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Method of preparing natural human thymosin a1 using series expression mode

A thymosin, a natural technology, applied in the field of biological polypeptide preparation, can solve the problems of high cost, complicated operation, environmental pollution, etc., and achieve the effect of simple operation, low cost and simple process

Active Publication Date: 2006-01-25
广东暨大基因药物工程研究中心有限公司
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the common method for preparing thymus α1 in China is to extract it from the thymus of animals. The disadvantage of this method is that the operation is complicated, the purity of the extracted Tα1 is not high, and the source is limited by the number of substances.
The commonly used method for preparing thymosin α1 abroad is chemical synthesis. The disadvantage of this method is that it is expensive and easy to cause environmental pollution.

Method used

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Examples

Experimental program
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Effect test

specific Embodiment 1

[0030] The method for preparing natural human thymosin α1 in a tandem expression mode in specific example 1. The previous preparatory work at first comprises the following steps:

[0031] (1) Translate the 28 amino acids contained in natural human thymosin α1 into the following sequence according to the customary codons of Escherichia coli:

[0032] TCTGATGCTGCTGTAGATACTTCTTCTGAGATTACTACTAAAGACCTAAAGGAGAAGAAGGAAGTTGTCGAAGAGGCTGAGAAC

[0033] AGACTACGACGACATCTATGAAGAAGACTCTAATGATGATTTCTGGATTTCCTCT

[0034] TCTTCCTTCAACAGCTTTCTCGACTCTTG;

[0035] (2) Add the corresponding nucleotides of nucleic acid restriction endonuclease Hind III, methionine, nucleic acid restriction endonuclease BamH I, asparagine, glycine in front of the natural thymosin α1 sequence; The nucleotides corresponding to glycine, nucleic acid restriction endonuclease Bgl II, terminator, nucleic acid restriction endonuclease Xho I are added behind the sequence;

[0036] (3) The two fragments are annealed and e...

specific Embodiment 2

[0083] Except that the cleavage conditions in step (13) were adding hydroxylamine to make the final concentration to 1.0 M, adjusting the pH to 6.0, and reacting at 37° C. for 60 h, the others were the same as in Example 1.

specific Embodiment 3

[0084] Except that the cleavage conditions in step (13) are adding hydroxylamine to make the final concentration to 2.0 M, adjusting the pH to 7.0, and reacting at 45° C. for 72 hours, the others are the same as in Example 1.

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PUM

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Abstract

The invention discloses a method to manufacture biology polypeptide, especially a method to make natural human thymosin by gene series express method. It contains compounding two polynucleotide section, compounding double enzyme cut, T alpha 1 gene three times connecting in series, constructing high efficiency expression, constructing engineering fungus, expressing T alpha 1 polypeptide six series bodies in engineering fungus, purifying and cracking. The invention could abundantly express aim albumen, and it has simple technology, easy to operate and low cost.

Description

technical field [0001] The invention relates to a method for preparing biological polypeptides, in particular to a method for preparing natural human thymosin α1 by means of gene serial expression. Background technique [0002] Thymosin α1 (Thymosin-α1, Tα1) is a polypeptide composed 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. Its amino acid sequence 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 is an immune enhancer targeting T lymphocytes. It can make T cells mature and differentiate, and can promote mature T cells and NK cells to secrete various lymphokines such as interleukin-2 and γ-interferon. -2 receptor (IL-2) production. [0003] As an immune enhancer, Tα1 is mainly used for immunodeficiency and immunosuppressive diseases. When a patient is infected with certain viruses su...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/12C12N15/63C12N15/66C12P21/02
Inventor 龚兴国周亮曾冬云谢毅
Owner 广东暨大基因药物工程研究中心有限公司
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