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Human thymosin β4 triploid protein, coding gene and separation and purification method thereof

A technology for separation and purification and thymosin, which is applied in the field of genetic engineering technology research, can solve the problems of increasing separation and purification process steps and costs, and achieve the effects of easy separation and purification, improving yield and promoting hair regeneration.

Inactive Publication Date: 2016-09-07
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Increased separation and purification process steps and costs

Method used

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  • Human thymosin β4 triploid protein, coding gene and separation and purification method thereof
  • Human thymosin β4 triploid protein, coding gene and separation and purification method thereof
  • Human thymosin β4 triploid protein, coding gene and separation and purification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] This example is the construction of engineering bacteria of recombinant human thymosin β4 tandem triploid.

[0058] (1) Entrust Dalian Baobio Bioengineering Co., Ltd. to synthesize the gene sequence containing 3 copies of human thymosin β4, clone the synthesized sequence in the cloning vector pUC18 and provide E.coli cells (JM109) for recombining the vector;

[0059] (2) Plasmid preparation: Culture the E.coli bacterial cells of the recombinant target gene carrier provided by the company at 37°C and 200rpm / min for 12-16h, centrifuge at 12000rpm / min for 30-60s to collect the bacterial cells, and use commercially available plasmids Extract the kit to prepare high-purity plasmid DNA, and adjust the DNA concentration to 1 μg / μL;

[0060] (3) Nucleic acid restriction endonuclease digestion: In the 50 μL reaction system, add 5 μL nucleic acid restriction endonuclease (NcoI and XhoI 2.5 μL each), 5 μL 10× nucleic acid restriction endonuclease reaction buffer, 40 μL previous st...

Embodiment 2

[0076] This example uses the engineered bacteria constructed in Example 1 to produce the human thymosin β4 triploid protein of the present invention. The affinity tag used in this example is a 6-histidine tag (ie His-tag). This tag was introduced when constructing the expression vector, directly using the His-tag carried by the vector. The column material is Ni chelated dextran.

[0077] (1) Induced expression of recombinant human thymosin β4 tandem triploid in engineering bacteria: inoculate activated recombinant human thymosin β4 tandem triploid engineering bacteria (or freshly transformed In the fermentation medium of mL antibiotics, cultivate to OD at 37℃, 250rpm / min shaker 600 When it reaches above 0.4, add the inducer IPTG, continue culturing on a shaker at 30°C and 250rpm / min for 2-4h, collect the bacteria by centrifugation at 8000rpm / min at room temperature, resuspend the bacteria in an appropriate volume of protein loading buffer, boil water Bath for 5-10 minutes, ...

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Abstract

The present invention relates to human thymosin β4 triploid protein, encoding genes and methods for isolating and purifying the same. The amino acid sequence of the human thymosin β4 triploid protein involved is shown in SED ID NO: 1; the coding gene sequence involved is shown in SED ID NO: 2; the separation and purification method involved is to use an affinity chromatography column Carry out separation and purification. The recombinant human thymosin β4 tandem triploid of the present invention can significantly improve the immune response performance of T-lymphocytes as detected by the rosette experiment; the mouse fur growth experiment shows that the recombinant human thymosin β4 tandem triploid can significantly promote hair removal agent treatment in mice Growth in the fur.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology research, and specifically relates to a production method for producing recombinant human thymosin β4 tandem triploids by using genetic engineering technology. Background technique [0002] The thymus is an important immune organ in the human immune system. It can secrete dozens of thymosins to regulate the immune function of the body. It is named after it was first extracted from the calf thymus. [0003] Dozens of thymosins recognized by humans are classified into three categories of thymosin α, β, and γ due to their different isoelectric points, and each category contains multiple subtypes. β group thymosin (thymosin-β) is a group of thymosin with an isoelectric point between pI5.0-7.0, with a size of 40-44 amino acid residues, a relative molecular mass of about 5kD, and a highly conserved sequence structure. There are more than ten kinds of β thymosins found in different species,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/575C07K1/22C12N15/16
CPCC07K14/57581
Inventor 陈凯季佳菲李红民陈富林王阳阳崔继红
Owner NORTHWEST UNIV
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