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Method for preparing thymosin polypeptide by using interin

A technology of thymosin and intein, which is applied in the field of preparation of thymosin polypeptides, can solve problems such as fragmentation and inhomogeneity of polypeptide products, and achieve the effects of simple process, easy operation and industrialization, and simple reaction conditions

Inactive Publication Date: 2012-09-19
SHANGHAI ENTS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a method for preparing thymosin polypeptide, which is used to express thymosin and intein fusion, so as to overcome the difficulty of expressing the polypeptide in a commonly used prokaryotic expression system such as Escherichia coli because of its too small molecular weight, and even if it can The expression is also caused by the hydrolysis of proteases in the host, which leads to the problem of inhomogeneity of polypeptide products, and the later purification only needs to change the pH of the fusion protein solution and other conditions, which can break the intein and thymosin and release thymosin

Method used

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  • Method for preparing thymosin polypeptide by using interin
  • Method for preparing thymosin polypeptide by using interin
  • Method for preparing thymosin polypeptide by using interin

Examples

Experimental program
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Effect test

Embodiment 1

[0033] 1. Construction of pET32a / intein / Thymosin α-1 expression plasmid.

[0034] Synthesize thymosin alpha 1 (Thymosin α-1) gene, which contains LguI at the 5' end and XholI restriction site at the 3' end, and clones the synthesized gene into the pMD18-T vector (the above steps are all completed by the gene synthesis company ).

[0035] The resulting pMD18-T Vector / Thymosinα-1 plasmid was transformed into Escherichia coli DH5α competent cells. The transformation method is as follows: take 1 μL plasmid and add it to 100 μL E. coli DH5α competent cells, place on ice for 30 minutes; then place in a water bath at 42°C for 90 seconds, and then return to ice for 3-5 minutes; then add 800 μL LB Culture medium (Luria-Bertani medium), incubate at 37°C for 45min; then centrifuge at 2500rpm for 5min to collect the bacteria, discard 700 μL of medium, and spread the remaining 200 μL of medium on an agarose plate containing 50 μL / mL ampicillin for culture overnight. Pick the single clo...

Embodiment 2

[0046] 1. Construction of pTwin-1 / Thymosin α-1 expression plasmid.

[0047] As described in Example 1, the pMD18-T Vector / Thymosin α-1 plasmid was purchased from a gene synthesis company, transformed into Escherichia coli DH5α competent cells, and then the plasmid was extracted with a plasmid extraction kit, and Thymosin α-1 was obtained by PCR amplification Gene, the obtained target gene fragment is 90bp.

[0048] Digest the pTwin-1 plasmid with LguI and PstI. The reaction system is: 10 μL of pMD18-T Vector / Thymosin α-1 plasmid, 2 μL of digestion buffer, 1 μL of NcoI and XholI enzymes, add water to 20 μL, and incubate at 37°C React for 15 minutes. The double-digested product was purified by agarose gel electrophoresis and recovered with a gel extraction kit. The fragment size was 7375bp.

[0049] Ligate the recovered target gene fragment and the pTwin-1 vector digested with LguI and PstI (treated with CIAP to remove the blunt-end phosphate group) with T4-DNA ligase to for...

Embodiment 3

[0053] Example 3: In vitro acetylation of recombinant thymosin 28 peptide.

[0054] The molecular weight of thymosin α-1 is 3108Da, and the molecular weight of unacetylated thymosin is 42 smaller than it, which is 3066. See image 3 shown.

[0055] Use acetic acid to adjust the pH of the thymosin α-1 28 peptide solution obtained in Example 1 or Example 2 to 2-3, then add acetic anhydride at a rate of 2 μL of acetic anhydride per 10 mL of solution every 10 min, and keep on ice Stir the reaction. The reaction was monitored by RP-HPLC, and the reaction was stopped when acetylated thymosin was no longer accumulated. The natural structure thymosin α-1 was purified by RP-HPLC. see Figure 4As shown, Ⅰ, Ⅱ, Ⅲ, and Ⅳ in the figure respectively represent fusion protein cleavage, fusion protein cleavage purified by DEAE, purified thymosin 28 peptide subjected to in vitro acetylation reaction, and RP-HPLC purification after acetylation reaction peaks 1, 2, and 3 represent unacetylate...

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Abstract

The invention discloses a method for preparing thymosin polypeptide by using interin. The method comprises the following steps of: 1, synthesizing a thymosin alpha-1 gene fragment; 2, fusing a synthesized thymosin alpha-1 gene into a purification tag gene and an interin gene by using nucleate endonuclease and ligase to obtain a fusion protein recombinant gene and introducing into a host cell; 3, fermenting the host cell to obtain thymosin fusion protein; 4, purifying the fusion protein obtained by the step 3 by using a purification tag; 5, cutting the fusion protein obtained by the step 4 to obtain a thymosin 28 peptide monomer; 6, acetylating the 28 peptide monomer to obtain thymosin alpha-1 with a natural structure; and 7, purifying the thymosin alpha-1 with the natural structure. According to the method for preparing thymosin polypeptide provided by the invention, polypeptide can be easily expressed in the host cell; and the method has the advantages of simple process, low cost, easiness in operation and industrialization and no environmental pollution.

Description

technical field [0001] The present invention relates to a method for preparing thymosin polypeptide, in particular to a method for fused expression of thymosin α-1 and intein, and the fusion protein is cleaved under the mediation of intein to release thymosin polypeptide. A method for preparing a thymosin polypeptide containing a peptide. Background technique [0002] Thymosin alpha 1 (Thymosin α-1) is a polypeptide consisting of 28 amino acid residues, with an isoelectric point of 4.2, no secondary structure and no disulfide bond, and the only modification is N-terminal acetylation. The amino acid sequence of thymosin α-1 is: N-Ac-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-C, which is an immune enhancer for T lymphocytes, can make T cells mature and differentiate, and can promote mature T cells, NK cells secrete various lymphokines such as interleukin-2 and γ-interferon, and can also promote the product...

Claims

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Application Information

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IPC IPC(8): C12N15/16C12N15/62C07K14/575C07K1/22C07K1/20
Inventor 周亮祝君洪亮
Owner SHANGHAI ENTS BIOTECH
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