Simple method for extracting low-endotoxin bowman acinetobacter capsular polysaccharide

A technology of Acinetobacter baumannii and capsular polysaccharide, which is applied in antibacterial drugs, pharmaceutical formulations, bacterial antigen components, etc., and can solve the problems of weak removal of bacterial endotoxin, failure to meet clinical needs, and low capsular expression , to achieve the effect of high-efficiency endotoxin content, high cost and low cost

Inactive Publication Date: 2017-12-19
JIANGSU UNIV
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AI Technical Summary

Problems solved by technology

The difficulty in isolating and purifying capsular polysaccharide from Acinetobacter baumannii is that its capsular expression is low, which is far less than that of Haemophilus influenzae, Neisseria meningitidis, Salmonella typhi and Streptococcus pneumoniae. If conventional Sepharose 4B column purification method (reversed high pressure liquid chromatography technology), its output is very low and cannot meet clinical needs
However, although ethanol precipitation or cetyltrimethylammonium bromide (Cetavlon, CTAB) precipitation can remove bacterial nucleic acid alone, it has a weak effect on bacterial endotoxin removal, and its purified product will cause severe endotoxin reactions when injected into the human body

Method used

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  • Simple method for extracting low-endotoxin bowman acinetobacter capsular polysaccharide

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1: Acinetobacter baumannii capsular polysaccharide extraction

[0024] (1) Bacterial culture: resuscitate Acinetobacter baumannii, inoculate the bacteria on a sheep blood agar plate (BD Difco, USA), and culture at 37°C for 16 h to 24 h to form colonies visible to the naked eye. baumannii colonies, diluted to OD with sterile phosphate (PBS) buffer (pH 7.4) 600 =1. The above Acinetobacter baumannii suspension was evenly spread on 40 sheep blood agar plates, each plate was inoculated with 10 μL of bacterial solution, and cultured in a constant temperature incubator (37°C) for 16 h to 24 h until a lawn was formed.

[0025] (2) Bacteria collection: Add 30 mL of sterilized PBS buffer dropwise to each sheep blood agar plate to wash the bacteria repeatedly by blowing and blowing, and transfer all the washed bacteria liquid to 4 sterilized round-bottomed centrifuge tubes (50 mL) , centrifuged at 8 000 rpm for 10 min to collect the bacterial pellet, and discarded the...

Embodiment 2

[0029] Example 2: Detection of Capsular Polysaccharide Concentration of Acinetobacter baumannii

[0030] (1) Establish a standard curve: Accurately weigh 200 g of analytically pure glucose dried at 105°C to constant weight, dilute to 500 mL with three-distilled water, and absorb 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, For 1.2 mL, 1.4 mL, 1.6 mL and 1.8 mL, add an appropriate amount of triple-distilled water to make up the volume to 2.0 mL. Using triple-distilled water as the blank control, add 1.0 mL of 6% phenol solution and 5.0 mL of concentrated sulfuric acid to each tube to fully Shake well and react at room temperature for 40 min. After the reaction, the absorbance at 490nm of each tube was detected by a 722-type spectrophotometer, and the standard curve was drawn with the concentration of glucose in each tube as the abscissa and the absorbance value as the ordinate.

[0031] (2) Test the absorbance of the sample: draw 1.0 mL of the extracted capsular polysaccharide solu...

Embodiment 3

[0035] Example 3: Capsular polysaccharide endotoxin detection

[0036] The endotoxin content of the extracted capsular polysaccharide of Acinetobacter baumannii was detected by an endotoxin detector (product of Xiamen Limulus Reagent Experimental Factory Co., Ltd.) and its supporting reagents. The detection reaction adopts the endpoint method, and the endotoxin concentration is calculated according to the absorbance at 545 nm of the reaction system. After multiple tests, the endotoxin content of the purified capsular polysaccharide is lower than 0.01 EU / mL, which is lower than the standard of no more than 0.03 EU / mL for human injection, and can be directly used for human injection, such as Further coupled reverse-phase high-performance liquid chromatography purification can improve reverse-phase high-performance liquid chromatography purification efficiency and greatly reduce costs.

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Abstract

The invention relates to a simple method for extracting low-endotoxin bowman acinetobacter capsular polysaccharide, which belongs to the technical field of biomaterial preparation. According to the invention, the method employs a lysate and a ultrasonic method for co-cracking the bowman acinetobacter, uses a CTAB solution for primary removal of endotoxins such as lipopolysaccharide, ethanol with concentration being 25% and ethanol with concentration being 80% are respectively used for depositing the obtained bowman acinetobacter capsular polysaccharide, the impurities such as lipopolysaccharide and nucleic acid can be removed, and the low-endotoxin bowman acinetobacter capsular polysaccharide can be finally obtained. The method has the advantages of simpleness, rapidity and low cost, the endotoxin is low, and the method can be used for in-vivo tests and researches.

Description

technical field [0001] The invention relates to a simple low-endotoxin Acinetobacter baumannii capsular polysaccharide extraction method, which belongs to the technical field of biological material preparation. Background technique [0002] Acinetobacter baumannii ( Acinetobacter baumannii ) is the main pathogenic bacteria causing nosocomial infection, which can cause ventilator-associated pneumonia, urinary tract infection, sepsis and other multi-system infections in patients. In the past, Acinetobacter baumannii was not valued by clinicians, but with the significant increase in the proportion of clinically isolated multidrug-resistant and pan-drug-resistant Acinetobacter baumannii in recent years, coupled with the strong acquired drug resistance of the bacteria and clonal transmission ability, currently Acinetobacter baumannii has become a common pathogenic bacteria that is widely prevalent and highly drug-resistant around the world. Due to the prevalence of multidrug-re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C08B37/00A61K39/02A61P31/04
CPCC08B37/0003A61K39/0208
Inventor 吴亮王廷廷陈盛霞阴晴姜旭淦卢叶
Owner JIANGSU UNIV
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