CN109321602B - PKD2 recombinant overexpression vector and construction method and application thereof - Google Patents
PKD2 recombinant overexpression vector and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a PKD2 recombinant overexpression vector and a construction method and application thereof, wherein the PKD2 recombinant overexpression vector is obtained by inserting a target gene PKD2 into an adeno-associated virus vector, and the adeno-associated virus vector at least comprises the following operably connected sequence elements: EF1 promoter, MCS region, CMV promoter sequence, EGFP gene sequence. The PKD2 recombinant overexpression vector can efficiently express PKD 2. Through intensive research of the inventor, the PKD2 recombinant overexpression vector can relieve the myocardial infarction symptom of mice. The adeno-associated virus carrying the gene is injected into the myocardial infarction mouse body, so that the cardiac function of the mouse can be effectively recovered, and the myocardial infarction area is obviously reduced, which indicates that the technical scheme of the invention is suitable for myocardial infarction treatment.
Description
Technical Field
The invention relates to a PKD2 recombinant overexpression vector and a construction method and application thereof, belonging to the technical field of biology.
Background
The reference sequence of the gene standard of PKD2 is GeneBank NM-000297.3, PKD2 is located in 4q21-23, the gene length is 68kb, 15 exons are contained, and the coding region length is 2907 bp.
The prevalence of cardiovascular diseases in our country is in a continuously rising stage, with about 200 million patients with myocardial infarction per year, and increasing at a rate of 10.42% per year, becoming one of the leading causes of the rise in heart failure and total mortality of cardiovascular diseases and one of the intractable diseases seriously harming the health of people in the art, and there is a constant effort to develop a technology capable of effectively treating or preventing myocardial infarction. The problem in the prior art is the lack of means and methods that can effectively treat and prevent myocardial infarction.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a PKD2 recombinant overexpression vector, a construction method and a use thereof, and the technical problem to be solved by the present invention is to provide an adeno-associated viral vector which can significantly improve myocardial infarction.
In order to achieve the above and other related objects, the present invention provides an AQP5 recombinant overexpression vector, wherein the AQP5 recombinant vector is obtained by inserting a target gene AQP5 into an adeno-associated viral vector, and the adeno-associated viral vector comprises at least the following operably linked sequence elements:
5 'terminal inverted repeat sequence, TBG promoter sequence, MCS region, P2A sequence, ZsGreen gene sequence and 3' terminal inverted repeat sequence.
Adeno-associated virus is a single-stranded non-enveloped DNA virus. It enters the cell through receptor-mediated endocytosis, and then the adeno-associated viral genome is transferred into the nucleus, remains extrachromosomal, and does not integrate into the host cell genome.
The accession number of PKD2 gene in NCBI is 5311.
The invention obtains a recombinant overexpression vector by utilizing natural adeno-associated virus through gene modification.
The term "operably linked" or "operably linked" refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region is placed at a specific location relative to a nucleic acid sequence of a gene of interest such that transcription of the nucleic acid sequence is directed by the promoter, and thus, the promoter region can be "operably linked" to the nucleic acid sequence.
The "elements" refer to a number of functional nucleic acid sequences useful for expression of proteins, and in the present invention, are systematically constructed to form an expression construct. The "element" sequences are those which can be provided in the present invention, and also include their variants, as long as the functions of the "elements" are retained in these elements, which are obtained by inserting or deleting some bases, or random or site-directed mutagenesis, etc.
The upstream and downstream positions of each element may further comprise restriction sites for restriction enzymes, which facilitate organic linkage of each element.
Further, the AQP5 recombinant overexpression vector has a sequence shown in SEQ ID NO.1, and specifically comprises the following steps:
ttcatcaataatataccttattttggattgaagccaatatgataatgagggggtggagtttgtgacgtggcgcggggcgtgggaacggggcgggtgacgtagtagtgtggcggaagtgtgatgttgcaagtgtggcggaacacatgtaagcgacggatgtggcaaaagtgacgtttttggtgtgcgccggtgtacacaggaagtgacaattttcgcgcggttttaggcggatgttgtagtaaatttgggcgtaaccgagtaagatttggccattttcgcgggaaaactgaataagaggaagtgaaatctgaataattttgtgttactcatagcgcgtaatatttgtctagggccgcggggactttgaccgtttacgtggagactcgcccaggtgtttttctcaggtgttttccgcgttccgggtcaaagttggcgttttattattatagtcagntctagactcgagaaggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaacgggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgaagcttcgaggggctcgcatctctccttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactctacgtctttgtttcgttttctgttctgcgccgttacagatccaagctgtgaccggcgcctacgaattcatggtgaactccagtcgcgtgcagcctcagcagcccggggacgccaagcggccgcccgcgccccgcgcgccggacccgggccggctgatggctggctgcgcggccgtgggcgccagcctcgccgccccgggcggcctctgcgagcagcggggcctggagatcgagatgcagcgcatccggcaggcggccgcgcgggaccccccggccggagccgcggcctccccttctcctccgctctcgtcgtgctcccggcaggcgtggagccgcgataaccccggcttcgaggccgaggaggaggaggaggaggtggaaggggaagaaggcggaatggtggtggagatggacgtagagtggcgcccgggcagccggaggtcggccgcctcctcggccgtgagctccgtgggcgcgcggagccgggggcttgggggctaccacggcgcgggccacccgagcgggaggcggcgccggcgagaggaccagggcccgccgtgccccagcccagtcggcggcggggacccgctgcatcgccacctccccctggaagggcagccgccccgagtggcctgggcggagaggctggttcgcgggctgcgaggtctctggggaacaagactcatggaggaaagcagcactaaccgagagaaataccttaaaagtgttttacgggaactggtcacatacctcctttttctcatagtcttgtgcatcttgacctacggcatgatgagctccaatgtgtactactacacccggatgatgtcacagctcttcctagacacccccgtgtccaaaacggagaaaactaactttaaaactctgtcttccatggaagacttctggaagttcacagaaggctccttattggatgggctgtactggaagatgcagcccagcaaccagactgaagctgacaaccgaagtttcatcttctatgagaacctgctgttaggggttccacgaatacggcaactccgagtcagaaatggatcctgctctatcccccaggacttgagagatgaaattaaagagtgctatgatgtctactctgtcagtagtgaagatagggctccctttgggccccgaaatggaaccgcttggatctacacaagtgaaaaagacttgaatggtagtagccactggggaatcattgcaacttatagtggagctggctattatctggatttgtcaagaacaagagaggaaacagctgcacaagttgctagcctcaagaaaaatgtctggctggaccgaggaaccagggcaacttttattgacttctcagtgtacaacgccaacattaacctgttctgtgtggtcaggttattggttgaattcccagcaacaggtggtgtgattccatcttggcaatttcagcctttaaagctgatccgatatgtcacaacttttgatttcttcctggcagcctgtgagattatcttttgtttctttatcttttactatgtggtggaagagatattggaaattcgcattcacaaactacactatttcaggagtttctggaattgtctggatgttgtgatcgttgtgctgtcagtggtagctataggaattaacatatacagaacatcaaatgtggaggtgctactacagtttctggaagatcaaaatactttccccaactttgagcatctggcatattggcagatacagttcaacaatatagctgctgtcacagtattttttgtctggattaagctcttcaaattcatcaattttaacaggaccatgagccagctctcgacaaccatgtctcgatgtgccaaagacctgtttggctttgctattatgttcttcattattttcctagcgtatgctcagttggcataccttgtctttggcactcaggtcgatgacttcagtactttccaagagtgtatcttcactcaattccgtatcattttgggcgatatcaactttgcagagattgaggaagctaatcgagttttgggaccaatttatttcactacatttgtgttctttatgttcttcattcttttgaatatgtttttggctatcatcaatgatacttactctgaagtgaaatctgacttggcacagcagaaagctgaaatggaactctcagatcttatcagaaagggctaccataaagctttggtcaaactaaaactgaaaaaaaataccgtggatgacatttcagagagtctgcggcaaggaggaggcaagttaaactttgacgaacttcgacaagatctcaaagggaagggccatactgatgcagagattgaggcaatattcacaaagtacgaccaagatggagaccaagaactgaccgaacatgaacatcagcagatgagagacgacttggagaaagagagggaggacctggatttggatcacagttctttaccacgtcccatgagcagccgaagtttccctcgaagcctggatgactctgaggaggatgacgatgaagatagcggacatagctccagaaggaggggaagcatttctagtggcgtttcttacgaagagtttcaagtcctggtgagacgagtggaccggatggagcattccatcggcagcatagtgtccaagattgacgccgtgatcgtgaagctagagattatggagcgagccaaactgaagaggagggaggtgctgggaaggctgttggatggggtggccgaggatgaaaggctgggtcgtgacagtgaaatccatagggaacagatggaacggctagtacgtgaagagttggaacgctgggaatccgatgatgcagcttcccagatcagtcatggtttaggcacgccagtgggactaaatggtcaacctcgccccagaagctcccgcccatcttcctcccaatctacagaaggcatggaaggtgcaggtggaaatgggagttctaatgtccacgtaggatcccgaccggtccatcgatggggtaccccgacgcgtagcggccgcgactacaaggatgacgatgacaaggattacaaagacgacgatgataaggactataaggatgatgacgacaaataaatgcattagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgctagcatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacgggcacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcga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further, the sequence of the EF1 promoter is shown as 467-1012 th bits in SEQ ID NO. 1;
furthermore, the sequence of the gene PKD2 is shown as 1019-3922 in SEQ ID NO. 1;
furthermore, the sequence of the CMV promoter is shown as 4112-4619 th site in SEQ ID NO. 1;
furthermore, the EGFP gene sequence is shown as 4648-5367 in SEQ ID NO. 1.
In another aspect of the invention, the PKD2 recombinant overexpression vector is used for preparing myocardial infarction treatment drugs.
In another aspect of the invention, there is provided a cell comprising a recombinant overexpression vector for PKD2 as described above.
In another aspect of the invention, there is provided a kit comprising a recombinant overexpression vector of PKD2 as described above and a pharmaceutically acceptable carrier, or a cell as described above and a pharmaceutically acceptable carrier.
Another aspect of the present invention provides a method for constructing the above PKD2 recombinant overexpression vector, the method at least comprising the steps of:
(1) constructing an adeno-associated virus shuttle plasmid;
(2) and (4) packaging the adeno-associated virus.
Further, the sequence of the forward primer used in the step (1) is shown as SEQ ID NO.2, and the sequence of the reverse primer is shown as SEQ ID NO. 3.
SEQ ID NO.2atggtgaactccagtcgcgtgcagcct
SEQ ID NO.3tacgtggacattagaactcccatttcc
As mentioned above, the PKD2 overexpression adeno-associated virus vector, the construction method and the application thereof have the following beneficial effects:
the adeno-associated virus packaged by the technical scheme of the invention can effectively reduce the increase of the volume of myocardial cells caused by heart failure by about 30 percent and reduce the myocardial fibrosis caused by the heart failure by about 40 percent, which indicates that the technical scheme of the invention is particularly suitable for producing the adeno-associated virus capable of treating the heart failure.
Drawings
FIG. 1 shows a schematic view of the structure of the carrier used in example 1.
FIG. 2 shows a gel map of a fragment of PKD2 prepared in step 3 of example 1 (Lane 1: h-PKD2PCR product,
Lane 2：DNA Marker)。
FIG. 3 shows a gel diagram of the PCR product for the monoclonal identification of PKD2 obtained in step 6 of example 1. (wherein
Lane1DNAmarker, Lane 2-5: single clone identification of the PCR product by h-PKD 2).
FIG. 4 shows a schematic structure of the recombinant overexpression vector constructed in example 1.
FIG. 5 is a graph showing statistical results of the improvement in sham and MI ejection fraction in example 2.
FIG. 6 is a chart showing the statistics of myocardial infarction area of two groups of experimental mice in example 2.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art.
TABLE 1 relevant instruments for this experiment
Example 1 adeno-associated viral vector construction and transformation
1. The pHBAd-EF1-MCS-CMV-EGFP vector (shown in figure 1) is cut by BamHI and EcoRI, and the cutting system is as follows:
2. and recovering after the carrier enzyme digestion is finished.
3. PCR recovery of PKD2 fragment
The PKD2 sequence was PCR amplified in a 50. mu.l system, in which the sequence of the forward primer is shown in SEQ ID NO.2,
the sequence of the reverse primer is shown as SEQ ID NO. 3.
TABLE 2
PCR procedure: TABLE 3
The obtained target fragment is subjected to gel electrophoresis, wherein the gel electrophoresis of the PCR product is shown in FIG. 2.
4. The treated target fragment was ligated to the vector reaction system (20. mu.l):
TABLE 4
The reaction system is placed at 50 ℃ and is subjected to warm bath for 20min, after the reaction is finished, if the subsequent operation can not be carried out immediately, the reaction sample can be temporarily stored at-20 ℃, and for detailed operation, refer to the Hanheng HB-infusion seamless cloning kit instruction.
5. Transformation (competent cells: DH5 a).
Resistance Amp, 37 ℃, cultured overnight.
6. And (3) selecting transformed PKD2 flat plate bacteria, shaking the bacteria at 37 ℃ at 250 rpm for 14 hours, carrying out PCR identification on the bacteria liquid, and sending the positive clone bacteria liquid to a sequencing company for sequencing.
And (3) monoclonal PCR identification result: (the PCR identification primer is a universal primer on the carrier, so the size of the band is about 300bp larger than the theoretical size) the sequencing result of the obtained PKD2 overexpression carrier is shown as SEQ ID NO.1, the gel electrophoresis is shown as a figure 3, and the structure is shown as a figure 4.
Example 2
Myocardial infarction symptom improvement of mice by injecting adeno-associated virus recombinant vector into myocardial infarction model mice
1. Experimental Material
C57BL/6 mice (available from Shanghai Spiker laboratory animals, Inc.).
2. Experimental methods
2.1 mouse Heart Stem modeling
24 mice were removed, and 12 of them were used in the surgical group to expose the chest cavity by lateral dissection of the skin between the second and third ribs. Muscle between the second and third ribs is cut open, a rib spreader is placed, the heart is exposed, the heart is fixed by the left hand, the auricle is exposed, the needle holder is held by the right hand, 8-0 nylon thread is used for ligation at the LAD position of 3-5mm below the auricle, and the other 12 artificial operation groups only open the thoracic cavity and do not perform operation.
2.2 Tail intravenous injection of HBAAV-EF1-PKD2-CMV-EGFP and control adeno-associated Virus
Using a 5-gauge needle syringe, each mouse was injected with 1X1011v.g, the control adeno-associated virus was AAV-GFP (adeno-associated virus with green fluorescent protein).
3. Results of the experiment
The HBAAV-EF1-PKD2-CMV-EGFP adeno-associated virus can obviously improve the cardiac function after myocardial infarction and reduce the infarct size.
As shown in FIG. 5, sham was the sham group and MI was the myocardial infarction group. As can be seen from the figure, in the Sham test group, the improvement of the symptoms of myocardial hypertrophy was not significant. In the MI experimental group, compared with the control group, the cardiac ejection fraction was recovered by about 20% in the experimental group using the recombinant adeno-associated virus of the present invention, indicating that the cardiac function was significantly improved.
As shown in fig. 6, the experimental group using the recombinant adeno-associated virus of the present invention showed a significant improvement in myocardial infarct size reduced by about 20% as compared to the control group.
This example compares the myocardial infarction index of a normal control myocardial infarction mouse and a pHBAAV-EF1-PKD2-CMV-EGFP injected myocardial infarction mouse (a mouse highly expressing the PKD2 gene). Experiments show that the damage caused by the myocardial infarction of mice can be effectively reduced by highly expressing the PDK2 gene through the adeno-associated virus. All cardiac functions and other indexes of the mouse are effectively improved, which shows that the technical scheme of the invention is suitable for treating myocardial infarction diseases.
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Sequence listing
<110> Henan bioscience technology (Shanghai) Co., Ltd
<120> PKD2 recombinant overexpression vector and construction method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8264
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ttcatcaata atatacctta ttttggattg aagccaatat gataatgagg gggtggagtt 60
tgtgacgtgg cgcggggcgt gggaacgggg cgggtgacgt agtagtgtgg cggaagtgtg 120
atgttgcaag tgtggcggaa cacatgtaag cgacggatgt ggcaaaagtg acgtttttgg 180
tgtgcgccgg tgtacacagg aagtgacaat tttcgcgcgg ttttaggcgg atgttgtagt 240
aaatttgggc gtaaccgagt aagatttggc cattttcgcg ggaaaactga ataagaggaa 300
gtgaaatctg aataattttg tgttactcat agcgcgtaat atttgtctag ggccgcgggg 360
actttgaccg tttacgtgga gactcgccca ggtgtttttc tcaggtgttt tccgcgttcc 420
gggtcaaagt tggcgtttta ttattatagt cagntctaga ctcgagaagg atctgcgatc 480
gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 540
gaggggtcgg caattgaacg ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 600
atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 660
tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc 720
gaggggctcg catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc 780
ggttgagtcg cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct 840
aggtaagttt aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct tggagcctac 900
ctagactcag ccggctctcc acgctttgcc tgaccctgct tgctcaactc tacgtctttg 960
tttcgttttc tgttctgcgc cgttacagat ccaagctgtg accggcgcct acgaattcat 1020
ggtgaactcc agtcgcgtgc agcctcagca gcccggggac gccaagcggc cgcccgcgcc 1080
ccgcgcgccg gacccgggcc ggctgatggc tggctgcgcg gccgtgggcg ccagcctcgc 1140
cgccccgggc ggcctctgcg agcagcgggg cctggagatc gagatgcagc gcatccggca 1200
ggcggccgcg cgggaccccc cggccggagc cgcggcctcc ccttctcctc cgctctcgtc 1260
gtgctcccgg caggcgtgga gccgcgataa ccccggcttc gaggccgagg aggaggagga 1320
ggaggtggaa ggggaagaag gcggaatggt ggtggagatg gacgtagagt ggcgcccggg 1380
cagccggagg tcggccgcct cctcggccgt gagctccgtg ggcgcgcgga gccgggggct 1440
tgggggctac cacggcgcgg gccacccgag cgggaggcgg cgccggcgag aggaccaggg 1500
cccgccgtgc cccagcccag tcggcggcgg ggacccgctg catcgccacc tccccctgga 1560
agggcagccg ccccgagtgg cctgggcgga gaggctggtt cgcgggctgc gaggtctctg 1620
gggaacaaga ctcatggagg aaagcagcac taaccgagag aaatacctta aaagtgtttt 1680
acgggaactg gtcacatacc tcctttttct catagtcttg tgcatcttga cctacggcat 1740
gatgagctcc aatgtgtact actacacccg gatgatgtca cagctcttcc tagacacccc 1800
cgtgtccaaa acggagaaaa ctaactttaa aactctgtct tccatggaag acttctggaa 1860
gttcacagaa ggctccttat tggatgggct gtactggaag atgcagccca gcaaccagac 1920
tgaagctgac aaccgaagtt tcatcttcta tgagaacctg ctgttagggg ttccacgaat 1980
acggcaactc cgagtcagaa atggatcctg ctctatcccc caggacttga gagatgaaat 2040
taaagagtgc tatgatgtct actctgtcag tagtgaagat agggctccct ttgggccccg 2100
aaatggaacc gcttggatct acacaagtga aaaagacttg aatggtagta gccactgggg 2160
aatcattgca acttatagtg gagctggcta ttatctggat ttgtcaagaa caagagagga 2220
aacagctgca caagttgcta gcctcaagaa aaatgtctgg ctggaccgag gaaccagggc 2280
aacttttatt gacttctcag tgtacaacgc caacattaac ctgttctgtg tggtcaggtt 2340
attggttgaa ttcccagcaa caggtggtgt gattccatct tggcaatttc agcctttaaa 2400
gctgatccga tatgtcacaa cttttgattt cttcctggca gcctgtgaga ttatcttttg 2460
tttctttatc ttttactatg tggtggaaga gatattggaa attcgcattc acaaactaca 2520
ctatttcagg agtttctgga attgtctgga tgttgtgatc gttgtgctgt cagtggtagc 2580
tataggaatt aacatataca gaacatcaaa tgtggaggtg ctactacagt ttctggaaga 2640
tcaaaatact ttccccaact ttgagcatct ggcatattgg cagatacagt tcaacaatat 2700
agctgctgtc acagtatttt ttgtctggat taagctcttc aaattcatca attttaacag 2760
gaccatgagc cagctctcga caaccatgtc tcgatgtgcc aaagacctgt ttggctttgc 2820
tattatgttc ttcattattt tcctagcgta tgctcagttg gcataccttg tctttggcac 2880
tcaggtcgat gacttcagta ctttccaaga gtgtatcttc actcaattcc gtatcatttt 2940
gggcgatatc aactttgcag agattgagga agctaatcga gttttgggac caatttattt 3000
cactacattt gtgttcttta tgttcttcat tcttttgaat atgtttttgg ctatcatcaa 3060
tgatacttac tctgaagtga aatctgactt ggcacagcag aaagctgaaa tggaactctc 3120
agatcttatc agaaagggct accataaagc tttggtcaaa ctaaaactga aaaaaaatac 3180
cgtggatgac atttcagaga gtctgcggca aggaggaggc aagttaaact ttgacgaact 3240
tcgacaagat ctcaaaggga agggccatac tgatgcagag attgaggcaa tattcacaaa 3300
gtacgaccaa gatggagacc aagaactgac cgaacatgaa catcagcaga tgagagacga 3360
cttggagaaa gagagggagg acctggattt ggatcacagt tctttaccac gtcccatgag 3420
cagccgaagt ttccctcgaa gcctggatga ctctgaggag gatgacgatg aagatagcgg 3480
acatagctcc agaaggaggg gaagcatttc tagtggcgtt tcttacgaag agtttcaagt 3540
cctggtgaga cgagtggacc ggatggagca ttccatcggc agcatagtgt ccaagattga 3600
cgccgtgatc gtgaagctag agattatgga gcgagccaaa ctgaagagga gggaggtgct 3660
gggaaggctg ttggatgggg tggccgagga tgaaaggctg ggtcgtgaca gtgaaatcca 3720
tagggaacag atggaacggc tagtacgtga agagttggaa cgctgggaat ccgatgatgc 3780
agcttcccag atcagtcatg gtttaggcac gccagtggga ctaaatggtc aacctcgccc 3840
cagaagctcc cgcccatctt cctcccaatc tacagaaggc atggaaggtg caggtggaaa 3900
tgggagttct aatgtccacg taggatcccg accggtccat cgatggggta ccccgacgcg 3960
tagcggccgc gactacaagg atgacgatga caaggattac aaagacgacg atgataagga 4020
ctataaggat gatgacgaca aataaatgca ttagttatta atagtaatca attacggggt 4080
cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 4140
ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 4200
taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 4260
acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg 4320
gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc 4380
agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca 4440
atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca 4500
atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg 4560
ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctg 4620
gtttagtgaa ccgtcagatc cgctagcatg gtgagcaagg gcgaggagct gttcaccggg 4680
gtggtgccca tcctggtcga gctggacggc gacgtaaacg ggcacaagtt cagcgtgtcc 4740
ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc 4800
ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc 4860
ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa 4920
ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc 4980
gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc 5040
aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc 5100
tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac 5160
atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac 5220
ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac 5280
cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact 5340
cacggcatgg acgagctgta caagtaagag ctcgtcgact tcgagcaact tgtttattgc 5400
agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 5460
ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat 5520
cgtctagcat cgaagatcca ataacttcgt atagcataca ttatacgaag ttataagtag 5580
cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 5640
acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 5700
actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 5760
gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 5820
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 5880
tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 5940
gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 6000
ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 6060
aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 6120
tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 6180
ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 6240
gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 6300
tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 6360
caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 6420
ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 6480
cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 6540
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 6600
cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 6660
gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 6720
aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 6780
acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 6840
gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 6900
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 6960
cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 7020
tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 7080
cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 7140
gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 7200
cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 7260
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 7320
gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caacacggga 7380
taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 7440
gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 7500
acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 7560
aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 7620
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 7680
atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 7740
gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 7800
cactctaggc aaaatagcac cctcccgctc cagaacaaca tacagcgctt cacagcggca 7860
gcctaacagt cagccttacc agtaaaaaag aaaacctatt aaaaaaacac cactcgacac 7920
ggcaccagct caatcagtca cagtgtaaaa aagggccaag tgcagagcga gtatatatag 7980
gactaaaaaa tgacgtaacg gttaaagtcc acaaaaaaca cccagaaaac cgcacgcgaa 8040
cctacgccca gaaacgaaag ccaaaaaacc cacaacttcc tcaaatcgtc acttccgttt 8100
tcccacgtta cgtaacttcc cattttaaga aaactacaat tcccaacaca tacaagttac 8160
tccgccctaa aacctacgtc acccgccccg ttcccacgcc ccgcgccacg tcacaaactc 8220
caccccctca ttatcatatt ggcttcaatc caaaataagg tata 8264
<210> 2
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atggtgaact ccagtcgcgt gcagcct 27
<210> 3
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tacgtggaca ttagaactcc catttcc 27
Claims (1)
- The application of a PKD2 recombinant overexpression vector in the preparation of myocardial infarction treatment drugs, wherein the PKD2 recombinant overexpression vector is obtained by inserting a target gene PKD2 into an adeno-associated viral vector, and the adeno-associated viral vector at least comprises the following operably linked sequence elements: the kit comprises an EF1 promoter, an MCS region, a CMV promoter sequence and an EGFP gene sequence, wherein the PKD2 recombinant overexpression vector sequence is shown as SEQ ID NO.1, and the EF1 promoter sequence is shown as 467-1012 th sites in the SEQ ID NO. 1; the sequence of the target gene PKD2 is shown as 1019-3922 in SEQ ID NO. 1; the sequence of the CMV promoter is shown as 4112-4619 th site in SEQ ID NO. 1; the EGFP gene sequence is shown as 4648-5367 in SEQ ID NO. 1.
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| WO2001068850A2 (en) * | 2000-03-14 | 2001-09-20 | Aventis Pharmaceuticals Products Inc. | Akt nucleic acids, polypeptides, and uses thereof |
| CN104877971A (en) * | 2015-06-02 | 2015-09-02 | 汉恒生物科技（上海）有限公司 | MG53 gene carried adeno-associated virus vector with function of relieving heart failure symptom of mouse |
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| MXPA05012619A (en) * | 2003-05-21 | 2006-02-08 | Univ Texas | Inhibition of protein kinase c-mu (pkd) as a treatment for cardiac hypertrophy and heart failure. |
| CA2535585A1 (en) * | 2003-08-21 | 2005-03-03 | Osaka University | Pharmaceutical composition for preventing or remedying cardiac hypertrophy and cardiocascular disease caused thereby |
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| WO2001068850A2 (en) * | 2000-03-14 | 2001-09-20 | Aventis Pharmaceuticals Products Inc. | Akt nucleic acids, polypeptides, and uses thereof |
| CN104877971A (en) * | 2015-06-02 | 2015-09-02 | 汉恒生物科技（上海）有限公司 | MG53 gene carried adeno-associated virus vector with function of relieving heart failure symptom of mouse |
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