CN100591771C - Hypoxia response elements gene treating plasmid and its constructing method - Google Patents
Hypoxia response elements gene treating plasmid and its constructing method Download PDFInfo
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- CN100591771C CN100591771C CN200710018226A CN200710018226A CN100591771C CN 100591771 C CN100591771 C CN 100591771C CN 200710018226 A CN200710018226 A CN 200710018226A CN 200710018226 A CN200710018226 A CN 200710018226A CN 100591771 C CN100591771 C CN 100591771C
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Abstract
The present invention provides a hypoxic reaction element gene treatment plasmid and a constructing method thereof. Five copied hypoxic reaction element core sequences and CMV minimum prompter sequences are prepared by a manual DNA synthesis method; a directional clone method is used for replacing the SV40 promoter of the plasmid vector of pGL3-Promoter Vector towards the copied hypoxic reaction element core sequences and CMV minimum prompter sequences segments to construct a pGL3-5HRE-CMVmp plasmid; a pEGFP-N1 eukaryotic expression vector is used as a template, FCR amplifies and prepares fora EGFP segment, the directional clone method is used for replacing the prepared EGFP segment into a Luciferase segment of the pGL3-5HRE-CMVmp plasmid, so as to construct a pEGFP-5HRE-CMVmp vector. A plasmid promoter sequence obtained by the constructing method of the present invention can ensure that the expression of a target gene carried by the vector is controlled by the regulation of tissue intracellular oxygen signal; the target gene carried by the vector expresses inside specific tissue or cells; the plasmid promoter sequence can be integrated with the target gene to express, which is convenient for detecting the transfection efficiency and intracellular positioning of the target gene.
Description
Technical field
The present invention relates to a kind of plasmid and construction process thereof that carries out gene therapy in ischemic/anaerobic environment that be used in.
Background technology
Gene therapy is a kind of novel method for the treatment of disease.It is that foreign gene is imported host cell, makes it to express under specific environment, thereby reaches the purpose of regulating cell function or influencing the cell growth.
Transgenosis is the gordian technique of gene therapy.When carrying out gene therapy, not only will be with transgenosis to target cell, and want the expression of render transgenic to be subjected to the regulation and control of cell internal and external environment variation.
According to the used carrier difference, the method for transgenosis can be divided into non-virus-mediated and virus-mediated two big classes substantially.The former comprises physics method, chemical method, receptor-mediated method etc.; The latter comprises adenovirus (AV), retrovirus (RV), adeno-associated virus (AAV), hsv (HSV) etc.In addition, the novel method that above-mentioned two kinds of methods is combined formation is also arranged.Plasmid DNA and cationic-liposome transgenosis belong to non-virus-mediated gene transfer method, it is simple and easy to do, plasmid DNA can not put in order and go in the karyomit(e) and is free on outside the karyomit(e), can mutagenesis and tumour take place, be a kind of safer gene therapy method.
At present, the carrier that carries out transgenosis adopts the promotor of virus more, and the effect of these promotors is stronger, when using these carriers and carrying out transgenosis, unmanageable therapeutic gene is crossed expression can cause some side reactions, thereby reduces the specificity and the validity of gene therapy greatly.
Hypoxic-ischemic is a numerous disease common pathophysiological change.When hypoxic-ischemic took place tissue, we expected that the gene that shifts begins to express or genetically modified expression obviously increases; When the tissue oxygen/blood supply recovers just often, we require genetically modified expression to stop or obviously the reduction.But the carrier of existing simple employing viral promotors is difficult to satisfy above-mentioned requirements.
Summary of the invention
The objective of the invention is to solve the problem that the control therapeutic gene is expressed when hypoxic-ischemic, provide the expression of a render transgenic only to be limited under the hypoxic-ischemic pathological and physiological condition, the unlatching that render transgenic is expressed or stop to depend on in-house oxygen signal, hypoxia response elements gene treating plasmid and construction process thereof that genetically modified expression only starts in needs.
For achieving the above object, the present invention is achieved by following technique means: at first, the core sequence and the CMV minimal promoter sequence that prepare the hypoxia response elements of 5 copies with the artificial DNA synthesis method, SV40 promotor so that the directed cloning method is replaced plasmid vector pGL3-Promoter Vector with the core sequence and the CMV minimal promoter tract of the hypoxia response elements of copy is built into the pGL3-5HRE-CMVmp plasmid; Secondly, be template with the pEGFP-N1 carrier for expression of eukaryon, adopt pcr amplification to prepare the EGFP fragment, with the directed cloning method EGFP fragment of preparation is replaced the Luciferase fragment of plasmid pGL3-5HRE-CMVmp, thereby be built into the pEGFP-5HRE-CMVmp carrier.
The core sequence and the CMV minimal promoter sequence of the hypoxia response elements of 5 copies of DNA synthesis method preparation of the present invention, be core sequence 35bp and the synthetic 5 * HRE core sequence associating of CMV minimal promoter sequence CMV minimal promoter sequence according to HRE, insert Kpn I and two restriction enzyme sites of Hind III respectively in 5 * HRE core sequence associating CMV minimal promoter sequence upstream and downstream, total length 307bp, sequence SEQ ID NO.2 is as follows:
ggtaccactagtccacagtgcatacgtgggctccaacaggtcctcttggtcgaccccacagtgcatacg
tgggctccaacaggtcctcttgcggccgcccacagtgcatacgtgggctccaacaggtcctcttccatg
gccacagtgcatacgtgggctccaacaggtcctcttcggatccgccacagtgcatacgtgggctccaac
aggtcctcttccggaattccggggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagt
gaaccgtcagatcaccggtacgcgtaagctt
Said is template with the pEGFP-N1 carrier for expression of eukaryon, adopts the pcr amplification legal system to be equipped with the EGFP fragment, is that primer sequence is as follows: P1:5 '-AAGCTTCGAATTCTGCAGT-3 ' according to pEGFP-N1 sequences Design synthetic pcr primer thing; P2:5 '-GCTCTAGAGTCGCGGCCGCTTTACT-3 '.With the pEGFP-N1 plasmid is template clone EGFP total length 795bp, contains HindIII and Xba I site, and sequence SEQ IDNO.3 is as follows:
aagcttcgaattctgcagtcgacggtaccgcgggcccgggatccaccggtcgccaccatggtgagcaag
ggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaag
ttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcacc
accggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagc
cgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggag
cgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacacc
ctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctg
gagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaac
ttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacaccccc
atcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagac
cccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatg
gacgagctgtacaagtaaagcggccgcgactctaga。
Hypoxia response elements gene treating plasmid of the present invention, carrier for expression of eukaryon pEGFP-5HRE-CMVmp are gone up promotor and the enhanced green fluorescent protein EGFP encoding sequence of being made up of the core sequence associating CMV minimal promoter of the hypoxia response elements of 5 copies.
Promoter region and the base between multiple clone site that the base sequence of carrier for expression of eukaryon pEGFP-5HRE-CMVmp of the present invention and SEQ ID NO.1 form at the core sequence associating CMV of the hypoxia response elements of 5 copies minimal promoter can not change, and the base sequence of rest part is identical more than 99%; The base sequence of carrier for expression of eukaryon pEGFP-5HRE-CMVmp is SEQ ID NO.1:
1 ggtaccacta?gtccacagtg?catacgtggg?ctccaacagg?tcctcttggt?cgaccccaca
61 gtgcatacgt?gggctccaac?aggtcctctt?gcggccgccc?acagtgcata?cgtgggctcc
121?aacaggtcct?cttccatggc?cacagtgcat?acgtgggctc?caacaggtcc?tcttcggatc
181 cgccacagtg?catacgtggg?ctccaacagg?tcctcttccg?gaattccggg?gtaggcgtgt
241 acggtgggag?gtctatataa?gcagagctcg?tttagtgaac?cgtcagatca?ccggtacgcg
301 taagcttcga?attctgcagt?cgacggtacc?gcgggcccgg?gatccaccgg?tcgccaccat
361 ggtgagcaag?ggcgaggagc?tgttcaccgg?ggtggtgccc?atcctggtcg?agctggacgg
421 cgacgtaaac?ggccacaagt?tcagcgtgtc?cggcgagggc?gagggcgatg?ccacctacgg
481 caagctgacc?ctgaagttca?tctgcaccac?cggcaagctg?cccgtgccct?ggcccaccct
541 cgtgaccacc?ctgacctacg?gcgtgcagtg?cttcagccgc?taccccgacc?acatgaagca
601 gcacgacttc?ttcaagtccg?ccatgcccga?aggctacgtc?caggagcgca?ccatcttctt
661 caaggacgac?ggcaactaca?agacccgcgc?cgaggtgaag?ttcgagggcg?acaccctggt
721 gaaccgcatc?gagctgaagg?gcatcgactt?caaggaggac?ggcaacatcc?tggggcacaa
781 gctggagtac?aactacaaca?gccacaacgt?ctatatcatg?gccgacaagc?agaagaacgg
841 catcaaggtg?aacttcaaga?tccgccacaa?catcgaggac?ggcagcgtgc?agctcgccga
901 ccactaccag?cagaacaccc?ccatcggcga?cggccccgtg?ctgctgcccg?acaaccacta
961 cctgagcacc?cagtccgccc?tgagcaaaga?ccccaacgag?aagcgcgatc?acatggtcct
1021?gctggagttc?gtgaccgccg?ccgggatcac?tctcggcatg?gacgagctgt?acaagtaaag
1081?cggccgcgac?tctagagtcg?gggcggccgg?ccgcttcgag?cagacatgat?aagatacatt
1141?gatgagtttg?gacaaaccac?aactagaatg?cagtgaaaaa?aatgctttat?ttgtgaaatt
1201?tgtgatgcta?ttgctttatt?tgtaaccatt?ataagctgca?ataaacaagt?taacaacaac
1261?aattgcattc?attttatgtt?tcaggttcag?ggggaggtgt?gggaggtttt?ttaaagcaag
1321?taaaacctct?acaaatgtgg?taaaatcgat?aaggatccgt?cgaccgatgc?ccttgagagc
1381?cttcaaccca?gtcagctcct?tccggtgggc?gcggggcatg?actatcgtcg?ccgcacttat
1441?gactgtcttc?tttatcatgc?aactcgtagg?acaggtgccg?gcagcgctct?tccgcttcct
1501?cgctcactga?ctcgctgcgc?tcggtcgttc?ggctgcggcg?agcggtatca?gctcactcaa
1561?aggcggtaat?acggttatcc?acagaatcag?gggataacgc?aggaaagaac?atgtgagcaa
1621?aaggccagca?aaaggccagg?aaccgtaaaa?aggccgcgtt?gctggcgttt?ttccataggc
1681?tccgcccccc?tgacgagcat?cacaaaaatc?gacgctcaag?tcagaggtgg?cgaaacccga
1741?caggactata?aagataccag?gcgtttcccc?ctggaagctc?cctcgtgcgc?tctcctgttc
1801?cgaccctgcc?gcttaccgga?tacctgtccg?cctttctccc?ttcgggaagc?gtggcgcttt
1861?ctcatagctc?acgctgtagg?tatctcagtt?cggtgtaggt?cgttcgctcc?aagctgggct
1921?gtgtgcacga?accccccgtt?cagcccgacc?gctgcgcctt?atccggtaac?tatcgtcttg
1981?agtccaaccc?ggtaagacac?gacttatcgc?cactggcagc?agccactggt?aacaggatta
2041?gcagagcgag?gtatgtaggc?ggtgctacag?agttcttgaa?gtggtggcct?aactacggct
2101?acactagaag?aacagtattt?ggtatctgcg?ctctgctgaa?gccagttacc?ttcggaaaaa
2161?gagttggtag?ctcttgatcc?ggcaaacaaa?ccaccgctgg?tagcggtggt?ttttttgttt
2221?gcaagcagca?gattacgcgc?agaaaaaaag?gatctcaaga?agatcctttg?atcttttcta
2281?cggggtctga?cgctcagtgg?aacgaaaact?cacgttaagg?gattttggtc?atgagattat
2341?caaaaaggat?cttcacctag?atccttttaa?attaaaaatg?aagttttaaa?tcaatctaaa
2401?gtatatatga?gtaaacttgg?tctgacagtt?accaatgctt?aatcagtgag?gcacctatct
2461?cagcgatctg?tctatttcgt?tcatccatag?ttgcctgact?ccccgtcgtg?tagataacta
2521?cgatacggga?gggcttacca?tctggcccca?gtgctgcaat?gataccgcga?gacccacgct
2581?caccggctcc?agatttatca?gcaataaacc?agccagccgg?aagggccgag?cgcagaagtg
2641?gtcctgcaac?tttatccgcc?tccatccagt?ctattaattg?ttgccgggaa?gctagagtaa
2701?gtagttcgcc?agttaatagt?ttgcgcaacg?ttgttgccat?tgctacaggc?atcgtggtgt
2761?cacgctcgtc?gtttggtatg?gcttcattca?gctccggttc?ccaacgatca?aggcgagtta
2821?catgatcccc?catgttgtgc?aaaaaagcgg?ttagctcctt?cggtcctccg?atcgttgtca
2881?gaagtaagtt?ggccgcagtg?ttatcactca?tggttatggc?agcactgcat?aattctctta
2941?ctgtcatgcc?atccgtaaga?tgcttttctg?tgactggtga?gtactcaacc?aagtcattct
3001?gagaatagtg?tatgcggcga?ccgagttgct?cttgcccggc?gtcaatacgg?gataataccg
3061?cgccacatag?cagaacttta?aaagtgctca?tcattggaaa?acgttcttcg?gggcgaaaac
3121?tctcaaggat?cttaccgctg?ttgagatcca?gttcgatgta?acccactcgt?gcacccaact
3181?gatcttcagc?atcttttact?ttcaccagcg?tttctgggtg?agcaaaaaca?ggaaggcaaa
3241?atgccgcaaa?aaagggaata?agggcgacac?ggaaatgttg?aatactcata?ctcttccttt
3301?ttcaatatta?ttgaagcatt?tatcagggtt?attgtctcat?gagcggatac?atatttgaat
3361?gtatttagaa?aaataaacaa?ataggggttc?cgcgcacatt?tccccgaaaa?gtgccacctg
3421?acgcgccctg?tagcggcgca?ttaagcgcgg?cgggtgtggt?ggttacgcgc?agcgtgaccg
3481?ctacacttgc?cagcgcccta?gcgcccgctc?ctttcgcttt?cttcccttcc?tttctcgcca
3541?cgttcgccgg?ctttccccgt?caagctctaa?atcgggggct?ccctttaggg?ttccgattta
3601?gtgctttacg?gcacctcgac?cccaaaaaac?ttgattaggg?tgatggttca?cgtagtgggc
3661?catcgccctg?atagacggtt?tttcgccctt?tgacgttgga?gtccacgttc?tttaatagtg
3721?gactcttgtt?ccaaactgga?acaacactca?accctatctc?ggtctattct?tttgatttat
3781?aagggatttt?gccgatttcg?gcctattggt?taaaaaatga?gctgatttaa?caaaaattta
3841?acgcgaattt?taacaaaata?ttaacgctta?caatttgcca?ttcgccattc?aggctgcgca
3801?actgttggga?agggcgatcg?gtgcgggcct?cttcgctatt?acgccagccc?aagctaccat
3961?gataagtaag?taatattaag?gtacgggagg?tacttggagc?ggccgcaata?aaatatcttt
4021?attttcatta?catctgtgtg?ttggtttttt?gtgtgaatcg?atagtactaa?catacgctct
4081?ccatcaaaac?aaaacgaaac?aaaacaaact?agcaaaatag?gctgtcccca?gtgcaagtgc
4141?aggtgccaga?acatttctct?atcgata。
Therefore the plasmid that obtains according to construction process of the present invention has following three characteristics: 1, comprise the core sequence of the hypoxia response elements of 5 copies in the promoter sequence, can make the expression of the goal gene that carrier carries be controlled by the adjusting of oxygen signal in the histocyte; 2, three multiple clone site that comprise in the promoter sequence can be inserted the promotor of tissue or cell-specific, thereby can make goal gene that carrier carries at specific tissue or cell inner expression; 3, comprise enhanced green fluorescent protein (EGFP) encoding sequence, can with the goal gene amalgamation and expression, be convenient to location in the transfection efficiency of testing goal gene and the cell.
Description of drawings
Fig. 1 is a pEGFP-5HRE-CMVmp support element of the present invention, 4167bp.
1) 5HRE-CMVmp promoter region: 1-302
2) multiple clone site: 1-7; 290-302
3) enhanced green fluorescent protein (EGFP) ORF:359-1078
4)SV40?late?poly(A)signal：1121-1342
5)RVprimer4?binding?region?1410-1429
6)ColE1-derived?plasmid?replication?origin?1667
7)β-lactamase?gene(Ampr)2429-3289
8)f1?origin?3421-3876
9)Upstream?poly(A)signal?4007-4160
10)RVprimer3?binding?region?4109-4128
Embodiment
The present invention adopts on the basis of the U.S. pGL3 of Promega company plasmid vector (pGL3 Promoter vector) and the pEGFP-N1 of Clontech company carrier for expression of eukaryon and reconstructs.Can from
Http:// www.promega.comWith
Http:// www.clontech.comOn obtain its structure and sequence information respectively.
1) structure of plasmid of the present invention is with the core sequence and the CMV minimal promoter sequence of the hypoxia response elements of 5 copies of artificial DNA synthesis method preparation, SV40 promotor so that the directed cloning method is replaced plasmid vector pGL3-Promoter Vector with the core sequence and the CMV minimal promoter tract of the hypoxia response elements of copy is built into the pGL3-5HRE-CMVmp plasmid;
2) secondly, adopting the pEGFP-N1 carrier for expression of eukaryon is template, pcr amplification prepares enhanced green fluorescent protein EGFP fragment, with the directed cloning method EGFP fragment of preparation is replaced the Luciferase fragment of plasmid pGL3-5HRE-CMVmp, thereby is built into the pEGFP-5HRE-CMVmp carrier.The base sequence of the plasmid that makes up is SEQ ID NO.1
1 ggtaccacta?gtccacagtg?catacgtggg?ctccaacagg?tcctcttggt?cgaccccaca
61 gtgcatacgt?gggctccaac?aggtcctctt?gcggccgccc?acagtgcata?cgtgggctcc
121 aacaggtcct?cttccatggc?cacagtgcat?acgtgggctc?caacaggtcc?tcttcggatc
181 cgccacagtg?catacgtggg?ctccaacagg?tcctcttccg?gaattccggg?gtaggcgtgt
241 acggtgggag?gtctatataa?gcagagctcg?tttagtgaac?cgtcagatca?ccggtacgcg
301 taagcttcga?attctgcagt?cgacggtacc?gcgggcccgg?gatccaccgg?tcgccaccat
361 ggtgagcaag?ggcgaggagc?tgttcaccgg?ggtggtgccc?atcctggtcg?agctggacgg
421 cgacgtaaac?ggccacaagt?tcagcgtgtc?cggcgagggc?gagggcgatg?ccacctacgg
481 caagctgacc?ctgaagttca?tctgcaccac?cggcaagctg?cccgtgccct?ggcccaccct
541 cgtgaccacc?ctgacctacg?gcgtgcagtg?cttcagccgc?taccccgacc?acatgaagca
601 gcacgacttc?ttcaagtccg?ccatgcccga?aggctacgtc?caggagcgca?ccatcttctt
661 caaggacgac?ggcaactaca?agacccgcgc?cgaggtgaag?ttcgagggcg?acaccctggt
721 gaaccgcatc?gagctgaagg?gcatcgactt?caaggaggac?ggcaacatcc?tggggcacaa
781 gctggagtac?aactacaaca?gccacaacgt?ctatatcatg?gccgacaagc?agaagaacgg
841 catcaaggtg?aacttcaaga?tccgccacaa?catcgaggac?ggcagcgtgc?agctcgccga
901 ccactaccag?cagaacaccc?ccatcggcga?cggccccgtg?ctgctgcccg?acaaccacta
961 cctgagcacc?cagtccgccc?tgagcaaaga?ccccaacgag?aagcgcgatc?acatggtcct
1021?gctggagttc?gtgaccgccg?ccgggatcac?tctcggcatg?gacgagctgt?acaagtaaag
1081?cggccgcgac?tctagagtcg?gggcggccgg?ccgcttcgag?cagacatgat?aagatacatt
1141?gatgagtttg?gacaaaccac?aactagaatg?cagtgaaaaa?aatgctttat?ttgtgaaatt
1201?tgtgatgcta?ttgctttatt?tgtaaccatt?ataagctgca?ataaacaagt?taacaacaac
1261?aattgcattc?attttatgtt?tcaggttcag?ggggaggtgt?gggaggtttt?ttaaagcaag
1321?taaaacctct?acaaatgtgg?taaaatcgat?aaggatccgt?cgaccgatgc?ccttgagagc
1381?cttcaaccca?gtcagctcct?tccggtgggc?gcggggcatg?actatcgtcg?ccgcacttat
1441?gactgtcttc?tttatcatgc?aactcgtagg?acaggtgccg?gcagcgctct?tccgcttcct
1501?cgctcactga?ctcgctgcgc?tcggtcgttc?ggctgcggcg?agcggtatca?gctcactcaa
1561?aggcggtaat?acggttatcc?acagaatcag?gggataacgc?aggaaagaac?atgtgagcaa
1621?aaggccagca?aaaggccagg?aaccgtaaaa?aggccgcgtt?gctggcgttt?ttccataggc
1681?tccgcccccc?tgacgagcat?cacaaaaatc?gacgctcaag?tcagaggtgg?cgaaacccga
1741?caggactata?aagataccag?gcgtttcccc?ctggaagctc?cctcgtgcgc?tctcctgttc
1801?cgaccctgcc?gcttaccgga?tacctgtccg?cctttctccc?ttcgggaagc?gtggcgcttt
1861?ctcatagctc?acgctgtagg?tatctcagtt?cggtgtaggt?cgttcgctcc?aagctgggct
1921?gtgtgcacga?accccccgtt?cagcccgacc?gctgcgcctt?atccggtaac?tatcgtcttg
1981?agtccaaccc?ggtaagacac?gacttatcgc?cactggcagc?agccactggt?aacaggatta
2041?gcagagcgag?gtatgtaggc?ggtgctacag?agttcttgaa?gtggtggcct?aactacggct
2101?acactagaag?aacagtattt?ggtatctgcg?ctctgctgaa?gccagttacc?ttcggaaaaa
2161?gagttggtag?ctcttgatcc?ggcaaacaaa?ccaccgctgg?tagcggtggt?ttttttgttt
2221?gcaagcagca?gattacgcgc?agaaaaaaag?gatctcaaga?agatcctttg?atcttttcta
2281?cggggtctga?cgctcagtgg?aacgaaaact?cacgttaagg?gattttggtc?atgagattat
2341?caaaaaggat?cttcacctag?atccttttaa?attaaaaatg?aagttttaaa?tcaatctaaa
2401?gtatatatga?gtaaacttgg?tctgacagtt?accaatgctt?aatcagtgag?gcacctatct
2461?cagcgatctg?tctatttcgt?tcatccatag?ttgcctgact?ccccgtcgtg?tagataacta
2521?cgatacggga?gggcttacca?tctggcccca?gtgctgcaat?gataccgcga?gacccacgct
2581?caccggctcc?agatttatca?gcaataaacc?agccagccgg?aagggccgag?cgcagaagtg
2641?gtcctgcaac?tttatccgcc?tccatccagt?ctattaattg?ttgccgggaa?gctagagtaa
2701?gtagttcgcc?agttaatagt?ttgcgcaacg?ttgttgccat?tgctacaggc?atcgtggtgt
2761?cacgctcgtc?gtttggtatg?gcttcattca?gctccggttc?ccaacgatca?aggcgagtta
2821?catgatcccc?catgttgtgc?aaaaaagcgg?ttagctcctt?cggtcctccg?atcgttgtca
2881?gaagtaagtt?ggccgcagtg?ttatcactca?tggttatggc?agcactgcat?aattctctta
2941?ctgtcatgcc?atccgtaaga?tgcttttctg?tgactggtga?gtactcaacc?aagtcattct
3001?gagaatagtg?tatgcggcga?ccgagttgct?cttgcccggc?gtcaatacgg?gataataccg
3061?cgccacatag?cagaacttta?aaagtgctca?tcattggaaa?acgttcttcg?gggcgaaaac
3121?tctcaaggat?cttaccgctg?ttgagatcca?gttcgatgta?acccactcgt?gcacccaact
3181?gatcttcagc?atcttttact?ttcaccagcg?tttctgggtg?agcaaaaaca?ggaaggcaaa
3241?atgccgcaaa?aaagggaata?agggcgacac?ggaaatgttg?aatactcata?ctcttccttt
3301?ttcaatatta?ttgaagcatt?tatcagggtt?attgtctcat?gagcggatac?atatttgaat
3361?gtatttagaa?aaataaacaa?ataggggttc?cgcgcacatt?tccccgaaaa?gtgccacctg
3421?acgcgccctg?tagcggcgca?ttaagcgcgg?cgggtgtggt?ggttacgcgc?agcgtgaccg
3481?ctacacttgc?cagcgcccta?gcgcccgctc?ctttcgcttt?cttcccttcc?tttctcgcca
3541?cgttcgccgg?ctttccccgt?caagctctaa?atcgggggct?ccctttaggg?ttccgattta
3601?gtgctttacg?gcacctcgac?cccaaaaaac?ttgattaggg?tgatggttca?cgtagtgggc
3661?catcgccctg?atagacggtt?tttcgccctt?tgacgttgga?gtccacgttc?tttaatagtg
3721?gactcttgtt?ccaaactgga?acaacactca?accctatctc?ggtctattct?tttgatttat
3781?aagggatttt?gccgatttcg?gcctattggt?taaaaaatga?gctgatttaa?caaaaattta
3841?acgcgaattt?taacaaaata?ttaacgctta?caatttgcca?ttcgccattc?aggctgcgca
3801?actgttggga?agggcgatcg?gtgcgggcct?cttcgctatt?acgccagccc?aagctaccat
3961?gataagtaag?taatattaag?gtacgggagg?tacttggagc?ggccgcaata?aaatatcttt
4021?attttcatta?catctgtgtg?ttggtttttt?gtgtgaatcg?atagtactaa?catacgctct
4081?ccatcaaaac?aaaacgaaac?aaaacaaact?agcaaaatag?gctgtcccca?gtgcaagtgc
4141?aggtgccaga?acatttctct?atcgata
Concrete grammar is as follows:
According to core sequence 35bp and the synthetic 5 * HRE core sequence associating of the CMV minimal promoter sequence CMV minimal promoter sequence of HRE, the downstream is inserted Kpn I and two restriction enzyme sites of Hind III (total length 307bp) respectively thereon.Sequence SEQID NO.2 is as follows:
ggtaccactagtccacagtgcatacgtgggctccaacaggtcctcttggtcgaccccacagtgcatacg
tgggctccaacaggtcctcttgcggccgcccacagtgcatacgtgggctccaacaggtcctcttccatg
gccacagtgcatacgtgggctccaacaggtcctcttcggatccgccacagtgcatacgtgggctccaac
aggtcctcttccggaattccggggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagt
gaaccgtcagatcaccggtacgcgtaagctt
With the directed cloning method this fragment is replaced the SV40 promotor of pGL3 promoter vector, thereby be built into the pGL3-5HRE-CMVmp carrier.Sequencing result shows, the SV40 fragment that 5 * HRE core sequence associating CMV minimal promoter sequence of synthetic has been replaced pGL3 promoter vector.
According to pEGFP-N1 sequences Design synthetic pcr primer thing.Primer sequence is as follows: P1:5 '-AAGCTTCGAATTCTGCAGT-3 '; P2:5 '-GCTCTAGAGTCGCGGCCGCTTTACT-3 '.With the pEGFP-N1 plasmid is template clone EGFP total length (795bp contains HindIII and Xba I site).Sequence SEQID NO.3 is as follows:
aagcttcgaattctgcagtcgacggtaccgcgggcccgggatccaccggtcgccaccatggtgagcaag
ggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaag
ttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcacc
accggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagc
cgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggag
cgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacacc
ctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctg
gagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaac
ttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacaccccc
atcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagac
cccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatg
gacgagctgtacaagtaaagcggccgcgactctaga
According to single HindIII in EGFP both sides and Xba I site, with the directed cloning method this fragment is replaced the Luciferase fragment of pGL3-5HRE-CMVmp, thereby be built into pEGFP-5HRE-CMVmp.Sequencing result shows that EGFP has replaced the Luciferase fragment of pGL3-5HRE-CMVmp.
The amplification of pEGFP-5HRE-CMVmp plasmid, extraction and purification
The plasmid that successfully constructs is transformed the DH5a competent cell, choose the clone and use PCR method and enzyme cutting method respectively, cut with HindIII and Xba I or Kpn I and Hind III enzyme and identify.With conventional molecular biology method amplification pEGFP-5HRE-CMVmp plasmid, and extraction and purification.Concrete operations see document " modern molecular biology experimental technique " (Lu Shengdong. second edition, Beijing: press of China Concord Medical Science University).
Cytologic experiment
The Hela cell containing in the RPM1640 nutrient solution of 100ml/LFBS (adding penicillin 100u/ml, Streptomycin sulphate 100u/ml), is put 37 ℃, 5%CO
2Cultivate in the condition incubator, went down to posterity 1 time in per 3 days.Before the transfection with the Hela cell with 0.5 * 10
5/ hole is inoculated in 24 orifice plates, when treating cytogamy to 50% left and right sides, adopts liposome Lipofectamine2000 transfection plasmid pEGFP and pEGFP-5HRE-CMVmp, cultivates 3-6 hour with serum free medium before and after the transfection.Changed perfect medium continuation cultivation in 3 hours after the transfection, give anoxic and normal oxygen respectively and handle 48h.Obtain fluorescence parameter analysis with flow cytometer.Each sample collection 1 * 10
6Individual cell excites down at wavelength 488nm, detects EGFP fluorescence at the 530nm place.With the Hela cell climbing sheet of transfection plasmid with fixing 20min under the 4% Paraformaldehyde 96 room temperature, under fluorescent microscope, to observe luciferase expression after the buffering glycerine mounting.Found that, the Hela cell hypoxia of pEGFP-5HRE-CMVmp transfection is handled normal oxygen processing green fluorescence intensity and is eager to excel, flow cytometry analysis shows that the Hela cell EGFP expression level that transfection plasmid pEGFP and the normal oxygen of pEGFP-5HRE-CMVmp are handled is respectively 24.80% and 25.14%, no significant difference between the expression level, and the Hela cell EGFP expression level of anaerobic treatment is respectively 21.25% and 49.19%, there is notable difference (p＜0.05) between the expression level, the Hela cell high level expression EGFP of transfection pEGFP-5HRE-CMVmp plasmid.
Plasmid construction of the present invention can not adopt above-mentioned commercial plasmid, and adopts the promotor of the multiple clone site upstream of directly reconstructing other eukaryon expression plasmids and the mode of enhancer sequence and introducing enhanced green fluorescent protein (EGFP) encoding sequence to obtain.
What the present invention adopted is plasmid, and therefore pass of the present invention key sequence can be operationally connected to some virus vector and be used for damage of histocyte hypoxic-ischemic or genetic treatment of tumor through genetically engineered.
Plasmid of the present invention can be used for the treatment of histocyte hypoxic-ischemic damage after the restriction enzyme site in promotor downstream inserts therapeutic gene.And can with the other drug combined utilization, comprise and make compound preparation.
Claims (3)
1, a kind of construction process of hypoxia response elements gene treating plasmid is characterized in that:
1) at first, the core sequence and the CMV minimal promoter sequence that prepare the hypoxia response elements of 5 copies with the artificial DNA synthesis method, SV40 promotor so that the directed cloning method is replaced plasmid vector pGL3-Promoter Vector with the core sequence and the CMV minimal promoter tract of the hypoxia response elements of copy is built into the pGL3-5HRE-CMVmp plasmid;
The core sequence and the CMV minimal promoter sequence of the hypoxia response elements of 5 copies of said DNA synthesis method preparation, total length 307bp, shown in sequence SEQ ID NO.2:
ggtaccactagtccacagtgcatacgtgggctccaacaggtcctcttggtcgaccccacagtgcatacg
tgggctccaacaggtcctcttgcggccgcccacagtgcatacgtgggctccaacaggtcctcttccatg
gccacagtgcatacgtgggctccaacaggtcctcttcggatccgccacagtgcatacgtgggctccaac
aggtcctcttccggaattccggggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagt
gaaccgtcagatcaccggtacgcgtaagctt
2) secondly, with the pEGFP-N1 carrier for expression of eukaryon is template, adopt pcr amplification to prepare the EGFP fragment, with the directed cloning method EGFP fragment of preparation is replaced the Luciferase fragment of plasmid pGL3-5HRE-CMVmp, thereby be built into the pEGFP-5HRE-CMVmp carrier.
2, the construction process of hypoxia response elements gene treating plasmid according to claim 1 is characterized in that:
Said is template with the pEGFP-N1 carrier for expression of eukaryon, adopts the pcr amplification legal system to be equipped with the EGFP fragment, is that primer sequence is as follows: P1:5 '-AAGCTTCGAATTCTGCAGT-3 ' according to pEGFP-N1 sequences Design synthetic pcr primer thing; P2:5 '-GCTCTAGAGTCGCGGCCGCTTTACT-3 ';
With the pEGFP-N1 plasmid is template clone EGFP total length 795bp, contains HindIII and Xba I site, and sequence SEQ ID NO.3 is as follows:
aagcttcgaattctgcagtcgacggtaccgcgggcccgggatccaccggtcgccaccatggtgagcaag
ggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaag
ttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcacc
accggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagc
cgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggag
cgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacacc
ctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctg
gagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaac
ttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacaccccc
atcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagac
cccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatg
gacgagctgtacaagtaaagcggccgcgactctaga。
3, a kind of hypoxia response elements gene treating plasmid that obtains according to the described construction process of claim 1 is characterized in that: the base sequence of said carrier for expression of eukaryon pEGFP-5HRE-CMVmp is that SEQ ID NO.1 is as follows:
1 ggtaccacta?gtccacagtg?catacgtggg?ctccaacagg?tcctcttggt?cgaccccaca
61 gtgcatacgt?gggctccaac?aggtcctctt?gcggccgccc?acagtgcata?cgtgggctcc
121?aacaggtcct?cttccatggc?cacagtgcat?acgtgggctc?caacaggtcc?tcttcggatc
181?cgccacagtg?catacgtggg?ctccaacagg?tcctcttccg?gaattccggg?gtaggcgtgt
241?acggtgggag?gtctatataa?gcagagctcg?tttagtgaac?cgtcagatca?ccggtacgcg
301?taagcttcga?attctgcagt?cgacggtacc?gcgggcccgg?gatccaccgg?tcgccaccat
361?ggtgagcaag?ggcgaggagc?tgttcaccgg?ggtggtgccc?atcctggtcg?agctggacgg
421?cgacgtaaac?ggccacaagt?tcagcgtgtc?cggcgagggc?gagggcgatg?ccacctacgg
481?caagctgacc?ctgaagttca?tctgcaccac?cggcaagctg?cccgtgccct?ggcccaccct
541?cgtgaccacc?ctgacctacg?gcgtgcagtg?cttcagccgc?taccccgacc?acatgaagca
601?gcacgacttc?ttcaagtccg?ccatgcccga?aggctacgtc?caggagcgca?ccatcttctt
661?caaggacgac?ggcaactaca?agacccgcgc?cgaggtgaag?ttcgagggcg?acaccctggt
721?gaaccgcatc?gagctgaagg?gcatcgactt?caaggaggac?ggcaacatcc?tggggcacaa
781?gctggagtac?aactacaaca?gccacaacgt?ctatatcatg?gccgacaagc?agaagaacgg
841?catcaaggtg?aacttcaaga?tccgccacaa?catcgaggac?ggcagcgtgc?agctcgccga
901?ccactaccag?cagaacaccc?ccatcggcga?cggccccgtg?ctgctgcccg?acaaccacta
961?cctgagcacc?cagtccgccc?tgagcaaaga?ccccaacgag?aagcgcgatc?acatggtcct
1021?gctggagttc?gtgaccgccg?ccgggatcac?tctcggcatg?gacgagctgt?acaagtaaag
1081?cggccgcgac?tctagagtcg?gggcggccgg?ccgcttcgag?cagacatgat?aagatacatt
1141?gatgagtttg?gacaaaccac?aactagaatg?cagtgaaaaa?aatgctttat?ttgtgaaatt
1201?tgtgatgcta?ttgctttatt?tgtaaccatt?ataagctgca?ataaacaagt?taacaacaac
1261?aattgcattc?attttatgtt?tcaggttcag?ggggaggtgt?gggaggtttt?ttaaagcaag
1321?taaaacctct?acaaatgtgg?taaaatcgat?aaggatccgt?cgaccgatgc?ccttgagagc
1381?cttcaaccca?gtcagctcct?tccggtgggc?gcggggcatg?actatcgtcg?ccgcacttat
1441?gactgtcttc?tttatcatgc?aactcgtagg?acaggtgccg?gcagcgctct?tccgcttcct
1501?cgctcactga?ctcgctgcgc?tcggtcgttc?ggctgcggcg?agcggtatca?gctcactcaa
1561?aggcggtaat?acggttatcc?acagaatcag?gggataacgc?aggaaagaac?atgtgagcaa
1621?aaggccagca?aaaggccagg?aaccgtaaaa?aggccgcgtt?gctggcgttt?ttccataggc
1681?tccgcccccc?tgacgagcat?cacaaaaatc?gacgctcaag?tcagaggtgg?cgaaacccga
1741?caggactata?aagataccag?gcgtttcccc?ctggaagctc?cctcgtgcgc?tctcctgttc
1801?cgaccctgcc?gcttaccgga?tacctgtccg?cctttctccc?ttcgggaagc?gtggcgcttt
1861?ctcatagctc?acgctgtagg?tatctcagtt?cggtgtaggt?cgttcgctcc?aagctgggct
1921?gtgtgcacga?accccccgtt?cagcccgacc?gctgcgcctt?atccggtaac?tatcgtcttg
1981?agtccaaccc?ggtaagacac?gacttatcgc?cactggcagc?agccactggt?aacaggatta
2041?gcagagcgag?gtatgtaggc?ggtgctacag?agttcttgaa?gtggtggcct?aactacggct
2101?acactagaag?aacagtattt?ggtatctgcg?ctctgctgaa?gccagttacc?ttcggaaaaa
2161?gagttggtag?ctcttgatcc?ggcaaacaaa?ccaccgctgg?tagcggtggt?ttttttgttt
2221?gcaagcagca?gattacgcgc?agaaaaaaag?gatctcaaga?agatcctttg?atcttttcta
2281?cggggtctga?cgctcagtgg?aacgaaaact?cacgttaagg?gattttggtc?atgagattat
2341?caaaaaggat?cttcacctag?atccttttaa?attaaaaatg?aagttttaaa?tcaatctaaa
2401?gtatatatga?gtaaacttgg?tctgacagtt?accaatgctt?aatcagtgag?gcacctatct
2461?cagcgatctg?tctatttcgt?tcatccatag?ttgcctgact?ccccgtcgtg?tagataacta
2521?cgatacggga?gggcttacca?tctggcccca?gtgctgcaat?gataccgcga?gacccacgct
2581?caccggctcc?agatttatca?gcaataaacc?agccagccgg?aagggccgag?cgcagaagtg
2641?gtcctgcaac?tttatccgcc?tccatccagt?ctattaattg?ttgccgggaa?gctagagtaa
2701?gtagttcgcc?agttaatagt?ttgcgcaacg?ttgttgccat?tgctacaggc?atcgtggtgt
2761?cacgctcgtc?gtttggtatg?gcttcattca?gctccggttc?ccaacgatca?aggcgagtta
2821?catgatcccc?catgttgtgc?aaaaaagcgg?ttagctcctt?cggtcctccg?atcgttgtca
2881?gaagtaagtt?ggccgcagtg?ttatcactca?tggttatggc?agcactgcat?aattctctta
2941?ctgtcatgcc?atccgtaaga?tgcttttctg?tgactggtga?gtactcaacc?aagtcattct
3001?gagaatagtg?tatgcggcga?ccgagttgct?cttgcccggc?gtcaatacgg?gataataccg
3061?cgccacatag?cagaacttta?aaagtgctca?tcattggaaa?acgttcttcg?gggcgaaaac
3121?tctcaaggat?cttaccgctg?ttgagatcca?gttcgatgta?acccactcgt?gcacccaact
3181?gatcttcagc?atcttttact?ttcaccagcg?tttctgggtg?agcaaaaaca?ggaaggcaaa
3241?atgccgcaaa?aaagggaata?agggcgacac?ggaaatgttg?aatactcata?ctcttccttt
3301?ttcaatatta?ttgaagcatt?tatcagggtt?attgtctcat?gagcggatac?atatttgaat
3361?gtatttagaa?aaataaacaa?ataggggttc?cgcgcacatt?tccccgaaaa?gtgccacctg
3421?acgcgccctg?tagcggcgca?ttaagcgcgg?cgggtgtggt?ggttacgcgc?agcgtgaccg
3481?ctacacttgc?cagcgcccta?gcgcccgctc?ctttcgcttt?cttcccttcc?tttctcgcca
3541?cgttcgccgg?ctttccccgt?caagctctaa?atcgggggct?ccctttaggg?ttccgattta
3601?gtgctttacg?gcacctcgac?cccaaaaaac?ttgattaggg?tgatggttca?cgtagtgggc
3661?catcgccctg?atagacggtt?tttcgccctt?tgacgttgga?gtccacgttc?tttaatagtg
3721?gactcttgtt?ccaaactgga?acaacactca?accctatctc?ggtctattct?tttgatttat
3781?aagggatttt?gccgatttcg?gcctattggt?taaaaaatga?gctgatttaa?caaaaattta
3841?acgcgaattt?taacaaaata?ttaacgctta?caatttgcca?ttcgccattc?aggctgcgca
3801?actgttggga?agggcgatcg?gtgcgggcct?cttcgctatt?acgccagccc?aagctaccat
3961?gataagtaag?taatattaag?gtacgggagg?tacttggagc?ggccgcaata?aaatatcttt
4021?attttcatta?catctgtgtg?ttggtttttt?gtgtgaatcg?atagtactaa?catacgctct
4081?ccatcaaaac?aaaacgaaac?aaaacaaact?agcaaaatag?gctgtcccca?gtgcaagtgc
4141?aggtgccaga?acatttctct?atcgata。
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| EP3922721A4 (en) * | 2019-03-12 | 2023-01-11 | Chongqing Precision Biotech Company Limited | Hypoxia-regulated promoter and application thereof |
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Non-Patent Citations (4)
| Title |
|---|
| a noninvasive approach for assessing tumor hypoxiainxenografts:developing a urinary marker for hypoxia. Daniel W.Nelson et al.cancer res.,Vol.65 No.14. 2005 |
| a noninvasive approach for assessing tumor hypoxiainxenografts:developing a urinary marker for hypoxia. Daniel W.Nelson et al.cancer res.,Vol.65 No.14. 2005 * |
| pGL3-EPO-HRE报告基因质粒的构建及功能鉴定. 苏燕等.基础医学与临床，第26卷第11期. 2006 |
| pGL3-EPO-HRE报告基因质粒的构建及功能鉴定. 苏燕等.基础医学与临床，第26卷第11期. 2006 * |
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