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Preparation and application of low-toxicity Kdo2-monophosphoryl lipid A containing five fatty acid chains

A monophosphoric acid lipid and fatty acid chain technology, applied in the field of bioengineering, can solve the problems of cumbersome extraction process, no extraction and purification method, large molecular weight, etc., and achieve the effect of simple extraction process

Active Publication Date: 2015-08-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the very low water solubility of lipid A molecules, they cannot exhibit effective biological activity in the aqueous phase. Therefore, in recent decades, crude samples of LPS are still used in basic research or clinical practice to test the corresponding lipid A derivatives. active research
However, due to the large molecular weight of LPS, the structural diversity of core sugar and O antigen, it cannot be detected and quickly identified by mass spectrometry or nuclear magnetic resonance, and there is no standardized extraction and purification method for the time being.
In addition, in the actual production of lipid A vaccine adjuvant, Salmonella currently used is a pathogenic bacterium, which has certain safety hazards, and Salmonella produces a variety of lipid A with different structures at the same time, and the extraction process is cumbersome and energy-consuming.

Method used

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  • Preparation and application of low-toxicity Kdo2-monophosphoryl lipid A containing five fatty acid chains
  • Preparation and application of low-toxicity Kdo2-monophosphoryl lipid A containing five fatty acid chains
  • Preparation and application of low-toxicity Kdo2-monophosphoryl lipid A containing five fatty acid chains

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Experimental program
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Effect test

Embodiment 1

[0027] The Kdo produced by the genetic engineering bacteria BW002 of embodiment 1 2 - Extraction and structural analysis of lipid A

[0028] LPS produced by W3110 and containing long sugar chains cannot be directly extracted, and its structure cannot be directly analyzed by thin layer chromatography (TLC) and ESI / MS analysis due to its large mass and low solubility in the organic phase. Therefore, we selected Escherichia coli WBB06 (W3110waaCF::tet6), the heptose-deficient mutant of W3110 as a control for structural analysis. WBB06 was confirmed to be able to directly synthesize Kdo with six fatty acid chains and bisphosphoric acid in lipid A part 2 - Lipid A.

[0029] 1. Kdo 2 - Extraction of Lipid A

[0030] Kdo produced by control bacteria WBB06 and genetically engineered bacteria BW001 2 -Lipid A was extracted using a chloroform / methanol / water mixed phase extraction method. The specific operation is as follows: the seed bacteria liquid cultured overnight was 600 =0....

Embodiment 2

[0035] The five-chain Kdo produced by the genetic engineering bacteria BW002 of embodiment 2 2 - Toxicity analysis of monophospholipid A

[0036] Kdo extracted directly 2 -Lipid A sample is mixed with various membrane phospholipids, so it needs to be purified to remove phospholipid interference before performing cytotoxicity analysis. Kdo 2 - The purification of lipid A is mainly accomplished by gradient elution on a DEAE fiber column. In the cell stimulation experiment, the purified sample of lipopolysaccharide (LPS) from the wild-type strain W3110 was used as a positive control, and PBS was used as a hard control; an attenuated strain produced by genetically engineered bacteria BW001 (E.coli W3110△waaCF△lacIlacZ::FnlpxE) was also introduced. , monophosphate six fatty acid chains Kdo 2 - Lipid A for comparison.

[0037] 1. Kdo 2 - DEAE fiber column purification of lipid A

[0038] The genetically engineered bacteria BW002 and the control engineered bacteria BW001 were ...

Embodiment 3

[0045] Embodiment 3 five-chain Kdo with low toxicity 2 -Analysis of hydrophobicity and self-aggregation ability of genetically engineered bacteria BW002 producing monophosphate lipid A

[0046] 1. Comparison of hydrophobicity between genetically engineered bacteria BW002 and wild-type Escherichia coli W3110

[0047] The method for measuring the hydrophobicity of the bacterial strain is as follows: overnight cultured bacterial liquid is centrifuged (7000g, 10min) to collect the bacterial cells; the bacterial cells are washed twice with PBS7.4; then the bacterial cells are suspended with PBS7.4 to make the OD 600 =1.0 (marked as A 0 , keep 3 decimal places); Finally, mix 2mL bacterial suspension with 800μL xylene, vortex for 2min, and measure the OD of the aqueous phase after standing at room temperature for 1h 600 (recorded as A, keep 3 decimal places), calculate cell surface hydrophobicity: H%=(A 0 -A) / A 0 *100.

[0048] Since LPS is the main macromolecule on the surface ...

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Abstract

The invention discloses preparation and application of a low-toxicity Kdo2-monophosphoryl lipid A containing five fatty acid chains, belonging to the field of bioengineering. By modifying Escherichia coli, a special-structure low-toxicity five-chain Kdo2-monophosphoryl lipid A is synthesized, and the Kdo2-lipid A molecule is not connected with the core polysaccharide long chain, and only composed of two 2-one-3-desoxyoctoic acids, five fatty acid chains and a C4' site single phosphoric acid. The Kdo2-lipid A is amphiphilic, is convenient for separation, extraction and purification, can be detected and directly identified, maintains the biological immunocompetence of the lipid A part, has obvious toxicity attenuation effect as compared with wild type W3110 LPS, and is a vaccine adjuvant with great development potential. Meanwhile, the method for extracting and purifying the Kdo2-lipid A is simple in steps and easy to operate.

Description

technical field [0001] The invention relates to a Kdo with low toxicity and five fatty acid chains 2 - Preparation and application of monophosphoric acid lipid A, belonging to the field of bioengineering. Background technique [0002] Lipopolysaccharide (LPS), the bacterial endotoxin, is the main component that constitutes the outer layer of the outer membrane of most Gram-negative bacteria, mainly composed of Kdo 2 - Lipid A (Kdo 2 -Lipid A), core polysaccharide (Core) and O-antigen (O-antigen) are composed of three parts; where Kdo 2 - Lipid A is the hydrophobic end of LPS. Its structure is very conservative. It connects the hydrophilic core polysaccharide and O-antigen and assists the two to adhere to the cell surface to form a complete cell wall. The core polysaccharide and O-antigen have heterogeneity , Different Gram-negative strains and even the same strain have various combinations of glycosyl types, numbers, and positions. Gram-negative bacteria will release LPS...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H13/04C07H1/06C12N1/21C12P19/26A61K39/39C12R1/19
Inventor 王小元王碧雯韩亚宁李烨李颜颜
Owner JIANGNAN UNIV
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