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Polyethylene glycol modified human thymosin beta 4 tandem repeat protein, as well as preparation method and application thereof

A technology of polyethylene glycol and methoxypolyethylene glycol, which is applied in the field of preparation of human thymosin β4 dimer protein, which can solve the problems of unfavorable prokaryotic expression system expression and purification, inability to produce on a large scale, and synthesis efficiency Low-level problems, to achieve the effect of promoting wound healing, promoting the proliferation of cardiomyocytes, and preserving the activity

Inactive Publication Date: 2020-04-07
HARBIN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The human thymosin β4 used at this stage is mainly derived from chemical synthesis; however, the chemical synthesis of a polypeptide with a length of 43 amino acids has disadvantages such as low synthesis efficiency, high cost, serious environmental pollution, and inability to produce on a large scale.
The genetic engineering product of human thymosin β4 is not conducive to the expression and later purification of prokaryotic expression system because of its small molecular weight

Method used

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  • Polyethylene glycol modified human thymosin beta 4 tandem repeat protein, as well as preparation method and application thereof
  • Polyethylene glycol modified human thymosin beta 4 tandem repeat protein, as well as preparation method and application thereof
  • Polyethylene glycol modified human thymosin beta 4 tandem repeat protein, as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Preparation of Human Thymosin β4 Ditandem Protein (rTβ4)

[0069] 1. Construction of pET-28a(+)-rTβ4

[0070] Fusion of human thymosin β4 with HKDCDI gene sequence, GS linker gene sequence, 6*His tag gene sequence, and after codon preference optimization, a new gene sequence (shown in SEQ ID NO.1) is formed. The connection sequence is: HKDCDI gene sequence-Thymosin β4 gene sequence-GS Linker GSGSG-Thymosin β4 gene sequence-6*His tag gene sequence, the gene sequence is chemically synthesized with NcoI (enzyme cutting site CCATG / G to avoid When the frameshift gene is synthesized, the base G) and XhoI (C / TCGAG) are removed as the restriction site and cloned into the expression vector pET-28a(+) by homologous recombination to obtain the recombinant plasmid pET-28a(+)-rTβ4. The gene sequence synthesized by codon optimization after recombination is:

[0071]

[0072] among them:

[0073] NcoI and XhoI restriction sites are CCATG, CTCGAG

[0074] The HKCDI gene sequence i...

Embodiment 2

[0093] Example Preparation of di-polyethylene glycol modified human thymosin β4 distrand protein (PEG-rTβ4)

[0094] The PEG-rTβ4 (n=2000, n=12000, n=20000) protein prepared in the present invention includes the following steps:

[0095] 1. Combine the obtained rTβ4 protein with methoxy polyethylene glycol maleimide (hereinafter referred to as mPEG 2000 -MAL, mPEG 12000 -MAL, mPEG 20000 -MAL) and tris(2-carboxyethyl)phosphine (hereinafter referred to as TCEP) according to 1:1:4, 1:15:6, 1:40:8 (w:w:w) dissolved in PBS (pH8.0 ), each milliliter of PBS solution contains rTβ4 protein, mPEG n- MAL and TCEP are respectively 1mg, 1mg, 4mg, 1mg, 15mg, 6mg, 1mg, 40mg, 8mg, 8℃ shaker (200rpm / min) reaction overnight;

[0096] 2. The reaction product was replaced with PBS (pH 8.0) by a dialysis device, and PEG-rTβ4 protein was purified with AKTA Prime Plus; firstly, the gel column was fully equilibrated with PBS (pH 8.0) with 5 column volumes, and then The protein solution prepared in the prev...

Embodiment 3

[0102] Example 3 MALDI-TOF molecular weight determination of rTβ4 protein and PEG-rTβ4 (n=2000) protein

[0103] In this experiment, the freeze-dried powder of rTβ4 protein and PEG-rTβ4 protein was mailed to Shanghai Zhongke New Life Biotechnology Co., Ltd. for MALDI-TOF molecular weight determination. The results showed that the determination results were consistent with the expected molecular weight and purity of rTβ4 protein and PEG-rTβ4 protein. Higher (see Picture 10 , Picture 11 ).

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Abstract

The invention discloses a polyethylene glycol modified human thymosin beta 4 tandem repeat protein (PEG-rTbeta4), as well as a preparation method and application thereof. The PEG-rTbeta4 is obtained after recombinant human thymosin beta 4 tandem repeat protein is subjected to specific chemical modification with polyethylene glycol, wherein the rTbeta4 has gene sequence codes connected in the following sequence: a HKCDI gene sequence, a thymosin beta4 gene sequence, a GS Linker GSGSG-thymosin beta4 gene sequence and a 6*His label gene sequence. Furthermore, the invention discloses a method forpreparing the protein. The preparation method can be used for preparing target protein having the purity of 90 percent or more, has the advantages of low cost, high bioactivity and the like, and is simple and quick. Experiments prove that the PEG-rTbeta4 protein has the effects of promoting proliferation and migration of myocardial cells, resisting apoptosis, promoting hair growth and angiogenesisand accelerating wound healing. Therefore, the PEG-rTbeta4 protein has a wide application prospect in heart function recovery after myocardial infarction, hair growth promotion and wound healing acceleration.

Description

Technical field [0001] The present invention relates to a polyethylene glycol-modified human thymosin β4 distrand protein (PEG-rTβ4 for short), and also relates to a preparation method and application of the protein. The invention belongs to the field of medical biotechnology. Background technique [0002] Human Thymosin β4 is a small acidic peptide composed of 43 amino acids, with a molecular weight of 5053 and an isoelectric point of 5.1. It has fewer hydrophobic amino acids and exists and functions as a single chain. It is highly conserved in species and is widely distributed in most cells except red blood cells. It participates in many important life activities of the human body, can promote wound healing, promote hair growth, anti-inflammatory, promote tissue repair, anti-endotoxin shock, promote angiogenesis, maintain the dynamic balance of cytoskeleton, etc.; especially in the field of cardiovascular diseases. Is widely concerned. [0003] Polyethylene glycol (hereinafter...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C07K1/113C07K1/16A61K38/22A61K47/60A61P17/02A61P17/14A61P9/10C12R1/19
CPCA61K38/2292A61K47/60A61P9/10A61P17/02A61P17/14C07K14/57581C07K2319/21C12N15/70C12N2800/22
Inventor 彭海生杜智敏柴彦群李明慧刘肖莹张文远
Owner HARBIN MEDICAL UNIVERSITY
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