Human recombinant amelogenin and preparation method thereof

A technology of amelogenin and fusion protein, which is applied in the protein field to achieve the effect of promoting expression, increasing solubility, and increasing protein stability

Inactive Publication Date: 2009-10-28
SHANGHAI NINTH PEOPLES HOSPITAL SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no artificially synthesized single-component human Am in China.

Method used

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  • Human recombinant amelogenin and preparation method thereof
  • Human recombinant amelogenin and preparation method thereof
  • Human recombinant amelogenin and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] A kind of recombinant human amelogenin preparation

[0039] I. Materials and methods for cloning human amelogenin mature peptide gene hAm encoded by X chromosome

[0040] 1. Main experimental materials

[0041] 1. Human tooth germ tissue: The laboratory of the Ninth People's Hospital Affiliated to Shanghai Second Medical University.

[0042] 2. Main reagents and solutions

[0043] (1) Total RNA Extraction Kit (Baicai Biotechnology Co., Ltd.)

[0044] (2) c DNA First Strand Synthesis Kit (Baicai Biotechnology Co., Ltd.)

[0045] (3) Small amount of glue recovery kit (Tiangen Company)

[0046] (4) PCR reagents: Taq DNA polymerase, 10× amplification buffer, MgCL 2 , 10mmol / L 4×dNTP (Pro omeg company)

[0047] (5) DEPC (Boca Biotechnology Co., Ltd.)

[0048] (6) DNA markers

[0049] (7) 6× agarose electrophoresis sample solution: consists of the following components: 2.5g / L bromophenol blue (adjusted to dark blue pH=8.0 with 0.5mol / L NaOH), 400g / L sucrose, 10mmol / L ...

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Abstract

The invention relates to protein, in particular to human recombinant amelogenin and a preparation method thereof. The human recombinant amelogenin is prepared by the following method: a, cloning human amelogenin mature peptide gene hAm coded by the X chromosome; b, constructing recombinant prokaryotic expression plasmid PGEX4T1-hAm containing the human amelogenin mature peptide gene; c, transfecting the recombinant prokaryotic expression plasmid PGEX4T1-hAm obtained from the step b into an expression strain E.coli.Rosetta; d, inducing the cloning expression strain obtained from the step c to express fusion protein GST-hAm; and e, purifying the fusion protein by GST affinity chromatography. The invention firstly obtains the encoding sequence of human amelogenin mature peptide from human deciduous dental germ through cloning, and successfully expresses the fusion protein in bacillus through the prokaryotic expression plasmid; and the artificially synthesized recombinant amelogenin can be obtained through the purification by the GST affinity chromatography.

Description

technical field [0001] The invention relates to a protein, in particular to a recombinant human amelogenin and a preparation method thereof. Background technique [0002] Enamel matrix proteins (enamel matrix proteins, EMPs) are a series of extracellular matrix proteins synthesized and secreted by ameloblasts differentiated from inner enamel epithelial cells during tooth development to the bell-shaped stage. Since it was first proposed in 1975 that enamel-associated proteins from the Hertwig's epithelial root sheath can induce the formation of acellular cementum in the tooth root, in vitro experiments and clinical treatments have widely confirmed that EMPs can effectively promote the periodontal ligament, cementum, tooth Alveolar bone regenerates, forming an arrangement similar to normal periodontal tissue, achieving true periodontal regeneration. Amelogenin (Am), the most important component of developing EMPs, is also the main active component in EMPs that promotes period...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/70
Inventor 束蓉程岚
Owner SHANGHAI NINTH PEOPLES HOSPITAL SHANGHAI JIAO TONG UNIV SCHOOL OF MEDICINE
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