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A kind of melioidosis polysaccharide and its preparation method and application

A technology of melioidosis and bacterial polysaccharide, which is applied in the field of melioidosis polysaccharide and its preparation, can solve the problems of unclear biological function of polysaccharide antigen type and the like

Active Publication Date: 2022-04-19
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to reports in prior art literature, clinical isolates of melioidosis from the United States or Thailand can express a variety of different LPS antigens and CPS antigens, but up to now, there is no relevant report on the surface polysaccharides of clinical isolates of melioidosis in my country. The types of polysaccharide antigens and their biological functions are still unclear. Therefore, it is urgent to establish a method for isolating the surface polysaccharides of melioidosis in my country to promote further research.

Method used

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  • A kind of melioidosis polysaccharide and its preparation method and application
  • A kind of melioidosis polysaccharide and its preparation method and application
  • A kind of melioidosis polysaccharide and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1 Extraction of polysaccharides of class 1 rhinoplasma

[0044] 1. Culture and inactivation of bacteria

[0045] The -80 °C preservation of rhinodermid bacteria BPC006 (domestic standard strain) (rhinodermid bacteria originated from Sanya People's Hospital, isolated and cultured from the patient's blood, sputum, pus and urine, identified by the applicant as rhinodermid bacteria, sequencing results have been uploaded to the NCBI database) using a three-line method of resuscitation in LB solid plates, after 48h incubation in a incubator incubator, pick up a single colony in 400mL LB liquid medium overnight bacteriological culture, the next day, centrifuge the bacterial fluid (8000rpm, 5min) Discard the supernatant, add PBS buffer (0.01M, pH 7.4) to the bacterial pellet, resuspend the bacterial body and then centrifuge again (8000 rpm, 5min) to discard the supernatant, and finally add 10mL PBS buffer to the bacterial pellet, mix well and then treat it in a water bath a...

Embodiment 2

[0052] Example 2 Purification of class 2 rhinoplasma polysaccharides

[0053] First, the crude polysaccharide sample BPC006-BPPI extracted in the previous step was dissolved in primary water (except bubbles) so that the final concentration was 7mg / mL, and the supernatant was collected after centrifugation (12000 rpm, 5 min) and the supernatant was collected using the ion exchange chromatography column (DEAESepharose Fast Flow) (GE Corporation of the United States) according to the difference in charge, and the sample was elutioned in a linear gradient using 0~1.5M NaCl solution to collect the eluate. According to the phenol sulfuric acid method (400 μL sample + 400 μL 6% phenol solution + 2 mL concentrated sulfuric acid → A 490 Plot the elution curve and collect the corresponding elution peaks for each component, BPC006-BPPI-a and BPC006-BPPI-b ( Figure 1 ), the eluent is lyophilized and prepared after dialysis and concentration under reduced pressure.

[0054] Then the main polysa...

Embodiment 3

[0056] Example 3 GC-MS analysis of class 3 rhinoplasma polysaccharides

[0057] Accurately weigh 1 mg of BPC006-BPPI-b1 polysaccharide component in a clean sample flask, add 1 mL of 2mol / L alcohol to make standard titrant (Xiamen Haibiao Technology Co., Ltd.) and charge N 2 Seal the tube, carry out methanol lysis reaction at 80 °C for 8h, after the sample is restored to room temperature, repeatedly blow dry with an air pump, add 1mL 2mol / L trifluoroacetic acid (TFA) (Aladdin Biochemical Technology Co., Ltd.), perform hydrolysis reaction at 120 °C for 1h, and repeatedly add anhydrous ethanol to evaporate the residual TFA after the sample is restored to room temperature. Add 1 mL20 mg / mL of NaBH 4 (Tianjin Kemiou Chemical Reagent Factory) solution, stirred at room temperature to reduce 8h, then added 50μL of 50% acetic acid to adjust the pH to the entire system is neutral, and then add cation exchange resin (Shanghai McLean Biochemical Technology Co., Ltd.) to remove the sodium ions...

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Abstract

The present invention provides a kind of melioidosis polysaccharide, containing polymerized cross-linked pentasaccharide repeating unit [3‑(a‑D‑Manp‑1→3‑a‑D‑Manp) 4 ‑2Me‑a‑L‑6dTalp‑1→]. The invention also provides a preparation method and application of the melioid polysaccharide. The polysaccharide can be used as an antigen to specifically bind to the serum of a melioid patient, but does not react with the serum of a healthy donor, which proves that the polysaccharide antigen can recognize the specific antibody in the serum of a melioid patient, and can be used to prepare a drug for the diagnosis or prevention of melioid infection drug.

Description

Technical field [0001] The present invention relates to the field of bioengineering and biomedical technology, in particular to a kind of rhinomycete polysaccharide and preparation method and application thereof. Background [0002] Burkholderia pseudomallei (BP), referred to as epistaxis, is a facultative intracellular gram-negative bacterium that can exist in water sources and soil, mainly causing a rhino-like disease characterized by multiple abscesses of liver, lungs and other organs. The disease is mainly distributed in Southeast Asia and northern Australia, and coastal cities and regions such as Hainan, Guangdong, Taiwan, and Hong Kong in China have gradually become endemic areas. Humans and animals can be infected with the bacteria through skin abrasions, inhalation or ingestion, etc., and its clinical manifestations are diverse, including asymptomatic infection, subclinical infection, severe sepsis, etc., with a mortality rate of about 40%, and the recurrence rate is als...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C08B37/00A61K39/02A61P31/04
CPCC08B37/0003C08B37/006A61K39/0208A61P31/04
Inventor 闫晶敏章美娟李倩李骁饶承龙杨文波毛旭虎
Owner ARMY MEDICAL UNIV