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Preparation method of gene-recombination human thymosin beta 4

A gene recombination and thymosin technology, applied in the field of bioengineering, can solve the problems of high production cost and low purity of thymosin β4

Active Publication Date: 2012-09-12
SHAANXI HUIKANG BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to overcome the above-mentioned shortcomings of high production cost and low purity of thymosin β4, and provide a simple and effective preparation method of recombinant human thymosin β4 with high purity and low production cost

Method used

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  • Preparation method of gene-recombination human thymosin beta 4
  • Preparation method of gene-recombination human thymosin beta 4
  • Preparation method of gene-recombination human thymosin beta 4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] A recombinant human thymosin β4 fusion protein that uses type I human collagen as a leader peptide to guide the expression of human thymosin β4. The nitrogen terminal is a leader peptide consisting of 600 amino acids of type I human collagen, and the carbon terminal is 43 Amino acids, an enterokinase cleavage site, glutamic acid and phenylalanine for connection are added between the two amino acid sequences, after secreted expression, the recombinant human thymosin β4 is obtained by cutting with enterokinase in vitro, and the recombinant human The amino acid sequence of thymosin β4 is as described above.

[0127] The preparation method of the above-mentioned gene recombinant human thymosin β4 comprises the following steps:

[0128] 1. Acquisition of genes

[0129] Total RNA was extracted from isolated neonatal placental tissue, and the GeneCopoeia First-Strand cDNA synthesis Kit kit was used for reverse transcription according to the instructions in the instructions to...

Embodiment 2

[0241] In the purification step 6 of the genetically recombinant human thymosin β4 in Example 1, the fusion protein was cleaved by the enterokinase cleavage method. The enterokinase cleavage method was: the protein concentration was determined by the Coomassie brilliant blue method, diluted to 5 mg / ml, and added Recombinant enterokinase, cut 5 mg of fusion protein per unit of recombinant enterokinase, pH 6.0, cut at 16°C for 18 hours.

[0242] Ultrafiltration with an ultrafiltration membrane with a molecular weight of 10000D, collecting the filtrate, and desalting with an ultrafiltration membrane with a molecular weight of 1000D to obtain human thymosin β4 crude protein, other steps in this step are the same as in Example 1. The other steps were the same as in Example 1, and the recombinant human thymosin β4 was prepared.

Embodiment 3

[0244] In the purification step 6 of the genetically recombinant human thymosin β4 in Example 1, the fusion protein was cleaved by the enterokinase cleavage method. The enterokinase cleavage method was: the protein concentration was determined by the Coomassie brilliant blue method, diluted to 5 mg / ml, and added Recombinant enterokinase, cut 5 mg of fusion protein per unit of recombinant enterokinase, pH 7.0, cut at 16°C for 18 hours.

[0245] Ultrafiltration with an ultrafiltration membrane with a molecular weight of 40000D, collecting the filtrate, and desalting with an ultrafiltration membrane with a molecular weight of 1000D to obtain human thymosin β4 crude protein, other steps in this step are the same as in Example 1. The other steps were the same as in Example 1, and the recombinant human thymosin β4 was prepared.

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Abstract

The invention discloses a preparation method of gene-recombination human thymosin beta 4. The preparation method comprises the following steps of: acquisition of genes, connection of a carrier p PIC (positive-impedance converter) 9k and human-like collagen I and human thymosin beta 4 through pichia pastoris electrotransformation, selection of multi-copy insertion recombinants, fermentation of fusion protein of the gene-recombination human thymosin beta 4 with the pichia pastoris, and purification of the gene-recombination human thymosin beta 4. According to the method, the characteristic of high expression of the human-like collagen I in the pichia pastoris is utilized to guide the stable and efficient expression of the human thymosin beta 4 in the pichia pastoris. As enterokinase cuttingsites are introduced between leading peptide of the human-like collagen I of the fusion protein and the human thymosin beta 4, the problems, caused by small molecular weight, of the human thymosin beta 4 in the process of expression and purification are solved, the expression index is increased, the purification procedure is simplified, and the extraction and purification efficiency of the product is improved. The preparation method can be used for preparing the gene-recombination human thymosin beta 4.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a preparation method of recombinant human thymosin β4 expressed by Pichia pastoris. Background technique [0002] Thymosins are a lymphatic growth factor produced by the thymus, which was first discovered by Goldstein and White in 1966 from the extract of fetal bovine thymus protein. It is a group of small molecular polypeptides containing more than 40 components. According to the position of these polypeptides on the isoelectric focusing electrophoresis analysis map, they can be divided into three types: α, β, and γ: PI (α)<5, 5<PI (β)<7, PI (γ)>7. More than 20 β-thymosin isomers have been identified, and there are mainly three types of thymosin β4, thymosin β10 and thymosin β15 in the human body, among which thymosin β4 has the highest content. Thymosin β4 is a water-soluble polypeptide composed of 43 amino acids with a highly conserved structure. The N-te...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/16C12N15/81C07K14/575C07K1/34
Inventor 侯增淼高恩李晓颖李哲赵金礼
Owner SHAANXI HUIKANG BIO TECH CO LTD
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