CN102586257A - Sika deer IGF (Insulin-like Growth Factor)-1 polypeptide and preparation method thereof - Google Patents
Sika deer IGF (Insulin-like Growth Factor)-1 polypeptide and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a gene of sika deer IGF (Insulin-like Growth Factor)-1 mature peptide. An EcoR I restriction enzyme cutting site and a base sequence of code Asn are added at the end 5 of a forward primer; a Hind III restriction enzyme cutting site and a sequence of a termination codon are added at the end 5 of a reverse primer; a base sequence 234bp gene for expressing the sika deer IGF-1 mature peptide is cloned; an expression vector, namely pET-32a-IGF-1 is constructed and is induced and expressed in Escherichia coli Rosetta to obtain the sika deer IGF-1 mature peptide. The EcoR I restriction enzyme cutting site and the Hind III restriction enzyme cutting site are introduced and an Asn is added in front of natural IGF-1, so that a specific cracking part of hydroxylamine is formed, and further the cutting cost of protein is reduced, the influence on protein renaturation caused by extra amino acid sequence is reduced and the state of the protein in a natural state is kept. The pET-32a as an expression vector is expressed in the Escherichia coli Rosetta, so that the protein content can reach over 50 percent of soluble protein of thallus; and through the detection by adopting an MTT (Methyl Thiazolyl Tetrazolium) method, the multiplication rate of N1H3T3 cells can be increased.
Description
Technical field
The invention belongs to biological technical field, is spotted deer IGF-1 polypeptide and preparation method thereof specifically.
Background technology
Pilose antler is the secondal sexual character of deer, is the sclerotin sex organization that goes out deer frontal bone minister.Because periodically grow every year, growing of it possibly receive the specific molecule mechanism regulation.The unique distinction that is pilose antler just is to upgrade its every year, and this is unique in Mammals, thereby it becomes the living model of ideal research organization neomorph.The fast of antler growth speed also is that other any mammalian tissues is incomparable, can reach 1-2cm every day.Therefore people infer the active substance that wherein possibly have special promotion osseous tissue growth, can stimulate the quick growth of pilose antler.(insulin-like growthfactor is a kind of multi-functional cell proliferation regulatory factor IGF) to rhIGF-1, because of its chemical structure and proinsulin is similar gains the name.Wherein IGF-1 is made up of 70 amino-acid residues, has the single chain polypeptide of internal secretion, autocrine and paracrine characteristic, the mainly synthetic and release by liver cell of the IGF-1 in the blood.IGF-1 is playing crucial effect aspect cell normal growth, fetal development, nerve growth and the energy metabolism, wherein clearer and more definite biological effect mainly contains two big types, i.e. promoting mitosis effect and para-insulin effect.IGF-1 is the key link of growth of animal axle, in the growth of animal regulation and control, plays an important role.The growth rate height correlation of discovering IGF-1 and pilose antler of Suttie etc., growth factor raises in the antler growth period deer blood plasma, this means that IGF-1 stimulates antler growth probably.Researchs such as Elliott show that a large amount of IGF-1 acceptors are arranged at the top of pilose antler, and further indirect proof IGF-1 has participated in the possibility that the antler growth growth is regulated.At present, from people and many animal body tissues, separated obtaining IGF-1, as on people, pig, chicken, promoting that about IGF-1 Study on Growth is more.But the expression and the activity research of similar northeast spotted deer IGF-1 mature peptide are fewer at home and abroad.
At present, people IGF-1 (hIGF-1) has been used to treat mellitus clinically, Regular Insulin supports multiple diseases such as syndrome, nanism and nervous system disorders, and has obtained good effect.Existingly both at home and abroad obtain the report of hIGF-1 through mammalian cell and escherichia coli expression, but exist expression amount low with purifying than problems such as difficulties.
The correlated series that the inventor has delivered according to GenBank designs spotted deer IGF1 gene specific primer and clones the IGF1 gene; Spotted deer IGF1 gene DNA sequence has been submitted GenBank, and utilized the quantitative Real-time PCR of relative fluorescence method to detect the transcriptional expression abundance of IGF1 different sites tissue on the pilose antler top.Successfully obtain spotted deer antler and organized IGFI full length gene coding region cDNA (the GenBank accession number is HQ890468); This gene length 465bp; The polypeptide of coding 154aa length; Carry out homology analysis with red deer, ox, sheep, horse, pig, people and mouse IGF1 gene and show that IGF1 gene and red deer, ox and sheep homology are the highest, nucleotide sequence is respectively 99.78%, 98.28% and 97.85%; Aminoacid sequence is respectively 99.35%, 98.05% and 97.40%.The variance analysis of relative fluorescence quantitative PCR finds, the IGF1 gene on the pilose antler top different sites organize expression all arranged.Wherein, at the expression level of precartilage and cartilaginous tissue apparently higher than corium and mescenchymal tissue (see " Northeast Forestry University's journal " 2011 the 39th volume o. 11ths, spotted deer IGF1 full length cDNA clone and in the expression of pilose antler tissue).
The pET serial carrier is in the present prokaryotic expression, uses expression system comparatively widely.Be connected with T7 phage re-reading system in the pET serial carrier, the resultant velocity of its mRNA is colibacillary 5 times, and then realizes efficiently expressing of target protein.Wherein, The pET-32a carrier contains the base sequence of a Trx; After connecting the purpose fragment, together express, not only can promote the formation of target protein disulfide linkage with the mode and the target protein of fusion rotein; Make target protein recover correct three-dimensional structure, but also can protect target protein to avoid the degraded of Bacillus coli cells endoproteinase.In addition, also contain histidine-taggedly in this carrier, can combine with metals ions such as cobalt or nickel specifically, improve protein purification efficient.Although contain the special cleavage site of enteropeptidase and zymoplasm in the pET-32a expression product; But enzyme reagent costs an arm and a leg; Be unfavorable for suitability for industrialized production; And the target protein upper reaches can have tens to tens extra aminoacid sequences after the cutting, and are very big for the renaturation influence of small molecular weight protein, directly influence protein-active.
Summary of the invention
The objective of the invention is, a kind of gene, mature peptide and plum blossom IGF-1 mature peptide preparation method thereof of spotted deer IGF-1 mature peptide is provided.
The gene of spotted deer IGF-1 mature peptide, its nucleotide sequence is shown in sequence table SEQ ID NO.1;
Spotted deer IGF-1 mature peptide, its aminoacid sequence is shown in sequence table SEQ ID NO.2.
The preparation method of plum blossom IGF-1 mature peptide, this method comprises:
1) use primer:
P1：TTCGAATTCAACGGACCCGAGACCCTCTG
P2：CGCAAGCTTCTAGGCCGCCTTGGTGG；
Extract total RNA of spotted deer antler tissue, reverse transcription becomes double-stranded cDNA, is template amplification;
2) amplified production inserts among the expression vector pET-32a, obtains expression vector pET-32a-IGF-1, in its transfection Escherichia coli Rosetta competent cell; The IPTG abduction delivering; Collect purified fusion protein;
3) with hydroxylamine cleavage liquid crack fusion protein, protein renaturation liquid renaturation, impurity elimination gets plum blossom IGF-1 mature peptide;
Described IPTG abduction delivering is that concentration is that the IPTG of 0.6mmol/L induces, and expresses 4 hours;
Described hydroxylamine cleavage liquid is the 2mol/L azanol, 200mmol/L CHES, 8mol/L urea, pH9.5;
Described protein renaturation liquid is 0.5mmol/L NaCl, 1mmol/L EDTA, and 20mmol/L Tris-HCl, 1%Gly, 1mmol/LGSH, 3mmol/L GSSG, pH 8.0.
The invention provides a kind of gene of spotted deer IGF-1 mature peptide; And add the base sequence of EcoRI restriction enzyme site and coding Asn at upstream primer 5 ends; Downstream primer 5 ' end adds Hind III restriction enzyme site and termination codon subsequence, has cloned the base sequence 234bp gene of expressing spotted deer IGF-1 mature peptide, has made up expression vector pET-32a-IGF-1; Abduction delivering in intestinal bacteria Rosetta has obtained spotted deer IGF-1 mature peptide.Introduce EcoRI, Hind III restriction enzyme site; Utilize the characteristics of first amino acid of spotted deer IGF-1 for Gly; Add an Asn in natural IGF-1 front, form the special cracking position (Asn-Gly peptide bond) of an azanol, reduced the albumen cutting cost; Reduce of the influence of additional amino acid sequence, keep the state of albumen under natural condition protein renaturation; Adopt pET-32a to make to express carrier and in intestinal bacteria Rosetta, express, protein content can reach more than 50% of thalline soluble protein, detects through mtt assay to show; Increase along with IGF-1 concentration; The NIH3T3 cell proliferation rate is and increases progressively trend, between 0ng/ml～200ng/ml, presents concentration dependent, finds through the analysis of flow cytometer cell cycle; Compare G with the cell of handling without IGF-1
0Phase and G
1The phase cell count reduces to some extent, and S phase cell count increases to some extent, proves that spotted deer IGF-1 has important effect to the quick growth of pilose antler, and there is confidential relation in the regeneration of IGF1 and pilose antler.
Description of drawings
Fig. 1 is the detection of IGF-1 gene PCR amplified production, 1.DNA Marker (DL2000); 2, the 3.PCR product.
The SDS-PAGE electrophoretic analysis of Fig. 2 pET32a-IGF-1 expression product, 1-6. is respectively 0.1,0.2,0.4,0.6,0.8,1.0mmol/L IPTG inductive expression product; 7. inductive pET32a-IGF-1 not; 8. protein relative molecular weight standard.
The SDS-PAGE electrophoretic analysis of Fig. 3 pET32a-IGF-1 cracking and purified product, 1. protein relative molecular weight standard; 2. hydroxylamine cleavage product; 3. purified product.
NIH3T3 cell cycle mutation analysis behind Fig. 4 IGF-1 processing 48h.
The NIH3T3 cell cycle mutation analysis that Fig. 5 handles without IGF-1.
Embodiment
The clone of embodiment 1 spotted deer IGF-1 mature peptide gene
Extract total RNA of spotted deer (from Jilin Agriculture University test deer field) pilose antler tissue, reverse transcription becomes double-stranded cDNA, according to spotted deer IGF-1 gene order in the GenBank DB (accession number: HQ890468), design I GF-1 mature peptide primer.Upstream primer: TTC
GAATTCAACGGACCCGAGACCCTCTG adds the EcoRI restriction enzyme site and the base sequence of the Asn that encodes at 5 ends; Downstream primer: CGC
AAGCTTCTAGGCCGCCTTGGTGG adds Hind III restriction enzyme site and termination codon subsequence at 5 ends; CDNA with the spotted deer antler tissue is that template is carried out pcr amplification; The PCR reaction conditions is: 94 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s and carry out 35 circulations, last 72 ℃ of extension 10min.Utilize dna gel to reclaim test kit and reclaim the PCR product, will reclaim fragment and be connected on the pMD-18T carrier, change in the escherichia coli DH5a, coated plate and in 37 ℃ of cultivations.Picking transforms bacterium colony, is inoculated in the LB liquid nutrient medium, and 37 ℃ of shaking culture are spent the night, and adopts alkaline lysis to extract plasmid in a small amount and will identify that also correct recombinant plasmid send the order-checking of TaKaRa company.
IGF-1 mature peptide gene PCR amplified production obtains a specific band about 234bp through electrophoresis, conforms to expected results (Fig. 1).Extract recombinant plasmid,, can obtain the electrophoretic band 234bp that conforms to purpose band size, show and clone successfully with EcoRI and Hind III double digestion plasmid pMD-18T-IGF-1.Analyze demonstration through BLAST, IGF-1 mature peptide gene sequencing is correct.
The structure and the evaluation of embodiment 2 recombinant expression vectors
With EcoRI and the Hind III double digestion pET-32a carrier pMD-IGF-I plasmid correct with order-checking; After reclaiming respectively, spend the night in 16 ℃ of connections, obtain recombinant plasmid with the T4DNA ligase enzyme; After in its transformed into escherichia coli Rosetta competent cell, coat the LB flat board, 37 ℃ of overnight cultures.Select positive colony and carry out the double digestion evaluation, send the order-checking of TaKaRa company, the clone's called after pET-32a-IGF-1 that checks order correct after evaluation is correct.
With EcoR I and Hind III double digestion prokaryotic expression carrier pET-32a-IGF-1, obtain size and be about the 234bp specific band, consistent with the expection size.The result shows that spotted deer IGF-1 gene successfully inserts the pET-32a carrier, and prokaryotic expression carrier pET32a-IGF-1 makes up successfully.
The abduction delivering of embodiment 2 recons and the purifying of fusion rotein
With identifying that correct pET-32a-IGF-1 recon is inoculated in the LB substratum; 37 ℃ be cultured to OD600 and be 0.4～0.6 after; 37 ℃ are down carried out abduction delivering with different IP TG concentration (final concentration be respectively 0.1,0.2,0.4,0.6,0.8,1.0mmol/L), and collection bacterium liquid carries out the SDS-PAGE electrophoresis detection behind the 4h; Inductive reorganization Rosetta does not confirm best IPTG concentration for contrast.
Recombinant expressed bacterium is inoculated in the LB substratum, after 37 ℃ of shaking culture are 0.4～0.6 to OD600, induces 4h with the optimum concn IPTG that confirms.12000g, 4 ℃ of centrifugal 1min collect thalline, add an amount of bacterial lysate; Ultrasonic disruption 2min precipitates with Tris-HCl (20mmol/L) imidazoles (10mmol/L); NaCl (0.5mol/L) solution flushing 2 times, the gained deposition is inclusion body behind the centrifugal 20min of 12000g.Inclusion body is dissolved in Tris-HCl (20mmol/L), urea (8mol/L), imidazoles (5mmol/L), NaCl (0.5mol/L) solution, dissolving inclusion body 1h in ice-water bath, the centrifugal 20min of 10000g gets supernatant with 0.45 μ m membrane filtration.Again after the Ni-Agarose post affinity chromatography after the balance, with Tris-HCl (20mmol/L), urea (8mol/L), imidazoles (0.5mol/L), NaCl (0.5mol/L) eluant solution is collected elution peak according to the 280nm ultraviolet absorption value.
With pET32a-IGF-1 plasmid transformation escherichia coli Rosetta expression strain, abduction delivering conforms to prediction fusion protein molecule quality size (Fig. 2) after SDS-PAGE electrophoresis detection result shows that have differential protein to take out of at about 28kDa place existing; When the IPTG final concentration was 0.6mmol/L, expressing quantity was the highest simultaneously.The SDS-PAGE electrophoresis detection shows behind the recombinant protein purification, at about 28kDa place 1 protein band is arranged, and consistent with fusion rotein pET32a-IGF-1 molecular mass, target protein content all can reach more than 50% of thalline soluble protein, and purity is higher.
Western blotting hybridization analysis
Behind detected sample SDS-PAGE electrophoresis, 60V, 0.5h electrotransfer are to pvdf membrane, and the skim-milk with 5% seals 2h.After the TBST washes clean, use the anti-people IGF-1 one of rabbit anti-(dilution in 1: 200), 37 ℃ are bred 1.5h, TBST washing 3 times; The goat anti-rabbit igg of HRP mark is two anti-(dilutions in 1: 500), and 37 ℃ are bred 1h, use the TBST washes clean at last, DAB colour developing post analysis result.Western blotting detected result can detect the differential protein band at about 28kDa place, conforms to expression of results, explains that the fusion rotein sequence of expressing is correct and has the antigenic activity of IGF-1.
Cutting, purifying and the protein renaturation of embodiment 3 fusion roteins
With hydroxylamine cleavage liquid (the 2mol/L azanol, 200mmol/LCHES, 8mol/L urea, pH9.5) fusion rotein of above-mentioned acquisition being diluted to concentration is 1.0mg/ml, 45 ℃ of reaction 5h are with crack fusion protein.With HCl solution is adjusted to the pH8.0 termination reaction, after Ni-Agarose post affinity chromatography, collects effluent again according to the 280nm ultraviolet absorption value.The effluent of collecting is slowly joined renaturation solution (0.5mmol/L NaCl, 1mmol/L EDTA, 20mmol/L Tris-HCI, 1%Gly; 1mmol/L GSH, 3mmol/L GSSG, pH 8.0) in; Making recombinant protein concentration is 0.15mg/ml, dialyses behind 4 ℃ of renaturation 12h, removes other impurity.Recombinant protein pET32a-IGF-1 after the cutting of hydroxylamine cleavage liquid can detect the differential protein band through the SDS-PAGE electrophoresis showed at about 7.5kDa place; Again behind affinitive layer purification, SDS-PAGE detects demonstration, and purified product has 1 protein band at about 7.5kDa place, conforms to nearly 100% (Fig. 3) of purity with expression of results.
The BA of embodiment 4 recombinant proteins detects
NIH3T3 cell with after the DMEM culture medium culturing recovery that contains 10% calf serum after waiting to grow into 80%, passes in 6 orifice plates, continues to cultivate 24h.After cultivating NIH3T3 cell 24h with the DMEM substratum that contains 2% calf serum is hungry, add the IGF-1 recombinant protein, make that its final concentration is respectively 0,100,200,400ng/ml.Continue to cultivate 24h, 48h, behind the 72h, mtt assay detects the propagation situation of cell.Select recombinant protein the best use of time and concentration according to MTT result, utilize flow cytometer to detect the variation of cell cycle.
Show through the mtt assay check and analysis: after adding different concns IGF-1, the NIH3T3 cell proliferation rate increases to some extent.And along with increasing progressively of IGF-1 concentration, propagation degree more obvious (table 1).Flow cytometer detects the cell cycle result and shows: after IGF-1 (concentration is 200ng/ml) handles NIH3T3 cell 48h, compare G with the control group of handling without IGF-1
1And G
0Phase cell per-cent is reduced to 45.4% (P＜0.05) by 76.0%, and S phase cell per-cent increases to 31.9% (P＜0.05) (Fig. 4 and Fig. 5) by 16.1%.
Table 1MTT method detects the influence of IGF-1 to NIH3T3 cell proliferation
Table1?The?influence?of?cell?proliferation?by?IGF-1
Annotate:
*P＜0 05,
*P＜0 01.
Claims (6)
1. the gene of spotted deer IGF-1 mature peptide, its nucleotide sequence is shown in sequence table SEQ ID NO.1.
2. spotted deer IGF-1 mature peptide, its aminoacid sequence is shown in sequence table SEQ ID NO.2.
3. the preparation method of plum blossom IGF-1 mature peptide, this method comprises:
1) use primer:
P1：TTCGAATTCAACGGACCCGAGACCCTCTG
P2：CGCAAGCTTCTAGGCCGCCTTGGTGG；
With total RNA of spotted deer antler tissue, reverse transcription becomes double-stranded cDNA, is template amplification;
2) amplified production inserts among the expression vector pET-32a, obtains expression vector pET-32a-IGF-1, in its transfection Escherichia coli Rosetta competent cell; The IPTG abduction delivering; Collect purified fusion protein;
3) with hydroxylamine cleavage liquid crack fusion protein, protein renaturation liquid renaturation, impurity elimination gets plum blossom IGF-1 mature peptide.
4. the preparation method of plum blossom IGF-1 mature peptide according to claim 3 is characterized in that: described IPTG abduction delivering is that concentration is that the IPTG of 0.6mmol/L induces, and expresses 4 hours.
5. the preparation method of plum blossom IGF-1 mature peptide according to claim 4 is characterized in that: described hydroxylamine cleavage liquid is the 2mol/L azanol, 200mmol/L CHES, 8mol/L urea, pH9.5.
6. according to the preparation method of claim 3,4 or 5 described plum blossom IGF-1 mature peptides, it is characterized in that: described protein renaturation liquid is 0.5mmol/L NaCl, 1mmol/L EDTA, 20mmol/L Tris-HCl, 1%Gly, 1mmol/L GSH, 3mmol/L GSSG, pH 8.0.
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| CN110305936A (en) * | 2019-08-06 | 2019-10-08 | 吉林农业大学 | The specificity amplification primer of sika deer microsatellite locus M009 a kind of and its application |
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| CN110305936A (en) * | 2019-08-06 | 2019-10-08 | 吉林农业大学 | The specificity amplification primer of sika deer microsatellite locus M009 a kind of and its application |
| CN110305936B (en) * | 2019-08-06 | 2022-02-22 | 吉林农业大学 | Specificity amplification primer of sika deer microsatellite locus M009 and application thereof |
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Application publication date: 20120718 |