaaagaaataaataagcaagcaa
gtaagcaagcaggcagtttcttggtgccttgtacccctgtggcctgtgtggtatacaagtaacagctgatccatgtctcagtc
atgtttccccctcagactacctttcctgccccatctctccctttgacataattggaaaaacaaattcagaattttgtcccactacc
tttcttgctagctctgtggccttgggaaagctatttattgcctctgagcctctaattttcatctgcaccaaggattaataaaaagg
agaggataagatgaattacttatattaatatttattgaaccagatactgtgctaggcactcttaaataaattagcttgagtgata
gtcatagtatcctggtgagacagattttttttttccttttatggttgcacgtgcaacatatggaagttcctgggctggggtcgaat
tggagctgcaggtgcttgcctatgccacagccatggcaacatcatatacaaaccgcacctgtgacctacaccacagattgc
agcaacgctggatccttcacccaaggagcaaggccaggaatcaaatgtgcatcctcacaaacactatgtccggtttttaac
ccgctgagccacaccaggaactccatggcgagacagattttatactctgtctacagaagaggaaagtgaagctcagaatg
gttaggtaggtaacttggccaagatcaaaaaattcaaagaagatttggggcaagtggtgatatcatggcagcattagaaaa
aataaagaagcatccacttgttttccaacactgaacaactgagattttcttactctcacagctttttccagcttcatatccaagga
cagacgctctgccattttcccatcagaccaatatttgctgaacactgcacctttacttttaggtccaagtcaccaggggttttcc
cagtttgctcctacagattctgacactatctccacattttttttgcacctttattttaaagcattttttatacctgtcataccttgctaga
taaatgggaaggaatgaatcttcccatttatggtgagaaaattgaggttcaaagtgactcaccaaaagtcatatagcatca
ctcctcaacaggaggacagcagtccccaccagagggtaacatgtccatggagcctagtggacacatttttctaactgactg
ggaagcagcagagtggtattgtgaagggggaatcataggtatatcaaacagacttaggttctgatccgagctattctgcttg
caaacaaccatagttcaatttaaaaaaaaaaaagaaagaaagaaagaaagaaaggagcccccatcctggtgcagtggta
acaaattcaactaggaactgtgaggttgtgggttcgatccctggccttgctcagtgggttaaggatctggcgttgccatgag
ccgtggtgtaggttgcagactcaactcagatctggcgttgctgtgactgtggctgtgatgtaggctggcagctgtaactccg
gttagaccccagcctgggaacctccatatgcaacctccatatgcggtgggtgtggccctaaaaagaaaaaaaaaaaaaa
aagaggaattcccttatggctcagcaggttaaggatctggtattgtcactgctgtggctctagttacagccatagtgcaggtt
caatccctggcccaggaacgtctgcatcccacaggtgtggccaaaaaagaaagaaaggaaggagttctgttgtggcaca
ataggattggcaacatcttaggagtactgggacacaggttcaatccctggcccagcacagtgggtaaggagccagtgttg
ctggtcaaaaaagaaaagaaaaagtaccatagttagagtaaatctgttttaggagctattctttggggcagaacagagagat
caggagctccttgagagcagaaacttacctttacatccctcgtgcctagcacggttctaggggcatacctggtatttaataaa
tatagccaactggataggggattggaaggaaagagcaggggagggaacttgagtgagttgaaaaattgagaatccaaa
ggggagacagcctagaaagagtaggtccaagaaagagatcccaggcatttgtggccctggttccctttttccaagccatg
aggaaatcctcagaggaacagagtgctgtggctttaaatgacttcagcgttgtcaatgaatctgctcggctaaaagagttat
cctcttgctccttcgcttgtcctccccctcctctcagctccccaaacccttctcggctgctgtgatgggataattagatgcgag
agctcagcacagatgatgctccagttgcctagcaactaatggtttccatggagaccgcaaagcacagcctccagagcag
ccagtgagcagctcggcagggcagggagaagacgcaactctcagctcctccagaaacctggggagggccaggagtg
gggaagaagggggggatcggagggcttaaaggcacaggcccctcttatcctcttaaaatctggtcagagctctgccctc
ccctcccctactctgtcccactcataatttcagatggagttgggggcttaggagtggacccaacacaacctaccctgcaata
aacccaaccttctttctgcttctggtttgtggctgaaaatggnaaaagaaatctcccaagtgcaagtgtaaacancntcctg
ggttggcaatgggatctgaagagtactaagatccctcagacctggaattccaccatttagtctttccctctctccaaagttctc
aatgtgcaaaagatcctctttcagtttgcagagcaatgataggatcttctaaaaggagacaaaagccaaggtgcaggaaaa
atagaattcagttcttcacccaaaggcagcctgtcctgggagacaggggtgaaacacttggtcctgatctccatcagagga
tccagagtgtgtgtgtttgttgctggggagggggacacaatatagagcatctggtgactcaaagtatgtgcctcccagagt
agcatcaatcaatgttacctggaagcttgttagaaatgcagaatttcaggcttcacctcagacccactgaatcagaaactgc
atcttaacaagatccctcatgattcatacgcacattaaatttggagaagcgctgacctgagaccctcctcctctctgcttggg
cccatagttctacctttattgtcacctcgtctcacctcgtgctcataccccaggctttgagcctacccttccccccatggggaa
aggacacaaggccaccagcccctcacttccctaccaggaccctggccctcctctgggactggagaaggacaaagagga
ccccctctgtggaggtctacgacctctcctgaccaagtagtccactcaccacaagtggctctacctctctgagtctcagtttc
cacatccacaaaaggtggccaatgctatctgccacccagaatggctgtgagggtggagcaggcaaagcctctgtgccat
cagagaaattgtgtctctttttcattttctcccagtgggtttctttctcgtctttattcttttttttttttttttttcctgtctgttgtatttttag
ggccgtgcctgtggcatacggaagttcccagggtaggggtccaatgggagctgtagccccgggcctacgccacagcca
cagcaatgtgggatctgagccacgtctgcaacctacaccacagctcacggcaacaccagatccttaacccactgagcaa
ggccagggatcgagcccacgtcctcatggatgctagttgggttcgttaaccgctgagccatgatgataactcctctttctatt
ctttagtcacaaacagtcaacaaaggttgctgaccaaggctgatcgtgcccaccccccagccccccagactgggccagt
gcccaccccttgggtctctctggaaatcctgcccagcatcaattggctccactctccaggaggatgggaagccctgtggc
ccctgggactcacacccctctgcatctcccagagtgcaggacctggtcttcaggagacaccaagaactggctcccccgg
ctctgctgcccccaccccctactaccagtttctctcccattcctgcccagtccaggccccctggggttactctcctctctctgt
acaccagtgcaacctcagaacctgcttccctcctgggaacacccactaccacgtgggagaaggggtcgtctaggggttg
ggccccagatacacttgtaagcaggaacacacgagcccttacatgtgggtgtcccggaagaagggggttttccaccccc
cgctttagtcaccctgcccctctgcagctgcctgagccaccaagacccagccaaggtctcctgccttctggcctgagggc
cagctccccatcctgaaaaacctgtctgggggcctcccctgaggctgtagggcccaaggcctcccctgaggctgtaggg
cccaaggggcaggttgaacaggattcccctctggcccctcctacccccaggacaaaaccagagccccaggacagggc
ctcacttgcctcaggaaaccacagcttgccagcacccagcccagcaccagcccagct
In addition, pig uroplakin II promotor of the present invention can be selected from and have a place in the SEQ ID NO:1 base sequence or many places fracture (disruption) sudden change, deletion mutantion, insertion sudden change, point mutation, replacement sudden change, nonsense mutation, missense mutation, polymorphism sudden change or reset the function equivalent of sudden change.
In another embodiment, the invention provides and contain all or part of expression vector of promotor.
Expression vector of the present invention preferably comprises promotor and holds the base sequence of coding target protein in promotor 3 '.
In another embodiment, the invention provides the animal that transforms with the zygote that has imported expression vector.
In another embodiment, the invention provides the scale operation method of useful proteins, it comprises from transgenic animal collects urine, and separation and purifying are expressed in the target protein in the urine.
Promotor of the present invention is positioned at 5 ' end of pig uroplakin II gene, regulation and control pig uroplakin II expression of gene.
Promotor of the present invention can be separated by screening the pig genomic library in the following manner.
For the part base sequence that obtains pig uroplakinII gene is used as the screening probe, the uroplakin II base sequence that will have other animals of known base sequence compares mutually, with reference to the part structure primer sets (forward primer: SEQ ID NO:2 and reverse primer SEQ ID NO:3) of high conservative between the species.Then, the total RNA that uses Vesica sus domestica carries out RT-PCR as template with primer sets.
After RT-PCR reaction acquisition part uroplakin II fragment, use the part that obtains as probe screening pig genomic library.As shown in Figure 2, being used for probe of the present invention is two probes, is made up of probe A that contains exon 2-5 part in the uroplakin II gene and the probe B that contains exon 1-2 part in the uroplakin II gene.
As shown in Figure 2, library screening has obtained to contain the clone of uroplakin II gene or promotor.By the base sequence between relatively cloning, finally determine the base sequence of promotor, thereby obtain the full base sequence of pig uroplakin II promotor.
The promotor total length of Huo Deing is 8847bp like this, shows high G+C content in base sequence, the feature of house-keeping gene, and contain various Sp1 elements, comprise AP2 and GATA box.
In various porcine tissues, promotor of the present invention is only expressed target protein specifically in bladder body.For pig uroplakin II gene, it is expressed in total bladder cell of 8-14%, and active propagation especially on urothelium in the base portion cell, and shows high expression level in the umbrella shape cell of fragmentation (segmented).
Therefore, because the bladder of the efficient induced protein of promotor of the present invention is specific expressed, use promotor of the present invention to allow to generate the expression vector of expressing exogenous target protein in bladder specificity mode.
When generating expression vector of the present invention, with promotor of the present invention be inserted into be used for protein expression existing carrier as basic framework, the base sequence of coding target protein is inserted into 3 ' end of promotor, thereby produces carrier of the present invention.
Can be the suitable carrier that is selected from expression vector commonly used as the carrier of basic framework in the generation of expression vector of the present invention, its example comprises pBluescript SK carrier and the reverse transcription carrier with a plurality of cloning sites, as pLNCX.
Expression vector of the present invention can be expressed all proteins as the medical medicine activeconstituents, and these proteinic examples comprise erythropoietin (EPO), aldosterone, suprarenal gland-thyroliberin (adreno-corticotropin), thrombin, gonad-stimulating hormone, Regular Insulin, prolactin and vassopressin.
If desired, expression vector of the present invention can also contain regulator (regulator), (5 '-UTR), 3 '-UTR, polyadenylation signal, ribosome binding sequence, can be inserted into the base sequence in the genome specific site and the intron of appropriate position as another kind of promotor, enhanser, selective marker, non-translational region.
The invention provides can be under the regulation and control of pig uroplakin II promotor the expression vector pUP2/hEPO (Fig. 3) of expressing human EPO.Expression vector pUP2/hEPO is the preferred example that contains the expression vector of uroplakin II promotor.
In expression vector pUP2/hEPO of the present invention, pBluescript SK (-) carrier is as basic framework, gene (the Lin F.K.et al. of coding people EPO, Proc.Natl Acad.Sci, USA, Clonging and expression of the human erythropoietin gene, 82:7580-7584,1985; SEQ ID NO:4) being fused to 3 ' of uroplakinII promotor of the present invention holds.Expression vector pUP2/hEPO on October 17th, 2002 be deposited in Korea S's bio-science and biotechnology research Korea S typical case culture collection center (KCTC), preserving number is KCTC 10352BP.
If desired, expression vector pUP2/hEPO of the present invention can also contain neomycin resistance gene, insulator or alpine marmot hepatitis virus post-transcriptional control element (WPRE), thereby the foundation of render transgenic clone is easy to carry out, expression level maximization with target protein guarantees the stability that target protein is expressed.
Neomycin resistance gene is the gene that used G418 reagent table revealed resistance during pair cell system set up, and can serve as the effective as selective mark when expressing proteinic animal cell line under the control that is based upon the UPII promotor.Neomycin resistance gene has SEQ ID NO:5 base sequence.
[SEQ?ID?NO：5]：
gcggccgcgcgcgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaataca
ttcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtcctgaggcggaa
agaaccagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcat
gcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctc
aattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccc
catggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggctt
ttttggaggcctaggcttttgcaaagatcgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgca
cgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccg
ccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaag
acgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgg
gaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatcc
atcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcat
cgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgcc
agccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttg
ccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggac
atagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccg
ctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgac
caagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttcc
gggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccctagggggaggctaactga
aacacggaaggagacaataccggaaggaacccgcgctatgacggcaataaaaagacagaataaaacgcacggtgttg
ggtcgtttgttcataaacgcggggttcggtcccagggctggcactctgtcgataccccaccgagaccccattggggccaa
tacgcccgcgtttcttccttttccccaccccaccccccaagttcgggtgaaggcccagggctcgcagccaacgtcggggc
ggcaggccctgccatagcctcaggttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtga
agatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtccgatcg
Insulator is the factor that promotes the regulator effect adjacent with promotor, also promotes site dependency expression, makes target protein stably express under the control of UPII promotor.Insulator has SEQ ID NO:6 base sequence.
[SEQ?ID?NO：6]：
tcgactctagagggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgctaggggca
gcagcgagccgcccggggctccgctccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacag
cccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagcctgcagacacctgggg
ggatacggggaaaaagctttaggctgaaagagagatttagaatgacagaatcatagaacggcctgggttgcaaaggagc
acagtgctcatccagatccaaccccctgctatgtgcagggtcatcaaccagcagcccaggctgcccagagccacatcca
gcctggccttgaatgcctgcagggatggggcatccacagcctccttgggcaacctgttcagtgcgtcaccaccctctggg
ggaaaaactgcctcctcatatccaacccaaacctcccctgtctcagtgtaaagccattcccccttgtcctatcaagggggag
tttgctgtgacattgttggtctggggtgacacatgtttgccaattcagtgcatcacggagaggcagatcttggggataagga
agtgcaggacagcatggacgtgggacatgcaggtgttgagggctctgggacactctccaagtcacagcgttcagaaca
gccttaaggataagaagataggatagaaggacaaagagcaagttaaaacccagcatggagaggagcacaaaaaggcc
acagacactgctggtccctgtgtctgagcctgcatgtttgatggtgtctggatgcaagcagaaggggtggaagagcttgcc
tggagagatacagctgggtcagtaggactgggacaggcagctggagaattgccatgtagatgttcatacaatcgtcaaat
catgaaggctggaaagcctccaagatccccaagaccaaccccaacccacccaccgtgcccactggccatgtccctcagt
gccacatccccacagttcttcatcacctccagggacggtgacccccccacctccgtgggcagctgtgccactgcagcac
cgctctttggagaaggtaaatcttgctaaatccagcccgaccctcccctggcactacgtaaggccattatctctcatccaac
tccaggacggagtcagtgaggatggggctctagagggacagcccccccccaaagcccccagggatgtaattacgtccc
tcccccgctaggggcagcagcgagccgcccggggctccgctccggtccggcgctccccccgcatccccgagccggc
agcgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagc
ctgcagacacctggggggatacggggaaaaagctttaggctgaaagagagatttagaatgacagaatcatagaacggc
ctgggttgcaaaggagcacagtgctcatccagatccaaccccctgctatgtgcagggtcatcaaccagcagcccaggct
gcccagagccacatccagcctggccttgaatgcctgcagggatggggcatccacagcctccttgggcaacctgttcagt
gcgtcaccaccctctgggggaaaaactgcctcctcatatccaacccaaacctcccctgtctcagtgtaaagccattccccct
tgtcctatcaagggggagtttgctgtgacattgttggtctggggtgacacatgtttgccaattcagtgcatcacggagaggc
agatcttggggataaggaagtgcaggacagcatggacgtgggacatgcaggtgttgagggctctgggacactctccaa
gtcacagcgttcagaacagccttaaggataagaagataggatagaaggacaaagagcaagttaaaacccagcatggag
aggagcacaaaaaggccacagacactgctggtccctgtgtctgagcctgcatgtttgatggtgtctggatgcaagcagaa
ggggtccatgtccctcagtgccacatccccacagttcttcatcacctccagggacggtgacccccccacctccgtgggca
gctgtgccactgcagcaccgctctttggagaaggtaaatcttgctaaatccagcccgaccctcccctggcacaacgtaag
gccattatctctcatccaactccaggaacggagtcagtgag
Thereby WPRE gives mRNA stability to increase target protein synthetic regulator, can make target protein great expression under the regulation and control of UPII promotor.WPRE has SEQ ID NO:7 base sequence.
[SEQ?ID?NO：7]：
accaggttctgttcctgttaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgct
ccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataa
atcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaa
cccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcgga
actcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcgggga
agctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggcc
ctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacg
agtcggatctccctttgggccgcctccccgcctgtttcgcctcgggctcctcgag
The invention provides I/pUP2/hEPO carrier, pUP2/hEPO (WPRE) carrier and I/pUP2/hEPO (WPRE) carrier, as the preferred example of the expression vector that also contains regulator.
By neomycin resistance gene being inserted in the pUP2/hEPO carrier of the present invention, then WPRE being inserted into 3 ' end of EPO gene or insulator is inserted into 5 ' of UPII promotor and bring in this carrier of generation.
For instance, the animal that can transform expression vector of the present invention comprises the animal that all are urinated, as pig, mouse, ox, poultry, sheep and goat animal.
The method of using expression vector of the present invention to produce transgenic animal is carried out according to conventional methods.That is to say, collect zygote, expression vector of the present invention is imported in the zygote from the healthy individual of the animal that will transform.Use vasectomized mouse to obtain the false pregnancy mouse then, zygote is implanted in the uterine tube as surrogate mother's false pregnancy mouse.Then to screening the individuality that transforms the offspring who obtains from the surrogate mother.
Subsequently, from the screening individuality that has confirmed to transform, collect urine, from the urine of collecting, separate and the purifying target protein, thereby produce useful protein.
In producing useful proteins the inventive method, can be by routine techniques as the separation and the purifying process that filter or chromatography is urinated.
The transgenic animal of the present invention of Chan Shenging are expressed target protein in the specific mode of bladder as mentioned above, and in urine, to express target protein far above the concentration of existing method.
For example, transform the high EPO expression level that the mouse that expression vector pUP2/hEPO of the present invention is arranged shows 0.5-1mg/ml.Though EPO is a protein beyond expression of words, because it causes embryo's early stage death, the expression level of EPO is than at least 1,000 times of protein expression level height in the urine that uses existing uroplakin promotor in the animal of the present invention.
In addition, the protein of producing from transgenic animal of the present invention shows than the better physiologically active of the commercially available protein of same type.
For example, the EPO that obtains from the mouse that transforms expression vector pUP2/hEPO of the present invention keeps the survival rate of EPO dependency hepatic cell line with the level higher than commercially available EPO.
Therefore, the expression vector of promotor of the present invention and this promotor of use and transgenic animal can be advantageously utilised in and be difficult to mass-produced useful proteins production field.
Description of drawings
Fig. 1 explanation is used to separate the probe structure of pig uroplakin II promotor of the present invention and the clone who obtains by probe;
Fig. 2 illustrates the structure of expression vector pUP2/hEPO among the present invention;
Fig. 3 illustrates that the bladder of pig uroplakin II mRNA is specific expressed;
Fig. 4 illustrates that the proteic urothelium of pig uroplakin II is specific expressed;
Fig. 5 illustrates that the proteic expression level of pig uroplakin II is specific expressed with this proteinic umbrella cells in the bladder cell;
Fig. 6 illustrates that the bladder that transforms EPO mRNA in the mouse that expression vector pUP2/hEPO of the present invention is arranged is specific expressed;
Fig. 7 illustrates the expression that transforms epo protein in the mouse that expression vector pUP2/hEPO of the present invention is arranged;
Fig. 8 illustrates the structure of expression vector I/pUP2/hEPO of the present invention;
Fig. 9 illustrates the structure of expression vector pUP2/hEPO of the present invention (WPRE);
Figure 10 illustrates the structure of expression vector I/pUP2/hEPO of the present invention (WPRE);
Figure 11 illustrates the comparison between the EPO gene expression dose of expression vector of the present invention;
Figure 12 illustrates the comparison between the EPO gene expression dose of expression vector of the present invention.
Preferred forms
To the present invention be described in more detail by embodiment below.Should be realized that the present invention is not limited to embodiment or is subjected to the restriction of embodiment.
Embodiment 1: separate pig uroplakin II promotor of the present invention
In order to separate pig uroplakin II promotor of the present invention, carry out following test.
1) by RT-PCR (reverse transcription-polymerase chain reaction) preparation probe
Because the base sequence of pig uroplakin II gene is unknown, and known mouse of base sequence and ox uroplakin II cDNA are compared mutually.With reference to the part of high conservative between two species, generate the degenerate primer group of the pig uroplakin II cDNA that is used to increase.The base sequence of forward and reverse primer is respectively shown in SEQ ID NO:2 and SEQ ID NO:3.
Use primer sets, with the MuMLV reversed transcriptive enzyme total RNA of Vesica sus domestica is carried out the RT reaction, use Taq polysaccharase carries out PCR to the cDNA of gained.The DNA base sequence of amplification read the integral part that the DNA that shows amplification is a uroplakin II gene.DNA with the amplification of pGEM T-easy carrier cloning.
In order to generate the probe that is used to separate uroplakin II promotor, the DNA that 50 μ g are cloned boiled 3 minutes, and cooling makes it sex change in ice then.With the DNA of sex change join contain primer, dNTP, [α-
32P] (3000Ci/nmol in reaction buffer NEN), adds the Klenow fragment, in 37 ℃ of reactions 1 hour to dCTP then in solution.The probe that obtains is formed (Fig. 1) by the probe A of a part in the exon 2 that comprises uroplakin II gene-5 and the probe B that comprises a part in the exons 1-2 of uroplakin II gene.
Then, use Sephadex G-50 post that reaction soln is carried out purifying, thus preparation
32Dna probe A and probe B the P mark, that be used for the detection of pig uroplakin II promotor.
2) library screening
In order to separate pig uroplakin II promotor, the pig genomic library is screened.In this embodiment, used the pig genomic library that is inserted in the λ FixII phage vector (Stratagene).
Preparation transduction as described below has the host bacteria in library.
Make the bacterial colony of 5ml LB culture medium inoculated that contains 0.2% maltose, and in 37 ℃ of overnight incubation.1% culture medium is transferred in the fresh LB substratum that 50ml contains 0.2% maltose, and cultivated 2.5 hours.When the absorbancy at 600nm place reaches about 0.5 the time, with culture solution with 2, centrifugal 10 minutes of 500rpm.The cell precipitation of gained is suspended in the aseptic Adlerika of 10ml, to final concentration be 1 * 10
10Cell/ml, and stand-by in 4 ℃ of preservations.
In order to measure titre, with the library with the different concns serial dilution in SM solution.The flat board that will contain solid LB substratum is incubated in 37 ℃ of incubators, with the top-agar dissolving, and places the water-bath that remains on 48 ℃.10 μ l are mixed with the host bacteria of the above-mentioned preparation of 100 μ l with each phage solution of different concns dilution, use the phage-infect host bacteria in 37 ℃.
The host bacteria that has infected phage is joined in the top-agar, shake up, and pour on the above-mentioned ready LB substratum.After 15 minutes, make the plate overnight incubation in 37 ℃ incubator that faces down.Form plaque on the plate culture medium of overnight incubation, be used for subsequent step, in 4 ℃ of coolings at least 1 hour, described plaque explanation library DNA was bred the back phage with the host bacteria cracking in host bacteria with plate.
NC with sequence number is provided filter membrane, the library DNA flat board of above-mentioned preparation is covered with filter membrane in some way, so that the middle part of filter membrane at first contacts.With direction filter membrane is pricked the hole with pin,, after 1 minute, carefully filter membrane and substratum are separated with mark position perpendicular to filter membrane.
Every filter membrane is immersed in sex change liquid, neutralizer and the 2 * SSC solution continuously, and every kind of solution 1 minute placed 80 ℃ of baking boxs then 2 hours, thereby the library DNA of transfer is completely fixed on the filter membrane.
Every fixed filter membrane is swum in 2 * SSC solution make it moistening, prehybridization in containing the culture dish of prehybridization solution slowly rocked 1 hour in 68 ℃ then.Behind the prehybridization, every filter membrane add embodiment 1 the 1st) probe of preparation in the part, slowly rock in 68 ℃ and to hybridize in 18 hours.After the hybridization, in containing 2 * SSC solution of 0.1%SDS, soak, repeat twice in 65 ℃ of processes of rocking 10 minutes filter wash films.After the washing, that filter membrane is air-dry and carry out radioautograph.
By comparing between radioautograph result and flat board, picking shows the plaque of positive sign.Plaque is placed 500 μ l SM solution, and add a chloroform and with this solution thorough mixing, mixture is stored in 4 ℃.This screening process repeats 3 times, the clone that final acquisition shows positive sign.Use Qiagenlambda mini test kit purifying is contained in the DNA among each clone.
Use ABI 377DNA sequenator (Applied Biosystem) to carry out reading of DNA base sequence, use CAP2 arrangement set (assembly) system processing sequencing result, use BLAST, SMART, PROSITE etc. to carry out sequence relatively, use Clustal W program to carry out the motif analysis.
As a result, when screening, clone shown in Figure 11 and 2 have been obtained with probe A.When probe B is used to screen, clone shown in Figure 13 and 4 have been obtained.Because each this clone contains pig uroplakinII gene or structure gene, the complete base sequence that pig uroplakinII promotor relatively is provided between the clone at 3 ' end.
The total length of pig uroplakin II promotor of the present invention is 8847bp, and its base sequence is shown in SEQ IDNO:1.
3) verify the protein expression pattern of under promoter regulation of the present invention, expressing
In order to verify the protein expression pattern of expressing under promoter regulation of the present invention, the expression checking of pig uroplakin II is as follows.
3-1) verify that the proteinic bladder of expressing is specific expressed under promoter regulation of the present invention
In order to verify whether expressed protein is expressed in bladder specificity mode under promoter regulation of the present invention, has carried out the Northern analysis.
Use embodiment 1 the 2nd) the pig uroplakin II cDNA that obtains in the part is as probe, provide simultaneously with constant level be expressed in the Actin muscle probe in a organized way organize in contrast.In order to use probe to confirm whether the expression of pig uroplakin II mRNA is present in the tissue of each boar health, the total RNA to the tissue that comprises bladder, heart, liver, lung, uterus and spleen as described below carries out electrophoresis.
The 0.7g agarose is placed 250ml Erlenmeyer flask, add the distilled water of 58ml, make it dissolving fully in electronics water-bath (electronic rang), and in the water-bath of 60 ℃ of insulations, cool off.When the temperature of sepharose transfers to 60 ℃, carefully add 7ml 10 * race glue damping fluid, rock simultaneously, the formaldehyde that adds 11.9ml again runs the glue damping fluid to prepare 1 * formaldehyde.Solution is placed predefined electrophoresis system, leave standstill and made the generation gel in 20 minutes.
With 6 μ l RNA, 2.5 μ l 10 * race glue damping fluid, 4 μ l formaldehyde and 12.5 μ l methane amide thorough mixing in centrifuge tube, in 65 ℃ the heating 5 minutes, then in cooled on ice.Add 2.5 μ l gel sample-loading buffers, and with the sample thorough mixing, be added to on 5 minutes the gel of 5V prerunning.With the material of gained 1 * run in the glue damping fluid with the 120V/cm electrophoresis.Behind the electrophoresis, gel is placed about 10 minutes of the sodium hydroxide solution of 0.05N, part is downcut RNA, and the efficient of follow-up transfer process is increased.
Gel is placed 0.1M Tris solution (pH 7.5) 30 minutes, placed then in 20 * SSC solution (pH 7.3 for 3M sodium-chlor, 0.3M Trisodium Citrate) about 30 minutes, use positively charged film that RNA is transferred on the gel then.Fix in order to carry out RNA, the film that shifts was left standstill 2 hours in 80 ℃.
Film is placed the vinyl bag of the minimum volume hybridization solution that contains the complete submergence film of energy.Then, bag was preserved 1 hour in 68 ℃ of vibration incubators at least, then solution is taken out, be changed to the hybridization solution that 15ml contains probe, in 68 ℃ of vibration incubators, spend the night.
After the hybridization, (2 * SSC 0.1%SDS) washed 30 minutes under room temperature, changed washing soln, and (0.2 * SSC 0.1%SDS) in 55 ℃ of washings 30 minutes, changes washing soln 2 to use washing soln 2 then with washing soln 1.Make film behind complete drying under the room temperature,, whether expressed pig uroplakin IImRNA with checking to its radioautograph.The result as shown in Figure 3.
Shown in Fig. 3 a, express unanimity in institute in a organized way as the Actin muscle mRNA of internal contrast group.On the other hand, shown in Fig. 3 b, the uroplakin II mRNA that expresses under promoter regulation of the present invention only is expressed in (Fig. 3 b) in the Vesica sus domestica specifically.
The visible promotor of the present invention of result is with bladder specificity mode marking protein.
3-2) verify that the proteinic urothelium of expressing is specific expressed under the regulation and control of promotor of the present invention
Whether express the immunohistochemical staining that carried out as described below in the arbitrary cell at bladder body in order to verify therebetween, in expressed protein under the promoter regulation of the present invention.
The paraffin section (fragment) of Vesica sus domestica tissue is provided, in Histoclear solution, has kept 10 minutes to remove deparaffnize.Section is dipped in the aqueous ethanol solution that concentration reduces gradually dewatering, and is dipped into the methyl alcohol that contains 3% sodium hydroxide and contains in the 0.1% pepsic 0.05N hydrochloric acid, with the unspecific staining that prevents to cut into slices.
With twice washing of TBS damping fluid (0.05M Tris, pH 7.4,0.85% sodium-chlor) 5 minutes, carrying out capping with notmal horse sera in the TBS of 1: 5 dilution proportion then.
With section soaked overnight in resisting with the TBS of 1: 500 dilution proportion with of sealing, at this moment, use can be anti-as one with pig uroplakin II protein-specific bonded polyclonal antibody, and a horse serum in the use ABC test kit is as negative control group.
The section of having carried out an anti-reaction is washed 5 times with TBS, each 5 minutes,, make it and combine two anti-reactions 30 minutes of vitamin H then to remove excessive antibody.Then, will cut into slices with TBS washing 3 times, and with ABC reagent react 30 minutes.To cut into slices and wash once more, wash 30 seconds with the PBS that contains 1%Triton-X 100, then with 0.05M Tris damping fluid (pH 7.6) reaction solution that contains 0.5% diaminobenzidine (DAB) and 0.01% hydrogen peroxide with TBS.
After the color reaction, section is washed with water, and be placed into its coloured moiety of observation under the opticmicroscope.The result as shown in Figure 4.
Shown in Fig. 4 a, control group does not show any positive signal.But, shown in Fig. 4 b, the proteic bladder body reaction and display of antibody and uroplakin II: promoter regulation uroplakin II albumen of the present invention, thus make this protein only specific expressed in the Vesica sus domestica epithelium, especially in the endochylema of last base portion cell, express.3-3) verify the protein expression level of under promoter regulation of the present invention, expressing
Because the active mammary gland that protein synthesis takes place of the known ratio of Urothelial Cell has lower protein synthesis capacity, in the following manner the proteinic actual expression level of expressing under promoter regulation of the present invention by laser scanning cell art (hereinafter being called " LSC ") checking.
Separate the Vesica sus domestica tissue meticulously, join in the DMEM/F12 substratum (Gibco) that contains 1mg/ml type i collagen enzyme (Sigma), 0.51mg/ml Unidasa (Sigma) and 50 μ g/ml gentamicins, carried out digestion reaction 1 hour in 37 ℃.
The material of gained uses 60 μ m nylon membranes (Milipore) to filter out big quantity of material with after the PBS washing, make suspension unicellular be attached to the Lab-Tek slide glass that is coated with 0.1% gelatin (chamber slide, Nunc) in.The cell that is attached on the slide glass washs with cold PBS, and fixes 15 minutes in cold methyl alcohol, handles 10 minutes in 0.1%Triton-X 100 solution then.
The fixed cell sealed 1 hour in containing the PBS of 1%BSA, and and embodiment 1 3-2) in the part 1: 100 solution of the uroplakin II polyclonal antibody of preparation in room temperature reaction 2 hours.After washing with PBS, cell and anti-mouse IgG two anti-(Cappel Laboratories) reaction that combines FITC.At this moment, preparation only resists the groups of reaction as negative control group with two.
After cell is given a baby a bath on the third day after its birth time with the PBS that contains 0.1%Tween-20, with the dyeing of 50 μ g/ml iodate third ingots (PI), thus the mensuration total cellular score.When LSC analyzes,, FITC is used the 530nm spectral filter, uses the 570nm spectral filter to observe luciferase expression to PI with the argon laser emitting fluorescence of 488nm.The result as shown in Figure 5.Shown in Fig. 5 a, the result that the cell of expressing uroplakin II in the bladder cell is analyzed is shown in Fig. 5 a to the analytical results of negative control group, and the result that the immunophenotype of the bladder cell of expressing uroplakin II is analyzed is shown in Fig. 5 c.
The cell expressing uroplakin II of about 8-14% from visible all the bladder cells of Fig. 5 b.From the visible most cells of Fig. 5 c is the umbrella cells of active propagation and division (cleaved).Consider that protein level is generally the very low-level of 5-25mg/l in the urine, the expression level of above-mentioned uroplakin II is very high.In addition, think that also the use bladder body can make protein to be separated and purifying than the higher efficient of use mammary tissue.
The visible promotor of the present invention of result can make target protein efficiently express in bladder.
Embodiment 2: generate expression vector pUP2/hEPO of the present invention
Use isolating promotor of the present invention among the embodiment 1, be created on the carrier of the following EPO of expression of regulation and control of this promotor by following method.
Select pBluescript SK (-) as the basic framework carrier, insert embodiment 1 the 2nd) isolating promotor of the present invention in the part.Then, the gene (SEQ ID NO:4) of coding people EPO is inserted into 3 ' end of promotor.
The expression vector of gained has structure shown in Figure 2, expresses EPO under the regulation and control of uroplakin II promotor of the present invention.This carrier is called " pUP2/hEPO ", on October 17th, 2002 be deposited in Korea S's bio-science and biotechnology research Korea S typical case culture collection center (KCTC), preserving number is KCTC 10352BP.
Embodiment 3: generate and import the fertilized egg cell that expression vector pUP2/hEPO of the present invention is arranged
Generation as described below imports the fertilized egg cell that the expression vector pUP2/hEPO of the present invention that generates among the embodiment 2 is arranged.
1) collects zygote
3 days the time, PMSG was administered in the abdominal cavity of female mice before collecting zygote, two days later, afternoon, 5 clockwise female mices were used hCG, were fertilized with the male mice mating then.In second day morning of mating fertilization, observe in the female mice whether generated plug (plug), whether conceived with the checking female mice.
With the mouse that the confirms conceived execution of craning one, cut off the abdominal cavity with operating scissors, the reticular tissue part of separating uterus.Part between uterine tube and the uterus is torn with tweezers, then the part between ovary and the uterine tube is cut with scissors.Then, downcut and tear the part Uterus wall, and separate uterine tube with tweezers.
Isolating uterine tube is placed the M2 substratum, and be placed on the insulcrete, descend to prevent its temperature.The uterine tube ampulla is cut open with the 1ml syringe needle at microscopically, collected the embryo.The embryo who collects is placed the hyaluronic acid enzyme solution that is exposed to room temperature, leave standstill cumulus cell and disperse.
The solution of gained is washed 2-3 time with the M2 substratum, with 13, the centrifugal 5min of 000rpm, and wash 2-3 time with the M2 substratum again.The zygote of screening is washed 2-3 time in the M16 substratum that covers paraffin oil, shift then and be stored in 37 ℃ of incubators.
2) microinjection of DNA in zygote
Use micrurgy, expression vector pUP2/hEPO of the present invention is expelled in the zygote of above-mentioned collection.
Embodiment 4: preparation generates the transgenic mice of people EPO under promoter regulation of the present invention
Use the zygote that generates among the embodiment 3, be created on the transgenic mice that generates people EPO under the promoter regulation of the present invention in the following manner.
1) prepares vasectomized mouse
Be used to cause the vasectomized mouse of surrogate mother false pregnancy to produce as follows.
Select and anaesthetize the ICR mouse in 6 ages in week, use tweezers and scissors then, cut epidermis apart from about 1cm at about 1.5cm place, pubis top along pubis.Keeping left or keep right, it is overlapping to prevent sheared edge to place, and cuts off the flesh layer, and the testis below the scrotum is moved on in the abdominal cavity.With tweezers testis, epididymis and vas deferens are separated from each other, separate vas deferens (speraduct) film on every side, with the tweezers cutting-out vas deferens of heating with tweezers.After confirming to have separated vas deferens, the flesh layered suture is closed, mouse is placed the greenhouse, up to reviving.
2) generation is as surrogate mother's false pregnancy mouse
Before test day, will be verified have estrous ICR female mice and embodiment 3 the 1st) the vasectomized mouse mating fertilization that produces in the part.In test day morning, observe in the female mice whether generated plug, with checking female mice false pregnancy.
3) embryo transfers in the uterine tube
Make embodiment 2 the 2nd) part in the preparation zygote in micropipet, arrange.Slight cutting is taken out the top of ovary, uterine tube and horn of uterus with tweezers as the epidermis and the flesh layer of surrogate mother's anesthesia female mice in body.Locate ovary in a certain way, make through the ovarian bursa exposed portions to face up.Then, use hemostasis device to insert fatty tissue with fixing ovary.Under stereoscopic microscope, remove the film of ovarian bursa, take out uterine tube and ovary then to observe cilium (fimbrae).Then, the forward tip of transplanting pipettor is inserted into 2-3mm in the uterine tube, and zygote carefully is transplanted in the uterine tube together with substratum.Whether first bubble of observing in the pipettor thing that serves as a mark in two bubbles has been inserted in the uterine tube, has been transplanted in the uterine tube really with checking zygote.
From the female mouse of scapegoat, obtained the offspring.In order therefrom to screen transgenic mice, use the exons 1 and 2 of EPO to carry out the Northern analysis as probe, analytical results shows has 12 to have to transform in 76 mouse.
Verified the expression pattern of epo protein in the transgenic mice, the result shows that epo protein expresses in bladder specificity mode.
Embodiment 6: generate people EPO from transgenic mice of the present invention
1) expression level of EPO in the checking transgenic mice of the present invention urine, urine obtains from transgenic mice, filters and carries out HPLC and analyze.Form for the protein of verifying each fraction, carry out electrophoresis and western and analyze, the result as shown in Figure 7.
Western analytical results among electrophoresis result from Fig. 7 a and Fig. 7 b is obviously as seen: the urine that obtains from transgenic mice contains the EPO of high density.
The concentration of EPO is calculated as the expression level of 0.5-1mg/ml in the urine, and it is apparently higher than the protein expression level from existing transgenic animal milk.
Therefore, the transgenic animal of using promotor of the present invention to produce can efficiently generate target protein in urine.
2) checking is from the physiologically active of the EPO of transgenic mice acquisition of the present invention
In order to verify the physiologically active of the EPO that obtains from transgenic mice of the present invention, with embodiment 3 the 1st) EPO that obtains in the part joins in the EPO dependency liver cell and cultivated.At this moment, control group adds commercially available EPO.The survival rate of each time point determining cell of 24,48 and 72 hours after cultivation, the result is as shown in table 1.
Table 1:
As shown in table 1, as seen in all time periods from transgenic mice of the present invention urine isolating EPO all show the physiologically active higher than commercially available EPO.
Therefore, the transgenic animal of using promotor of the present invention to produce can produce physiologically active and obtain proteinic protein far above existing method.
Embodiment 6: generation contains the expression vector of the present invention of regulator and verifies its efficient
1) makes up the expression vector that contains regulator
Generate maximized carrier system in order to set up the EPO that can make under the UPII promoter regulation of the present invention, selective marker and regulator are introduced in the pUP2/hEPO carrier in the following manner, to produce a series of improvement carrier.
1-1) make up the pUPII/hEPO-Neo carrier
When foundation can be expressed proteinic clone under the regulation and control of UPII promotor,, introduce neomycin resistance gene in the following manner to produce the pUP2/hEPO-Neo carrier for the effective choice mark is inserted in the carrier.
In order to obtain neomycin resistance gene, use pEGFP-N1 carrier (Clontech) as template, and forward primer (SEQ ID NO:8) and reverse primer (SEQ ID NO:9) carry out PCR and react.
5’-GCGGCCGCGCGCGTCAGGTGGCAC-3’(SEQ?ID?NO：8)
5’-CGATCGGACGCTCAGTGGAACGAAAACTC-3’(SEQ?ID?NO：9)
The 1.9-kb PCR product of gained is inserted in the pGEM T-easy carrier, and with NotI restriction enzyme digestion the neomycin resistance gene part that is used to clone with preparation.
By digesting the ampicillin resistance gene site of removing in the pUP2/hEPO carrier of the present invention with NotI and SalI restriction enzyme, the carrier that is used to clone with preparation.
The neomycin resistance gene of above-mentioned preparation is cloned in the carrier, thereby the generation neomycin resistance gene has been inserted into the pUP2/hEPO-Neo carrier in the existing pUP2/hEPO carrier.
1-2) make up the I/pUP2/hEPO carrier
In order to obtain the protein expression carrier under can stably express UPII promoter regulation, in the following manner the insulator gene is imported in the pUP2/hEPO-Neo carrier, thereby produce the I/pUP2/hEPO carrier.
In order to obtain the insulator gene, use the pBC1 carrier (Invitrogen) that contains chicken B-globin insulator gene as template, and forward primer (SEQ ID NO:10) and reverse primer (SEQ ID NO:11) carry out PCR and react.In order to increase PCR efficient, two copies have increased.
5’-TCGACTCTAGAGGGACAG-3’(SEQ?ID?NO：10)
5’-CTCACTGACTCCGTTCCT-3’(SEQ?ID?NO：11)
The 2.4-kb PCR product of gained is inserted in the pGEM T-easy carrier, and with NotI restriction enzyme digestion the insulator gene that is used to clone with preparation.
Insulator gene and top 1-1 with above-mentioned preparation) carrier of part is connected to each other by the NotI site, thereby produces I/pUP2/hEPO carrier (Fig. 8).
1-3) make up pUP2/hEPO (WPRE) carrier
For obtain can be under the control of UPII promotor great expression protein expression carrier, in the following manner the WPRE gene is incorporated in the pUP2/hEPO-Neo carrier, to produce pUP2/hEPO (WPRE) carrier.
In order to clone the WPRE gene, use forward primer (SEQ ID NO:12) and reverse primer (SEQ IDNO:13) to carry out the PCR reaction.
5’-ACCAGGTTCTGTTCCTGTTAATCAACCTC-3’(SEQ?ID?NO：12)
5’-CTCGAGGAGCCCGAGGCGAAACAGGCG-3’(sEQ?ID?NO：13)
The 0.6-kb PCR product of gained is inserted in the pGEM T-easy carrier, and is inserted into present embodiment 1-1) in the NcoI restriction site of the pUP2/hEPO-Neo that produces in the part.With the gained carrier with the digestion of BspHI restriction enzyme, the WPRE gene that is used to clone with preparation.
Therebetween, with the rear portion of EPO gene in the pUP2/hEPO carrier of the present invention with the digestion of NcoI restriction enzyme, the carrier that is used to clone with preparation.
With the WPRE gene clone of above-mentioned preparation in carrier, to produce pUP2/hEPO (WPRE) carrier (Fig. 9).
1-4) make up I/pUP2/hEPO (WPRE) carrier
In order to produce the effective cell that can satisfy expression level, expression stability and be based upon under the UPII promoter regulation is all maximized expression vector, has produced I/pUP2/hEPO (WPRE) carrier in the following manner.
With present embodiment 1-2) the insulator gene of part preparation is by NotI site and present embodiment 1-3) carrier in the part is connected, thus generation I/pUP2/hEPO carrier (Figure 10).
2) efficient of checking expression vector of the present invention
Verify the efficient of the expression vector that is produced among the embodiment 6 in the following manner.
2-1) expression vector of the present invention is carried out pcr analysis
In order to verify the EPO expression of gene level due to the expression vector of the present invention, PCR in real time is carried out as follows.
(Effectene Qiagen) four expression vectors of the present invention that produce among the embodiment 6 are imported among the bladder clone RT4, and subculture is to set up stable clone to use the transfection reagent box.Extract genomic dna from the clone of each gained, whether the performing PCR of going forward side by side has correctly carried out transfection with checking.
In order to verify EPO expression of gene level, from four clones, extract total RNA, and carry out RT-PCR with amplification cDNA.Use above-mentioned cDNA to carry out PCR as the forward and the reverse primer of the template and the EPO exon region that can increase.
In order to verify the expression level in each clone, use the house-keeping gene GAPDH that in cell, expresses to organize 3 these processes of repetition in contrast with constant level.Test result is carried out statistical treatment with the SAS program, is shown in (pUP2=pUP2/hEPO carrier among Figure 11; The 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 dose of expression vector of the present invention increases gradually with pUP2/hEPO carrier, I/pUP2/hEPO carrier, pUP2/hEPO (WPRE) carrier and I/pUP2/hEPO (WPRE) carrier order.
Particularly, I/pUP2/hEPO (WPRE) carrier that contains WPRE and insulator shows high about 50 times expression level (Figure 11 b) than the pUP2/hEPO that does not contain isolated insulation.
Therefore, the carrier of the present invention that comprises I/pUP2/hEPO (WPRE) carrier can be advantageously used in and generate EPO.
2-2) Western of expression vector of the present invention analyzes
In order to verify the expression level of the epo protein due to the expression vector of the present invention, the Western that carries out as described below analyzes.
Will be by importing the 2-1 of embodiment 6) clone set up of each expression vector of the present invention in the part places the lysis buffer that contains NP-40, and supersound process is to extract protein from clone.
With each 40 μ l protein on the SDS-PAGE gel through electrophoresis, transfer on the pvdf membrane, and with the EPO antibody treatment with checking IPO proteic expression level.For the expression level of quantitative epo protein, use actin antibody to organize twice this process of repetition in contrast.Result's SAS routine processes is shown in (pUP2=pUP2/hEPO carrier among Figure 12; The IUP2=I/pUP2/hEPO carrier; PW=pUP2/hEPO (WPRE) carrier; IW=I/pUP2/hEPO (WPRE) carrier).
As shown in figure 12, the epo protein expression level of expression vector of the present invention increases gradually with pUP2/hEPO carrier, I/pUP2/hEPO carrier, pUP2/hEPO (WPRE) carrier and I/pUP2/hEPO (WPRE) carrier order.
2-1 among this result and the embodiment 6) result is consistent shown in the part.
Therefore, the carrier of the present invention that comprises I/pUP2/hEPO (WPRE) carrier can be advantageously used in and generate EPO.
Commercial Application
As mentioned above, promoter of the present invention induces the bladder of destination protein specific expressed, in urine to express destination protein far above existing methodical concentration.
The animal that the expression vector that is made up of promoter of the present invention and the destination protein that is subjected to this promoter regulation transforms is with the efficient secretion destination protein higher than existing transgenic animals. In addition, the protein that obtains from transgenic animals of the present invention shows the better physiologically active of existing protein than same type.
Therefore, promoter of the present invention, the expression vector that uses this promoter and transgenic animals can be advantageously used in the useful proteins production field of pharmaceutical value.
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〉be used to the to increase forward primer of pig uroplakin II gene
<400>2
gatcctgatt?ctgctggctb 20
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase reverse primer of pig uroplakin II gene
<400>3
atggtggtca?tcacrgtgct 20
<210>4
<211>3602
<212>DNA
＜213〉mankind
<300>
<301>Lin，F.K.
＜223〉be used to the to increase reverse primer of neomycin resistance gene
<400>9
cgatcggacg?ctcagtggaa?cgaaaactc 29
<210>10
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase forward primer of chicken B-globin insulator
<400>10
tcgactctag?agggacag 18
<210>11
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase reverse primer of chicken B-globin insulator
<400>11
ctcactgact?ccgttcct 18
<210>12
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase forward primer of alpine marmot hepatitis virus post-transcriptional control element
Claims (8)
1. the pig uroplakin II gene promoter of forming by SEQ ID NO:1 base sequence.
2. contain the expression vector that right requires promotor base sequence in 1 and is positioned at the base sequence of described promotor 3 ' end coding target protein.
3. the expression vector of claim 2, wherein target protein is erythropoietin (EPO).
4. the expression vector of claim 3, it is that preservation registration number is the expression vector pUP2/hEPO of KCTC 10352BP preservation, this carrier structure is as shown in Figure 2.
5. the expression vector of claim 3, 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, this carrier structure is as shown in Figure 8.
6. the expression vector of claim 3, it is pUP2/hEPO (WPRE) carrier that contains the SEQ ID NO:7 alpine marmot hepatitis virus post-transcriptional control element of holding as the SEQ ID NO:5 neomycin resistance gene and the EPO gene 3 ' of selective marker (WPRE), and this carrier structure as shown in Figure 9.
7. the expression vector of claim 3, it is to contain the SEQ ID NO:5 neomycin resistance gene as selective marker, the SEQ ID NO:6 insulator of UP2 promotor 5 ' end and I/pUP2/hEPO (WPRE) carrier of the SEQ ID NO:7WPRE that EPO gene 3 ' is held, and this carrier structure as shown in figure 10.
8. method that obtains to modify fertilised non-human eggs, it comprises expression vector any in the claim 3 to 7 is imported fertilised non-human eggs.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
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| KR10-2002-0067856 | 2002-11-04 | ||
| KR20020067856 | 2002-11-04 | ||
| KR1020020067856 | 2002-11-04 | ||
| KR10-2003-0077256 | 2003-11-03 | ||
| KR1020030077256A KR100552634B1 (en) | 2002-11-04 | 2003-11-03 | Porcine uroplakin ? promoter and the production method of useful proteins using said promoter |
| KR1020030077256 | 2003-11-03 | ||
| PCT/KR2003/002339 WO2004042062A1 (en) | 2002-11-04 | 2003-11-04 | Porcine uroplakin ii promoter and the production method of useful proteins using said promoter |
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| KR100769291B1 (en) * | 2006-02-13 | 2007-10-24 | 조아제약주식회사 | Mammary gland-specific human erythropoietin expression vector transgenic animal and method for producing human erythropoietin using same |
| CN101012460B (en) * | 2006-12-12 | 2010-05-19 | 浙江大学 | Pig production trait related gene CACNA2D1 order and uses thereof |
| US20100205680A1 (en) * | 2007-08-08 | 2010-08-12 | Jin Hoi Kim | Mammary gland- specific human erytheropoietin expression vector, transgenic animal and method for producing human erythropoietin using same |
| AU2008359017B2 (en) | 2008-06-30 | 2013-01-17 | Cho-A Pharm Co., Ltd. | A gene of porcine beta-casein, a promoter of the same and the use thereof |
| US9738694B2 (en) | 2008-06-30 | 2017-08-22 | Cho-A Pharm. Co., Ltd. | Gene of porcine alpha-s1 casein, a promoter of the same and use thereof |
| CN101886075B (en) * | 2010-07-02 | 2011-12-21 | 东北农业大学 | Porcine ROSA26 promoter and application thereof |
| CN101979547B (en) * | 2010-09-02 | 2012-04-25 | 华中农业大学 | Identification of isolation cloning and core region of promoters suitable for gene expression of skeletal muscles in pigs |
| CN102127545A (en) * | 2010-11-24 | 2011-07-20 | 山东农业大学 | Skeletal muscle specificity CKM (Creatine Kinase Muscle) promoter and applications thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6001646A (en) * | 1995-06-05 | 1999-12-14 | New York University | Method and vector for expression and isolation of biologically active molecules in urine |
| US6339183B1 (en) * | 1995-06-05 | 2002-01-15 | New York University | Transgenic mammals expressing heterologous DNA in urothelium and isolation of biologically active molecules from urine |
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2003
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6001646A (en) * | 1995-06-05 | 1999-12-14 | New York University | Method and vector for expression and isolation of biologically active molecules in urine |
| US6339183B1 (en) * | 1995-06-05 | 2002-01-15 | New York University | Transgenic mammals expressing heterologous DNA in urothelium and isolation of biologically active molecules from urine |
Non-Patent Citations (2)
| Title |
|---|
| KWON, D.N.等.CLONING, SEQUENCING, AND EXPRESSION ANALYSISOF THE PORCINE UROPLAKIN ii GENE.BIOCHEM.BIOPHYS.RES.COMMUN293 2.2002,293(2),862-869. |
| KWON, D.N.等.CLONING, SEQUENCING, AND EXPRESSION ANALYSISOF THE PORCINE UROPLAKIN ii GENE.BIOCHEM.BIOPHYS.RES.COMMUN293 2.2002,293(2),862-869. * |
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