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Recombinant bacteria expressed by escherichia coli genome N-acetylase by control of heterogenous promoter, and use thereof

A technology of acetyltransferase and recombinant bacteria, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of restricting wide application, high product price, low yield, etc., achieve obvious application prospects, increase yield, The effect of easy separation

Active Publication Date: 2014-04-23
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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  • Recombinant bacteria expressed by escherichia coli genome N-acetylase by control of heterogenous promoter, and use thereof
  • Recombinant bacteria expressed by escherichia coli genome N-acetylase by control of heterogenous promoter, and use thereof
  • Recombinant bacteria expressed by escherichia coli genome N-acetylase by control of heterogenous promoter, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Recombinant bacteria co-expressed with acetyltransferase rimL and prothymosin α using T7 promoter

[0061] Using Red recombination technology, the T7 promoter (derived from the PET22b plasmid, which was purchased from Novagen) with the lactose operon 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 open reading frame (ORF) of the rimL gene 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 the overexpression of the N-acetyltran...

Embodiment 2

[0098] Embodiment 2, BL21 (DE3) (T7rimL / proTα (Thr 13 )) Preparation of N-acetylated prothymosin alpha

[0099] 1. Crude protein extract

[0100] BL21(DE3)(T7rimL / proTα(Thr 13 )) and control bacteria BL21(DE3)(proTα(Thr 13 )) were respectively inoculated in 50ml LB liquid medium, cultured on a shaker at 37°C overnight, and then transferred to 500ml medium (yeast extract 10g / L, tryptone 10g / L, sodium dihydrogen phosphate 20mM, hydrogen phosphate Disodium 30mM) in a 3L shake flask, after culturing at 37°C for 2h, add 1.25ml of 0.2mol / L IPTG (0.5mM) for induction, continue to cultivate for 6h, centrifuge the fermentation broth, and collect the thalli.

[0101] Resuspend the collected bacteria with water (add 10ml of water per gram of bacteria), break the wall with ultrasonic waves, collect the supernatant by centrifugation, add glacial acetic acid to the supernatant to adjust the pH to 4.5, let stand for 30 minutes, and collect the supernatant by centrifugation. It is the cru...

Embodiment 3

[0112] Example 3. Recombinant bacteria co-expressed with acetyltransferase rimJ of T7 promoter and prothymosin α

[0113] This example is similar to the method in Example 1. Red recombination technology is also used to insert the T7 promoter (derived from the PET22b plasmid, which was purchased from Novagen) with the lactose operon into the N- The upstream of the open reading frame (ORF) of the acetyltransferase rimJ gene, the T7 RNA polymerase gene controlled by the lactose promoter in the genome of the bacteria. When lactose or its analogues, 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 open reading frame (ORF) of the rimJ gene At the same time, the lactose operon downstream of the T7 promoter is derepressed to control the high-efficiency transcription of the N-acetyltransferase rimJ gene, thereby realizin...

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Abstract

The invention discloses recombinant bacteria expressed by escherichia coli genome N-acetylase by control of a heterogenous promoter, and a use thereof. The recombinant bacteria provided by the invention are obtained through the process that heterogenous inducible promoters are introduced into upstream sequences of acetylase coding genes of host bacteria to start the expression of the acetylase coding genes of the host bacteria. A result of an experiment of the invention shows that the recombinant bacteria are obtained by co-expression of N-acetylase coding genes and thymosin alpha coding genes. Thymosins alpha obtained by fermentation of the recombinant bacteria comprise mainly N-acetylated thymosin alpha. The recombinant bacteria have obvious application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant bacterium which utilizes a heterologous promoter to control the expression of N-acetyltransferase in Escherichia coli genome and its application. 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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/16C12N15/63C12P21/02C12R1/19
Inventor 刘波吴军唱韶红巩新马清钧
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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