Recombinant thymin alpha-1 and its prepn

A technology of thymosin and pgex-4t-1, applied in the field of medical bioengineering, can solve the problems of high chemical synthesis cost and environmental pollution

Inactive Publication Date: 2004-11-24
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The present invention adopts genetic engineering technology to prepare recombinant thymosin α1 to overcome the disadvantages of high chemical synthesis cost and environmental pollution

Method used

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  • Recombinant thymin alpha-1 and its prepn

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0007] Example 1: Construction of fusion expression vector pGEX-4T-1 / Tα1

[0008] 1. Reagents and materials

[0009] The vector plasmid pGEX-4T-1 was donated by Dr. Pan Xinghua, Department of Medical Genetics, Yale University, USA

[0010] Escherichia coli E.coli BL21(DE3)Ply S strain was purchased from Stratagene Company

[0011] Human Placenta Marathon-Roady TM -cDNA was purchased from Clontech Company

[0012] Restriction endonuclease and T4 DNA ligase were purchased from Shanghai Huamei Company

[0013] PCR primers were synthesized by Shanghai Jikang Biotechnology Co., Ltd.

[0014] Gel recovery kit was purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0015] 2. Method

[0016] (1) Design and synthesis of oligonucleotide primers: designed according to GenBank (accession number: S38640)

[0017] Upstream primer 5′-GG GGATCC GACGCAGCCGTAGAC-3′

[0018] Downstream primer 5′-GG GAATTC TTATTTTCTGCCTCTTCCAC-3′

[0019] The upstream primer contains a BamHI rest...

Embodiment 2

[0024] Embodiment 2: Fermentation of Escherichia coli engineering bacteria DE3 / pGEX-4T-1 / Tα1

[0025] 1. Take a small amount of Escherichia coli engineering bacteria DE3 / pGEX-4T-1 / Tα1 in 20ml LB medium containing 100μg / ml ampicillin (peptone 10g / L, yeast powder 5g / L, NaCl 5g / L) and shake at 37°C Cultivate overnight as the primary seed solution.

[0026] 2. Get the primary seed solution, inoculate it with 4% inoculum in 200ml LB medium containing 100 μg / ml ampicillin, and cultivate it with shaking at 37° C. for 6 hours in a Erlenmeyer shaker flask as the secondary seed solution.

[0027] 3. According to the 4% inoculum size of the working volume of the fermenter, insert the secondary seed liquid into the semi-synthetic medium [(every liter contains: tryptone 5g, yeast extract 5g, glycerol 10g, KH 2 PO 4 2g,K 2 HPO 4 4g, Na 2 HPO 4 12H 2 O 7g, (NH 4 ) 2 SO 4 1.2g, NH 4 Cl 0.2g)] and trace element solution (every liter containing: MnSO 4 ·5H 2 O 0.001g, CoCl 2 ·6...

Embodiment 3

[0030] Example 3: Preparation and purification of Gly-Tα1 protein:

[0031] 1. Centrifuge the fermented bacteria liquid to collect the bacteria, and use phosphate buffer solution (PBS: NaCl 140mmol / L KCl2.7mmol / L Na 2 HPO 4 10mmol / L KH 2 PO 4 1.8mmol / L (pH7.3) was centrifuged and washed once, and then 10ml of PBS was added for each gram of wet weight of bacteria to suspend the bacteria to precipitate.

[0032] 2. Ultrasonically break the bacteria in an ice bath, centrifuge, take the supernatant and pass it through the Glutathione Sepharose 4B column, discard the passing peak, and use 50mmol / L Tris-HCl, 10mmol / L reduced glutathione (pH8.0) Elution is carried out, and the elution peak is collected to obtain GST-Tα1 fusion protein.

[0033] 3. Add GST-Tα1 fusion protein to PBS or 50mmol / L Tris-HCl, 2.5mmol / L CaCl 2 , 150mmol / L NaCl (pH8.0) dialysis, add thrombin at a rate of 50-500μg fusion protein per unit of thrombin digestion, react at 25-30°C for 2-16 hours, and genera...

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Abstract

The present invention relates to the field of medicinal bioengineering technology and is a recombinant thymin alpha-1, that is Gly-T alpha-1, and its preparation method. The gene engineering process to prepare Gly-T alpha-1 includes PCR to amplify gene segment with 5'-GGGGATCCGACGCAGCCGTAGAC-3' as upstream primer, 5'-GGGAATTCTTATTTTCTGCCTCTTCCAC-3' as downstream primer and human placenta CDNA as template, EcoRI / BamHI double enzyme incision for directional cloning of destination gene to pGEX-4T-1 to constitue fused expression vector pGEX-4T-1 / T alpha-1. The prepared engineering bacterium can express glutathion S-transferase- thymin alpha-1 fusion protein and affinity chromatography purified fusion protein, and thrombase incision releases Gly-T alpha-1, which is finally purified by the affinity chromatography and ion exchange chromatography.

Description

Technical field: [0001] The invention relates to the technical field of medical bioengineering, and relates to a recombinant thymosin α1 and a preparation method thereof. Background technique: [0002] Thymosin α1 (thymosin α1, referred to as Tα1) is a polypeptide composed of 28 amino acid residues, which has immune activity and is mostly used clinically to treat viral hepatitis. The amino acid sequence of native Tα1 is NH 2 -Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu -Ala-Glu-Asn-COOH, N-terminus is acetylated [Goldstein A L, Low TL, McAdoo M, McClure J, Thurman GB, Rossio J, Lai C Y, Chang D, Wang S S, Harvey C, Ramel A H, Meienhofer. J. Thymosin alpha 1: isolation and sequence analysis of an immunological active thymic peptide. Proc Natl Acad Sci USA, 1977, 74(2): 725-729]. At present, there is only one source of Tα1 monomer, which is fully synthesized by solid phase synthesis, which is costly and easy to cause e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/32A61P37/02C07K14/575C12N15/16
Inventor 郭葆玉苗红章杰袁鹏群道书艳邱磊张冉
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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