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Sika deer IGF (Insulin-like Growth Factor)-1 polypeptide and preparation method thereof

A technology of IGF-1 and sika deer, applied in the biological field, can solve the problems of expensive enzyme reagents, affecting protein activity, and greatly affecting the refolding of small molecular weight proteins.

Inactive Publication Date: 2012-07-18
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the pET-32a expression product contains specific cleavage sites for enterokinase and thrombin, the enzyme reagents are expensive and unfavorable for industrial production, and after cleavage, there will be dozens to dozens of additional amino acid sequences upstream of the target protein , which has a great influence on the renaturation of small molecular weight proteins and directly affects protein activity

Method used

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  • Sika deer IGF (Insulin-like Growth Factor)-1 polypeptide and preparation method thereof
  • Sika deer IGF (Insulin-like Growth Factor)-1 polypeptide and preparation method thereof
  • Sika deer IGF (Insulin-like Growth Factor)-1 polypeptide and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Cloning of sika deer IGF-1 mature peptide gene

[0026] The total RNA of sika deer antler tissue (from Jilin Agricultural University Experimental Deer Farm) was extracted and reverse-transcribed into double-stranded cDNA. According to the sequence of sika deer IGF-1 gene in GenBank database (accession number: HQ890468), IGF-1 mature peptide primers were designed. Upstream primer: TTC GAATTCAAC GGACCCGAGACCCTCTG, add EcoRI restriction site and base sequence encoding Asn at the 5 end; downstream primer: CGC AAGCTTCTA GGCCGCCTTGGTGG, add a Hind III restriction site and a stop codon sequence at the 5-end; use the cDNA of sika deer antler tissue as a template for PCR amplification; the PCR reaction conditions are: 94°C pre-denaturation for 5 minutes, 94°C denaturation for 30 seconds, and 55°C annealing 30s, 72°C extension for 30s for 35 cycles, and finally 72°C extension for 10min. The PCR product was recovered using a DNA gel recovery kit, and the recovered fr...

Embodiment 2

[0028] Construction and identification of embodiment 2 recombinant expression vector

[0029]Digest the pET-32a vector and the correctly sequenced pMD-IGF-I plasmid with EcoRI and Hind III, recover them separately, and connect them with T4 DNA ligase overnight at 16°C to obtain recombinant plasmids, which are transformed into Escherichia coli Rosetta competent cells Spread on LB plates and incubate overnight at 37°C. Positive clones were selected for double-enzyme digestion identification, and then sent to TaKaRa Company for sequencing after correct identification. The clone with correct sequencing was named pET-32a-IGF-1.

[0030] The prokaryotic expression vector pET-32a-IGF-1 was digested with EcoR I and Hind III to obtain a specific band with a size of about 234bp, which was consistent with the expected size. The results showed that the sika deer IGF-1 gene was successfully inserted into the pET-32a vector, and the prokaryotic expression vector pET32a-IGF-1 was successful...

Embodiment 3

[0037] Example 3 Cutting, purification and protein renaturation of fusion protein

[0038] The fusion protein obtained above was diluted to a concentration of 1.0 mg / ml with hydroxylamine lysate (2mol / L hydroxylamine, 200mmol / LCHES, 8mol / L urea, pH9.5), and reacted at 45°C for 5h to cleave the fusion protein. The solution was adjusted to pH 8.0 with HCl to terminate the reaction, and after Ni-Agarose column affinity chromatography, the effluent was collected according to the 280 nm ultraviolet absorption value. The collected effluent was slowly added to the refolding solution (0.5mmol / L NaCl, 1mmol / L EDTA, 20mmol / L Tris-HCl, 1%Gly, 1mmol / L GSH, 3mmol / L GSSG, pH 8.0), so that The concentration of the recombinant protein was 0.15 mg / ml, renaturation at 4°C for 12 hours and then dialyzed to remove other impurities. SDS-PAGE electrophoresis of the recombinant protein pET32a-IGF-1 cut with hydroxylamine lysate showed that a specific protein band could be detected at about 7.5kDa; ...

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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 [0001] The invention belongs to the field of biological technology, in particular to sika deer IGF-1 polypeptide and a preparation method thereof. Background technique [0002] Antler is the second sex characteristic of deer, and it is a bony tissue that grows from the frontal bone of deer. Since it grows periodically every year, its growth and development may be regulated by special molecular mechanisms. That is, the uniqueness of velvet antler lies in its annual renewal, which is unique in mammals, so it becomes an ideal biological model for studying tissue and organ regeneration. The growth rate of velvet antler is unmatched by any other mammalian tissue, which can reach 1-2cm per day. Therefore, people speculate that there may be special active substances that promote the growth of bone tissue, which can stimulate the rapid growth of deer antler. Insulin-like growth factor (insulin-like growth factor, IGF) is a multifunctional cell proliferation regula...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/475C12N15/70
Inventor 胡薇孟星宇李婷李沐胡锐王健李雨婷田玉华刘宁
Owner JILIN AGRICULTURAL UNIV
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