CN102206677B - Construction and application of overexpression vector of mouse adiponectin gene - Google Patents
Construction and application of overexpression vector of mouse adiponectin gene Download PDFInfo
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- CN102206677B CN102206677B CN2011100882709A CN201110088270A CN102206677B CN 102206677 B CN102206677 B CN 102206677B CN 2011100882709 A CN2011100882709 A CN 2011100882709A CN 201110088270 A CN201110088270 A CN 201110088270A CN 102206677 B CN102206677 B CN 102206677B
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Abstract
The invention provides a construction method of an overexpression vector of a mouse adiponectin gene, comprising the following steps of: cloning to obtain mouse adiponectin fragments through an RT-PCR (Reverse Transcription-Polymerase Chain Reaction) technology, and connecting the mouse adiponectin fragments to an expression vector system of a pTarget mammal; transforming colon bacillus DH5a; and constructing to obtain a recombinant plasmid of the pTarget/ADP (Adiponectin) overexpression vector, wherein the constructed pTarget/ADP (Adiponectin) overexpression vector has very good effect of increasing gene expression levels. Through adopting the construction method provided by the invention, an ideal researching the regulation and the control of animal fat metabolism and gene functions at present is developed; the used overexpression vector convenience and easiness for obtaining and can specifically and efficiently enhance the expression of the ADP gene and be applied to prepare drugs for treating fat and metabolic syndromes or research the regulation of ADP on sugar and fat metabolism and influence of ADP on the growth and the development of muscle fibers by building an animal model; and the construction method of the overexpression vector is simple and feasible.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of construction process and purposes of mouse adiponectin gene overexpression vector.
Background technology
Adiponectin (Adiponectin) is a kind ofly also to claim Acrp30, GBP28 or AdipoQ mainly by the adipocyte excretory adipocyte factor, is the 30 kDa protein relevant with complement.The adiponectin of mouse is made up of 247 amino acid, comprises N-end secreting signal peptide, the non-helical functional zone of aminoterminal, collagen spline structure territory and C-end globosity territory (gAcrp), and posttranslational modification is 8 kinds of different homologous proteins.After the trypsinase cracking, obtain the globosity territory, activity is far longer than adiponectin, and has structural homology with C1q and TNF-α family.Adiponectin connects into tripolymer through 3 globosity territory monomers, and 4～6 tripolymers are connected to form oligomer or higher structure through the collagen structure territory, and total length and spherical two kinds of circulation forms are arranged.Adiponectin globosity territory can more effectively be improved insulin resistant and increase Fatty Acid Oxidation than total length structural domain.Quite high concentration (5～30 μ g/ml) is arranged in blood plasma.Discover that Adiponectin (ADP) and insulin receptor (Insulin receptor) have close ties; Anti-inflammatory, antiatherogenic effect are arranged; In animal tallow deposition and muscle development, whether play the regulating and controlling effect of key, become one of research focus of the fat-derived factor in recent years.Therefore, carrying out the functional study of adiponectin in Animal lipid metabolism and muscle development has great importance.
The gene overexpression is exactly the expression amount that adopts controlling element enhancing gene such as strong promoter and enhanser, can be special, express specific gene efficiently, become a kind of strong instrument of studying gene function.Because the structure of overexpression vector pTarget/ADP the adiponectin expression amount is strengthened, so overexpression vector pTarget/AdDP can be used as a kind of research strong tool that is used for functional genome.
Summary of the invention
The purpose of this invention is to provide the construction process of a kind of mouse adiponectin (ADP) gene overexpression vector, realize through following steps:
(1) RNA in the normal ICR male mice white adipose tissue of extraction, reverse transcription is cDNA, obtains mouse ADP dna fragmentation, sequence is shown in SEQ ID NO:1;
(2) RT-PCR amplification:
The CDNA that obtains with (1) is a template, with
Adiponectin-F（SEQ ID NO: 2）:5'-ATGCTACTGTTGCAAGCTCT-3'
Adiponectin-R（SEQ ID NO: 3）:5'-TCAGTTGGTATCATGGTAGA-3'
Be the upstream and downstream primer, carry out the PCR reaction with the pfu archaeal dna polymerase.Amplification condition is after 93 ℃ of 3 preparatory sex change of min, 94 ℃ of 50 S, 56 ℃ of 45 S-2min, 72 ℃ of 30s totally 30 circulations. last 1 takes turns circulation accomplish after again 72 ℃ extend 10 min.After reaction finishes, with 1% agarose gel electrophoresis inspection amplification;
(3) expression vector establishment:
The target DNA fragment that reclaims is connected to the pTarget carrier. in transformed into escherichia coli-TOP10 competent cell, coating contains on the LB flat board of 100mg/mL Ampicillin Trihydrate (Ampicillin) incubated overnight.Picking list bacterium colony is to the liquid medium that contains same concentration Amp (LB). the extracting plasmid, use the EcoRI digested plasmid, and 1% agarose gel electrophoresis is identified.
Further carry out sequential analysis to inserting fragment.It is consistent with the cDNA sequence that NM_009605.4 GI:87252710 provides to insert the fragments sequence result.
Another object of the present invention provides the application of this mouse adiponectin (ADP) gene overexpression vector in preparation treatment obesity and metabolic syndromes (mellitus) medicine.Or carry out ADP to sugar and the growth of lipometabolic adjusting and myofiber, the application during the influence of growth is studied setting up animal model.
Purposes as mouse ADP overexpression vector of the present invention, realize through following steps:
(1) will contain the intestinal bacteria enlarged culturing of mouse ADP overexpression vector after, utilize and to go the intracellular toxin test kit to extract high-purity transfection level plasmid;
High-purity transfection level plasmid that (2) will contain mouse ADP overexpression vector carries out the experiment of mouse body inner fluid effect method overexpression, and hydrodynamic effect method parameter is: plasmid 15ug, tail vein injection overexpression vector volume: 0.1ml/ body weight (g), the time is 10s.The hydrodynamic effect method gets into rapidly in the body through big quantity of fluid, produces instantaneous high pressure, causes hepatic vein hole endothelial cell membrane to produce a large amount of apertures and gets in the cell by plasmid; This method is simple, and effect is obvious;
(3) mouse tail vein injection overexpression vector; After 1 day; Analyze of the influence of pTarget/ADP overexpression vector to metabolism of fat key function gene triglyceride lytic enzyme (ATGL), hormone-sensitive lipase (HSL), LPL (LPL), yeast mating type silent message regulatory factor 1 (SIRT1), auxiliary activation factor-1 α (PGC-1 α) of oxide compound enzyme body proliferator activated receptor γ, jaw albumen O3a (FOXO3a) genetic expression; After 1 week; The same analysis assisted activation factor-1 α (PGC-1 α), jaw albumen O3a (FOXO3a) to fat and the above-mentioned metabolism of fat key function of muscle gene triglyceride lytic enzyme (ATGL), hormone-sensitive lipase (HSL), LPL (LPL), yeast mating type silent message regulatory factor 1 (SIRT1), oxide compound enzyme body proliferator activated receptor γ; And the influence of myofiber somatotype myoglobulin heavy chain I type (MyHCI), myoglobulin heavy chain II a type (MyHCIIa), myoglobulin heavy chain II b (MyHCIIb), myoglobulin heavy chain IIx (MyHCIIx) genetic expression in the muscle, the function of research ADP.
The present invention has following beneficial effect: the present invention utilizes gene overexpression technology, has opened up to zoologize the new approaches of metabolism of fat regulation and control and gene functional research at present.The used overexpression vector of this method conveniently is easy to get, and carrier construction method is simple, feasible, and the overexpression vector that makes up simultaneously can strengthen the ADP expression of gene special, efficiently, is a kind of strong investigative technique of research ADP function.The present invention can be used for studying the influence of mouse ADP gene pairs metabolism of fat and the generation of myofiber somatotype function, and for further the function of research ADP gene and the action pathway of regulation and control metabolism of fat and myofiber somatotype lay the first stone.
Description of drawings
Fig. 1 is that the pTarget/ADP overexpression vector makes up collection of illustrative plates.
Fig. 2 is that the enzyme after the pTarget/ADP overexpression vector makes up is cut the evaluation collection of illustrative plates.
Fig. 3 is pTarget/ADP carrier ADP genetic expression figure behind liver expression.
Fig. 4 is pTarget/ADP carrier protein level detection figure behind liver expression.
Fig. 5 is the variation of pTarget/ADP carrier mice serum ADP concentration behind liver expression.
The variation of mouse food ration in Fig. 6 was the pTarget/ADP carrier behind liver expression 1 week.
Fig. 7 is that pTarget/ADP carrier 1 all weight of mice behind liver expression change.
Fig. 8 is that pTarget/ADP carrier 1 all mouse white adiposes behind liver expression change.
Fig. 9 is that the pTarget/ADP carrier is in liver expression mouse white adipose lytic enzyme ATGL, HSL, variation of LPL mRNA expression level after 1 day.
Figure 10 is that the pTarget/ADP carrier changes at 1 week of liver expression back mouse white adipose lytic enzyme ATGL, HSL, LPL mRNA expression level.
Figure 11 is the variation of pTarget/ADP carrier at liver expression mouse fat associated transcription factor SIRT1, PGC-1 α and FOXO3a after 1 day.
Figure 12 is the variation of pTarget/ADP carrier at 1 week of liver expression back mouse fat associated transcription factor SIRT1, PGC-1 α and FOXO3a.
Figure 13 is that the pTarget/ADP carrier changes at 1 week of liver expression back mouse gastrocnemius muscle lytic enzyme ATGL, HSL, LPL mRNA expression level.
Figure 14 is that the pTarget/ADP carrier changes at 1 week of liver expression back mouse musculus extensor digitorum longus pedis lytic enzyme ATGL, HSL, LPL mRNA expression level.
Figure 15 is the variation of pTarget/ADP carrier at 1 week of liver expression back mouse gastrocnemius muscle fat associated transcription factor SIRT1, PGC-1 α and FOXO3a.
Figure 16 is the variation of pTarget/ADP carrier at 1 week of liver expression back mouse musculus extensor digitorum longus pedis fat associated transcription factor SIRT1, PGC-1 α and FOXO3a.
Figure 17 is pTarget/ADP carrier mouse gastrocnemius muscle MyHC-I, MyHC-IIa, MyHC-IIb, MyHC-IIx mRNA changes of expression level behind liver expression.
Figure 18 is pTarget/ADP carrier mouse musculus extensor digitorum longus pedis MyHC-I, MyHC-IIa, MyHC-IIb, MyHC-IIx mRNA changes of expression level behind liver expression.
Embodiment
The present invention combines accompanying drawing and specific embodiment to do further explain.
Embodiment 1:
The structure of pTarget/ADP overexpression vector
Be the function of checking ADP gene, we must will build overexpression vector earlier.The overexpression vector synoptic diagram that Fig. 1 builds for us.We extract the RNA in the normal ICR male mice white adipose tissue, and reverse transcription is CDNA, obtains mouse ADP dna fragmentation, and is connected to it on the pTarget expression vector, like Fig. 1.Fig. 2 EcoR I enzyme is cut the result and is shown that the purpose fragment is connected on the expression vector.M--DNA marker among the figure, 1-pTarget/ADP EcoR I enzyme is cut 2-pTarget/ADP.The junction fragment sequencing result of Fig. 3 shows that it is consistent with the cDNA sequence that NM_009605.4 GI:87252710 provides to insert fragment.
Embodiment 2:
PTarget/ADP in liver, expresses back ADP mRNA and the ADP protein level changes
For detecting the whether over-expresses in liver of pTarget/ADP carrier, ADP mRNA in the mouse liver that imports the pTarget/ADP carrier through the hydrodynamic effect method is expressed for we and protein level utilizes RT-PCR and Western blotting to detect.Experimentize: the ICR in age in 5-6 week is 10 of male mices, is divided into 2 groups at random, 5 every group, is respectively control group and experimental group.Adopt hydrodynamic effect method tail vein injection 15ugpTarget/ADP, dissect after mouse was fed 1 day.Get liver 50 mg and measure concentration with Trizol reagent extracted total RNA and with NanoDrop ND-1000 spectrophotometer, with 2.5
total RNA is RT and reacts and synthesize cDNA.With cDNA is that template is passed through the PCR reaction.The PCR reaction conditions: the sex change in 50 seconds of 94 degree, the annealing in 45 seconds of 56 degree, 72 degree prolong 30 circulations.Agarose gel electrophoresis with 1 %.The Auele Specific Primer of Adiponectin: forward: 5'-ATGCTACTGTTGCAAGCTCT-3' (SEQ ID NO:2); Oppositely: 5'-TCAGTTGGTATCATGGTAGA-3' (SEQ ID NO:3).Get liver 100 mg with lysate (1% triton X-100,0.5% sodium deoxycholate, 0.1% SDS, and 2 mM EDTA) collecting cell.SDS-PAGE with 13% forwards albumen albumen sepn to pvdf membrane on then, incubates mouse antibodies, and then incubates and be connected with the two anti-of cured px.(Tokyo Japan) develops for Amersham, GE Healthcare with test kit.
The result is referring to Fig. 4: RT-PCR result shows that the liver ADP mRNA expression level of test group is obviously high than control group, and size is 744bp.It is more eager to excel in whatever one does than control group for the ADP protein-specific band of 31kDa that immune protein trace (Western Blotting) result observes the liver size of test group, and size is 31kDa.After this explanation pTarget/ADP imports mouse, in liver, successfully transcribe and synthetic ADP albumen.
Embodiment 3:
The pTarget/ADP overexpression vector is expressed the influence of back to ADP in the serum in mouse liver
After detecting pTarget/ADP entering liver cell; Whether synthetic ADP albumen can secrete entering blood; To the mouse that imports with the hydrodynamic effect method through empty carrier and pTarget/ADP, respectively at injection back the 1st day, the 3rd day and the 7th day; Take the serum of control group and test group mouse, with the concentration change of ADP in the mouse ELISA kit measurement mice serum.
The result is referring to Fig. 5: behind the injection plasmid the 1st day, the concentration of ADP was obviously than control group high (P < 0.01), the 3rd day in the test group mice serum; The test group mice serum is also high than control group, significant difference (P 0.05), by the 7th day; The test group mice serum is also high than control group; Difference is (P < 0.05) still significantly, this result show pTarget/>ADP import liver after a large amount of rapidly synthetic ADP justacrines get into blood, make that ADP concentration peaks in the blood; But the synthetic time length weak point of ADP in the liver, ADP concentration returns to normal level in all inner bloods.* * representes P < 0.01, difference is extremely remarkable among Fig. 5; * < 0.05, difference is not remarkable to represent P.
Embodiment 4:The pTarget/ADP carrier imports behind the mouse influence to mouse food ration and body weight
Be to detect the influence of ADP, carry out the observation of 7 days by a definite date food rations and body weight through empty carrier and pTarget/ADP with the mouse of hydrodynamic effect method importing mouse food ration, body weight.The result is referring to Fig. 6, and 7: behind the hydrodynamic effect method injection ADP overexpression plasmid, compare with control group, the mouse food ration of test group had the significant difference except back in plasmid imports body in the 1st day and the 6th day, all do not have difference with control group in At All Other Times.The back is the 1st, 2,3 days in plasmid imports body, body weight (P < 0.01) obviously in rising trend.* * representes P < 0.01, difference is extremely remarkable among Fig. 6; * represent P 0.05, significant difference.* * representes P < 0.01, difference is extremely remarkable among Fig. 7; * represent P 0.05, significant difference.
Embodiment 5:The pTarget/ADP carrier imports behind the mouse influence to mouse fat
Feed a week with the mouse that the hydrodynamic effect method imports through empty carrier and pTarget/ADP, get mouse white adipose tissue and weigh.
The result is referring to Fig. 8: injection plasmid the 7th day, dissect mouse, and find that the experimental mice white adipose does not have significant difference (P>0.05).
Embodiment 6:ADP is to the influence of fatty deposits genes involved in the white adipose
Be to detect the influence of ADP to white fatty deposits genes involved, experimentize: the ICR in 5-6 age in week is 20 of male mices, is divided into 2 groups at random, 10 every group, is respectively control group and experimental group.Adopt hydrodynamic effect method tail vein injection 15ug pTarget/ADP expression vector, dissect 5 for every group after mouse was fed 1 day, remaining 1 week back dissection.With RT-PCR fatty sample is analyzed.Employed Auele Specific Primer is following:
ATGL Sense, 5'-AAC ACC AGC ATC CAG TTC AA-3'(SEQ ID NO: 4)
Antsense, 5'-GGT TCA GTA GGC CAT TCC TC-3'(SEQ ID NO: 5)
HSL Sense, 5'-TGA GAT GGT AAC TGT GAG CC-3'(SEQ ID NO: 6)
Antsense, 5'-ACT GAG ATT GAG GTG CTG TC-3'(SEQ ID NO: 7)
LPL Sense, 5'-CTG GGC TAT GAG ATC AAC AAG GT-3'(SEQ ID NO: 8)
Antsense, 5'-AGG GCA TCT GAG AGC GAG TCT-3'(SEQ ID NO: 9)
SIRT1 Sense, 5'-CAG ACC CTC AAG CCA TGT TT-3'(SEQ ID NO: 10)
Antisense, 5'-ACA CAG AGA CGG CTG GAA CT-3'(SEQ ID NO: 11)
PGC-1α Sense, 5'-CCG AGA ATT CAT GGA CA AT-3'(SEQ ID NO: 12)
Antisense, 5'-GTG TGA GGA GGG TCA TCG TT-3'(SEQ ID NO: 13)
Foxo3a Sense, 5'-ATG GGA GCT TGG AAT GTG AC-3'(SEQ ID NO: 14)
Antisense, 5'-CCA CAT TCA AAC CAA CAA CG-3'(SEQ ID NO: 15)
18s rRNA Sense, 5'-GTA ACC CGT TGA ACC CCA TT-3'(SEQ ID NO: 16)
Antisense, 5'-CCA TCC AAT CGG TAG TAG CG-3'(SEQ ID NO: 17)。
The result: behind the injection pTarget/ADP overexpression plasmid the 1st day, the test group white adipose
ATGL, LPL mRNA expression level are higher than control group, significant difference (be respectively: 54.36%, 146.14%, P 0.05) (see figure 9); After 1 week, test group white adipose ATGL mRNA expression level is higher than control group, significant difference (51.79%, P < 0.05), and HSL, LPL mRNA expression level are higher than control group, and difference is (39.15%, 32.49%, P < 0.01) (see figure 10) extremely significantly.
Behind the injection pTarget/ADP overexpression plasmid the 1st day, test group white adipose SIRT1, PGC-1 α, FOXO3a mRNA expression level is higher than control group, difference extremely remarkable (be respectively: 89.21%, 173.1%, 122.36%, P 0.01) (seeing Figure 11); After 1 week, test group white adipose SIRT1 mRNA expression level is higher than control group, significant difference (76.77%, P < 0.05), and PGC-1 α mRNA expression level is higher than control group, and difference is (171.04%, P < 0.01) (seeing Figure 12) extremely significantly.
Embodiment 8:ADP is to the influence of fatty deposits genes involved in the muscle
For detecting the influence of ADP to fatty deposits genes involved in the muscle, (seeing embodiment 6) experimentizes.The result shows:
Injection pTarget/ADP overexpression plasmid is after 1 week; Test group gastrocnemius muscle ATGL mRNA expression level is higher than control group, and difference is (64.84%, P < 0.01) (seeing Figure 13) extremely significantly; HSL, LPL mRNA expression level are higher than control group; Significant difference (be respectively 23.62%, 83.81, P 0.05).After 1 week, test group musculus extensor digitorum longus pedis ATGL, HSL, LPL mRNA expression level are higher than control group, significant difference (be respectively 20.12%, 67.87%, 44.45%, P 0.05) (seeing Figure 14).
Injection pTarget/ADP overexpression plasmid is after 1 week; FOXO3a mRNA expression level is higher than control group in the test group gastrocnemius muscle, and difference is (76.25%, P < 0.01) extremely significantly; SIRT1, PGC-1 α mRNA expression level are higher than control group; Significant difference (be respectively 29.30%, 271.32%, P 0.05) (seeing Figure 15); After 1 week, test group musculus extensor digitorum longus pedis PGC-1 α mRNA expression level is higher than control group, significant difference (246.26%, P < 0.01), and SIRT1, FOXO3a mRNA expression level are higher than control group, significant difference (27.78%, 38.97%, P 0.05) (seeing Figure 16).
Embodiment 9:ADP is to the influence of myofiber somatotype
For detecting the influence of ADP to the myofiber somatotype, (seeing embodiment 6) experimentizes.The result shows:
Test group gastrocnemius muscle MyHC-I, MyHC-IIa, MyHC-IIx mRNA expression level expression level are higher than control group, significant difference (be respectively 61.35%, 73.25%, 80.49%, P 0.05) (seeing Figure 17).Musculus extensor digitorum longus pedis MyHC-I, MyHC-IIa, MyHC-IIx mRNA expression level are higher than control group, significant difference (be respectively: 104.81%, 44.41%, 75.53%, P 0.05) (seeing Figure 18).
Among Figure 17 * represent P 0.05, significant difference; Among Figure 18 * represent P 0.05, significant difference.
PCR reaction primer is following:
MyHC-I: Sense 5＇-ATA GGG GAC CGT AGC AAG AAG-3＇(SEQ ID NO: 18)
Ant-sense 5＇-TCC TCT CAG CCT TTA GCT GGA-3＇(SEQ ID NO: 19)
MyHC-II a: Sense 5＇-CGA TGA TCT TGC CAG TAA TG-3＇(SEQ ID NO: 20)
Ant-sense 5＇-ATA ACT GAG ATA CCA GCG-3＇(SEQ ID NO: 21)
MyHC-IIx：Sense 5＇-GGA CCC ACG GTC GAA GTT G-3＇(SEQ ID NO: 22)
Ant-sense 5＇-GGC TGC GGG CTA TTG GTT-3＇(SEQ ID NO: 23)
MyHC-IIb：Sense 5＇-CAA TCA GGA ACC TTC GGA ACA C-3＇(SEQ ID NO: 24)
Ant-sense 5＇-GTC CTG GCC TCT GAG AGC AT-３＇(SEQ ID NO: 25)。
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
< 110>Zhejiang University
< 120>structure of mouse adiponectin gene overexpression vector and purposes
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< 223>the specificity upstream primer of MyHC-IIx
<440> 22
gga ccc acg gtc gaa gtt g
<210> 23
<211> 18
<212> DNA
< 213>artificial sequence
<220>
< 223>the specificity downstream primer of MyHC-IIx
<440> 23
ggc tgc ggg cta ttg gtt
<210> 24
<211> 22
<212> DNA
< 213>artificial sequence
<220>
< 223>the specificity upstream primer of MyHC-IIb
<440> 24
caa tcta gaa acc ttc gga aca c
<210> 25
<211> 20
<212> DNA
< 213>artificial sequence
<220>
< 223>the specificity downstream primer of MyHC-IIb
<440> 25
gtc ctg gcc tct gag agc at
Claims (1)
1. the construction process of a mouse adiponectin gene overexpression vector is characterized in that, realizes through following steps:
(1) RNA in the normal ICR male mice white adipose tissue of extraction, reverse transcription is cDNA, obtains the mouse adiponectin dna fragmentation, sequence is shown in SEQ ID NO:1;
(2) RT-PCR amplification: the cDNA that obtains with (1) is a template, and the upstream and downstream primer does,
SEQ ID NO:2 5'-ATGCTACTGTTGCAAGCTCT-3'
SEQ ID NO:3 5'-TCAGTTGGTATCATGGTAGA-3'
Carry out the PCR reaction with the pfu archaeal dna polymerase; After amplification condition is preparatory sex change in 93 ℃, 3 minutes; 94 ℃ 50 seconds, 56 ℃ 45 seconds-2 minutes, 72 ℃ totally 30 circulations in 30 seconds; Last 1 takes turns circulation accomplish after again 72 ℃ extended 10 minutes, after reaction finishes, check amplification with 1% agarose gel electrophoresis;
(3) expression vector establishment:
The target DNA fragment that reclaims is connected to the pTarget carrier; In transformed into escherichia coli-TOP10 competent cell, coating contains on the LB flat board of 100mg/mL Ampicillin Trihydrate incubated overnight; Picking list bacterium colony is to the liquid medium that contains same concentration Amp; The extracting plasmid is used the EcoRI digested plasmid, and 1% agarose gel electrophoresis is identified.
2. the application of a kind of mouse adiponectin gene overexpression vector in preparation treatment obesity and metabolic syndrome medicament that makes up according to the said method of claim 1.
3. a kind of mouse adiponectin gene overexpression vector that makes up according to the said method of claim 1 carries out the application of adiponectin during sugar and lipometabolic adjusting and myofiber growth, the influence of growing are studied setting up animal model.
4. according to claim 2 or 3 described application, it is characterized in that said purposes realizes through following steps:
(1) will contain the intestinal bacteria enlarged culturing of mouse adiponectin overexpression vector after, utilize and to go the intracellular toxin test kit to extract high-purity transfection level plasmid;
High-purity transfection level plasmid that (2) will contain the mouse adiponectin overexpression vector carries out the experiment of mouse body inner fluid effect method overexpression, and hydrodynamic effect method parameter is: plasmid 15ug, and tail vein injection overexpression vector volume: the 0.1ml/ body weight, the time is 10 seconds;
(3) mouse tail vein injection overexpression vector; After 1 day; Analyze of the influence of pTarget/ adiponectin overexpression vector to metabolism of fat key function Gene A TGL, HSL, LPL, SIRT1, PGC-1 α, FOXO3a genetic expression; After 1 week; The same analysis, and the influence of myofiber somatotype MyHCI, MyHCIIa, MyHCIIb, MyHCIIx genetic expression in the muscle to fatty and the above-mentioned metabolism of fat key function of muscle Gene A TGL, HSL, LPL, SIRT1, PGC-1 α, FOXO3a, the function of research adiponectin.
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| CN102492713A (en) * | 2011-12-01 | 2012-06-13 | 浙江大学 | Consruction and purposes of mouse pTarget/mutated-LPL super expression vector |
| CN109082440A (en) * | 2018-08-08 | 2018-12-25 | 华南农业大学 | Rapid Accumulation model and the preparation method and application thereof in a kind of foreign protein body |
| CN110724186A (en) * | 2019-10-29 | 2020-01-24 | 四川农业大学 | Method for prokaryotic expression of acipenser baerii globular adiponectin protein gene and application |
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| CN1737150A (en) * | 2005-07-22 | 2006-02-22 | 浙江大学 | Adiponectin-Glucagon-like peptide-1-like peptide recombinant protein expression vector and construction |
| CN1898260A (en) * | 2003-10-28 | 2007-01-17 | 普罗特米克斯发现有限公司 | Peptides with anti-obesity activity and other related uses |
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| CN1898260A (en) * | 2003-10-28 | 2007-01-17 | 普罗特米克斯发现有限公司 | Peptides with anti-obesity activity and other related uses |
| CN1737150A (en) * | 2005-07-22 | 2006-02-22 | 浙江大学 | Adiponectin-Glucagon-like peptide-1-like peptide recombinant protein expression vector and construction |
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| 张淼等.ADIPONECTIN基因转染对肌细胞糖原合成和葡萄糖氧化的影响.《中国病理生理杂志》.2005,第21卷(第12期), * |
| 张淼等.转染adiponectin基因对C2C12肌细胞糖原合成和葡萄糖氧化的影响.《中山大学学报(医学科学版)》.2004,第25卷(第5期), * |
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