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Production method of recombined human alpha1-thymus peptide and preparation thereof

A production method and technology of thymosin, applied in the production method and preparation field of recombinant human α1-thymosin, can solve the problems of high price, difficult synthesis, unbearable for ordinary patients, etc., and achieve the effect of low cost and guaranteed consistency

Inactive Publication Date: 2007-07-04
SHANGHAI HUAXIN HIGH BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In recent years, foreign α1-thymosin peptide drugs are mostly prepared by chemical synthesis. The advantage of chemical synthesis is that the product has high purity and activity. However, traditional peptide synthesis methods can only synthesize peptides with less than 10 amino acids. For α1-thymosin with 28 amino acids For thymosin, the synthesis is very difficult, the yield is low, and it is difficult to meet the needs of the market
For example, the thymosin drug named "Ridaxian" contains 1.6 mg per tube, and the price is more than 800 yuan, and a course of treatment is more than 50,000 yuan. The price is too expensive for ordinary patients to bear.

Method used

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  • Production method of recombined human alpha1-thymus peptide and preparation thereof
  • Production method of recombined human alpha1-thymus peptide and preparation thereof
  • Production method of recombined human alpha1-thymus peptide and preparation thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Construction of genetically engineered bacteria highly expressing human α1-thymosin:

[0032] 1. Acquisition of the full-length base sequence of human α1-thymosin gene: According to the amino acid sequence of human α1-thymosin, the following DNA fragments were synthesized:

[0033] 5'-CCT CTA GAA ATA ATT TTG TTT AAC TTT AAG AAG GAG ATA TACATA TGT CTG GAT CAG GTC ATC ATC ATC ATC ATC ATT CTT CTG GTA CCGATG ACG ACG ACA AGA GCG ATG CCG CCG TGG ATA CCA GCA GCG AAATTA CCA CCA AAG ATC TGA AAG AAA AAA AAG AAG TGG TGG AAG AAGCCG AAA ACT AAG ACT AGT GAA TTC AC-3'

[0034] The fragment sequentially includes SD sequence, purification tag His-tag, enterokinase restriction site, full-length human α1-thymosin gene and stop codon.

[0035] 2. Digest the fragment with XbaI / EoRI double enzymes in a 37°C water bath, recover from the gel, and connect it with the large fragment recovered from pET-32a(+) XbaI / EoRI double enzyme digestion to construct the expression vector pET-32a-Tα1 (as sho...

Embodiment 2

[0038] Fermentation of genetically engineered bacteria

[0039] 1. Take and inoculate the constructed genetically engineered bacteria into 100ml LB medium (containing 100ug / ml ampicillin), and culture in a shake flask at 37°C for 12-14h; absorb the bacterial liquid according to 10% inoculum size and add it to 500ml LB medium , 37°C, 230r / min shake flask culture for 6h, complete the preparation of seed solution;

[0040] 2. In a 30-liter fermenter (Switzerland Bioengineering Company), the seed solution was added to a fermenter equipped with 12L M9+LB medium by 5% inoculum, cultivated at 37° C. at 300 r / min, and the ventilation rate was 10 L / min. Control the pH value at about 7.0, and control the dissolved oxygen above 30%. When the OD600 is about 12, start to add the feed medium. When the OD600 is about 18, add IPTG induction, and the final concentration is 0.8mmol / L , control the dissolved oxygen curve and pH to be basically stable but slightly decreased, and the bacteria wer...

Embodiment 3

[0042] Purification of recombinant human α1-thymosin (see Figure 2)

[0043] 1. Collect the bacteria, centrifuge at 6000-7000g in continuous flow or in batches, and the centrifugation time for each batch is 8-10 minutes; use the bacteria-breaking buffer [30mmol / L imidazole-200mmol / L NaCl-20mmol / L PB (pH8 .0)] suspending, carrying out high-pressure homogenization to destroy bacteria, the operating pressure adopted during the destruction of bacteria is 70Mpa~80Mpa, and centrifuging to remove impurities after the completion of microscopic inspection of bacteria;

[0044] 2. Prepare a metal nickel ion chelating affinity chromatography column, take Chelating Sepharose FF 180mL (Amersham Biosciences company) to pack the column, wash 3 column volumes with 50mmol / L NiSO4 solution and hang Ni 2+ ; The above-mentioned centrifuged supernatant (or supernatant concentrated by ultrafiltration) is loaded, equilibrated with a buffer [30mmol / L imidazole-200mmol / L NaCl-20mmol / L PB (pH8.0)] unti...

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Abstract

The invention discloses a method for preparing human alpha 1- thymosin and the human alpha 1- thymosin agent produced with said method. The method comprises: constructing gene engineering bacteria for human alpha 1- thymosin, liquid culturing gene engineering bacteria and fermentating, and puring the recombined human alpha 1- thymosin. The method is characterized by no limit to raw material source, low cost, feasibility for large- scale production, and the product can be used as injection agent for preventing and curing and assistant curing for disease caused by low immune function.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a production method and preparation of recombinant human α1-thymosin. Background technique [0002] α1-thymosin (Tα1) was first discovered in thymus tissue by Goldstein et al. in 1977. It mainly exists in thymocytes, T-lymphocytes and thymus tissue in vivo, and in liver, kidney, heart, lung, spleen, etc. It is also distributed in organs. In the body, it is produced by enzymatic hydrolysis of the precursor α1-thymosin composed of 111 amino acids. Mature human α1-thymosin consists of 28 amino acids, molecular weight 3.108KD, isoelectric point 4.2, and its sequence is: [0003] 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. [0004] The normal concentration of human α1-thymosin in human serum is about 540-670pg / mL, and its main physiological function is to regulate the immune activity of the body, inclu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/12C12P21/02C07K14/435C12N15/70C12N1/21A61K38/17A61K9/19
Inventor 常志远杨忠王伟
Owner SHANGHAI HUAXIN HIGH BIOTECH
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