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Bacterium 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, DNA/RNA fragments, recombinant DNA technology, etc., can solve problems such as low yield and complicated process steps

Active Publication Date: 2019-06-04
KOREA INST OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the disadvantages of these methods are that they are complicated in process steps and have low yield

Method used

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  • Bacterium producing monophosphoryl lipid A and method of producing monophosphoryl lipid A by using bacterium
  • Bacterium producing monophosphoryl lipid A and method of producing monophosphoryl lipid A by using bacterium
  • Bacterium 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

[0051] Example 1, preparation of vectors comprising polynucleotides encoding Escherichia coli LpxL and Escherichia coli LpxM

[0052] 1.1. Preparation of pWSK29-EcLpxLEcLpxM

[0053]In order to obtain the polynucleotide encoding the E. coli LpxL polypeptide from the E. coli W3110 genome (GenBank accession number NC_000918.1, ATCC), the encoding was amplified by first polymerase chain reaction (PCR) using a pair of primers shown below. Polynucleotide (GenBank Accession No. AP009048.1 (c1118159.1117239, SEQ ID NO: 2) (see Figure 2A ).

[0054] LpxL forward primer P1: SEQ ID NO: 3

[0055] LpxL reverse primer P2: SEQ ID NO: 4

[0056] In order to obtain polynucleotides encoding E. coli LpxM polypeptides from the E. coli W3110 genome, a pair of primers shown below were used to amplify the EcLpxM polypeptides encoding EcLpxM including RBS (GenBank accession number BAA15663.1, SEQ ID NO: 5) polynucleotide (GenBank accession number AP009048.1 (c1941907.1940936, SEQ ID NO: 6) (se...

example 2

[0080] Example 2, Preparation of Escherichia coli KHSC003 (pWSK29-EcLpxLEcLpxM, kdtA::kan, ΔlpxT, ΔpagP, W3110) strain

[0081] 2.1. Preparation of Escherichia coli with the lpxT gene removed from the genome

[0082] As described in 1.1, the pCP20 plasmid (Kirill A. Datsenko and Barry L. Wanner PNAS (2000), Vol. 97, pp. 6640-6645) was transformed into the kanamycin gene expression cassette (kanamycin cassette) inserted into the coding The lpxT gene (SEQ ID NO: 21) in the E. coli genome of the LpxT polypeptide (SEQ ID NO: 20) was selected on LB-ampicillin solid medium in E. coli strain W3110, lpxT::kan . The selected Escherichia coli was inoculated on LB solid medium, and it was selected at a temperature of 42°C, thereby preparing an Escherichia coli strain from which expression cassettes of lpxT and kanamycin genes were removed, namely, ΔlpxT, W3110 ( Figure 2B Step 1) in .

[0083] 2.2. Preparation of Escherichia coli with pagP and lpxT genes removed from the genome

[...

example 3

[0090] Example 3. Test of lipids of Escherichia coli introduced with AaLpxE, HpLpxE or FnLpxE

[0091] 3.1. Lipid extraction from Escherichia coli W3110 transformed with pWSK29-FnLpxE by acid hydrolysis

[0092] For comparative experiments, Escherichia coli strain W3110 transformed with pWSK29-FnLpxE and Escherichia coli strain W3110 not transformed with pWSK29-FnLpxE were prepared.

[0093] Specifically, pWSK29-FnLpxE was prepared (Wang, X., Karbarz, M.J., McGrath, S.C., Cotter, R.J. and Raetz, C.R., J Biol Chem (2004), Vol. 279(47), pp. 49470-49478), It amplifies the polynucleotide (gb|CP000439.1|:414941-415660 Francisella novicidaU112, SEQ ID NO: 24) encoding FnLpxE polypeptide (gi|118422929|gb|ABK89319.1, Francisella novicida U112, SEQ ID NO: 24) ID NO: 25). The prepared pWSK29-FnLpxE was transformed into Escherichia coli strain W3110 by electroporation, and then the transformed Escherichia coli was selected on LB solid medium containing 50 μg / mL of ampicillin. The sele...

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Abstract

A bacterium producing monophosphoryl lipid A (MLA) comprising a genetic modification that increases expression of a gene encoding LpxE polypeptide and a method of producing MLA are provided. Accordingto the present invention, MLA may be produced in a simple manner without acid hydrolysis and / or base hydrolysis.

Description

technical field [0001] One or more embodiments relate to monophosphoryl lipid A (MLA)-producing bacteria and methods of using the bacteria to produce MLA. Background technique [0002] Lipopolysaccharide (LPS) is one of the components of the outer membrane surrounding peptidoglycan of 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. Lipid A, derivatives of lipid A or variants of lipid A can be used, for example, 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, or also for the prevention and treatment of dementia. In addition,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12P7/64C12P19/04C12N9/10C12N9/18
CPCC12N15/74C12N9/1029C12N9/16C12N15/52C12N15/70C12P19/12Y02A50/30C12N15/63C12P7/64C12P19/04
Inventor 郑学淑杨恩卿黄斗炫
Owner KOREA INST OF SCI & TECH
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