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Escherichia coli overexpressing riml and its application in the preparation of n-acetylated thymosin α

A thymosin, the technology in the sequence table, applied in the biological field, can solve the problems of high product price, limited wide application, low yield, etc., and achieves the effects of convenient separation, obvious application prospects, and improved yield

Active Publication Date: 2011-12-14
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, thymosin extracted from animals has complex components, low purity, mixed with animal-derived pollutants, and prone to allergic reactions.
Chemically synthesized N-acetylated thymosin α1 is a kind of N-acetylated thymosin α1 prepared by polypeptide chemical synthesis method. The Tα1 obtained by this method has high purity, high activity and no obvious side effects. For example, SciClone company The "Zidaxian" has been approved by many countries including my country for the treatment of viral hepatitis as an immunomodulatory drug; And the purification process is complex, the yield is low, so the price of the product is high, which limits the wide application of the drug

Method used

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  • Escherichia coli overexpressing riml and its application in the preparation of n-acetylated thymosin α
  • Escherichia coli overexpressing riml and its application in the preparation of n-acetylated thymosin α
  • Escherichia coli overexpressing riml and its application in the preparation of n-acetylated thymosin α

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, the construction of the Escherichia coli overexpressing RimL expressing thymosin α

[0059] 1. Construction of Escherichia coli overexpressing N-acetyltransferase RimL

[0060] 1. Construction of pOKBV plasmid

[0061] Due to the co-expression of RimL and Thymosin α, considering the incompatibility of the plasmid, the replicon and ampicillin resistance gene of pBV220 (purchased from Shanghai Jingjing Molecular Biotechnology Co., Ltd.) were replaced with the plasmid pOK12 (Jeffrey Vieira and Joachim Messing.New pUC-derived cloning vectors with different selectable markers and DNAreplication origins.Gene, 1991,100:189-194, can be constructed from the information provided by this document, and the construction method of the carrier has been disclosed in many documents (such as J . Sambrook et al., "Molecular Cloning Experiment Guide" Second Edition, Science Press, 1995; ) etc., so it can be obtained by using the method of gene total synthesis.) p15A replico...

Embodiment 2

[0084] Embodiment 2, recombinant bacterium BL21 (DE3) (pOKBV-RimL / proTα (Thr 13 )) expresses N-acetylated prothymosin α (Thr 13 )

[0085] 1. Induce recombinant bacteria

[0086] BL21(DE3)(pOKBV-RimL / proTα(Thr 13 )) and control bacteria BL21(DE3)(pOKBV / proTα(Thr 13 )) were inoculated into 5 mL No. I medium containing 50 μg / ml kanamycin and 100 μg / ml ampicillin (50 mM disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, 20 g / L yeast extract, 10 g / L tryptone, 2g / L glucose), cultivated overnight at 30°C, and transferred the overnight cultured bacterial solution to 500ml shake flasks containing 150mL No. 600nm At about 0.6, raise the temperature to 42°C to induce expression, and centrifuge after 12 hours of induction (the centrifugation time is 20 minutes, the centrifugation temperature is 4°C, and the centrifugal force is 10,000g;) to harvest the bacteria respectively.

[0087] 2. Preparation of N-acetylated thymosin α progen (Thr 13 )

[0088]1) Tris saturated ...

Embodiment 3

[0099] Example 3, using the T7 promoter inserted into the genome to construct a gene containing and expressing N-acetylated thymosin α 13 ) recombinant bacteria

[0100] Using Red recombination technology, the T7 promoter with the lactose operon (derived from the PET22b plasmid, which was purchased from Novagen) was inserted into the open reading frame (ORF) of the N-acetyltransferase RimL gene of Escherichia coli BL21 (DE3). ), the T7 RNA polymerase gene controlled by the lactose promoter in the genome of the bacterium. When lactose or its analogs, such as IPTG, are added to the medium, the T7 RNA polymerase gene expression controlled by the lactose promoter, the synthetic T7 RNA polymerase binds to the T7 promoter artificially inserted into the upstream of the RimL gene open reading frame (ORF) At the same time, the lactose operon downstream of the T7 promoter is derepressed to control the high-efficiency transcription of the N-acetyltransferase RimL gene, thereby realizing...

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Abstract

The invention discloses a colon bacillus for over-expressing RimL and an application on preparing N-extrasin alpha acetylate. The invention provides a recombinant strain and the recombinant strain is obtained by introducing a coding gene of the RimL and a coding gene of the extrasin alpha into host bacteria. The experiment provided by the invention shows that the invention constructs the recombinant strain which is obtained by co-expressing the coding genes of N-acetylase RimL and the extrasin alpha, the recombinant strain is fermented to obtain the extrasin alpha, and a large part of the extrasin alpha is the N-extrasin alpha acetylate; and the colon bacillus has a obvious application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an Escherichia coli overexpressing RimL and its application in preparing N-acetylated thymosin α. Background technique [0002] The N-terminal acetylation modification of proteins and polypeptides is a common modification in eukaryotic cells, and more than 50% of eukaryotic cytoplasmic proteins have this modification. Preliminary functional studies have shown that N-terminal acetylation can have an important impact on the spatial structure, ligand recognition, and degradation resistance of many proteins and polypeptides by changing the charge of the N-terminal and blocking the N-terminal. For example, the N-terminal acetylation modification of the small molecule GTPaes-Arl3P protein promotes its recognition with the membrane receptor Sys1p / hSys1 and localizes it to the membrane. N-terminal acetylation of fetal hemoglobin can increase the depolymerization ability of its tetramer by 3...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P21/02C12R1/19
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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