CN101993892A - Eukaryotic expression vector for expressing shRNA (short hairpin Ribonucleic Acid) in manner of targeting in cancer cells - Google Patents
Eukaryotic expression vector for expressing shRNA (short hairpin Ribonucleic Acid) in manner of targeting in cancer cells Download PDFInfo
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- CN101993892A CN101993892A CN2009100442218A CN200910044221A CN101993892A CN 101993892 A CN101993892 A CN 101993892A CN 2009100442218 A CN2009100442218 A CN 2009100442218A CN 200910044221 A CN200910044221 A CN 200910044221A CN 101993892 A CN101993892 A CN 101993892A
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Abstract
The invention provides a eukaryotic expression vector for expressing shRNA (short hairpin Ribonucleic Acid) in manner of targeting in cancer cells, comprising the structure as follows: (1) an expression cassette structure which is driven by a polII-type promoter and connects a fluorescent protein gene and a mirshRNA structure in series together; (2) a hTERT (human telomerase reverse transcriptase) promoter enhanced by a CMV (cytomegalovirus) enhancer and a SV40 (simian virus 40) enhancer; (3) mirshRNA structures based on mir30: mir30 left arm-shRNA-mir30 right arm, and multiple mirshRNA structures can be connected in series; (4) a Kan or Amp resistance selection marker; and (5) LR homologous recombination arms. By utilizing the method to construct the shRNA eukaryotic expression vector, one or more than one shRNA can be specifically expressed in the cancer cells in manner of targeting; normal cells are not influenced while the RNA interference and the gene therapy are carried out on the cancer cells, thereby solving the problem of non-specific interference during carrying out the gene therapy by utilizing the RNA interference and being beneficial to the research and the application of the RNA interference in the cancer gene therapy aspect.
Description
Technical field
The present invention relates to a kind ofly can express carrier for expression of eukaryon and the structure thereof of wall scroll or many shRNA the tumour cell tropism that hits.
Background technology
It is a kind of gene silencing that is brought out by double-stranded RNA (gene silelencing) that RNA disturbs (RNA interference).In this process, there is the messenger RNA(mRNA) (mRNA) of homologous sequence to be degraded with double-stranded RNA, thereby suppressed this expression of gene.The RNA perturbation technique is being detected gene function and treatment has broad application prospects aspect the human diseases.Scientific circles generally believe, by utilizing RNAi, the Yeast Nucleic Acid of manipulated cell (genetic messenger), disturb or the neutralizing target gene, to prevent that those protein that can cause disease from forming, might produce the pretty good medicine of prospect, be used for the treatment of the disease that comprises cancer, blind and acquired immune deficiency syndrome (AIDS).
At present, people have successfully used hair clip shape RNA or have generated siRNA sample transcript with plasmid DNA or virus as carrier in cell, suppress exogenous specifically or endogenous gene in mammal or the intracellular expression of people.With plasmid DNA or virus is that carrier can for a long time, stably generate siRNA in cell.This type of research will help the RNA perturbation technique is used for the treatment of human diseases.
Present stage, the shRNA expression cassette structure that most of shRNA carrier for expression of eukaryon are adopted is the structure of polIII type promotor (as hU6)+shRNA+ termination signal form.This structure, advantage are the wide spectrum expression, accurately stop; But because usefulness is polIII type promotor such as U6, so expression efficiency is not high, the quantity of the shRNA of generation is general, and shines into non-specific interference easily, purpose cells such as tumour cell being carried out the RNA interferential simultaneously, also can impact other normal cell.The problems referred to above have limited the application of RNA perturbation technique aspect gene therapy to a certain extent.
Along with the research to the microRNA in the microRNA, scientists finds that microRNA has three kinds of forms: the pri-microRNA that has original both arms; Remove the pre-microRNA of both arms; Sophisticated microRNA.Studies show that, the original both arms of microRNA are most important to the formation of the shearing of microRNA, ripe microRNA, and, under the prerequisite that keeps arm structure, the original microRNA sequence of intermediary is changed into other shRNA sequence, entire structure still can and be sheared by the intravital relevant enzymes identification of biology, thereby forms the siRNA of mature form.
Utilize above-mentioned principle, investigators' development and Design the shRNA expression cassette structure of mirshRNA form, i.e. the structure of polII type promotor (as the CMV promotor)+microRNA left arm+shRNA+microRNA right arm+PolyA form.In order to observe convenient, generally can be immediately following a fluorescence protein gene after polII type promotor.
The shRNA expression cassette structure of this mirshRNA form, advantage is a lot:
(1) owing to adopted polII type promotor efficiently such as CMV promotor, makes that the output of shRNA is more.
(2) owing to adopted the both arms of microRNA, make that the shear efficiency of shRNA is higher, can generate more siRNA, simultaneously, the jamming effectiveness of siRNA also has certain lifting.
(3) since fluorescence protein gene and shRNA expression structure closely link together, so the fluorescence intensity that observes can directly reflect the expression amount of shRNA.
(4) polII type promotor can change other organizing specific type promotor into, to reach the purpose at specific cells or tissue specific expression shRNA.
On the other hand, can the target expression of therapeutic shRNA has determined to utilize the RNA interference to carry out gene therapy successful, especially to genetic treatment of tumor.ShRNA is only expressed in tumour cell, killing tumor cell and protect healthy tissues to greatest extent, promptly to be gene therapy from fundamental research push one of problems that clinical treatment must overcome to the target problem.
In recent years discover that Telomerase is high expression level in the tumour cell more than 90%, and in normal somatocyte, do not express.Human telomerase has 3 kinds of components to be identified, promptly RNA template, Telomerase associated protein, reverse transcriptase of telomere (humantelomerase reverse transcriptase, hTERT).HTERT is the rate-limiting enzyme of Telomerase, the hTERT activity regulation mainly occurs in its transcriptional level, the hTERT gene is the height activated in tumour cell, and is repressed in most of normal cells, and specificity shRNA expresses in the tumour cell so the hTERT promotor can be used for.
At present, existing investigator is applied to the hTERT promotor in the RNA The Study of Interference, but all is directly to connect a shRNA sequence in hTERT promotor back.This shRNA expression structure, though can accomplish specific expressed shRNA in tumour cell, but because employing is the structure of polII type promotor+shRNA, can't be as accurate terminations of polIII type promotor such as U6, so the expression of shRNA and shearing all can be under some influence, thereby reduce the output of siRNA.
In addition, because the hTERT promotor lacks the TATA box, a little less than so its strong promoter such as expression regulation energy force rate CMV to downstream gene or shRNA is wanted, so, by various modifications, increasing some other functional element, to strengthen the activity of hTERT promotor, is an emphasis in research of hTERT promotor and the application.
Summary of the invention
For overcome the many disadvantages that above-mentioned shRNA expresses in tumour cell, strengthen the activity of hTERT promotor, reach tumour cell hit the tropism, express the purpose of wall scroll or many shRNA specifically, the invention provides a kind of technical scheme and be the tumour cell tropism that hits and express the carrier for expression of eukaryon of shRNA, comprise the placed in-line expression cassette structure of polII type promotor, fluorescence protein gene, mirshRNA structure and Poly A, with the placed in-line expression cassette structure construction of polII type promotor, fluorescence protein gene, mirshRNA structure and PolyA to the same carrier; Described polII type promotor is the hTERT promotor that the upstream has cmv enhancer and SV40 enhanser; Described mirshRNA structure is: microRNA left arm+shRNA+microRNA right arm to be expressed; Described fluorescence protein gene is positioned at hTERT promotor downstream, mirshRNA structure upstream.
Above-mentioned fluorescence protein gene is green fluorescence protein gene (EGFP), red fluorescent protein gene (RFP) or yellow fluorescence protein gene (YFP).
Can insert a shRNA in the described expression cassette, the more than one mirshRNA structure of perhaps connecting; Described mirshRNA structure is fundamental construction with mir30, adopts the both arms of mir30, inserts identical or different shRNA between mir30 left arm and mir30 right arm.
There is pair of L R homologous recombination arm described expression cassette both sides; Described carrier also comprises Kan or Amp resistance screening mark.
According to technique scheme, the present invention also provides a carrier for expression of eukaryon, and sequence is shown in SEQ NO 1.
HTERT promotor in the technical solution of the present invention in the expression cassette structure, owing to obtained the enhancing of cmv enhancer and SV40 enhanser, its activity is greatly improved; Adopted the structure of " strengthened hTERT promotor+mirshRNA+PolyA " form, compare with " polIII type promotor (as the U6 promotor)+shRNA " or structures such as " polII type promotors (as the hTERT promotor)+shRNA ", the expression amount of shRNA increases greatly, and its shear efficiency significantly improves, thereby has greatly promoted the output of siRNA; Because fluorescence protein gene and mirshRNA structure are linked together, and place hTERT promotor downstream jointly, on the one hand can be by observing fluorescence, the transfection efficiency during monitoring plasmid transfection cell; On the other hand, also can judge the activity of hTERT promotor and the expression amount of shRNA very intuitively by observing fluorescence intensity.
In addition, for the broadened application scope, the present invention has also inserted LR homologous recombination arm in hTERT promotor-fluorescence protein gene-mirshRNA-PolyA structure both sides, this carrier can be used as common shRNA carrier for expression of eukaryon on the one hand, on the other hand, can also be where necessary with it as the shuttle vectors in the adenovirus packaging system, utilize the BLOCK-iT Adenoviral System encapsidated adenovirus virus of Invitrogen company, thereby carry out RNA The Study of Interference or application better.
It is the application of above-mentioned carrier for expression of eukaryon in preparation treatment cancer drug that the present invention also provides a kind of technical scheme; And above-mentioned carrier for expression of eukaryon is used to prepare adenovirus.
The method of utilizing technical solution of the present invention to provide makes up above-mentioned shRNA carrier for expression of eukaryon, can tumour cell hit the tropism, express wall scroll or many shRNA specifically, when tumour cell being carried out the RNA interference, carrying out gene therapy, do not influence normal cell again, solved the non-specific interference difficult problem when utilizing the RNA interference to carry out gene therapy.
Description of drawings
Fig. 1 is the plasmid map (to express wall scroll shRNA, to select for use the EGFP green fluorescence to be labeled as example) that utilizes the shRNA carrier for expression of eukaryon pYrbio-hTERT-Mir30shRNA of method structure provided by the invention;
The fluorescence observation photo of the HepG2 cell of Fig. 2 has been transfection pYrbio-hTERT-Mir30-CD146 plasmid (A) and any plasmid of untransfected (B);
Fig. 3 is a CD146 expression of gene situation in interference group and the control group HepG2 cell, and A is the RT-PCR detected result, and B is the Western-blot detected result, and wherein the 1st swimming lane is a control group, and the 2nd swimming lane is the interference group;
Fluorescence observation photo in Fig. 4 HepG2 cell (A) and the HELF cell (B);
The green fluorescence photo of the HepG2 cell of Fig. 5 has been transfection pYrbio-nCnS-hTERT-Mir30shRNA carrier (A) and pYrbio-hTERT-Mir30shRNA carrier (B);
Fig. 6 is the green fluorescence photo of the HepG2 cell after the adenovirus infection;
The plasmid map of Fig. 7 commercialization carrier pYrbio-Mir30-shRNA.
Embodiment
The tumour cell tropism that hits expresses the structure of the carrier pYrbio-hTERT-Mir30shRNA of shRNA
Method: (Changsha Yingrun Biological Technology Co., Ltd. produces with commercialization carrier pYrbio-Mir30-shRNA, shown in SEQID2) be skeleton, by a series of structures that change, make up the tumour cell tropism that hits and express the carrier pYrbio-hTERT-Mir30shRNA of shRNA.
(1) synthetic hTERT promoter (about 385bp) is loaded into it on pMD19-T Simple Vector.In hTERT promoter fragment upstream, add BglII, EcoRI, PstI restriction enzyme site; In hTERT promoter fragment downstream, add the SpeI restriction enzyme site.The carrier called after TS-hTERTp that builds.
(2) go up pcr amplification SV40 Enhancer fragment from pCDNA3.1 (+), two ends add EcoRI and PstI restriction enzyme site.With EcoRI and PstI is the subclone site, with SV40 Enhancer fragment subclone to TS-hTERTp.The carrier called after TS-SV40E-hTERTp that builds.
(3) go up pcr amplification CMV Enhancer fragment from pCDNA3.1 (+), two ends add BglII and EcoRI restriction enzyme site.With BglII and EcoRI is the subclone site, with CMV Enhancer fragment subclone to TS-SV40E-hTERTp.The carrier called after TS-CMVE-SV40E-hTERTp that builds.
(4) be the subclone site with BglII and SpeI, with the CMVE-SV40E-hTERTp fragment subclone on the TS-CMVE-SV40E-hTERTp to pYrbio-Mir30-shRNA.The carrier called after pYrbio-hTERT-Mir30shRNA that builds.
The result: the tumour cell carrier pYrbio-hTERT-Mir30shRNA that the tropism expresses shRNA that hits successfully constructs, and Fig. 1 is the pYrbio-hTERT-Mir30shRNA plasmid map, is this carrier complete sequence in the sequence table.
Wherein, before and after the shRNA Insertion Site BsaI restriction enzyme site is arranged respectively, be used for inserting the shRNA fragment.
Conclusion: can successfully make up the pYrbio-hTERT-Mir30shRNA screening vector in this way.
Specificity is disturbed the CD146 expression of gene in the liver cancer cell
Method: in the shRNA of pYrbio-hTERT-Mir30shRNA carrier Insertion Site, insert shRNA, make up the pYrbio-hTERT-Mir30-CD146 carrier at people CD146 gene.
With pYrbio-hTERT-Mir30-CD146 carrier transfection hepatoma cell strain HepG2, collecting cell after 48 hours extracts RNA and protein respectively, carries out RT-PCR and Western-blot detection at goal gene CD146.HepG2 cell with any plasmid of untransfected is organized in contrast.
The result: the shRNA expression vector pYrbio-hTERT-Mir30-CD146 transfection that builds is to the HepG2 cell, can under fluorescent microscope, observe tangible green fluorescence after 24 hours, show that whole plasmid expression frame is working properly, the shRNA expression amount is higher.And do not see green fluorescence in the control group HepG2 cell.The fluorescence observation photo of interference group and control group HepG2 cell is seen Fig. 2.
RT-PCR detects and Western-blot detects all demonstrations, compares with control group, and the CD146 expression of gene in the interference group HepG2 cell has been subjected to obvious inhibition.RT-PCR and Western-blot detected result are seen Fig. 3.
Conclusion: utilize pYrbio-hTERT-Mir30-CD146 carrier that method provided by the invention makes up can be in hepatoma cell strain HepG2 works better, give expression in a large number shRNA, thereby effectively suppressed the CD146 expression of gene at target gene CD146.
HTERT promotor The specificity
Method:, under fluorescent microscope, observe fluorescence after 24 hours with pYrbio-hTERT-Mir30shRNA carrier difference transfection hepatoma cell strain HepG2 and people's normal fibroblast strain HELF.
The result: the transfection of pYrbio-hTERT-Mir30shRNA carrier can be observed tangible green fluorescence after 24 hours under fluorescent microscope to hepatoma cell strain HepG2 cell, show that whole plasmid expression frame is working properly.And the pYrbio-hTERT-Mir30shRNA transfection is not seen green fluorescence to people's normal fibroblast strain HELF.The fluorescence observation photo of HepG2 cell and HELF cell is seen Fig. 4.
Conclusion: the pYrbio-hTERT-Mir30shRNA carrier that utilizes method provided by the invention to make up, its hTERTpromoter-EGFP-Mir30shRNA-SV40 PolyA expression cassette structure can be in hepatoma cell strain HepG2 works better, and in people's normal fibroblast strain HELF, do not work, show that the expression cassette by the hTERT promoter regulation has good tumor cells expression specificity.
Embodiment 4
Cmv enhancer and SV40 enhanser are to the enhancement research of hTERT promoter activity
Method: with BglII and SpeI is the subclone site, with the hTERTp fragment subclone on the TS-hTERTp among the embodiment 1 to pYrbio-Mir30-shRNA, the carrier called after pYrbio-nCnS-hTERT-Mir30shRNA that builds, other structure of this carrier and pYrbio-hTERT-Mir30shRNA are basic identical, and difference is do not have CMV Enhancer and SV40 Enhancer before its hTERT promoter.
With pYrbio-nCnS-hTERT-Mir30shRNA and two carriers difference of pYrbio-hTERT-Mir30shRNA transfection hepatoma cell strain HepG2, under fluorescent microscope, observe fluorescence after 24 hours.
The result: two the shRNA expression vectors transfection that builds all can be observed green fluorescence after 24 hours under fluorescent microscope to hepatoma cell strain HepG2 cell.But the green fluorescence intensity that changes in the HepG2 cell of pYrbio-hTERT-Mir30shRNA obviously is better than the HepG2 cell that changes pYrbio-nCnS-hTERT-Mir30-shRNA over to.Transfection the green fluorescence photo of the HepG2 cell behind pYrbio-nCnS-hTERT-Mir30-shRNA and the pYrbio-hTERT-Mir30shRNA see Fig. 5.
Conclusion: cmv enhancer and SV40 enhanser have tangible enhancement to the hTERT promoter activity.
With the pYrbio-hTERT-Mir30shRNA carrier is shuttle vectors, encapsidated adenovirus virus
Method: with the pYrbio-hTERT-Mir30-CD146shRNA carrier is shuttle vectors, and itself and adenovirus skeleton carrier pAd/BL-DEST are mixed, and adds LR vitro recombination enzyme, recombinates one hour for 25 ℃, then transformed into escherichia coli.Picking the clone identify, will identify correct recombinant vectors PacI linearization for enzyme restriction, and transfection is carried out the adenovirus packing to the 293C packing cell behind the purifying.
With packaged adenovirus infection hepatoma cell strain HepG2, under fluorescent microscope, observe fluorescence after 24 hours.
The result: with the pYrbio-hTERT-Mir30shRNA carrier is shuttle vectors, utilizes the BLOCK-iT Adenoviral System of Invitrogen company successfully to pack out adenovirus.Behind the packaged adenovirus infection hepatoma cell strain HepG2 24 hours, can under fluorescent microscope, observe tangible green fluorescence.Fig. 6 is the green fluorescence photo of the HepG2 cell after the adenovirus infection.
Conclusion: with the pYrbio-hTERT-Mir30shRNA carrier is shuttle vectors, utilizes the BLOCK-iT Adenoviral System of Invitrogen company, can successfully pack out adenovirus.This adenovirus can infect cells such as HepG2, has expanded the range of application of this shRNA expression system.
Sequence table
＜110〉Changsha Yingrun Biological Technology Co., Ltd.
Easily, silver is husky
Lu, the beautiful woman
Grandson, woods forever
Yuan, the bright autumn
＜120〉the tumour cell tropism that hits expresses the carrier for expression of eukaryon of shRNA
<130>PLH09816
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<170>PatentIn?version?3.1
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acctacaccg?aactgagata?cctacagcgt?gagcattgag?aaagcgccac?gcttcccgaa 4860
gggagaaagg?cggacaggta?tccggtaagc?ggcagggtcg?gaacaggaga?gcgcacgagg 4920
gagcttccag?ggggaaacgc?ctggtatctt?tatagtcctg?tcgggtttcg?ccacctctga 4980
cttgagcgtc?gatttttgtg?atgctcgtca?ggggggcgga?gcctatggaa?aaacgccagc 5040
aacgcggcct?ttttacggtt?cctggccttt?tgctggcctt?ttgctcacat?gtt 5093
<210>2
<211>4625
<212>DNA
＜213〉artificial sequence
<400>2
ctttcctgcg?ttatcccctg?attctgtgga?taaccgtatt?accgcctttg?agtgagctga 60
taccgctcgc?cgcagccgaa?cgaccgagcg?cagcgagtca?gtgagcgagg?aagcggaaga 120
gcgcccaata?cgcaaaccgc?ctctccccgc?gcgttggccg?attcattaat?gcagctggca 180
cgacaggttt?cccgactgga?aagcgggcag?tgagcgcaac?gcaattaata?cgcgtaccgc 240
tagccaggaa?gagtttgtag?aaacgcaaaa?aggccatccg?tcaggatggc?cttctgctta 300
gtttgatgcc?tggcagttta?tggcgggcgt?cctgcccgcc?accctccggg?ccgttgcttc 360
acaacgttca?aatccgctcc?cggcggattt?gtcctactca?ggagagcgtt?caccgacaaa 420
caacagataa?aacgaaaggc?ccagtcttcc?gactgagcct?ttcgttttat?ttgatgcctg 480
gcagttccct?actctcgcgt?taacgctagc?atggatgttt?tcccagtcac?gacgttgtaa 540
aacgacggcc?agtcttaagc?tcgggcccca?aataatgatt?ttattttgac?tgatagtgac 600
ctgttcgttg?caacaaattg?atgagcaatg?cttttttata?atgccaactt?tgtacaaaaa 660
agcaggctcc?gcggccgccc?ccttcaccat?cgatagatct?ggagttccgc?gttacataac 720
ttacggtaaa?tggcccgcct?ggctgaccgc?ccaacgaccc?ccgcccattg?acgtcaataa 780
tgacgtatgt?tcccatagta?acgccaatag?ggactttcca?ttgacgtcaa?tgggtggagt 840
atttacggta?aactgcccac?ttggcagtac?atcaagtgta?tcatatgcca?agtacgcccc 900
ctattgacgt?caatgacggt?aaatggcccg?cctggcatta?tgcccagtac?atgaccttat 960
gggactttcc?tacttggcag?tacatctacg?tattagtcat?cgctattacc?atggtgatgc 1020
ggttttggca?gtacatcaat?gggcgtggat?agcggtttga?ctcacgggga?tttccaagtc 1080
tccaccccat?tgacgtcaat?gggagtttgt?tttggcacca?aaatcaacgg?gactttccaa 1140
aatgtcgtaa?caactccgcc?ccattgacgc?aaatgggcgg?taggcgtgta?cggtgggagg 1200
tctatataag?cagagctcgt?ttagtgaacc?gtcagatcgc?ctggagacgc?catccacgct 1260
gttttgacct?ccatagaaga?caccgactct?agaggatcca?ctagtccagt?gtggtggaat 1320
tgatcccttc?accatggtga?gcaagggcga?ggagctgttc?accggggtgg?tgcccatcct 1380
ggtcgagctg?gacggcgacg?taaacggcca?caagttcagc?gtgtccggcg?agggcgaggg 1440
cgatgccacc?tacggcaagc?tgaccctgaa?gttcatctgc?accaccggca?agctgcccgt 1500
gccctggccc?accctcgtga?ccaccctgac?ctacggcgtg?cagtgcttca?gccgctaccc 1560
cgaccacatg?aagcagcacg?acttcttcaa?gtccgccatg?cccgaaggct?acgtccagga 1620
gcgcaccatc?ttcttcaagg?acgacggcaa?ctacaagacc?cgcgccgagg?tgaagttcga 1680
gggcgacacc?ctggtgaacc?gcatcgagct?gaagggcatc?gacttcaagg?aggacggcaa 1740
catcctgggg?cacaagctgg?agtacaacta?caacagccac?aacgtctata?tcatggccga 1800
caagcagaag?aacggcatca?aggtgaactt?caagatccgc?cacaacatcg?aggacggcag 1860
cgtgcagctc?gccgaccact?accagcagaa?cacccccatc?ggcgacggcc?ccgtgctgct 1920
gcccgacaac?cactacctga?gcacccagtc?cgccctgagc?aaagacccca?acgagaagcg 1980
cgatcacatg?gtcctgctgg?agttcgtgac?cgccgccggg?atcactctcg?gcatggacga 2040
gctgtacaag?taaagcggcc?tcgagctcaa?gcttcgaatt?ctgcagtcga?ctagggataa 2100
cagggtaatt?gtttgaatga?ggcttcagta?ctttacagaa?tcgttgcctg?cacatcttgg 2160
aaacacttgc?tgggattact?tcttcaggtt?aacccaacag?aaggctaaag?aaggtaattg 2220
ctgttgacag?tgagcgagag?acccgggatc?cggtctcgtg?cctactgcct?cggacttcaa 2280
ggggctactt?taggagcaat?tatcttgttt?actaaaactg?aataccttgc?tatctctttg 2340
atacattttt?acaaagctga?attaaaatgg?tataaattaa?atcacttttt?tcaattggaa 2400
gactaatgcg?tttaaacacg?cggcgacgtt?ggatccaccg?gatctagata?actgatcata 2460
atcagccata?ccacatttgt?agaggtttta?cttgctttaa?aaaacctccc?acacctcccc 2520
ctgaacctga?aacataaaat?gaatgcaatt?gttgttgtta?acttgtttat?tgcagcttat 2580
aatggttaca?aataaagcaa?tagcatcaca?aatttcacaa?ataaagcatt?tttttcactg 2640
cattctagtt?gtggtttgtc?caaactcatc?aatgtatctt?aacgcgtaaa?ttgtaagcgt 2700
taatattttg?ttaaaattcg?cgttaaattt?ttgttaaatc?ggcgcgccga?cccagctttc 2760
ttgtacaaag?ttggcattat?aagaaagcat?tgcttatcaa?tttgttgcaa?cgaacaggtc 2820
actatcagtc?aaaataaaat?cattatttgc?catccagctg?atatccccta?tagtgagtcg 2880
tattacatgg?tcatagctgt?ttcctggcag?ctctggcccg?tgtctcaaaa?tctctgatgt 2940
tacattgcac?aagataaaaa?tatatcatca?tgaacaataa?aactgtctgc?ttacataaac 3000
agtaatacaa?ggggtgttat?gagccatatt?caacgggaaa?cgtcgaggcc?gcgattaaat 3060
tccaacatgg?atgctgattt?atatgggtat?aaatgggctc?gcgataatgt?cgggcaatca 3120
ggtgcgacaa?tctatcgctt?gtatgggaag?cccgatgcgc?cagagttgtt?tctgaaacat 3180
ggcaaaggta?gcgttgccaa?tgatgttaca?gatgagatgg?tcagactaaa?ctggctgacg 3240
gaatttatgc?ctcttccgac?catcaagcat?tttatccgta?ctcctgatga?tgcatggtta 3300
ctcaccactg?cgatccccgg?aaaaacagca?ttccaggtat?tagaagaata?tcctgattca 3360
ggtgaaaata?ttgttgatgc?gctggcagtg?ttcctgcgcc?ggttgcattc?gattcctgtt 3420
tgtaattgtc?cttttaacag?cgatcgcgta?tttcgtctcg?ctcaggcgca?atcacgaatg 3480
aataacggtt?tggttgatgc?gagtgatttt?gatgacgagc?gtaatggctg?gcctgttgaa 3540
caagtctgga?aagaaatgca?taaacttttg?ccattctcac?cggattcagt?cgtcactcat 3600
ggtgatttct?cacttgataa?ccttattttt?gacgagggga?aattaatagg?ttgtattgat 3660
gttggacgag?tcggaatcgc?agaccgatac?caggatcttg?ccatcctatg?gaactgcctc 3720
ggtgagtttt?ctccttcatt?acagaaacgg?ctttttcaaa?aatatggtat?tgataatcct 3780
gatatgaata?aattgcagtt?tcatttgatg?ctcgatgagt?ttttctaatc?agaattggtt 3840
aattggttgt?aacactggca?gagcattacg?ctgacttgac?gggacggcgc?aagctcatga 3900
ccaaaatccc?ttaacgtgag?ttacgcgtcg?ttccactgag?cgtcagaccc?cgtagaaaag 3960
atcaaaggat?cttcttgaga?tccttttttt?ctgcgcgtaa?tctgctgctt?gcaaacaaaa 4020
aaaccaccgc?taccagcggt?ggtttgtttg?ccggatcaag?agctaccaac?tctttttccg 4080
aaggtaactg?gcttcagcag?agcgcagata?ccaaatactg?tccttctagt?gtagccgtag 4140
ttaggccacc?acttcaagaa?ctctgtagca?ccgcctacat?acctcgctct?gctaatcctg 4200
ttaccagtgg?ctgctgccag?tggcgataag?tcgtgtctta?ccgggttgga?ctcaagacga 4260
tagttaccgg?ataaggcgca?gcggtcgggc?tgaacggggg?gttcgtgcac?acagcccagc 4320
ttggagcgaa?cgacctacac?cgaactgaga?tacctacagc?gtgagcattg?agaaagcgcc 4380
acgcttcccg?aagggagaaa?ggcggacagg?tatccggtaa?gcggcagggt?cggaacagga 4440
gagcgcacga?gggagcttcc?agggggaaac?gcctggtatc?tttatagtcc?tgtcgggttt 4500
cgccacctct?gacttgagcg?tcgatttttg?tgatgctcgt?caggggggcg?gagcctatgg 4560
aaaaacgcca?gcaacgcggc?ctttttacgg?ttcctggcct?tttgctggcc?ttttgctcac 4620
Claims (9)
1. the tumour cell tropism that hits expresses the carrier for expression of eukaryon of shRNA, comprise the placed in-line expression cassette structure of polII type promotor, fluorescence protein gene, mirshRNA structure and Poly A, it is characterized in that the placed in-line expression cassette structure construction of polII type promotor, fluorescence protein gene, mirshRNA structure and PolyA to same carrier; Described polII type promotor is the hTERT promotor that the upstream has cmv enhancer and SV40 enhanser; Described mirshRNA structure is: microRNA left arm+shRNA+microRNA right arm to be expressed; Described fluorescence protein gene is positioned at hTERT promotor downstream, mirshRNA structure upstream.
2. carrier for expression of eukaryon according to claim 1 is characterized in that, described fluorescence protein gene is green fluorescence protein gene (EGFP), red fluorescent protein gene (RFP) or yellow fluorescence protein gene (YFP).
3. carrier for expression of eukaryon according to claim 1 is characterized in that, there is pair of L R homologous recombination arm described expression cassette both sides.
4. carrier for expression of eukaryon according to claim 1 is characterized in that, the more than one mirshRNA structure of series connection in the described expression cassette; Insert identical or different shRNA to be expressed between described left arm and the right arm.
5. according to claim 1 or 4 described carrier for expression of eukaryon, it is characterized in that described mirshRNA structure is the structure based on mir30: mir30 left arm+shRNA+mir30 right arm to be expressed.
6. according to the described carrier for expression of eukaryon of one of claim 1-5, it is characterized in that described carrier also comprises Kan or Amp resistance screening mark.
7. carrier for expression of eukaryon according to claim 6 is characterized in that, described carrier for expression of eukaryon is shown in the SEQ NO1.
8. the application of the described carrier for expression of eukaryon of claim 1 to 7 in preparation treatment cancer drug.
9. the described carrier for expression of eukaryon of claim 1 to 7 is used to prepare adenovirus.
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| CN108203717A (en) * | 2016-12-20 | 2018-06-26 | 上海生博生物医药科技有限公司 | Her2 can be targeted and PD-1 is interfered to reduce the CAR-T carriers of tumor immune escape and its construction method and application |
| CN112105731A (en) * | 2018-03-30 | 2020-12-18 | 日内瓦大学 | micro-RNA expression constructs and uses thereof |
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| CN112011573A (en) * | 2020-07-21 | 2020-12-01 | 山西医科大学 | Lentiviral vector for transfecting primary neurons of mice and construction method |
| CN114875065A (en) * | 2022-05-06 | 2022-08-09 | 广东医科大学 | Design and application of eIF4E/c-Myc gene positive feedback loop self-control shRNA silencing vector |
| CN114875065B (en) * | 2022-05-06 | 2023-10-27 | 广东医科大学 | Design and application of eIF4E/c-Myc gene positive feedback loop self-control shRNA silencing vector |
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