CN108103102A - A kind of skeletal muscle specificity PCK1 expression vectors virus-mediated AAV1 and application thereof - Google Patents
A kind of skeletal muscle specificity PCK1 expression vectors virus-mediated AAV1 and application thereof Download PDFInfo
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Abstract
The present invention provides a kind of specific expressed phosphoenolpyruvate carboxykinase of musculature（phosphoenolpyruvatecarboxykinase,PEPCK）To reduce hyperlipidemia, anti-aging and the gene expression and the transfer vector that improve fecundity.The carrier contains improved people α skeletal actins（α‑skeletal actin）The promoter of gene, mouse parvovirus（minute virus of mice,MVM）Introne, improved people PCK1（phosphoenolpyruvatecarboxykinase 1,PCK1）122 target sequences of people miR of cDNA and 4 series connection of gene.The recombinant vector includes but not limited to 1 type adeno-associated virus by mediated by adeno-associated virus vector.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of skeletal muscle specificity PCK1 of AAV1 viral vectors mediation
Expression vector and application thereof, recombination expression unit and gene therapy mode comprising the carrier.
Background technology
Phosphoenolpyruvate carboxykinase（phosphoenolpyruvatecarboxykinase, PEPCK）It is catalysis
Gluconeogenesis（gluconeogenesis）One rate-limiting enzyme of reaction, i.e., be changed into phosphoric acid enol form propanone by oxaloacetic acid and GTP
Acid, GDP and CO2Reaction.Tricarboxylic acid cycle is carried out in mitochondria, and PEPCK is then online grain respectively there are two types of isodynamic enzyme
PEPCK-M in body（mitochondrial form）With the PEPCK-C in endochylema（cytosolic form）.PEPCK-C
Gene is PCK1 in people, plays an important role in gluconeogenesis.PCK1 has become first of liver gluconeogenesis and has clearly marked at present
Will object, the transcriptional level of the gene is the important indicator of clinically diabetes B evaluation in liver, i.e., if the mRNA tables of PCK1
Increase up to level, the rate of gluconeogenesis is just bound to increase.
The PCK1 gene codes protease of one 63kD, mainly there are three aspects for function：In liver and cortex renis
Expression, catalysis gluconeogenesis reaction；It is expressed in liver and white and brown adipose tissue, participates in the reaction of glycerine heteroplasia
（glyceroneogenesis）；It is reacted being lost in（cataplerosis）In work, remove tricarboxylic acid cycle in anion
To greatly increase the dynamics of tricarboxylic acid cycle.However transcriptional activities of the PCK1 in different tissues is different.Except mainly depositing
In liver, PCK1 is also widely present in the tissue of mammal simultaneously, including small intestine, colon, mammary gland, adrenal gland, lung
And muscle, but the metabolism in these tissues is also indefinite.In addition, major functions of the PCK1 in different tissues
Also it is different, so particular conditions knock out in different tissues or overexpression PCK1 will cause a variety of different phenotypes.Positive reason
Under condition, PCK1 is not expressed in skeletal muscle or expression quantity is very low.PCK1 is changed into one with the energy of 6 ATP to be catalyzed pyruvic acid
A glucose, and during the anerobic glycolysis of skeletal muscle, it often decomposes a glucose and but can be only generated 2 ATP.Triglycerides
Content in skeletal muscle is directly proportional to PCK1, is main energy source.
The transgenic mice of PEPCK-C is overexpressed in muscle（PEPCK-Cmus）There is infusive phenotype.First,
PEPCK-CmusThe locomitivity of transgenic mice is significantly larger than control mice, can run 5 kilometers with 20 ms/min of speed, and right
0.2 kilometer can only be run under similary speed according to mouse, this may be related with the increase of triglycerides in skeletal muscle.Secondly,
PEPCK-CmusThe service life of transgenic mice is considerably longer than control mice, and can also give birth to normal mouse at 30-35 months
Offspring, most of mouse just lost fecundity at 12-18 months（Hakimi P1, Yang J, Casadesus G, et
al.Overexpression of the cytosolic form of phosphoenolpyruvatecarboxykinase
(GTP) in skeletal muscle repatterns energy metabolism in the mouse.J Biol
Chem. 2007;282(45):32844-32855.）.
The transgene pig that PEPCK-C is overexpressed in skeletal muscle has also obtained the result similar with mouse.One kind is obtained
Similar with top beef characteristic, the high intramuscular fat content of low-fat content pork, obtains significantly on meat and mouthfeel
It is promoted, there is huge economic value.And PEPCK-C transgene pigs increase in fertility, therefore in excellent product
It has a wide range of applications in the breeding of kind.
Adeno-associated virus（Adeno-associated virus, AAV）The carrier safety barrier new as one kind,
Importance is just gradually recognized by researcher.It is parvovirus family member, is nonencapsulated linear ssdna virus, has
There is extensive host range, somatoblast can be infected but also infect non-dividing cell, and the long-term table of energy mediate foreign gene
It reaches.As important a member of viral vectors, AAV does not have apparent cytotoxic effect, and will not be as other viral vectors
The immune response for causing cell strong like that；Simultaneously when structure recombinates AAV viruses, the viral codings of itself of AAV are lacked completely
Sequence and only retain its both ends length be 145 bp terminal repeat（Inverted terminal repeat, ITR）, from
And the possibility of its restructuring and expression oneself protein is effectively reduced, security is made to be further enhanced.Therefore, AAV is as one
The preferable gene therapy vector of kind, intriguing more and more and concern（Lu Y. Recombinant
adeno-associated virus as delivery vector for gene therapy-a review.Stem
Cells Dev. 2004; 13(1):133-145; Daly TM.Overview of adeno-associated viral
vectors. Methods Mol Biol. 2004; 246:157-165.）.Senses of the AAV of different serotypes to different tissues
Dye ability slightly has difference, and wherein AAV1 is fine to the infection ability of skeletal muscle（Rebuffat A, et al. Comparison
of Adeno-Associated Virus Pseudotype 1, 2, and 8 Vectors Administered by
Intramuscular Injection in the Treatment of Murine Phenylketonuria.Hum Gene
Ther.2010;21(4):463-477.）, the mode that direct multi-point injection purpose skeletal muscle can be used effectively infected.
As previously mentioned, under normal physiological condition, PCK1 is primarily present in liver, thus it is low to design high expression according to this
The skeletal muscle specificity PCK1 expression vectors of toxicity.α-skeletal muscle of high expression people in skeletal muscle under normal physiological condition
Actin, therefore the promoter of the gene can be selected, and the introne of MVM is added to improve transcriptional efficiency to realize that PCK1 exists
Specificity overexpression in skeletal muscle.And point mutation is carried out to promoter sequence, has eliminated XhoI the and SalI enzymes in sequence
Enzyme site.The cDNA sequence of PCK1 is connected afterwards, and according to Codon degeneracy principle, the CDS sequences of PCK1 genes are carried out
Point mutation eliminates EcoRI and BglII restriction enzyme sites therein.PCK1 genes are imported in liver in order to reduce external source simultaneously
Expression is introduced into the miRNA of high expression in people's liver of 4 series connection again after PCK1 gene end codons --- miR-122's
Base complete complementary target sequence inhibits to import expression of the PCK1 genes in liver, virus is avoided to enter liver via blood circulation
Toxicity caused by the dirty middle excessively high institutes of expression PCK1 are possible.
Therefore, by the promoter of improved people α-skeletal muscle actin gene, MVM intrones, improved PCK1
The target sequence of cDNA and 4 series connection miR-122 of gene combines, and the recombination expression load of transport skeletal muscle is targeted with AAV1
System system be it is a kind of can realize skeletal muscle specificity expression PCK1 good method, treatment hyperlipidemia, slow down aging and
It improves and has broad application prospects in fecundity.
The content of the invention
In view of this, the present invention provides a kind of skeletal muscle specificity PCK1 expression vectors of AAV1 viral vectors mediation
And application thereof, recombination expression unit and gene therapy mode comprising the carrier.The recombinant gene expression vector and virus can have
Effect reduces blood lipid level so as to achieve the purpose that treat hyperlipidemia.At the same time also have both anti-aging and improve fecundity
Function.Recombinant virus provided by the invention has biological activity, promises to be while treats hyperlipidemia, delays to decline
Viral candidates that are old and improving fecundity.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The present invention provides a kind of recombinant vector expression units, which is characterized in that including：
（1）The nucleotide sequence of people α-skeletal muscle actin gene promoter as shown in SEQ ID No.1；And/or
（2）The nucleotide sequence of mouse small virus MVM intrones as shown in SEQ ID No.2；And/or
（3）The cDNA sequence of people's PCK1 genes as shown in SEQ ID No.3；And/or
（4）The target sequence of the people miR-122 of 4 series connection as shown in SEQ ID No.4.
The present invention also provides the construction method of the recombinant vector expression unit, base complete sequence such as SEQ ID
Shown in No.5.
The present invention provides the base complete sequences of recombinant vector expression unit, which is characterized in that has：
（Ⅰ）, nucleotide sequence as shown in SEQ ID No.5；Or
（Ⅱ）, nucleotide sequence as shown in SEQ ID No.5 complementary series；Or
（Ⅲ）And（Ⅰ）Or（Ⅱ）Nucleotide sequence coded same protein, but due to the degeneracy of genetic code with（Ⅰ）Or
（Ⅱ）The different sequence of nucleotide sequence；Or
（Ⅳ）And（Ⅰ）Or（Ⅱ）Or（Ⅲ）The sequence of the sequence at least 70% homology.
In some specific embodiments of the present invention, the construction method of the recombinant vector expression unit is will to transform
CDNA and 4 series connection of the promoter, MVM intrones, improved PCK1 genes of people α-skeletal muscle actin gene afterwards
The target sequence of people miR-122 combine and be connected in carrier, build expression plasmid carrier.
The present invention also provides the preparation methods of the recombinant plasmid vector and viral vectors：
Step 1：The promoter of improved people α-skeletal muscle actin gene, MVM intrones, improved PCK1 genes
People's miR-122 target sequences of cDNA and 4 series connection, which combine, to be connected in AAV plasmid vectors, builds expression plasmid carrier.
Step 2：By the expression vector with related plasmids cotransfection host cell, packaging, harvest, purifying obtain restructuring disease
Poisonous carrier.
In some specific embodiments of the present invention, carrier in the recombinant expression carrier construction method for plasmid or
Virus.
In some specific embodiments of the present invention, the virus in the recombinant expression carrier construction method is included but not
It is limited to 1 type adeno-associated virus.
The structure of the recombinant vector expression unit is ITR- α-skeletal actin promoter-MVM intron-
The AAV1 recombinant expression carriers of PCK1-4 × miR-122T-ITR, abbreviation HSA-PCK1.
The recombinant vector expression unit includes：ITR (adeno-associated virus 2 inverted
terminal repeat sequence)、α-skeletal actin promoter、MVM intron (minute virus
Of mice intron), PCK1 (phosphoenolpyruvatecarboxykinase 1), 4 × miR-122 target and
ITR (adeno-associated virus 2 inverted terminal repeat sequence)。
Wherein, the sequence of ITR sequence (Patent WO0220748) is as shown in SEQ ID No.6；It is improved
The sequence of people α-skeletal actin promoter is as shown in SEQ ID No.1；The sequence of MVM intron such as SEQ ID
Shown in No.2；The cDNA sequence of improved people PCK1 genes is as shown in SEQ ID No.3；The sequence of 4 × miR-122 target
Row are as shown in SEQ ID No.4.
The present invention also provides a kind of gene therapy modes, which is characterized in that the gene therapy mode is muscle multiple spot
Inject recombinant virus.
The present invention also provides the recombinant expression carriers and gene therapy mode to prepare treatment hyperlipidemia, delay to decline
Application in drug that is old and improving fecundity.
In the experiment of the present invention, a kind of skeletal muscle specificity PCK1 recombinant expression carriers are devised（Fig. 1）.In normal physiological
Under state, PCK1 is primarily present in liver, thus can design the skeletal muscle specificity PCK1 genes of high expression hypotoxicity according to this
Expression vector.α-skeletal actin of high expression people in skeletal muscle under normal physiological condition, therefore the gene can be selected
Promoter, and add the introne of MVM to improve transcriptional efficiency to realize specificity overexpressions of the PCK1 in skeletal muscle.So
Point mutation is carried out to promoter sequence afterwards, eliminates XhoI the and SalI restriction enzyme sites in sequence.The cDNA sequences of PCK1 are connected afterwards
Row, and according to Codon degeneracy principle, point mutation is carried out to the CDS sequences of PCK1 genes, eliminate EcoRI therein and
BglII restriction enzyme sites.Simultaneously in order to reduce expression of the PCK1 genes in liver, introduced again after PCK1 gene end codons
MiRNA --- the base complementrity target sequence of miR-122 of high expression in the liver of 4 series connection, avoids virus via blood circulation
Toxicity caused by the excessively high institutes of expression PCK1 are possible into liver.
Further according to document（Xiao X, et al. Production of High-Titer Recombinant
Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus.J
Virol.1998;72(3):2224-2232.）The method of report carries out packaging and the purifying of restructuring AAV carriers, quantifying PCR method
Measure the genome titer of virus.Specifically, AAV virus packagings are carried out to the recombinant vector using three plasmid co-transfection methods,
Cesium chloride density gradient centrifugation purifying is packaged to be restructuring AAV viruses, and SYBR Green quantifying PCR methods measure viral genome
Titre.Choosing has skeletal muscle the 1 type AAV viruses of more preferable compatibility.To hyperlipemia in mice in a manner of muscle multi-point injection
Injection of AAV 1-HSA and AAV1-HSA-PCK1 virus are distinguished in animal model, injection dosage is 1 × 1011Vg/ mouse, can be with
See and being compared with control group A AV1-HSA, total cholesterol level and triglycerides water in AAV1-HSA-PCK1 group mouse bloods
It is flat to be decreased obviously（Fig. 2）.Show that AAV1-HSA-PCK1 can effectively reduce blood fat in vivo, treat hyperlipidemia.
Further mouse naturally-aged situation is tested to assess works of the AAV1-HSA-PCK1 in anti-aging
With.In the life cycle of mouse, 16 ~ 20 months are aging early stage, and 22 ~ 24 months are the senescence phase.Respectively in mouse 0 month, 6
It is administered when the moon and 12 months, administering mode is muscle multi-point injection, and injection dosage is 1 × 1011 Vg/ mouse.The results show that
It is compared with control group, the service life of AAV1-HSA-PCK1 group mouse is obviously prolonged（Fig. 3）.Show to note in all ages and classes muscle
It can effectively slow down aging after penetrating AAV1-HSA-PCK1, extend the service life of mouse.
Further the fecundity of mouse is tested to assess works of the AAV1-HSA-PCK1 in fecundity is improved
With.Control mice has lost fecundity at 18 months, and AAV1-HSA-PCK1 groups mouse still had reproduction at 30 months
Ability（Fig. 4）.Show that intramuscular injection AAV1-HSA-PCK1 can effectively improve the fecundity of mouse.
The important Initial experiments material that the present invention uses is as follows：
PHelper plasmids, from AAV Helper Free System（Agilent Technologies, the U.S.）, by this
Company is purchased from AgilentTechnologies companies and preserves.The plasmid includes three plasmid co-transfection HEK293 cells and prepares weight
Required adenovirus source helper function genes E2A, E4 and VA RNA of group AAV viruses etc..
PAAV-R2C1 plasmids are built by our company and preserved.With AAV Helper Free System（Agilent
Technologies, the U.S.）In pAAV-RC plasmids for basic framework, with AAV1 genomes（GenBank ID：NC_
002077）Middle coat protein coding sequence Cap1（2223rd to 4433 bit sequence in genome）It replaces the in pAAV-RC plasmids
2013 to 4220 bit sequences are to get pAAV-R2C1 plasmids.Brief building process is to obtain pAAV-R2C1 according to foregoing thinking
Plasmid sequence information, HindIII is to sequence between PmeI restriction enzyme sites in artificial synthesized pAAV-R2C1 plasmids, using standard
Molecular cloning method replaces pAAV-RC plasmids with composition sequence and obtains pAAV-R2C1 plasmids.PAAV-R2C1 plasmids include complete
The cap genes of AAV1 and the rep genes of AAV2, three plasmid co-transfections pack Prepare restructuring AAV1 viruses in provide packaging institute
Necessary 4 kinds of Rep albumen（Rep78, Rep68, Rep52 and Rep40）With AAV1 coat protein.
PAAV2neo plasmids, our company's structure preserve, a kind of common AAV plasmid cloning vectors, containing there are two AAV2's
Inverted terminal repeat（Inverted terminal repeat, ITR）, human cytomegalovirus is included between two ITR
The elements such as early promoter, multiple cloning sites and bovine growth hormone polyA tailing signals.Plasmid construction process is referring to document
（Dong X, et al. Establishment of an AAV reverse infection-based array.PLoS
ONE. 2010; 5(10):e13479.）.It is used as the basic framework of clone's PCK1 gene expression units in the present invention.
Description of the drawings
It in order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is attached drawing needed in technology description to be briefly described.
Fig. 1 shows recombinant vector expression unit design drawing of the present invention.
Fig. 2 shows that AAV1-HSA-PCK1 treats the effect of hyperlipidemia in mouse；Mouse is given respectively with muscle injection mode
Injection dosage is 1 × 1011AAV1-HSA and the AAV1-HSA-PCK1 virus of vg/, lipid of mice are horizontal.
Fig. 3 shows the effect that AAV1-HSA-PCK1 slows down aging in mouse；Respectively at 0 month, 6 months and 12 months
Using muscle injection mode to mouse injection dosage as 1 × 1011 AAV1-HSA and the AAV1-HSA-PCK1 virus of vg/, mouse
Service life extend situation.
Fig. 4 shows that AAV1-HSA-PCK1 acts on the raising of mouse propagation ability；It is 1 × 10 through intramuscular injection dosage11 vg/
After AAV1-HSA and AAV1-HSA-PCK1 only, fecundity and the window phase variation of mouse.
Specific embodiment
The invention discloses the skeletal muscle specificity PCK1 expression vectors and its use of a kind of mediation of AAV1 viral vectors
On the way, recombination expression unit and gene therapy mode comprising the carrier.The recombinant gene expression vector and virus can be reduced effectively
Blood lipid level so as to achieve the purpose that treat hyperlipidemia.At the same time also have both anti-aging and improve the work(of fecundity
Energy.Those skilled in the art can use for reference present disclosure, be suitably modified technological parameter realization.It is in particular, it should be pointed out that all
Similar replacement and change is apparent to those skilled in the art, they are considered as being included in the present invention.This
The method of invention and application are described by preferred embodiment, and related personnel can be not substantially being departed from the present invention
Hold, method described herein and application be modified or suitably changed with combining in spirit and scope, to realize and using this
Inventive technique.
First of the present invention is designed to provide a kind of recombinant vector expression unit.
The recombinant vector expression unit includes improved people α-skeletal actin（α-skeletal actin）
The promoter of gene, mouse parvovirus（minute virus of mice, MVM）Introne, improved people PCK1
（phosphoenolpyruvatecarboxykinase 1, PCK1）The miR-122 target sequences of cDNA and 4 series connection of gene.
Second object of the present invention is to provide a kind of preparation method of recombinant vector expression unit, this recombinant vector table
It is ITR- α-skeletal actin promoter-MVM intron-PCK1-4 × miR-122T-ITR's up to cellular construction
AAV1 recombinant expression carriers, abbreviation HSA-PCK1.
The recombinant vector expression unit includes：ITR (adeno-associated virus 2 inverted
terminal repeat sequence)、α-skeletal actin promoter、MVM intron (minute virus
Of mice intron), PCK1 (phosphoenolpyruvatecarboxykinase 1), 4 × miR-122 target and
ITR (adeno-associated virus 2 inverted terminal repeat sequence)。
Wherein, the sequence of ITR sequence (Patent WO0220748) is as shown in SEQ ID No.6；It is improved
The sequence of people α-skeletal actin promoter is as shown in SEQ ID No.1；The sequence of MVM intron such as SEQ ID
Shown in No.2；The cDNA sequence of improved people PCK1 genes is as shown in SEQ ID No.3；The sequence of 4 × miR-122 target
Row are as shown in SEQ ID No.4.
The carrier is plasmid or virus, and the virus includes but not limited to 1 type adeno-associated virus.
Third object of the present invention is to provide a kind of gene therapy mode, and the gene therapy mode is muscle multiple spot
Inject recombinant virus.
Fourth object of the present invention is to provide the carrier of any of the above-described, virus or gene therapy mode in treatment height
Purposes in the drug of pionemia, anti-aging and raising fecundity.
In the present invention, using the modern biologies such as genetic engineering technology and method, provide including improved people α-bone
Flesh actin gene promotor, MVM intrones, improved people PCK1 gene cDNAs and 4 series connection miR-122 target sequences
Recombinate preparation, packaging and its application of AAV1 coexpression vectors and virus.
Skeletal muscle specificity PCK1 expression vectors the present invention provides the mediation of AAV1 viral vectors and application thereof, bag
Recombination expression unit and gene therapy mode containing the carrier.Wherein, unless otherwise specified, the various reactions involved in embodiment
Reagent can be commercially available by commercial channel；Unless otherwise specified, the concrete operations involved in embodiment referring to《Molecule gram
The grand experiment guide third edition》.
With reference to embodiment, the present invention is further explained：
The structure of 1 AAV1-HSA-PCK1 recombinant expression carriers of embodiment
People α-skeletal actin promoter sequence (Mol Cell Biol. 1987 are obtained according to the literature; 7(11):
4089-4099. J Biol Chem. 2007; 282(45):32844-32855.), and promoter sequence is mutated,
Eliminate XhoI the and SalI restriction enzyme sites in sequence.Then MVM intron sequences are added behind promoter, obtain expression PCK1
The complete promoter sequence that gene needs.This section of sequence designations are HSA.
The cDNA sequence of PCK1 genes is obtained according to GenBank databases, according to Codon degeneracy principle, to being obtained
PCK1 gene coded sequences be mutated, eliminate EcoRI and BglII restriction enzyme sites therein.Then in PCK1 gene ends
4 miR-122 are introduced after codon（High expression in liver）Complete complementary target sequence.This section of sequence designations for PCK1-4 ×
miR-122T。
Nanjing Jin Sirui Bioisystech Co., Ltd is transferred to synthesize two sections of sequences of HSA and PCK1-4 × miR-122T, institute
It obtains sequence and is cloned into pUC57 simple carriers, obtain pUC57-HSA and pUC57-PCK1-4 × miR-122T.With our company
The pAAV2neo plasmids of preservation（Dong X, et al. Establishment of an AAV reverse infection-
based array.PLoS ONE. 2010; 5(10):e13479.）To clone skeleton, first with XhoI and KpnI（NEB is beautiful
State）PUC57-HSA carriers are digested, the HSA segments that length is 2.3kb is obtained, Ago-Gel DNA QIAquick Gel Extraction Kits is used after electrophoresis
（Tiangen, Beijing, China）Recycling.Equally by pAAV2neo carriers XhoI and KpnI double digestions, coagulated after electrophoresis with agarose
Glue DNA QIAquick Gel Extraction Kits recycle.By HSA genetic fragments and pAAV2neo carrier segments T4 DNA ligases（Takara, greatly
Even, China）After connection, Transformed E coli DH5 α competent cells（Takara, Dalian, China）, picked clones, extraction plasmid
It is identified through XhoI and KpnI double digestions, obtains 2.3kb and 6.9kb segments to get to pAAV2neo-HSA carriers.Next, with
PAAV2neo-HSA is basic framework, with the double digested pUC57-PCK1-4 × miR-122T of KpnI and BglII, recycles length
For the segment of 2058bp, sequence between KpnI and BglII restriction enzyme sites is replaced in pAAV2neo-HSA carriers, is obtained
PAAV2neo-HAS-PCK1-4 × miR-122T carriers are identified correct through XhoI single endonuclease digestions（6915bp/2421bp/1118bp）,
Obtain pAAV2neo-HSA-PCK1-4 × miR-122T recombinant expression carriers, abbreviation HSA-PCK1.
Fig. 1 shows designed and structure recombinant vector expression unit.
The packaging of embodiment 2AAV1-HSA and AAV1-HSA-PCK1 recombinant virus and calibrating
Reference literature（Xiao X, et al. Production of High-Titer Recombinant Adeno-
Associated Virus Vectors in the Absence of Helper Adenovirus. J Virol. 1998;
72(3):2224-2232.）, restructuring AAV viruses are packed and purified using three plasmid packaging systems.Briefly, AAV vector plasmids
（PAAV2neo-HSA or pAAV2neo-HSA-PCK1-4 × miR-122T）, helper plasmid（pHelper）With the Rep of AAV1 and
Cap protein expression plasmid pAAV-R2C1 is according to 1:1:After 1 molar ratio mixing, using calcium phosphate procedure transfected HEK 293,
After transfecting 48h, cell and culture supernatant are harvested, restructuring AAV viruses are isolated and purified using cesium chloride density gradient centrifugation.Packaging
Purifying obtains AAV1-HSA（It is packaged to be by pAAV2neo-HSA plasmids）、AAV1-HSA-PCK1（By pAAV2neo-HAS-
PCK1-4 × miR-122T plasmids are packaged to be）Deng 2 kinds of recombinant viruses.
The genome titer of AAV viruses is prepared using quantifying PCR method measure.Detailed process is as follows：
Two primers HSA-Q-F and HSA-Q-R are designed in HSA promoters：
HSA-Q-F：5’-ATTTTTGGGATGAACTGCCATGATG-3’ (SEQ ID NO.7)
HSA-Q-R：5’-TGGGCCAGAACAGAATCACTCATTT-3’ (SEQ ID NO.8)
It is 177bp segments that HSA promoter length as primer specificity is expanded using HSA-Q-F and HSA-Q-R, using SYBR
Green dye binding methods, using the sample of the pAAV2neo-HSA plasmids of 1 μ g/ μ l and its 10 times of gradient dilutions as standard items, application
SYBR Premix Ex Taq II (TliRNaseH Plus) reagent（Takara, Dalian, China）, use quantitative fluorescent PCR
Instrument（Model：ABI 7500 fast, ABI）Detect viral genome titre.Operating process is referring to SYBR Premix Ex Taq
II (TliRNaseH Plus) reagent specification.The processing method of virus is referring to document（Ulrich-Peter R, et al.
Fast andreliable titration of recombinant adeno-associated virus type-2 using
quantitative real-time PCR. J Virol Methods. 2002; 106: 81-88.）.
The foundation of 3 hyperlipemia in mice animal model of embodiment
C57BL/6J Strains of Mouse 12, half male and half female, 18 ~ 22g of weight, in relative humidity 60 ± 10%, temperature 22 ± 1oC items
It is raised under part.After mouse gives the nursing of normal diet adaptability 3 days, high lipid food is changed to（Corn flour 33.5%, fish meal 5.0%, courage
A variety of nutriments such as sterol 2.0%, milk powder 4.0%, palm oil 10.0%, soya bean 16.7%）, continuously feed 16 weeks and be successfully established
Hyperlipemia model.Mouse is divided into control group and experimental group, every group of half male and half female immediately afterwards.Respectively to control group and experiment
Group muscle multi-point injection dosage is 1 × 1011AAV1-HSA and the AAV1-HSA-PCK1 virus of vg/, after continuing raising one month
It plucks eyeball and takes blood（12h is deprived of food but not water before taking blood）, 3000rpm centrifugation 10min take serum, serum is detected on Biochemical Analyzer
Middle T-CHOL（TC）, triglycerides（TG）Content.
Mouse is after injection of AAV 1-HSA-PCK1, T-CHOL and triglyceride situation of change in blood：
Fig. 2 shows mouse after injection of AAV 1-HSA-PCK1, compared with AAV1-HSA control groups, in mouse blood total cholesterol level and
Triglyceride levels are decreased obviously.
The foundation of 4 mouse naturally-aged model of embodiment and the observation of aging situation
ICR mouse 12, half male and half female, 18 ~ 22g of weight are raised under the conditions of relative humidity 60 ± 10%, 22 ± 1oC of temperature.
After mouse gives the nursing of normal diet adaptability 3 days, it is divided into three groups of control groups and experimental group, every group of half male and half female.Respectively at 0
Month, 6 months and to control group and experimental mice muscle multi-point injection dosage be 1 × 10 at 12 months11 The AAV1- of vg/ only
HSA and AAV1-HSA-PCK1 viruses, continue to raise, and observe mice age situation.
It can be seen that after in a manner of muscle multi-point injection to mouse injection of AAV 1-HSA-PCK1 viruses, whichever in year
The life cycle of age group mouse is all obviously prolonged, and plays an important role of to slow down aging.
Life cycle situation of change of the mouse after injection of AAV 1-HSA and AAV1-HSA-PCK1 virus：
Fig. 3 shows that the mouse of age groups is respectively provided with the effect of anti-aging after injection of AAV 1-HSA-PCK1, and life cycle is all bright
Fig. 3 is shown in aobvious extension.
5 mouse propagation ability of embodiment is observed
Female ICR mice 12, male ICR mouse 2,18 ~ 22g of weight, in relative humidity 60 ± 10%, temperature 22 ± 1oC items
It is raised under part.After mouse gives the nursing of normal diet adaptability 3 days, female mice is divided into control group and experimental group immediately, often
Group 6.It is respectively 1 × 10 to control group and experimental group muscle multi-point injection dosage11 The AAV1-HSA and AAV1-HSA- of vg/ only
PCK1 viruses, continue to raise.It mates, observes with male mice in female mice 3 months, 18 months and 30 months respectively
Mouse propagation capabilities might.
Mouse is after injection of AAV 1-HSA-PCK1, fecundity situation of change：
Fig. 4 shows mouse after injection of AAV 1-HSA-PCK1, and compared with AAV1-HSA control groups, the fecundity of mouse significantly improves,
Still there is fecundity at 30 months.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
SEQ ID
No.1
5'-CTCGAGAAGGCCCAAATGTAAGCTAGTCCCCTTACGTTACATGCAGCTCATTTGCTAAGTGGTTTTTTTC
TAGTATCTCCACTACTCGCTGACACAGGAGGACACAGGATGTTAAAAAGGAAATACAGTTCTGTCAATTATTCACTT
ACTCTCCAAAATACTTGGAAGAACTAAATATGGAACCATAGGAGACTTTATCCTCACCGCATAGTCCCTATACTAGT
CAAACTCCTTATTTTTTAATTGATCATTTTTAGGAAGGTAGCATTTTATTCACTAGAACATTTTTGTTAATACTTGT
TTATTTTTGGGATGAACTGCCATGATGTGGGCTACAGAGGAGGGTCGCATATGCTTCCATCCCCCTTTTAGAGAATC
CACACCTGTCCCAGTTGCTGGGTTCCACTACCAAAAGTGAATTGCAACTATTTTAGGAGCACTTAAGCACATCCGAA
AAATGAGTGATTCTGTTCTGGCCCACACCACATCACTGATGTACCCCCTTAAAGCATGTCCCTGAGTTCATCACAGA
AGACTGCTCCTCCTGTGCCCTCCACAAGGTTAGAACTGTCCTTGTCTTAGGGAAAAAGGAGAGAGAGAGAGAGAGAG
AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGGGACAGGCACCAACTGGGTAACCTCTGCTGACCCCCACTCTACTT
TACCATAAGTAGCTCCAAATCCTTCTAGAAAATCTGAAAGGCATAGCCCCATATATCAGTGATATAAATAGAACCTG
CAGCAGGCTCTGGTAAATGATGACTACAAGGTGGACTGGGAGGCAGCCCGGCCTTGGCAGGCATCATCCTCTAAATA
TAAAGATGAGTTTGTTCAGCCTTTGCAGAAGGAAAAACTGCCACCCATCCTAGAGTGCCGCGTCCTTGTCCCCCCAC
CCCCTCCAATTTATTGGGAGGAAGGACCAGCTAAGCCTCATCTAGGAAGAGCCCCTCACCCATCTCCACCTCCACTC
CAGGTCTAGCCAGTCCTGGGTTGTGACCCTTGTCTTTCAGCCCCAGGAGAGGGACACACATAGTGCCACCAAAGAGG
CTGGGGGAGGGCCTCAGCCCACCAAAACCTGGGGCCAGTGCGTCCTACAGGAGGGGAACCCTCACCCCTTCAATCCC
TTTAGGAGACCCAAGGGCGCTGCGCGTCCCTGAGGCGGACAGCTCCGTGTGCTCAGGCTTTGCGCCTGACAGGCCTA
TCCCCGGGAGCCCCCGCGCCTCCTCCCCGGCGCTCCGCCCTCGCCTCCCCCCGCCAGTTGTCTATCCTGCGACAGCT
GCGCGCCCTCCGGCCGCCGGTGGCCCTCTGTGCGGTGGGGGAAGGGGTTGACGTGGCTCAGCTTTTTGGATTCAGGG
AGCTCGGGGGTGGGAAGAGAGAAATGGAGTTCCAGGGGCGTAAAGGAGAGGGAGTTCGCCTTCCTTCCCTTCCTGAG
ACTCAGGAGTGACTGCTTCTCCAATCCTCCCAAGCCCACCACTCCACACGACTCCCTCTTCCCGGTAGTCGCAAGTG
GGAGTTTGGGGATCTGAGCAAAGAACCCGAAGAGGAGTTGAAATATTGGAAGTCAGCAGTCAGGCACCTTCCCGAGC
GCCCAGGGCGCTCAGAGTGGACATGGTTGGGGAGGCCTTTGGGACAGGTGCGGTTCCCGGAGCGCAGGCGCACACAT
GCACCCACCGGCGAACGCGGTGACCCTCGCCCCACCCCATCCCCTCCGGCGGGCAACTGGGTCGGGTCAGGAGGGGC
AAACCCGCTAGGGAGACACTCCATATACGGCCCGGCCCGCGTTACCTGGGACCGGGCCAACCCGCTCCTTCTTTGGT
CAACGCAGGGGACCCGGGCGGGGGCCCAGGCCGCGAACCGGCCGAGGGAGGGGGCTCTAGTGCCCAACACCCAAATA
TGGCTTGAGAAGGGCAGCGACATTCCTGCGGGGTGGCGCGGAGGGAATGCCCGCGGGCTATATAAAACCTGAGCAGA
GGGACAAGCGGCCACCGCAGCGGACAGCGCCAAGTGAAGCCTCGCTTCCCCTCCGCGGCGACCAGGGCCCGAGCCGA
GAGTAGCAGTTGTAGCTACCCGCCCAGGTAGGGCAGGAGTTGGGAGGGGACAGGGGGACAGGGCACTACCGAGGGGA
ACCTGAAGGACTCCGGGGCAGAACCCAGTCGGTTCACCTGGTCAGCCCCAGGCCTGCGCCCTGAGCGCTGTGCCTCG
TCTCCGGAGCCACACGCGCTGGTACC-3'
No.2
5'-GTAAGTTGGCGCCGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTTTTTTACAG-3'
No.3
5'-GGTACCGTAAGTTGGCGCCGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTTTTTTA
CAGGAATTCGCCACCATGCCTCCTCAGCTGCAAAACGGCCTGAACCTCTCGGCCAAAGTTGTCCAGGGAAGCCTGGA
CAGCCTACCCCAGGCAGTGAGGGAGTTTCTCGAGAATAACGCTGAGCTGTGTCAGCCTGATCACATCCACATCTGTG
ACGGCTCTGAGGAGGAGAATGGGCGGCTTCTGGGCCAGATGGAGGAAGAGGGCATCCTCAGGCGGCTGAAGAAGTAT
GACAACTGCTGGTTGGCTCTCACTGACCCCAGGGATGTGGCCAGGATCGAAAGCAAGACGGTTATCGTCACCCAAGA
GCAAAGAGACACAGTGCCCATCCCCAAAACAGGCCTCAGCCAGCTCGGTCGCTGGATGTCAGAGGAGGATTTTGAGA
AAGCGTTCAATGCCAGGTTCCCAGGGTGCATGAAAGGTCGCACCATGTACGTCATCCCATTCAGCATGGGGCCGCTG
GGCTCGCCTCTGTCAAAGATCGGCATCGAGCTGACGGATTCACCCTACGTGGTGGCCAGCATGCGGATCATGACGCG
GATGGGCACGCCCGTCCTGGAAGCAGTGGGCGATGGGGAGTTTGTCAAATGCCTCCATTCTGTGGGGTGCCCTCTGC
CTTTACAAAAGCCTTTGGTCAACAACTGGCCCTGCAACCCGGAGCTGACGCTCATCGCCCACCTGCCTGACCGCAGA
GAGATCATCTCCTTTGGCAGTGGGTACGGCGGGAACTCGCTGCTCGGGAAGAAGTGCTTTGCTCTCAGGATGGCCAG
CCGGCTGGCCAAGGAGGAAGGGTGGCTGGCAGAGCACATGCTGATTCTGGGTATAACCAACCCTGAGGGTGAGAAGA
AGTACCTGGCGGCCGCATTTCCCAGCGCCTGCGGGAAGACCAACCTGGCCATGATGAACCCCAGCCTCCCCGGGTGG
AAGGTTGAGTGCGTCGGGGATGACATTGCCTGGATGAAGTTTGACGCACAAGGTCATTTAAGGGCCATCAACCCAGA
AAATGGCTTTTTCGGTGTCGCTCCTGGGACTTCAGTGAAGACCAACCCCAATGCCATCAAGACCATCCAGAAGAACA
CAATCTTTACCAATGTGGCCGAGACCAGCGACGGGGGCGTTTACTGGGAAGGCATTGATGAGCCGCTAGCTTCAGGT
GTCACCATCACGTCCTGGAAGAATAAGGAGTGGAGCTCAGAGGATGGGGAACCTTGTGCCCACCCCAACTCGAGGTT
CTGCACCCCTGCCAGCCAGTGCCCCATCATTGATGCTGCCTGGGAGTCTCCGGAAGGTGTTCCCATTGAAGGCATTA
TCTTTGGAGGCCGTAGACCTGCTGGTGTCCCTCTAGTCTATGAAGCTCTCAGCTGGCAACATGGAGTCTTTGTGGGG
GCGGCCATGAGATCAGAGGCCACAGCGGCTGCAGAACATAAAGGCAAAATCATCATGCATGACCCCTTTGCCATGCG
GCCCTTCTTTGGCTACAACTTCGGCAAATACCTGGCCCACTGGCTTAGCATGGCCCAGCACCCAGCAGCCAAACTGC
CCAAGATTTTCCATGTCAACTGGTTCCGGAAGGACAAGGAAGGCAAATTCCTCTGGCCAGGCTTTGGAGAGAACTCC
AGGGTGCTGGAGTGGATGTTCAACCGGATCGATGGAAAAGCCAGCACCAAGCTCACGCCCATAGGCTACATCCCCAA
GGAGGATGCCCTGAACCTGAAAGGCCTGGGGCACATCAACATGATGGAGCTTTTCAGCATCTCCAAGGAGTTCTGGG
AGAAGGAGGTGGAAGACATCGAGAAGTATCTGGAGGATCAAGTCAATGCCGACCTCCCCTGTGAAATCGAGAGAGAG
ATCCTTGCCTTGAAGCAAAGAATAAGCCAGATGT-3'
No.4
5'-AAACACCATTGTCACACTCCAGATCCAAACACCATTGTCACACTCCATAGCCAAACACCATTGTCACACT
CCAGATCCAAACACCATTGTCACACTCCA-3'
No.5
5'-CTCGAGAAGGCCCAAATGTAAGCTAGTCCCCTTACGTTACATGCAGCTCATTTGCTAAGTGGTTTTTTTC
TAGTATCTCCACTACTCGCTGACACAGGAGGACACAGGATGTTAAAAAGGAAATACAGTTCTGTCAATTATTCACTT
ACTCTCCAAAATACTTGGAAGAACTAAATATGGAACCATAGGAGACTTTATCCTCACCGCATAGTCCCTATACTAGT
CAAACTCCTTATTTTTTAATTGATCATTTTTAGGAAGGTAGCATTTTATTCACTAGAACATTTTTGTTAATACTTGT
TTATTTTTGGGATGAACTGCCATGATGTGGGCTACAGAGGAGGGTCGCATATGCTTCCATCCCCCTTTTAGAGAATC
CACACCTGTCCCAGTTGCTGGGTTCCACTACCAAAAGTGAATTGCAACTATTTTAGGAGCACTTAAGCACATCCGAA
AAATGAGTGATTCTGTTCTGGCCCACACCACATCACTGATGTACCCCCTTAAAGCATGTCCCTGAGTTCATCACAGA
AGACTGCTCCTCCTGTGCCCTCCACAAGGTTAGAACTGTCCTTGTCTTAGGGAAAAAGGAGAGAGAGAGAGAGAGAG
AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGGGACAGGCACCAACTGGGTAACCTCTGCTGACCCCCACTCTACTT
TACCATAAGTAGCTCCAAATCCTTCTAGAAAATCTGAAAGGCATAGCCCCATATATCAGTGATATAAATAGAACCTG
CAGCAGGCTCTGGTAAATGATGACTACAAGGTGGACTGGGAGGCAGCCCGGCCTTGGCAGGCATCATCCTCTAAATA
TAAAGATGAGTTTGTTCAGCCTTTGCAGAAGGAAAAACTGCCACCCATCCTAGAGTGCCGCGTCCTTGTCCCCCCAC
CCCCTCCAATTTATTGGGAGGAAGGACCAGCTAAGCCTCATCTAGGAAGAGCCCCTCACCCATCTCCACCTCCACTC
CAGGTCTAGCCAGTCCTGGGTTGTGACCCTTGTCTTTCAGCCCCAGGAGAGGGACACACATAGTGCCACCAAAGAGG
CTGGGGGAGGGCCTCAGCCCACCAAAACCTGGGGCCAGTGCGTCCTACAGGAGGGGAACCCTCACCCCTTCAATCCC
TTTAGGAGACCCAAGGGCGCTGCGCGTCCCTGAGGCGGACAGCTCCGTGTGCTCAGGCTTTGCGCCTGACAGGCCTA
TCCCCGGGAGCCCCCGCGCCTCCTCCCCGGCGCTCCGCCCTCGCCTCCCCCCGCCAGTTGTCTATCCTGCGACAGCT
GCGCGCCCTCCGGCCGCCGGTGGCCCTCTGTGCGGTGGGGGAAGGGGTTGACGTGGCTCAGCTTTTTGGATTCAGGG
AGCTCGGGGGTGGGAAGAGAGAAATGGAGTTCCAGGGGCGTAAAGGAGAGGGAGTTCGCCTTCCTTCCCTTCCTGAG
ACTCAGGAGTGACTGCTTCTCCAATCCTCCCAAGCCCACCACTCCACACGACTCCCTCTTCCCGGTAGTCGCAAGTG
GGAGTTTGGGGATCTGAGCAAAGAACCCGAAGAGGAGTTGAAATATTGGAAGTCAGCAGTCAGGCACCTTCCCGAGC
GCCCAGGGCGCTCAGAGTGGACATGGTTGGGGAGGCCTTTGGGACAGGTGCGGTTCCCGGAGCGCAGGCGCACACAT
GCACCCACCGGCGAACGCGGTGACCCTCGCCCCACCCCATCCCCTCCGGCGGGCAACTGGGTCGGGTCAGGAGGGGC
AAACCCGCTAGGGAGACACTCCATATACGGCCCGGCCCGCGTTACCTGGGACCGGGCCAACCCGCTCCTTCTTTGGT
CAACGCAGGGGACCCGGGCGGGGGCCCAGGCCGCGAACCGGCCGAGGGAGGGGGCTCTAGTGCCCAACACCCAAATA
TGGCTTGAGAAGGGCAGCGACATTCCTGCGGGGTGGCGCGGAGGGAATGCCCGCGGGCTATATAAAACCTGAGCAGA
GGGACAAGCGGCCACCGCAGCGGACAGCGCCAAGTGAAGCCTCGCTTCCCCTCCGCGGCGACCAGGGCCCGAGCCGA
GAGTAGCAGTTGTAGCTACCCGCCCAGGTAGGGCAGGAGTTGGGAGGGGACAGGGGGACAGGGCACTACCGAGGGGA
ACCTGAAGGACTCCGGGGCAGAACCCAGTCGGTTCACCTGGTCAGCCCCAGGCCTGCGCCCTGAGCGCTGTGCCTCG
TCTCCGGAGCCACACGCGCTGGTACCGGTACCGTAAGTTGGCGCCGTTTAAGGGATGGTTGGTTGGTGGGGTATTAA
TGTTTAATTACCTTTTTTACAGGAATTCGCCACCATGCCTCCTCAGCTGCAAAACGGCCTGAACCTCTCGGCCAAAG
TTGTCCAGGGAAGCCTGGACAGCCTACCCCAGGCAGTGAGGGAGTTTCTCGAGAATAACGCTGAGCTGTGTCAGCCT
GATCACATCCACATCTGTGACGGCTCTGAGGAGGAGAATGGGCGGCTTCTGGGCCAGATGGAGGAAGAGGGCATCCT
CAGGCGGCTGAAGAAGTATGACAACTGCTGGTTGGCTCTCACTGACCCCAGGGATGTGGCCAGGATCGAAAGCAAGA
CGGTTATCGTCACCCAAGAGCAAAGAGACACAGTGCCCATCCCCAAAACAGGCCTCAGCCAGCTCGGTCGCTGGATG
TCAGAGGAGGATTTTGAGAAAGCGTTCAATGCCAGGTTCCCAGGGTGCATGAAAGGTCGCACCATGTACGTCATCCC
ATTCAGCATGGGGCCGCTGGGCTCGCCTCTGTCAAAGATCGGCATCGAGCTGACGGATTCACCCTACGTGGTGGCCA
GCATGCGGATCATGACGCGGATGGGCACGCCCGTCCTGGAAGCAGTGGGCGATGGGGAGTTTGTCAAATGCCTCCAT
TCTGTGGGGTGCCCTCTGCCTTTACAAAAGCCTTTGGTCAACAACTGGCCCTGCAACCCGGAGCTGACGCTCATCGC
CCACCTGCCTGACCGCAGAGAGATCATCTCCTTTGGCAGTGGGTACGGCGGGAACTCGCTGCTCGGGAAGAAGTGCT
TTGCTCTCAGGATGGCCAGCCGGCTGGCCAAGGAGGAAGGGTGGCTGGCAGAGCACATGCTGATTCTGGGTATAACC
AACCCTGAGGGTGAGAAGAAGTACCTGGCGGCCGCATTTCCCAGCGCCTGCGGGAAGACCAACCTGGCCATGATGAA
CCCCAGCCTCCCCGGGTGGAAGGTTGAGTGCGTCGGGGATGACATTGCCTGGATGAAGTTTGACGCACAAGGTCATT
TAAGGGCCATCAACCCAGAAAATGGCTTTTTCGGTGTCGCTCCTGGGACTTCAGTGAAGACCAACCCCAATGCCATC
AAGACCATCCAGAAGAACACAATCTTTACCAATGTGGCCGAGACCAGCGACGGGGGCGTTTACTGGGAAGGCATTGA
TGAGCCGCTAGCTTCAGGTGTCACCATCACGTCCTGGAAGAATAAGGAGTGGAGCTCAGAGGATGGGGAACCTTGTG
CCCACCCCAACTCGAGGTTCTGCACCCCTGCCAGCCAGTGCCCCATCATTGATGCTGCCTGGGAGTCTCCGGAAGGT
GTTCCCATTGAAGGCATTATCTTTGGAGGCCGTAGACCTGCTGGTGTCCCTCTAGTCTATGAAGCTCTCAGCTGGCA
ACATGGAGTCTTTGTGGGGGCGGCCATGAGATCAGAGGCCACAGCGGCTGCAGAACATAAAGGCAAAATCATCATGC
ATGACCCCTTTGCCATGCGGCCCTTCTTTGGCTACAACTTCGGCAAATACCTGGCCCACTGGCTTAGCATGGCCCAG
CACCCAGCAGCCAAACTGCCCAAGATTTTCCATGTCAACTGGTTCCGGAAGGACAAGGAAGGCAAATTCCTCTGGCC
AGGCTTTGGAGAGAACTCCAGGGTGCTGGAGTGGATGTTCAACCGGATCGATGGAAAAGCCAGCACCAAGCTCACGC
CCATAGGCTACATCCCCAAGGAGGATGCCCTGAACCTGAAAGGCCTGGGGCACATCAACATGATGGAGCTTTTCAGC
ATCTCCAAGGAGTTCTGGGAGAAGGAGGTGGAAGACATCGAGAAGTATCTGGAGGATCAAGTCAATGCCGACCTCCC
CTGTGAAATCGAGAGAGAGATCCTTGCCTTGAAGCAAAGAATAAGCCAGATGTGATAAGTCGACAAACACCATTGTC
ACACTCCAGATCCAAACACCATTGTCACACTCCATAGCCAAACACCATTGTCACACTCCAGATCCAAACACCATTGT
CACACTCCAAGATCT-3'
No.6
5'-GACGGCGCTAGGATCATCAACGAAACCCAGCATCTACACAATGTAGCTCAAGTATTCTGGTCACAGAATA
CAACGAAACCCAGCATCTACACAATGTAGCTCAAGATGATCCTAGCGCCGTCTT-3'
No.7
5'-ATTTTTGGGATGAACTGCCATGATG-3'
No.8
5'-TGGGCCAGAACAGAATCACTCATTT-3'
Claims (8)
1. a kind of recombinant vector expression unit, which is characterized in that including：
（1）The nucleotide sequence of people α-skeletal muscle actin gene promoter as shown in SEQ ID No.1；And/or
（2）The nucleotide sequence of mouse small virus MVM intrones as shown in SEQ ID No.2；And/or
（3）The cDNA sequence of people's PCK1 genes as shown in SEQ ID No.3；And/or
（4）People's miR-122 target sequences of 4 series connection as shown in SEQ ID No.4.
2. the construction method of recombinant vector expression unit according to claim 1, base complete sequence such as SEQ ID No.5
It is shown.
3. according to the recombinant vector expression unit described in claim 1 and claim 2, which is characterized in that have：
（Ⅰ）, nucleotide sequence as shown in SEQ ID No.5；Or
（Ⅱ）, nucleotide sequence as shown in SEQ ID No.5 complementary series；Or
（Ⅲ）And（Ⅰ）Or（Ⅱ）Nucleotide sequence coded same protein, but due to the degeneracy of genetic code with（Ⅰ）Or
（Ⅱ）The different sequence of nucleotide sequence；Or
（Ⅳ）And（Ⅰ）Or（Ⅱ）Or（Ⅲ）The sequence of the sequence at least 70% homology.
4. construction method according to claim 3, which is characterized in that the carrier is plasmid or virus.
5. construction method according to claim 4, which is characterized in that the virus includes but not limited to 1 type gland related diseases
Poison.
6. a kind of gene therapy mode, which is characterized in that wanted including such as claim 1, claim 2, claim 3, right
Seek the recombinant virus described in recombinant expression carrier and the claim 5 described in 4.
7. gene therapy mode according to claim 6, which is characterized in that the gene therapy mode is noted for muscle multiple spot
Penetrate recombinant virus.
8. gene therapy mode described in recombinant virus according to claim 5, claim 6 or 7 reduce hyperlipidemia,
Application in anti-aging and raising fecundity.
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