CN103709242B - A kind of recombinant protein based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene, preparation method and application - Google Patents
A kind of recombinant protein based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene, preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of recombinant protein based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene, preparation method and application, belong to biological technical field.The present invention, according to the cDNA encoding sequence of Fugu rubripes (Temmincket Schlegel) FoxO1 albumen, designs the primer pair for Fugu rubripes (Temmincket Schlegel) FoxO1 albumen coded sequence, carries out RT-PCR amplification after extracting Fugu rubripes (Temmincket Schlegel) liver total RNA with ISOGEN Reagent.RT-PCR product is connected to obtain recombinant plasmid with pGEM-T carrier, and after double digestion, goal gene is connected with expression vector pET-32a (+) again, obtains recombinant expression plasmid and is converted in intestinal bacteria Rosetta (DE3); The solubility expression of recombinant protein is achieved by IPTG induction.Fugu rubripes (Temmincket Schlegel) FoxO1 recombinant protein in the present invention can suppress the expression of peroxisome proliferator activated receptor PPAR γ gene, regulates Fugu rubripes (Temmincket Schlegel) body lipid metabolism balance.The recombinant products of purifying carries out Active MnO2 to fish or feed adds, and can improve the growth of cultured fishes and reduce the lipid content of fish, making delicious meat good to eat.
Description
Technical field
The present invention is specifically related to a kind of recombinant protein based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene, and the preparation method and application of this recombinant protein, belongs to biological technical field.
Background technology
Lipid is the main composition composition of organism, closely related with a lot of physiological function such as the storage of energy and Cellular Signaling Transduction Mediated approach.The major lipids deposition fraction of fish is liver and muscle, but the ratio of the existence of lipid is in both tissues also along with the difference of fingerling has very large difference, the lipid major storage of the fingerling such as globe fish and flatfish is liver (thin fish), and other fingerling such as the lipid of the fish such as porgy and eel is present in liver and muscle (fertile fish) simultaneously.Histological research shows that the muscle lipid of salmon flying fish is mainly deposited on the flesh diaphragm that namely adipocyte oppress.The adipocyte of porgy is by front fat Adipocyte Differentiation, and Peroxisome Proliferator-activated Receptors (peroxisome proliferator-activatedreceptors, PPARs) regulation and control are participated in, although its detailed mechanism participating in Adipocyte Differentiation is not clear, be likely that thin fish and fertile fish have different molecular regulation networks in the atomization of adipocyte.There are three member: PPAR α, PPAR β, PPAR γ in PPARs family, plays important regulating and controlling effect in lipid homeostasis.PPAR α and PPAR β mainly participates in Fatty acids oxidation, and PPAR γ mainly participates in steatogenesis.PPAR γ is regulated the expression of lipid metabolism target gene by ligand activation, promotes fatty deposits.
In Mammals and clone thereof, jaw transcription factor O subfamily 1(forkhead transcription factor groupO1, FoxO1) be the important regulating and controlling factor of preadipocyte differentiation process, affect cell proliferation, differentiation, apoptosis by many paths.FoxO1 can pass through to hinder the combination of PPAR γ functional complex and DNA thus the transcriptional regulatory activity suppressing PPAR γ.Study in rat fat tissue and also find that FoxO1 can suppress transcribing of PPAR γ gene, reduce the expression amount of PPAR γ.
So far, the research of the regulating effect played in mammal fat metabolism about FoxO1 has been carried out and has achieved many achievements in the Mammalss such as people, rat, mouse, but there is not yet the correlative study report about FoxO1 albumen balances for regulating Fish lipid metabolism.The research and development gene clone relevant to Fugu rubripes (Temmincket Schlegel) FoxO1 albumen and recombinant expressed there is extremely important effect.
Summary of the invention
The object of this invention is to provide a kind of recombinant protein based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene.
Meanwhile, the present invention also provides a kind of preparation method of the recombinant protein based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene.
Finally, the present invention also provides a kind of application of recombinant protein in the pharmaceutical composition or feed of preparation adjustment Fugu rubripes (Temmincket Schlegel) fatty tissue PPAR γ expression amount based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene.
In order to realize above object, the technical solution adopted in the present invention is:
Based on a recombinant protein for Fugu rubripes (Temmincket Schlegel) FoxO1 gene, it is containing, for example the aminoacid sequence shown in SEQ ID NO:4.
Nucleotide sequence coded by such as shown in SEQ ID NO:3 of described aminoacid sequence.
Based on a preparation method for the recombinant protein of Fugu rubripes (Temmincket Schlegel) FoxO1 gene, comprise the following steps:
(1) reverse transcription PCR (RT-PCR) increases Fugu rubripes (Temmincket Schlegel) fatty tissue total serum IgE, and amplified production is connected to obtain recombinant plasmid with pGEM-T carrier, and after double digestion, goal gene is connected with pET-32a (+) expression vector again, obtains recombinant expression plasmid;
(2) recombinant expression plasmid is transformed into Host Strains, adds inductor IPTG abduction delivering FoxO1 recombinant protein.
The primer pair P of described reverse transcription PCR amplification is:
Upstream primer P1:5 '-CG
gAATTCaTGGCGGAAGCAGCCCA-3 ' (see SEQ ID NO:1),
Downstream primer P2:5 '-CCG
cTCGAGcCCTGACACCCAGCTGTGC-3 ' (see SEQ ID NO:2).
In described step (1), the reaction conditions of reverse transcription PCR amplification is: 35 amplifications circulated carrying out 94 DEG C of sex change 40s, 61 DEG C of renaturation 50s, 72 DEG C of extension 2min after 94 DEG C of denaturation 5min, are stored in 4 DEG C after 72 DEG C of extension 10min.
The reaction system of reverse transcription PCR amplification is in described step (1): cDNA masterplate 1.0 μ l, 10 μMs of P11.0 μ l, 10 μMs of P21.0 μ l, 10 × PCR Buffer for KOD-Plus-Neo2.5 μ l, dNTP Mixture (each 2.5mM) 2.0 μ l, ddH2O17.0 μ l, 1U/ μ l KOD-Plus-Neo high-fidelity DNA polymerase 0.5 μ l, amount to 25 μ l.
Host Strains in described step (2) is E. coli expression strains Rosetta (DE3).
The cellular lysate after inducing culture is got in described step (2), centrifugal, get supernatant liquor purifying and namely obtain recombinant protein.
A kind of application of recombinant protein in the pharmaceutical composition or feed of preparation adjustment Fugu rubripes (Temmincket Schlegel) fatty tissue PPAR γ expression amount based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene.
Beneficial effect of the present invention:
The present invention, according to the cDNA encoding sequence of Fugu rubripes (Temmincket Schlegel) FoxO1 albumen, designs the primer pair for Fugu rubripes (Temmincket Schlegel) FoxO1 albumen coded sequence, carries out RT-PCR amplification after extracting Fugu rubripes (Temmincket Schlegel) liver total RNA with ISOGEN Reagent.RT-PCR product is connected to obtain recombinant plasmid with pGEM-T carrier, and after double digestion, goal gene is connected with expression vector pET-32a (+) again, obtains recombinant expression plasmid and is converted in intestinal bacteria Rosetta (DE3); The solubility expression of recombinant protein is achieved by IPTG induction.The fusion rotein that this expression plasmid is expressed is with histidine-tagged, facilitate qualification and the purifying of sample, avoid the denature and renature of sample, not only simple to operate, reduce cost, more importantly protein structure is not damaged, recombinant protein purity is good, yield is high and not easily degrade, and is adapted to suitability for industrialized production, for the function studying Fugu rubripes (Temmincket Schlegel) FoxO1 albumen further lays the foundation.
Fugu rubripes (Temmincket Schlegel) FoxO1 recombinant protein in the present invention can suppress the expression of peroxisome proliferator activated receptor PPAR γ gene, regulates Fugu rubripes (Temmincket Schlegel) body lipid metabolism balance.Specifically, there are two aspect advantages: the major lipids deposition fraction of (1) fish is liver and muscle, but the ratio of the existence of lipid is in both tissues also along with the difference of fingerling has very large difference, FoxO1 recombinant protein can suppress peroxisome proliferator activated receptor PPAR γ genetic expression, to maintain cultivation fish fats metabolic balance; (2) the Fugu rubripes (Temmincket Schlegel) FoxO1 recombinant protein regulating and controlling expression of fish lipid metabolism control gene in the present invention can be applicable in fish farming, reduces fatty,fiss deposition, improves meat, have important economic worth.
Fugu rubripes (Temmincket Schlegel) FoxO1 recombinant protein in the present invention has following application prospect: (1) Fugu rubripes (Temmincket Schlegel) FoxO1 recombinant protein is used for the research of fish lipid metabolic mechanism.Direct use restructuring Fugu rubripes (Temmincket Schlegel) FoxO1 albumen or this albumen prepare monoclonal antibody or polyclonal antibody, carry out the research of fish and other animal lipid metabolic mechanism.(2) Fugu rubripes (Temmincket Schlegel) FoxO1 recombinant protein is used as meat modifier.The recombinant products of purifying carries out Active MnO2 to fish or feed adds, and can improve the growth of cultured fishes and reduce the lipid content of fish, making delicious meat good to eat.
Accompanying drawing explanation
Fig. 1 is the double digestion electroresis appraisal figure of recombinant expression plasmid FoxO1-pET32 in the embodiment of the present invention 1;
Fig. 2 is the SDS-PAGE analysis chart of recombinant expression plasmid expression product in embodiment 4;
Fig. 3 is the western blot analysis figure of recombinant expression plasmid abduction delivering in embodiment 4;
Fig. 4 is the column diagram that in embodiment 5, FoxO1 recombinant protein expresses impact on Fugu rubripes (Temmincket Schlegel) PPAR γ.
Embodiment
Following embodiment is only described in further detail the present invention, but does not form any limitation of the invention.
Embodiment 1
The Construction and identification of pET32a-FoxO1 prokaryotic expression plasmid:
Fresh Fugu rubripes (Temmincket Schlegel) fatty tissue total serum IgE is extracted and the synthesis of the cDNA of the first chain is carried out in reverse transcription with ISOGEN Reagent.
According to the primer pair of Fugu rubripes (Temmincket Schlegel) FoxO1 protein gene conserved sequence design for FoxO1 albumen coded sequence, carry out pcr amplification with above-mentioned first chain cDNA, the nucleotides sequence of primer pair P is classified as:
Upstream primer P1:5 '-CG
gAATTC ecoR IaTGGCGGAAGCAGCCCA-3 ',
Downstream primer P2:5 '-CCG
cTCGAG xho IcCCTGACACCCAGCTGTGC-3 ';
RT-PCR reaction system is: cDNA masterplate 1.0 μ l, 10 μMs of P11.0 μ l, 10 μMs of P21.0 μ l, 10 × PCR Bufferfor KOD-Plus-Neo2.5 μ l, dNTP Mixture (each 2.5mM) 2.0 μ l, ddH2O17.0 μ l, 1U/ μ lKOD-Plus-Neo high-fidelity DNA polymerase 0.5 μ l, amount to 25 μ l; Reaction conditions is: 35 amplifications circulated carrying out 94 DEG C of sex change 40s, 61 DEG C of renaturation 50s, 72 DEG C of extension 2min after 94 DEG C of denaturation 5min, are stored in 4 DEG C after 72 DEG C of extension 10min.The dashed part of primer pair is respectively EcoR I restriction enzyme site and Xho I restriction enzyme site.With Fugu rubripes (Temmincket Schlegel) FoxO1 first chain cDNA for template, obtain goal gene with round pcr, obtain the band of single 2049bp size and reclaim.PCR electrophoresis reclaims product and is connected with cloning vector pGEM-T (Promaga company) and is transformed into competent escherichia coli cell JM109, cultivating by blue hickie method screening positive clone containing on the LB solid medium of acillin, and carrying out the extracting of plasmid;
With restriction enzyme EcoR I and Xho I plasmid that extracts of double digestion and expression vector pET-32a (+) respectively, 37 DEG C of effects are after 2 hours, digestion products QIAquick gel extraction kit (QIAGENE company) reclaims, pGEM-T-FoxO1 plasmid enzyme restriction product and carrier digestion products press 3:1 mixing, T4DNA ligase enzyme (promaga company) 16 DEG C of connections of spending the night, transformation of E. coli competent cell JM109 is in 37 DEG C of incubated overnight.Extract recombinant expression plasmid pET32a-FoxO1; With EcoR I and Xho I pair of recombinant expression plasmid double digestion qualification, as shown in Figure 1.Sequencing result shows, the row that check order high with other FoxO1 coding sequence homology, therefore tentatively confirm that the recombinant expression plasmid extracted is the encoding sequence of Fugu rubripes (Temmincket Schlegel) FoxO1.
Embodiment 2
The abduction delivering of FoxO1 recombinant protein:
Extracted recombinant expression plasmid is transformed in E. coli expression strains Rosetta (DE3), cultivate bacterial strain and obtain bacterium liquid, picking list colony inoculation 5mL Amp LB liquid nutrient medium incubated overnight, get the bacterium liquid inoculation 5mLAmp LB liquid nutrient medium of 50 μ L incubated overnight, 37 DEG C of 200rpm shake bacterium 2.5 hours, are 0.8 to OD600, it is lmM that the bacterium liquid that shakes adds inductor IPTG to final concentration, 37 DEG C were shaken bacterium after 5 hours, reclaimed thalline, namely obtained bacterial classification.
Collected bacterial classification is carried out enlarged culturing, collects thalline.SDS-PAGE detects contrast supernatant, precipitation.
Embodiment 3
The separation and purification of FoxO1 recombinant protein:
Thalline after a large amount of abduction delivering is through ultrasonic treatment thalline, and 4 DEG C of centrifugal 20min of 12000rpm, retain upper cleer and peaceful precipitation respectively.Supernatant fraction uses nickel post to carry out purifying, first go up low salt buffer balance columns material, then supernatant liquor upper prop, resuspended post material, ice bath 30min, discard effluent liquid, 15 times of column volume low salt buffer wash-outs, 10 times of column volume high-salt buffer wash-outs, 5 times of column volume low salt buffer wash-outs, 2 times of column volume elution, collect sample liquid.Western Blot detects purification result.
Embodiment 4
The qualification of Fugu rubripes (Temmincket Schlegel) restructuring FoxO1 albumen:
1.SDS-PAGE electrophoresis
The preparation separation gel of 8% and the concentrated glue of 5%.Sample after sample before induction and induction is respectively got 50 μ L add 20 μ L2 × SDS loading buffer respectively and mix, boiling water boiling 5 minutes, the centrifugal 10min of 12000rpm, leaves and takes supernatant and is SDS-PAGE.120V constant voltage electrophoresis 2h, coomassie brilliant blue R250 dye 2 hours, destainer decolouring.Observing has obvious band to occur at 105kDa place.As shown in Figure 2, M in figure: albumen Maker; Expressing protein after 1:pET32a (+) induction; Precipitation after 2:pET32a-FoxO1 inducing lysis; Supernatant after 3:pET32a-FoxO1 inducing lysis; Expressing protein after 4:pET32a-FoxO1 induction.
2.Western Blot identifies
Cut from SDS-PAGE glue the part needing transferring film, and shear 6 filter paper and 1 pvdf membrane.Pvdf membrane and filter paper are immersed in transfering buffering liquid, balance 5 minutes.Put correctly from anode to negative electrode by the order of paper, film, glue, paper, connect power supply, note positive and negative electrode.0.8mA/cm2 making current is pressed, electrotransfer 1.5h according to gel area.After transfer terminates, cut off the electricity supply, from top to bottom stripping assembly, remove each layer one by one.Pvdf membrane is marked.Gel is taken out and carries out antibody labeling.PVDF is placed in the plastics bag of sealing, inside adds the Anti-HisAntibody (GE Healthcare) of confining liquid and 1:1000 dilution, incubation 1h in 37 DEG C of shaking tables.Incubation 1h in 37 DEG C of shaking tables.Take out pvdf membrane, with PBS rinsing 3 times, each 10min.TBST rinsing 10min used again by filter membrane.
Pvdf membrane is placed in the plastics bag of sealing, inside adds the sheep anti mouse ELIAS secondary antibody of the HRP mark of confining liquid and 1:2000 dilution.Incubation 1h in 37 DEG C of shaking tables.Take out pvdf membrane, with PBS rinsing 4 times.Be placed on by pvdf membrane until there is band to occur (general 1-5min) in substrate solution, taking-up PVDF rinsing in water once, is placed in PBS.Observations, has a specific immunity band at 105KDa place.As shown in Figure 3, M in figure: albumen Marker; 1: the pET32a-FoxO1 after purifying.As can be seen from Figure 3, the recombinant protein after purifying and expection in the same size.
Embodiment 5
FoxO1 recombinant protein is on the impact of Fugu rubripes (Temmincket Schlegel) PPAR γ genetic expression:
Body cavity test injection is carried out to tail counterpoise 15-30 gram of Fugu rubripes (Temmincket Schlegel), fasting 24h before experiment, inject after Fugu rubripes (Temmincket Schlegel) light anaesthesia immediately, not inject any reagent for negative control group, to inject PBS for positive controls, to inject restructuring FoxO1 albumen for test group, test group injected dose is 3 μ g/g body weight, continuously injection three days, once a day, after testing one week, get three groups of Fugu rubripes (Temmincket Schlegel) fatty tissues and quantitative fluorescence analysis is carried out to PPAR γ expression.As shown in Figure 4, the test group Fugu rubripes (Temmincket Schlegel) fatty tissue PPAR γ expression amount of injection restructuring FoxO1 albumen significantly reduces than positive and negative control group concrete outcome.Positive and negative control group fin dongfang globe fish PPAR γ expression amount difference is not remarkable.Therefore inject FoxO1 recombinant protein and can reduce Fugu rubripes (Temmincket Schlegel) fatty tissue PPAR γ expression amount.
Claims (6)
1. based on a recombinant protein for Fugu rubripes (Temmincket Schlegel) FoxO1 gene, it is characterized in that: the aminoacid sequence as shown in SEQ ID NO:4.
2. the recombinant protein based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene according to claim 1, is characterized in that: nucleotide sequence coded by such as shown in SEQ ID NO:3 of described aminoacid sequence.
3., as claimed in claim 1 based on a preparation method for the recombinant protein of Fugu rubripes (Temmincket Schlegel) FoxO1 gene, it is characterized in that: comprise the following steps:
(1) reverse transcription PCR amplification Fugu rubripes (Temmincket Schlegel) fatty tissue total serum IgE, amplified production is connected to obtain recombinant plasmid with pGEM-T carrier, after double digestion goal gene again with the Pet-32a(+ after double digestion) expression vector is connected, acquisition recombinant expression vector; Wherein double digestion is for cut with restriction enzyme EcoR I and Xho I enzyme;
(2) recombinant expression plasmid is transformed into Host Strains, adds inductor IPTG abduction delivering FoxO1 recombinant protein;
The primer pair P of described reverse transcription PCR amplification is:
Upstream primer P1:5 '-CG
gAATTCaTGGCGGAAGCAGCCCA-3 ',
Downstream primer P2:5 '-CCG
cTCGAGcCCTGACACCCAGCTGTGC-3 ';
Wherein, in described step (1), the reaction conditions of reverse transcription PCR amplification is: 35 amplifications circulated carrying out 94 DEG C of sex change 40s, 61 DEG C of renaturation 50s, 72 DEG C of extension 2min after 94 DEG C of denaturation 5min, are stored in 4 DEG C after 72 DEG C of extension 10min;
The reaction system of reverse transcription PCR amplification is in described step (1): cDNA masterplate 1.0 μ l, 10 μMs of P1 1.0 μ l, 10 μMs of P2 1.0 μ l, 10 × PCR Buffer 2.5 μ l, 2.5mM dNTP Mixture 2.0 μ l, ddH
2o 17.0 μ l, 1 U/ μ l archaeal dna polymerase 0.5 μ l, amount to 25 μ l.
4. the preparation method of the recombinant protein based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene according to claim 3, is characterized in that: the Host Strains in described step (2) is E. coli expression strains Rosetta (DE3).
5. the preparation method of the recombinant protein based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene according to claim 3, is characterized in that: get the cellular lysate after inducing culture in described step (2), centrifugal, gets supernatant liquor purifying and namely obtains recombinant protein.
6. the application of recombinant protein in the pharmaceutical composition or feed of preparation adjustment Fugu rubripes (Temmincket Schlegel) fatty tissue PPAR γ expression amount based on Fugu rubripes (Temmincket Schlegel) FoxO1 gene as claimed in claim 1.
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