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Method for preparing genetic engineering N-acetylated thymosin alpha1

A technology of genetic engineering and thymosin, which is applied in the field of preparing genetic engineering N-acetylated thymosin α1, can solve the problems of affecting biological activity and immunogenicity, and achieve the effect of convenient purification and reduced complexity

Active Publication Date: 2010-06-16
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

It has been found that N-acetylated prothymosin alpha can be prepared by genetic engineering Escherichia coli (Wu J, Chang S, Gong X, Liu D, Ma.Q. Identification of N-terminal acetylation of recombinant human prothymosin alpha in Escherichia coli.BiochimBiophys Acta.2006; 1760(8): 1241-7.), and found that the N-acetylated thymosin α1 analog can be obtained by cutting N-acetylated thymosin α by hydroxylamine, but the N-acetylated thymosin The C-terminus of the α1 analog is modified by hydroxylamine, which is different from the free carboxyl terminus of the natural N-acetylated thymosin α1. This difference may cause immunogenicity when N-acetylated thymosin α1 is used as a drug , affecting biological activity and other issues

Method used

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  • Method for preparing genetic engineering N-acetylated thymosin alpha1
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  • Method for preparing genetic engineering N-acetylated thymosin alpha1

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1. Preparation of N-acetylated thymosin α1 with N-acetylated thymosin α progen

[0049] The pfu enzymes, endonucleases, ligases, and kits used in the experiment were purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd. and Beijing Biotech Biogene Technology Co., Ltd.

[0050] 1. Construction of genetically engineered bacteria expressing N-acetylated prothymosin α

[0051] Take the aborted human fetal thymus, use the total RNA preparation kit, and prepare total RNA according to the method provided by the kit; use the RT-PCR kit, and reverse-transcribe the mRNA into cDNA according to the method provided by the kit; use the cDNA as a template, Using Prot1: 5'-CCCATATGTCTGATGCAGCTGTAGATACC-3' and Prot2: 5'-CGGGATCCCTAGTCATCCACGTCGGTCTTCTG-3' as primers, the cDNA of thymosin was amplified by PCR. Add 1 μl cDNA, 3 μl each of 20 μmol / L Prot1 and Prot2 primers, 10 μl of 2 mmol / L dNTP, 10 μl of 10X reaction buffer, and 5 U of pfu DNA polymerase int...

Embodiment 2

[0064] Example 2, Preparation of N-acetylated thymosin α1 (Ile13) (sequence 2 in the sequence listing)

[0065] Using the recombinant plasmid pET-NproT constructed in the above-mentioned Example 1 as a template, using Prot3: 5'-cccatatgtctgatgcagctgtagataccagctccgaaatcaccatcaaggactta-3' and Prot2 as primers, PCR amplification was carried out, and the amino acid residue encoded by the deoxyribonucleotide sequence obtained was The gene was named NproT(Ile13) from prothymosin alpha prothymosin whose 13th amino-terminal amino acid is isoleucine.

[0066] NproT (Ile13) was inserted between the NdeI and BamHI restriction sites of the vector PET22b using the same method as in Example 1 above to obtain the recombinant plasmid pET-NproT (Ile13); then the recombinant plasmid pET-NproT (Ile13) was transferred into Escherichia coli BL21 (DE3) competent cells, adopt the screening method identical with above-mentioned embodiment 1 to obtain recombinant Escherichia coli BL21 (DE3) (pET-NproT...

Embodiment 3

[0068] Example 3, using natural genes and artificially synthesized genes to express C-terminal deletions and / or substitution of G from the 36th position of the amino terminal to A, and substitution of G to A from the 43rd position of the amino terminal to prothymosin α variants, Preparation of N-acetylated thymosin α1

[0069] 1. Construction of engineering bacteria expressing C-terminal deleted prothymosin α variants

[0070]Using the recombinant plasmid pET-NproT constructed in the above-mentioned Example 1 as a template, and using Prot1 and Prot4: 5'-gtggatccttaATCGACATCGTCATCCTCATC-3' as primers, the gene (NproT- C), its deoxyribonucleotide sequence is shown in sequence 11 in the sequence listing, and its coded amino acid sequence is shown in sequence 4 in the sequence listing.

[0071] Using the same method as in Example 1 above, NproT-C was inserted between the BamHI and NdeI restriction sites of the vector PET22b to obtain the recombinant plasmid pET-NproT-C; then the ...

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Abstract

The invention discloses a method for preparing a method for preparing genetic engineering N-acetylated thymosin alpha1. The method comprises the following steps of: 1) preparing precursor proteins or polypeptides containing a polypeptide sequence of the N-acetylated thymosin alpha1 by genetic engineering escherichia coli; and 2) performing restriction enzyme digestion of the precursor proteins or polypeptides containing the polypeptide sequence of the N-acetylated thymosin alpha1, obtained by the step 1, by endopeptidase, and then purifying the products to obtain the N-acetylated thymosin alpha1. The method has the advantages of obtaining an expression level of the N-acetylated thymosin alpha1, simplifying cutting, facilitating the purification of the N-acetylated thymosin alpha1, improving the production efficiency of the N-acetylated thymosin alpha1, having wide clinical application prospect, along with low cost and the like.

Description

technical field [0001] The invention relates to a method for preparing genetic engineering N-acetylated thymosin α1. Background technique [0002] Thymosin α is a family of immunomodulatory polypeptides with the same or similar N-terminal sequence, including prothymosin α, thymosin α1 and thymosin α11. Among them, thymosin α1 and thymosin α11 are the degradation products of prothymosin α in vivo. The N-terminal of thymosin α from natural sources has N-acetylation modification, and N-acetylation modification plays an important role in the stability of thymosin α1 in vivo. N-acetylated thymosin α1 synthesized by chemical method is already on the market, and its trade name is "Ridaxian", which is used for the treatment of viral hepatitis, etc. However, the chemical synthesis of N-acetylated thymosin α1 is costly and highly polluting, which restricts the widespread use of the drug. The use of genetically engineered Escherichia coli to prepare polypeptides has the advantages o...

Claims

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Application Information

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IPC IPC(8): C12N15/16C12N15/70C07K14/66A61K38/32A61P31/12A61P35/00A61P31/16A61P1/16
CPCC07K14/57581A61P1/16A61P31/12A61P31/16A61P35/00
Inventor 吴军刘波巩新唱韶红马清钧
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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