Application of heptose chain-containing oligosaccharide compound in preparation of helicobacter pylori vaccines
A Helicobacter pylori, heptose-containing technology, applied in the field of medicine, can solve the problems of lipopolysaccharide structure not specific enough, limiting vaccine therapeutic application, similar impurities, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0041] The synthesis of embodiment 1 antigen oligosaccharide compound:
[0042] Antigenic oligosaccharide compounds such as figure 1 shown.
[0043] Compounds 1, 2, 3, 4, 5, 6 and 7 were synthesized starting from the corresponding fully protected compounds 1*, 2*, 3*, 4*, 5*, 6* and 7* (these compounds It can be obtained by referring to patent CN201910156533.1, CN109776632A), remove all acyl groups under alkaline conditions, and use 10% Pd / C and hydrogen after purification to remove all benzyl groups and benzyloxycarbonyl groups to obtain full deprotection Target molecules 1, 2, 3, 4, 5, 6 and 7.
[0044] Specific test operation and steps:
[0045] Compound 1:
[0046]
[0047] Compound 1* (28 mg, 0.0143 mmol) was dissolved in THF / MeOH (1:1, v / v, 2.0 mL), CH 3 ONa (20mg), stirred at room temperature for 0.5h, then added NaOH (aq, 1M, 100μL), the reaction was stirred at room temperature for 12h, after TLC detected that the reaction was complete, added Amerlite IR 120 (H...
Embodiment 2
[0065] Embodiment 2: the cultivation of Helicobacter pylori O6 type, inactivation and the extraction of lipopolysaccharide
[0066] When cultivating Helicobacter pylori O6 type, the H. 2 Cultured on the blood agar plate under the condition of 72 hours, and then transferred to Brucella broth medium (ELITE-MEDIA) at 37 ° C, the gas environment is 7% CO 2 Cultivate under certain conditions for 72 hours to obtain a bacterial liquid.
[0067] When inactivating Helicobacter pylori O6 type, at 1.7×10 11 CFU / mL, 5mL of 4% paraformaldehyde was added to 50mL of bacterial solution to make the final concentration 0.4%, and incubated at 37°C for 72 hours. The cells were collected by centrifugation, washed twice with PBS (pH 7.4), and finally diluted to 5×10 with PBS (pH 7.4). 12 CFU / mL, stored at 4°C.
[0068] Helicobacter pylori type O6 lipopolysaccharide was extracted by the phenol-water method: the cells were collected by centrifugation, washed with phosphate buffer (PBS, pH7.4) twi...
Embodiment 3
[0074] Embodiment 3: Application of oligosaccharide compound in the preparation of Helicobacter pylori vaccine
[0075] Preparation of glycoprotein conjugates of oligosaccharide compounds and CRM-197:
[0076] Add triethylamine (12 μL, 86 μmol) to a DMSO / pyridine solution (1:1, 25 mL: 0.25 mL) of bis(p-nitrophenyl adipate) (PNP, 26.33 mg, 67.8 μmol) and stir at room temperature for 5 minutes . Add dropwise the oligosaccharide compound (1.6mg, 2.26μmol) that is dissolved in DMSO / pyridine (1 / 1, 0.1mL:0.1mL), stir and react at room temperature for 7 hours, TLC detection shows that the raw material is completely reacted, sugar stain (0.1% (v / v) 3-methoxyphenol, 2.5% (v / v) sulfuric acid-ethanol) showed product. The reaction mixture was lyophilized. The lyophilized solid was washed 6 times with chloroform (1 mL) to obtain oligosaccharide-PNP ester. CRM 197 Protein (1 mg, 0.017 μmol) was washed three times with sterile water (400 μL) in an ultrafiltration tube, and then washed ...
PUM
| Property | Measurement | Unit |
|---|---|---|
| molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More