CN1513859A - Preparation method of sulfuric acid polysaccharide kind substance 911 oligosaccharide fragment and its application - Google Patents
Preparation method of sulfuric acid polysaccharide kind substance 911 oligosaccharide fragment and its application Download PDFInfo
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- CN1513859A CN1513859A CNA031389708A CN03138970A CN1513859A CN 1513859 A CN1513859 A CN 1513859A CN A031389708 A CNA031389708 A CN A031389708A CN 03138970 A CN03138970 A CN 03138970A CN 1513859 A CN1513859 A CN 1513859A
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Abstract
A process for preparing the oligose fragment '911' for polyose sulfate includes degradation reaction by use of Fenton reagent, ultrasonic treating, centrifugal separation of deposit, and separation with gel column to obtain various oligose fragment components. Two oligose fragments (HexA)6(OSO3H)6 and (HexA)7(OSO3H)8 are determined as the anti-AIDS ones by SPR technique and virus infected cell model, so they can be used to prepare AIDS virus inhibitor.
Description
Technical field:
The present invention relates to a kind of manufacture method and the application aspect anti AIDS virus thereof of sulfated polysaccharide class material 911 oligose fragment.
Background technology:
Sulfated polysaccharide class material 911 (Sulfated Polymannuroguluronate, hereinafter to be referred as 911) be extraction separation and a kind of polyanion straight chain sulfated polysaccharide of obtaining through chemically modified from brown alga, molecular-weight average is 10000Da, its in vivo and in vitro the anti-immunodeficient disease cytotoxic activity of high-efficiency low-toxicity make it become the drug candidate of anti AIDS virus, and find, 911 anti-AIDS cytotoxic activities with its with gp120 molecule, CD4 molecule, TAT albumen, CypA molecule in conjunction with relevant.For from the research of 911 architecture basics its with the mechanism of above-mentioned protein molecular effect, 911 to be parent compound, it is very necessary having active minimum oligose fragment by part degraded preparation serial oligosaccharides, especially acquisition.But the 911st, semisynthetic polyuronide class sulfated polysaccharide does not also have available degrading enzyme up till now, and acid hydrolysis commonly used because condition is too violent, very easily causes losing of important activity group sulfate radical again.
Summary of the invention:
The purpose of this invention is to provide the preparation method and the application of 911 oligose fragment, it can overcome the above-mentioned shortcoming of prior art.
A kind of manufacture method of oligose fragment of sulfated polysaccharide class material 911 is characterized in that adopting Fenton reagent to carry out DeR, through supersound process, and the centrifugal precipitation of going, gel column separates, and obtains 911 oligose fragment components.
Marine sulfate polysaccharide class material 911 oligose fragment are used for the HIV (human immunodeficiency virus) infection inhibitor.
Advantage of the present invention has provided a kind of manufacture method of sulfated polysaccharide class material 911 oligose fragment, has determined its minimum active fragments, and their function and activity can be widely used.
Description of drawings and embodiment:
Accompanying drawing 1 is that 911 degradable components are to virus of AIDS gp120 molecule and 911 bonded restraining effect figure.
Accompanying drawing 2 is the MALDI-TOF-MS spectrogram of 911 degradable components 5.
Accompanying drawing 3 is the MALDI-TOF-MS spectrogram of 911 degradable components 6.
Embodiment:
Take by weighing 911 100mg and add in the reaction flask, add 5mL 2mM FeSO
4It is molten entirely that the aqueous solution makes it, and adds 5mL1%H again
2O
2The aqueous solution shakes up, and transfers pH to 7.0 with the 1M NaOH aqueous solution rapidly, in 37 ℃ of stirring reactions, when reaction proceeds to 1.5h, adds Na
2S
2O
3Termination reaction.Reaction mixture is through supersound process 10min, and the centrifugal precipitation of going is evaporated to 2mL, and (1.6 * 90cm) separate, and with the 0.5M sodium bicarbonate aqueous solution wash-out of pH6.5, flow velocity is 4mL/h, and substep is collected, the 2mL/ pipe with Sephadex G-10 post.Detect with the carbazole colorimetry, collect each component, outer water component is concentrated, continue to separate with Sephadex G-15 post, all the other components are concentrated freeze-dried.Water component separates with Sephadex G-25 post again outside Sephadex G-15 post is isolating, collects, and obtains 17 components altogether, and each component freeze-drying is preserved.
Determine the minimum active fragments of marine sulfate polysaccharide class material 911 anti-AIDS: 911 minimum active fragmentss are to determine by the activity detection of SPR technology and cell levels.Method is: the detection of SPR technology is to be connected with vitamin H by hexanediamine 911; Simultaneously Streptavidin is coupled to bio-sensing chip; Utilize the high-affinity of vitamin H and Streptavidin to link bio-sensing chip with biotinylated 911.Then, adopt the method for competitive inhibition, utilize bio-sensing chip technology (SPR) detect 911 each degradable component target spot gp120 molecule relevant, CD4 molecule, TAT albumen, CypA molecule with antiviral activity in conjunction with situation.911 each degradable component that are about to 1ug/ml were hatched 3 minutes jointly with certain density gp120 (3ug/ml), CD4 (17nM), TAT albumen (41nM), CypA (5ug/ml) respectively, mixtures incubated is flow through chip surface successively, flow velocity 5ul/min, sample size 10ul, write down its reacting value, observe 911 each degradable component gp120, CD4, TAT albumen, CypA and chip 911 bonded restraining effect.The result shows that 911 degradable component 1-4 are lower to gp120 molecule and 911 bonded inhibiting rates, the inhibiting rate of component 5 then extremely significantly increases, component 6 further increases, each component restraining effect slowly increases subsequently, to component 15, no longer be significantly increased, reach former 911 inhibition level (accompanying drawing 1) substantially.911 each degradable component are to CD4 molecule, TAT albumen, CypA molecule and 911 combine and have similar inhibition situation, illustrate that component 5 is 911 to combine required minimum oligosaccharides segment with gp120, CD4, TAT albumen, CypA equimolecular.
Minimum activity unit is carried out MALDI-TOF-MS to be analyzed: in order further 911 minimum active oligose fragment to be carried out Analysis and Identification, utilize MALDI-TOF-MS to measure its molecular weight, and determine the number of uronic acid and sulfate radical in view of the above.Mass spectrum is seen accompanying drawing 2, and the m/z of main molecules quasi-molecular ions is 1543.7 among this figure, is equivalent to [M+H]
+, thereby the molecular weight of 911 minimum active oligose fragment is: 1542.7, be one by six six uronic acids and six terminal reductive six sugar that sulfate radical is formed, its molecular formula is: (HexA)
6(OSO
3H)
6In addition, also measure the molecular weight of 911 high reactivities, six sugared units, seen accompanying drawing 3.Same definite its molecular formula is: (HexA)
7(OSO
3H)
8
Using Fenton reagent method can study the molecular mechanism of marine sulfate polysaccharide 911 anti-AIDS better to the 911 minimum active fragmentss that obtained of degrading.
In order further to determine the activity of 911 each degradable component, utilize the HIV cells infected to measure the antiviral activity of 911 each degradable component.Method is: the MT that gets the growth of subculture in vitro separately logarithmic phase
4Cell, the microscopically counting is got 5,000,000 cells and 10000TCID
50HIV-1 is hatched 90min, centrifuge washing twice, and adjusting cell concn is 5 * 10
5Individual/ml, 911 of every hole 100 μ l and 5ug/ml each degradable component 100 μ l co-cultivation in 96 well culture plates, every concentration are established three multiple holes, establish negative control hole simultaneously, cultivate six days, get supernatant according to P
24CAg Mieroelisa detection reagent cassette method is measured P
24The cAg amount is calculated the inhibiting rate of medicine to viral growth.The result shows that its inhibiting rate is consistent with 911 bonded situations with its inhibition gp120.Promptly 911 degradable component 1-4 are almost without any restraining effect, and the inhibiting rate of component 5 then extremely significantly increases, and component 6 further increases, and each component restraining effect slowly increases subsequently, no longer is significantly increased to component 15, reaches former 911 inhibition level substantially.Illustrate that component 5 is the required minimum oligosaccharide unit of 911 inhibition virus infectiones.
Claims (3)
1. the manufacture method of the oligose fragment of a sulfated polysaccharide class material 911 is characterized in that adopting Fenton reagent to carry out DeR, through supersound process, and the centrifugal precipitation of going, gel column separates, and obtains 911 oligose fragment components.
2. method according to claim 1 is characterized in that the molecular formula that can suppress the required minimum sugared unit of HIV (human immunodeficiency virus) infection cell is: (HexA)
6(OSO
3H)
6(HexA)
7(OSO
3H)
8
3. marine sulfate polysaccharide class material 911 oligose fragment are used for the HIV (human immunodeficiency virus) infection inhibitor.
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| CN 03138970 CN1234716C (en) | 2003-08-04 | 2003-08-04 | Preparation method of sulfuric acid polysaccharide kind substance 911 oligosaccharide fragment and its application |
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| CN 03138970 CN1234716C (en) | 2003-08-04 | 2003-08-04 | Preparation method of sulfuric acid polysaccharide kind substance 911 oligosaccharide fragment and its application |
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| CN1234716C CN1234716C (en) | 2006-01-04 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106084085A (en) * | 2016-07-19 | 2016-11-09 | 北京颐方生物科技有限公司 | A kind of preparation method and application of low-molecular-weight algal polysaccharide sulfate |
| CN110809474A (en) * | 2017-06-20 | 2020-02-18 | 加利福尼亚大学董事会 | Production of bioactive oligosaccharides |
| CN112051320A (en) * | 2020-08-24 | 2020-12-08 | 复旦大学 | Method for rapidly detecting bacterial activity based on laser analysis ionization mass spectrometry technology |
-
2003
- 2003-08-04 CN CN 03138970 patent/CN1234716C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106084085A (en) * | 2016-07-19 | 2016-11-09 | 北京颐方生物科技有限公司 | A kind of preparation method and application of low-molecular-weight algal polysaccharide sulfate |
| CN106084085B (en) * | 2016-07-19 | 2018-07-31 | 北京颐方生物科技有限公司 | A kind of preparation method and application of low-molecular-weight algal polysaccharide sulfate |
| CN110809474A (en) * | 2017-06-20 | 2020-02-18 | 加利福尼亚大学董事会 | Production of bioactive oligosaccharides |
| CN112051320A (en) * | 2020-08-24 | 2020-12-08 | 复旦大学 | Method for rapidly detecting bacterial activity based on laser analysis ionization mass spectrometry technology |
| CN112051320B (en) * | 2020-08-24 | 2021-09-24 | 复旦大学 | Method for rapidly detecting bacterial activity based on laser analysis ionization mass spectrometry technology |
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| CN1234716C (en) | 2006-01-04 |
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Granted publication date: 20060104 Termination date: 20100804 |