GP thymosin alpha 1 and preparation method

A technology of thymosin and Escherichia coli, which is applied in the field of medical bioengineering, can solve the problems of environmental pollution and high cost of chemical synthesis, and achieve the effect of simple preparation method, low cost and good biological activity

Inactive Publication Date: 2005-08-31
SUN YAT SEN UNIV
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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 GP-thymosin α1, and the GP-thymosin α1 prepared by this method overcomes the disadvantages of high chemical synthesis cost and environmental pollution, and has better biological activity than natural thymosin α1

Method used

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  • GP thymosin alpha 1 and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Construction of fusion expression plasmid pTRX-GP-Tα1

[0014] 1. Reagents and materials

[0015] The vector plasmid pTRX was constructed by Dr. Peng Lisheng from the School of Life Sciences, Sun Yat-sen University, and has applied for a national invention patent with application number 00124832; Escherichia coli BL21 (DE3) was purchased from Stratagene, and the thymosin α1 gene was synthesized by Shanghai Boya Biotechnology Company ; Restriction endonuclease and T4DNA ligase were purchased from New England Company, PCR primers were synthesized by Shanghai Boya Biotechnology Company, and gel recovery kit was purchased from Omega Biotechnology Company.

[0016] 2. Method

[0017] (1) Oligonucleotide primer:

[0018] Upstream primer: 5’-GG GGTACC TCTGATGCTGCGGTCGAT-3’

[0019] Downstream primer: 5’-GTCAT GCGGCCGC GTTCTCGGCTTCTTCCACAA-3’

[0020] The upstream primer contains Kpn I restriction site (single underline), PreScission Protease (3C) recognition site (do...

Embodiment 2

[0026] Example 2: Fermentation of Escherichia coli engineered strain DE3 / pTRX-GP-Tα1

[0027] 1. Engineering bacteria and plasmids: engineering bacteria BL21(DE3) / pTRX-GP-Tα1;

[0028] 2. Medium:

[0029] LB medium (g / L) for the activation of first-level seed bacteria: peptone 10g, yeast powder 5g, NaCl 10g;

[0030] 2YT medium for activation of secondary seed bacteria (g / L): peptone 16g, yeast powder 10g, NaCl 5g;

[0031] Fermentation tank fermentation medium (g / L): peptone 12g, yeast powder 12g, NaCl 5g, KH 2 PO 4 4g, MgSO 4 ·7H 2 O1g, K 2 HPO 4 5g, 2g glucose; mix the above medium during fermentation;

[0032] Feeding carbon source (400ml): 40g glucose, MgSO 4 ·7H 2 O 3g;

[0033] Feed nitrogen source (g / L): 37g peptone, 37g yeast powder;

[0034]3. Activation of seed bacteria: streak the engineered strains preserved in -70°C and 20% glycerol, culture at 37°C for about 16 hours, pick a single clone, and inoculate it in 200ml LB medium (containing 100μg / ml ampicillin) In an Erl...

Embodiment 3

[0042] Example 3: Preparation and purification of recombinant GP-Tα1 polypeptide:

[0043] 1. Centrifuge the fermentation broth to collect the bacteria, and resuspend the bacteria with 50mM Tris-HCl (pH8.0), 0.5M NaCl solution at the ratio of 10ml solution / g wet bacteria. Branson 450 ultrasonic cell crusher was used to break the bacteria, the probe model is 1 / 2", ultrasonic 15-25min (ultrasonic 8sec, intermittent 4sec) at 70% power in ice bath. Centrifuge at 4℃, 10,000rpm for 30min (BECKMAN AVANTI TM J-25), collect the supernatant.

[0044] 2. Supernatant Ni 2+ -Chelating Sepharose affinity chromatography column, discarding the passing peak; eluting with Tris-HCl (pH 7.0), 0.5M NaCl, 20 mM imidazole solution, until the plateau; using Tris-HCl (pH 7.0), 0.5M NaCl, 150mM imidazole solution was eluted, and the elution peaks were collected; the Sephadex G-25 column was replaced with 20mM Tris-HCl (pH 8.0), 20mM NaCl buffer, and then on the Q Sepharose Fast Flow column, with 50mM Tris-...

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Abstract

A GP-thymin alpha 1 (GP-T alpha 1) is prepared through chemically synthesizing thymin alpha 1 gene, cloning it to karyogamy expression carrier pTRX, configuring fusion expression plasmid pTRX-GP-T alpha 1, adding proteinase ReScission Protease (3C) recognition site between pTRX and Talpha 1, expressing thioredoxin-T alpha 1 fusion protein, purifying by chromatography, enzyme severing of Pre Scission Protease (3C) to release GP-Talpha 1, affinity chromatography, and HPLC purifying. It can be used to prepare the medicines for treating viral hepatitis, tumor and AIDS.

Description

Technical field [0001] The invention relates to the technical field of medical bioengineering, in particular to a GP-thymosin α1 and a preparation method thereof. Background technique [0002] Thymosin α1 (thymosin α1, Tα1 for short) is a polypeptide composed of 28 amino acid residues. It has immunological activity and has a wide range of clinical applications, such as the treatment of viral hepatitis and immune regulation. The amino acid sequence of natural Tα1 is NH2-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-terminal is acetylated (Goldstein AL, Low TL, McAdoo M, McClure J, Thurman GB, Rossio J, Lai CY, Chang D, Wang SS, Harvey C, Ramel AH, 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, Tα1 monomer has only one source, that is, it is fully synthesized by solid-phase synthesis, which...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/18A61P31/18A61P35/00C07K14/435C07K14/47C12N15/12C12N15/63C12P21/02
CPCY02A50/30
Inventor 徐安龙姜孝玉李靖于琳陈尚武王磊董美玲
Owner SUN YAT SEN UNIV
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