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Recombinant thymosin beta 4 two repeat protein and preparation thereof

A thymosin and protein technology, applied in the field of thymosin β4 dimer protein and its preparation, can solve the problems of complex purification process, unstable expression of expression products, increased cost, etc., achieve low immunogenicity and overcome the complexity of purification process costly effect

Inactive Publication Date: 2009-05-20
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Abstract
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Problems solved by technology

[0048] As mentioned above, although different expression vectors of thymosin β4 have been constructed to express and purify fusion or chimeric proteins, and have certain biological activities, they also have the following defects: the purification process is complicated and costly, and the expression product is unstable.
Fusion proteins with protein bridges need to be digested and purified again; the purification process of recombinant proteins with His tags requires expensive nickel column affinity purification, which greatly increases the cost and is not suitable for industrial production; and His tags affect recombinant proteins Applications in Clinical Trials

Method used

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  • Recombinant thymosin beta 4 two repeat protein and preparation thereof
  • Recombinant thymosin beta 4 two repeat protein and preparation thereof
  • Recombinant thymosin beta 4 two repeat protein and preparation thereof

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Embodiment Construction

[0079] The present invention utilizes Escherichia coli preferred codons to synthesize the full-length gene of human thymosin β4, combined with PCR technology to construct the human thymosin β4 distring gene expression vector pET-22b(+)-Tβ4②, and transform the recombinant plasmid into Escherichia coli BL21 competent Cells, after IPTG-induced expression, purified the recombinant protein by salting out, hydrophobic interaction chromatography and ion exchange interaction chromatography, and finally obtained high-purity human thymosin β4 dimer protein, and used Western blot and matrix-assisted laser desorption ionization It was identified by time-of-flight mass spectrometry (MALDI-TOF-MS), and its activity was determined by spleen lymphocyte proliferation assay.

[0080] Specifically follow the steps below:

[0081] 1) Construct thymosin β4 diplex gene expression vector or recombinant plasmid pET-22b(+)-Tβ4②

[0082] a) Construction of pET-22b(+)-Tβ4①

[0083] According to the pr...

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Abstract

The invention relates to recombinant thymosin Beta 4 two-repeat protein and a preparation method thereof. Human thymosin Beta 4 two-repeat gene expression vector pET-22b(+)-T Beta (2) is constructed through recombinant human thymosin Beta 4 full length cDNA combined with PCR technology, human thymosin Beta 4 that can not be expressed directly in colibacillus is highly actively expressed in colibacillus in the form of two-repeat and purified human thymosin Beta 4 two-repeat protein has biologic activity, can promote multiplication of lymphocyte of mice, has low immunogenicity and lays a foundation for the further research and wide application of human thymosin Beta 4.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the construction, prokaryotic expression, purification and identification of recombinant plasmids of target proteins, in particular to thymosin β4 (Tβ4) dimer protein and its preparation method. Background technique [0002] 1. Biochemical properties of thymosin β4 [0003] 1.1 Thymosin β family [0004] Thymosin is mainly produced by the thymus. It was originally prepared by Goldstein from the fetal bovine thymus protein extract. It contains more than 40 polypeptide components. According to the position of these polypeptides on the isoelectric focusing electrophoresis analysis map, they are divided into α, β, and γ. Three regions: pI<5 in the α region, pI in the β region between 5 and 7, and pI in the γ region>7, among which the polypeptide located in the β region is called the β group thymosin, and its structure is highly conserved, consisting of 40 to 44 Compose...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/66C12N15/16C12N15/70C07K1/20C07K1/18
Inventor 张英起韩苇李维娜张珍
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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