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Bacterium constitutively producing monophosphoryl lipid a and method of producing monophosphoryl lipid a by using bacterium

A monophosphoryl lipid and bacterial cell technology, applied in biochemical equipment and methods, recombinant DNA technology, acyltransferase, etc., can solve the problems of genetic engineering transformation efficiency and genetic engineering stability reduction

Active Publication Date: 2020-08-04
KOREA INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such natural mutations will lead to the reduction of genetic engineering transformation efficiency and genetic engineering stability

Method used

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  • Bacterium constitutively producing monophosphoryl lipid a and method of producing monophosphoryl lipid a by using bacterium
  • Bacterium constitutively producing monophosphoryl lipid a and method of producing monophosphoryl lipid a by using bacterium
  • Bacterium constitutively producing monophosphoryl lipid a and method of producing monophosphoryl lipid a by using bacterium

Examples

Experimental program
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Effect test

example 1

[0063] Example 1, preparation of vectors comprising polynucleotides encoding Escherichia coli LpxL and Escherichia coli LpxM

[0064] 1.1. Preparation of pWSK29-EcLpxLEcLpxM

[0065]In order to obtain polynucleotides encoding E. coli LpxL polypeptides from the E. coli W3110 genome (GenBank accession number NC_000918.1, ATCC), the following pair of primers were used to amplify the polynucleotides encoding the LpxL polypeptide including ribosomes by the first polymerase chain reaction (PCR). Polynucleotide (GenBank Accession No. AP009048.1 (c1118159.1117239, SEQ ID NO: 2):

[0066] LpxL forward primer P1:

[0067] 5'-CGCAGTCTAGAAAGGAGATATATTGATGACGAATCTACCCAAGTTCTC-3' (SEQ ID NO: 3)

[0068] LpxL reverse primer P2:

[0069] 5'-CGCTATTATTTTTTTTCGTTTCCATTGGTATATCTCTTTCTTATTAATAGCGTGAAGGAACGCCTTC-3' (SEQ ID NO: 4)

[0070] In order to obtain the polynucleotide encoding the E. coli LpxM polypeptide from the E. coli W3110 genome, the polynucleotide encoding the EcLpxM polypeptide...

example 2

[0104] Example 2, the preparation of escherichia coli bacterial strain

[0105] 2.1. Preparation of Escherichia coli KHSC0044 (pWSK29-EcLpxLEcLpxM, △lpxT, △pagP, bacA::HpLpxE, kdtA::kan, W3110) strain

[0106] 2.1.1. Preparation of Escherichia coli with the lpxT gene removed from the genome

[0107] Escherichia coli lpxT::kan, W3110 strain was prepared, wherein the kanamycin gene expression cassette was inserted into the lpxT gene (SEQ ID NO: 19) of the Escherichia coli genome, wherein the lpxT gene coded LpxT polypeptide (SEQ ID NO: 18).

[0108] Then, the pCP20 plasmid (Kirill A.Datsenko and Barry L.Wanner PNAS (2000), Vol. 97, pp. 6640-6645) was transformed into E. coli lpxT::kan, W3110 strain, and the transformation was selected on LB plates Escherichia coli. The selected Escherichia coli was inoculated on an LB plate, and selected at a temperature of 42° C., thereby preparing an E. coli △lpxT, W3110 strain in which the lpxT and kanamycin gene expression cassettes were ...

example 3

[0156] Example 3. Determination of Lipid Composition of Escherichia coli Strains KHSC0044, KHSC31, KHSC0045 and KHSC0055

[0157] 3.1. Culture of KHSC0044, KHSC0031 and KHSC0045

[0158] E. coli strains KHSC0044, KHSC0031 and KHSC0045 were prepared as described above in Sections 2.1.6, 2.2.3 and 2.3.3, respectively.

[0159] A stock solution of each E. coli strain was inoculated on LB plates containing 50 μg / mL ampicillin (EMD Microwell) and 1 mM isopropyl β-D-1-thiogalactoside (IPTG) (UBP Bio), Then cultivate. Select KHSC0044 (8 colonies) strains, KHSC0031 (7 colonies) strains and KHSC0045 (7 colonies) strains, and then inoculate them into 3 mL of LB liquid medium containing 50 μg / mL ampicillin (EMD microwell) and 1 mM IPTG , and then incubated overnight at approximately 30°C. Inoculate each resulting culture broth into 200 mL of fresh LB liquid medium, dilute to an OD of about 0.06 to about 0.1 600 , and then cultured overnight at about 30° C., the fresh LB liquid medium...

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Abstract

A bacterium that constitutively produces monophosphoryl lipid A (MLA) and a method of producing MLA by using the bacterium may simply produce MLA and a derivative thereof without acid hydrolysis, reduce a probability of natural mutation, and increase yields of MLA and a derivative thereof by constitutive expression of the MLA and derivative thereof.

Description

technical field [0001] One or more embodiments relate to a bacterium that constitutively produces monophosphoryl lipid A (MLA) and a method of producing MLA by using the bacterium. Background technique [0002] Lipopolysaccharide (LPS) is one of the components of the outer membrane surrounding peptidoglycan in Gram-negative bacteria. LPS is a molecule comprising lipid A and various polysaccharides bound to lipid A by covalent bonds. Among the components of LPS, lipid A (also known as endotoxin) is responsible for the virulence of Gram-negative bacteria. [0003] Lipid A is a very potent immune system stimulator, activating cells (eg, monocytes or macrophages) in picograms per milliliter. For example, derivatives of lipid A or variants of lipid A can be used as components of vaccines such as adjuvants. Monophosphoryl lipid A (MLA) is used as an adjuvant and for allergen-specific immunotherapy and immunotherapy of cancer, and has also been reported to be effective in the pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12P19/04C12N9/14C12N9/16C12P7/6436
CPCC12P7/6436C12P19/12C12N9/16C12Y301/03027C12N9/14C12Y306/01027C12Y301/03C12Y203/01Y02A50/30C12N15/74C12P19/04C12N15/63
Inventor 郑学淑池唯贤安镇守权翊赞杨恩卿
Owner KOREA INST OF SCI & TECH
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