CN104772121A - Preparation method of hepatitis B virus surface protein adsorbent - Google Patents
Preparation method of hepatitis B virus surface protein adsorbent Download PDFInfo
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- CN104772121A CN104772121A CN201510142632.6A CN201510142632A CN104772121A CN 104772121 A CN104772121 A CN 104772121A CN 201510142632 A CN201510142632 A CN 201510142632A CN 104772121 A CN104772121 A CN 104772121A
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Abstract
The invention discloses a preparation method of a hepatitis B virus surface protein adsorbent. The method uses amino acid treatment to connect adsorption microspheres of a ligand to make the ligand firmly combined, so the shedding probability of the hepatitis B virus surface protein adsorbent is reduced, and the application safety of the hepatitis B virus surface protein adsorbent is improved.
Description
Technical field
The present invention relates to biological technical field, specifically refer to a kind of preparation method of hepatitis B virus surface proteins adsorbent.
Background technology
Hepatitis type B virus (HBV) belongs to Hepadnaviridae (hepadnaviridae), and genome is about 3.2kb, is partially double stranded cyclic DNA.Because the situation of Chinese hepatitis B is serious, so country has formulated vaccine injection system.But research shows, and a lot of hepatitis B has the performance of " immunologic escape ", namely possess the ability infecting vaccine recipient, cause the application prospect of vaccine to allow of no optimist.
Research finds, the toxalbumin in blood is the major virulent factor of hepatitis B, and hepatitis B toxalbumin mediation hepatitis B is for hepatocellular infection.Sorbing material of a kind of hepatitis B antigen albumen and preparation method thereof is disclosed in prior art, the method adopts the polypeptide (aglucon) affine with hepatitis B virus surface proteins (HBsAg) to be connected with microballoon, by polypeptide in conjunction with hepatitis B virus surface proteins memebrane protein (HBsAg), thus reach hepatitis B toxalbumin in removing blood, reduce the effect of virus for hepatocyte infection.
Current preparation the type adsorbent the microballoon commonly used be agarose microbeads, polyvinyl alcohol microparticles, chitosan microball etc., the aglucon be connected with microballoon has polypeptide, albumen etc.When making microballoon, some microballoons can need first to use acid, oxygenation pretreatment, then adopt reagent activation microsphere surface, and what have is directly then direct activation; And then add crosslinking agent or coupling agent and connect microballoon and aglucon, the functional group on crosslinking agent (coupling agent) reacts as the amino on hydroxyl and aglucon, and aglucon is combined on microballoon.But these adsorbents in use, along with passage of time, the aglucon had in various degree comes off generation, and be easily subject to the ambient influnence such as temperature, current, may be come off because the combination between aglucon and crosslinking agent there occurs backward reaction, when in blood after aglucon comes off, may immune response be caused, there is certain security risk.
Summary of the invention
The object of the invention is to overcome aglucon on the hepatitis B virus surface proteins adsorbent prepared in prior art and hold caducous defect, provide a kind of simple to operate, the preparation method of aglucon in conjunction with the hepatitis B virus surface proteins adsorbent of firmness can be increased substantially.
For achieving the above object, the preparation method of the hepatitis B virus surface proteins adsorbent designed by the present invention, comprises the following steps:
1) prepare and the affine aglucon of hepatitis B virus surface proteins, and as the microballoon of carrier;
2) microsphere surface group is activated;
3) utilize crosslinking agent to make described aglucon be combined on microballoon, obtain adsorption microspheres;
4) under solution environmental, in adsorption microspheres, add amino acid, the weight ratio that amino acid adds weight and aglucon is greater than 2.5:1000, is attached to after on adsorption microspheres until amino acid, wash away unnecessary amino acid, obtain hepatitis B virus surface proteins adsorbent.
Preferably, one or more mixing in described amino acid selected from lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, valine, histidine, glycine.The stable amino acid of molecular properties all can adopt, and it is better that the amino acid effect of experimental result display mixing is better than the fixed effect of the unequal combination of amino acids of single amino acid, particularly size to aglucon.
Preferably, described amino acid is histidine: glycine: the mixture of lysine=1:2.1:0.5, and described aglucon is that sodium ion taurocholate cotransports polypeptide, and described microballoon is agarose microbeads.
Preferably, described step 4) in, add amino acid in adsorption microspheres after, the condition that amino acid is attached on adsorption microspheres is, in the environment of 25 ~ 40 DEG C, leaves standstill containing adsorption microspheres and amino acid whose solution more than 30 minutes.Suitable temperature can promote that amino acid is attached to microballoon and gets on.
Preferably, described step 4) in, washing away the cleaning solution that unnecessary amino acid adopts is PBS.
The principle of the invention: microballoon its upper group exposure after process activation is connected with aglucon by crosslinking agent, but this connection is by various factors, the amino acid added serves the effect of closed exposed group, covalently bound process is carried out to not participating in exposed group on the microballoon of reaction and crosslinking agent, also the binding site of functional group on aglucon and crosslinking agent may be have impact on, strengthen the combination between aglucon and crosslinking agent, make aglucon be not easy to come off from microballoon.In theory, amino acid is closed all effective to multiple aglucon.
Beneficial effect of the present invention: by the adsorption microspheres adopting amino acid process to be connected to aglucon, aglucon on it is combined more firm, reducing probability and speed that hepatitis B virus surface proteins adsorbent comes off, improve the security of hepatitis B virus surface proteins adsorbent when applying.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail, and following examples are explanation of the invention and the present invention is not limited to following examples.
Embodiment 1
The preparation method of hepatitis B virus surface proteins adsorbent, comprises the following steps:
1) preparation and the affine sodium ion taurocholate of hepatitis B virus surface proteins cotransport the agarose microbeads of polypeptide (NTCP) and carrier;
How NTCP and agarose microbeads are prepared as published prior art, do not repeat in the application.
2) activated agarose microsphere surface group: add agarose microbeads solution 600ml in the reactor of 2L, the NaOH aqueous solution 400ml of 2mol/L, mixing, after adding ethylene glycol bis glycidol ether 400ml, was placed in constant-temperature table, 37 DEG C of reactions 1 hour.Terminate reaction, by agarose microbeads filter, with a large amount of distilled water flushing to pH=7, the gel agarose microbeads activated is stored in 4 DEG C for subsequent use.
3) NTCP is connected in upper agarose microbeads by the method for chemical crosslinking:
Be added in the borate buffer 600ml of 0.3mol/L by agarose microbeads gel 200ml, pH value controls, in 8.0 ~ 10.0 scopes, to add 3g NTCP, 37 DEG C of reactions 12 hours, stop, reclaiming unreacted NTCP, obtain the adsorption microspheres solution combining aglucon.
4) in adsorption microspheres solution, add lysine, the weight ratio that lysine adds weight and aglucon is 1:200, leaves standstill 2h, use distilled water flushing again, wash away unnecessary amino acid, obtain hepatitis B virus surface proteins adsorbent 1, be kept in the phosphate buffer of 0.2mol/L pH=7.4.
Embodiment 2
The preparation method of hepatitis B virus surface proteins adsorbent, comprises the following steps:
1) preparation and the affine sodium ion taurocholate of hepatitis B virus surface proteins cotransport the agarose microbeads of polypeptide (NTCP) and carrier;
How NTCP and agarose microbeads are prepared as published prior art, do not repeat in the application.
2) activated agarose microsphere surface group: add agarose microbeads solution 600ml in the reactor of 2L, the NaOH aqueous solution 400ml of 2mol/L, mixing, after adding ethylene glycol bis glycidol ether 400ml, was placed in constant-temperature table, 37 DEG C of reactions 1 hour.Terminate reaction, by agarose microbeads filter, with a large amount of distilled water flushing to pH=7, the gel agarose microbeads activated is stored in 4 DEG C for subsequent use.
3) NTCP is connected in upper agarose microbeads by the method for chemical crosslinking:
Be added in the borate buffer 600ml of 0.3mol/L by agarose microbeads gel 200ml, pH value controls, in 8.0 ~ 10.0 scopes, to add 3g NTCP, 37 DEG C of reactions 12 hours, stop, reclaiming unreacted NTCP, obtain the adsorption microspheres solution combining aglucon.
4) in adsorption microspheres solution, add the mixture of phenylalanine and isoleucine, mixture is phenylalanine: isoleucine=1:1, the weight ratio that amino acid adds weight and aglucon is 3:1000,1 hour is left standstill on constant temperature 30 DEG C of water baths, PBS (0.01M pH8.0) is used to wash 3 times again, wash away unnecessary amino acid, obtain hepatitis B virus surface proteins adsorbent 2, be kept in the phosphate buffer of 0.2mol/L pH=7.4.
Embodiment 3
The preparation method of hepatitis B virus surface proteins adsorbent, comprises the following steps:
1) preparation and the affine sodium ion taurocholate of hepatitis B virus surface proteins cotransport the agarose microbeads of polypeptide (NTCP) and carrier;
How NTCP and agarose microbeads are prepared as published prior art, do not repeat in the application.
2) activated agarose microsphere surface group: add agarose microbeads solution 600ml in the reactor of 2L, the NaOH aqueous solution 400ml of 2mol/L, mixing, after adding ethylene glycol bis glycidol ether 400ml, was placed in constant-temperature table, 37 DEG C of reactions 1 hour.Terminate reaction, by agarose microbeads filter, with a large amount of distilled water flushing to pH=7, the gel agarose microbeads activated is stored in 4 DEG C for subsequent use.
3) NTCP is connected in upper agarose microbeads by the method for chemical crosslinking:
Be added in the borate buffer 600ml of 0.3mol/L by agarose microbeads gel 200ml, pH value controls, in 8.0 ~ 10.0 scopes, to add 3g NTCP, 37 DEG C of reactions 12 hours, stop, reclaiming unreacted NTCP, obtain the adsorption microspheres solution combining aglucon.
4) in adsorption microspheres solution, add the mixture of valine and histidine, mixture is valine: histidine=1:6, the weight ratio that amino acid adds weight and aglucon is 2.5:1000, leave standstill 3h, use distilled water flushing again, wash away unnecessary amino acid, obtain hepatitis B virus surface proteins adsorbent 3, be kept in the phosphate buffer of 0.2mol/L pH=7.4.
Embodiment 4
The preparation method of hepatitis B virus surface proteins adsorbent, comprises the following steps:
1) preparation and the affine sodium ion taurocholate of hepatitis B virus surface proteins cotransport the agarose microbeads of polypeptide (NTCP) and carrier;
How NTCP and agarose microbeads are prepared as published prior art, do not repeat in the application.
2) activated agarose microsphere surface group: add agarose microbeads solution 600ml in the reactor of 2L, the NaOH aqueous solution 400ml of 2mol/L, mixing, after adding ethylene glycol bis glycidol ether 400ml, was placed in constant-temperature table, 37 DEG C of reactions 1 hour.Terminate reaction, by agarose microbeads filter, with a large amount of distilled water flushing to pH=7, the gel agarose microbeads activated is stored in 4 DEG C for subsequent use.
3) NTCP is connected in upper agarose microbeads by the method for chemical crosslinking:
Be added in the borate buffer 600ml of 0.3mol/L by agarose microbeads gel 200ml, pH value controls, in 8.0 ~ 10.0 scopes, to add 3g NTCP, 37 DEG C of reactions 12 hours, stop, reclaiming unreacted NTCP, obtain the adsorption microspheres solution combining aglucon.
4) in adsorption microspheres solution, ispol is added, mixture is histidine: glycine: lysine=1:2.1:0.5, the weight ratio that amino acid adds weight and aglucon is 1:100,30 minutes are left standstill on constant temperature 30 DEG C of water baths, PBS (0.01M pH 8.0) is used to wash 3 times again, wash away unnecessary amino acid, obtain hepatitis B virus surface proteins adsorbent 4, be kept in the phosphate buffer of 0.2mol/L pH=7.4.
Embodiment 5
The preparation method of hepatitis B virus surface proteins adsorbent, comprises the following steps:
1) preparation and the affine sodium ion taurocholate of hepatitis B virus surface proteins cotransport polypeptide (NTCP) and the glass microsphere as carrier;
How NTCP and glass microsphere are prepared as published prior art, do not repeat in the application.
Glass microsphere surface preparation: boil with 65% red fuming nitric acid (RFNA) and wash average diameter 85 μm of glass microspheres 10 minutes, then with distillation washing (deionization washes 3 times, distillation washing 2 times, each 2 minutes), put into 37 DEG C of oven for drying.
2) activated glass microsphere surface group: get distilled water, acetone (analyzing pure), (proportions of γ aminopropyltriethoxy silane (APTES) 15:280:6 by volume becomes APTES activated solution to silanization coupling agent, then glass microsphere is immersed 2 hours; With acetone washing slide 5 times (5min/ time), then microsphere surface is dried.
3) NTCP is connected on upper glass microsphere by the method for chemical crosslinking:
1. the microballoon of activation is put into 0.2wt%PDC (Pyridinium dichromate) solution, be placed in baking oven 2 hours (holding temperature 37 DEG C); From PDC, take out microballoon, first use methanol wash 5 times (5min/ time), then wash 2 times with acetone, finally wash once with distilled water, dry;
2. NTCP is mixed with microballoon, with microsphere volume (mL): the ratio that NTCP weight ratio (mg) is 1:20 mixes, in 37 DEG C of water-bath 2h, obtain the adsorption microspheres solution combining aglucon.
4) in adsorption microspheres solution, ispol is added, mixture is methionine: leucine: isoleucine=1:1:1, the weight ratio that amino acid adds weight and aglucon is 1:100,30 minutes are left standstill on constant temperature 37 DEG C of water baths, on constant temperature 37 DEG C of water baths, microseism is swung lower PBS (0.01M pH 8.0) and is washed 3 times again, wash away unnecessary amino acid, obtain hepatitis B virus surface proteins adsorbent 5, then room temperature dry, for subsequent use.
Embodiment 6
The preparation method of hepatitis B virus surface proteins adsorbent, comprises the following steps:
1) preparation and the affine sodium ion taurocholate of hepatitis B virus surface proteins cotransport the chitosan microball of polypeptide (NTCP) and carrier;
How NTCP and chitosan microball are prepared as published prior art, do not repeat in the application.
2) chitosan microball surface group is activated: take the chitosan crosslinked microballoon of 10g in the beaker of sterilizing, add the certain density epoxychloropropane of 20mL, solvent pH 8 ~ 9 is regulated by 1M NaOH solution, then 40 DEG C, under rotating speed 80rpm, reaction 2h activates, after activation terminates, then with acetone, ethanol, wash away remaining epoxychloropropane, then use a large amount of distilled water drip washing microballoon, drain, obtain shitosan activation microballoon.
3) NTCP is connected on upper chitosan microball by the method for chemical crosslinking:
The solution of NTCP is added drop-wise in the chitosan microball after epoxy activation, in 37 DEG C of low-speed oscillation reaction 4h.After reaction terminates, wash away unnecessary NTCP with distilled water, suck dry moisture, obtain the viruses adsorption microspheres solution combining aglucon.
4) in viruses adsorption microspheres solution, ispol is added, mixture is histidine: glycine: lysine=1:1.5:3, the weight ratio that amino acid adds weight and aglucon is 1:100,30 minutes are left standstill on constant temperature 37 DEG C of water baths, on constant temperature 37 DEG C of water baths, microseism is swung lower PBS (0.01M pH 8.0) and is washed 3 times again, wash away unnecessary amino acid, obtain hepatitis B virus surface proteins adsorbent 6, then room temperature dry, for subsequent use.
Reference examples (not closing with amino acid)
The preparation method of hepatitis B virus surface proteins adsorbent, comprises the following steps:
1) preparation and the affine sodium ion taurocholate of hepatitis B virus surface proteins cotransport the agarose microbeads of polypeptide (NTCP) and carrier;
How NTCP and agarose microbeads are prepared as published prior art, do not repeat in the application.
2) activated agarose microsphere surface group: add agarose microbeads solution 600ml in the reactor of 2L, the NaOH aqueous solution 400ml of 2mol/L, mixing, after adding ethylene glycol bis glycidol ether 400ml, was placed in constant-temperature table, 37 DEG C of reactions 1 hour.Terminate reaction, by agarose microbeads filter, with a large amount of distilled water flushing to pH=7, the gel agarose microbeads activated is stored in 4 DEG C for subsequent use.
3) NTCP is connected in upper agarose microbeads by the method for chemical crosslinking:
Agarose microbeads gel 200ml is added in the borate buffer 600ml of 0.3mol/L, pH value controls in 8.0 ~ 10.0 scopes, add 3g NTCP, 37 DEG C of reactions 12 hours, stop, reclaim unreacted NTCP, obtain the adsorption microspheres solution combining aglucon, on constant temperature 37 DEG C of water baths, microseism is swung lower PBS (0.01M pH 8.0) and is washed 3 times, washes away unnecessary polypeptide, obtain hepatitis B virus surface proteins adsorbent (contrast), then room temperature dry, for subsequent use.
Test example
Get the hepatitis B virus surface proteins adsorbent 1 ~ 6 and reference examples that obtain in above-described embodiment, be added in physiological saline, then be placed on constant temperature 37 DEG C of water baths, slight concussion, respectively at 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, detect the dropping situations of NTCP peptide molecule on it, the NTCP come off adopts ultraviolet method to detect, then the original amount in conjunction with NTCP on the NTCP do not come off and adsorbent contrasted, result is as follows:
| Numbering | Contrast | Adsorbent 1 | Adsorbent 2 | Adsorbent 3 | Adsorbent 4 | Adsorbent 5 | Adsorbent 6 |
| 0.5h | 99％ | 99％ | 99％ | 99％ | 100％ | 99％ | 100％ |
| 1h | 93％ | 94％ | 96％ | 94.5％ | 97％ | 95％ | 97％ |
| 2h | 88％ | 92％ | 94％ | 92％ | 96％ | 93％ | 96％ |
| 4h | 72％ | 87％ | 88％ | 88％ | 90％ | 87％ | 89％ |
| 8h | 57％ | 78％ | 80％ | 84％ | 85％ | 75％ | 84％ |
| 12h | 52％ | 70％ | 75％ | 79％ | 80％ | 69％ | 78％ |
| 24h | 45％ | 64％ | 68％ | 69％ | 70％ | 62％ | 65％ |
Result shows, its firmness combined of the hepatitis B virus surface proteins adsorbent after amino acid process is utilized to rise to some extent, particularly there is no amino acid-treatedly littlely had the aglucon of about 30% to come off up to 4 hours these time periods to impinging upon 0.5, and the aglucon probability that comes off is reduced to about 10% on adsorbent after process, this security for hepatitis B virus surface proteins adsorbent promotes to some extent.
Claims (5)
1. a preparation method for hepatitis B virus surface proteins adsorbent, comprises the following steps:
1) prepare and the affine aglucon of hepatitis B virus surface proteins, and as the microballoon of carrier;
2) microsphere surface group is activated;
3) utilize crosslinking agent to make described aglucon be combined on microballoon, obtain adsorption microspheres;
4) under solution environmental, in adsorption microspheres, add amino acid, the weight ratio that amino acid adds weight and aglucon is greater than 2.5:1000, is attached to after on adsorption microspheres until amino acid, wash away unnecessary amino acid, obtain hepatitis B virus surface proteins adsorbent.
2. the preparation method of hepatitis B virus surface proteins adsorbent according to claim 1, is characterized in that: one or more mixing in described amino acid selected from lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, valine, histidine, glycine.
3. the preparation method of hepatitis B virus surface proteins adsorbent according to claim 1; it is characterized in that: described amino acid is histidine: glycine: the mixture of lysine=1:2.1:0.5; described aglucon is that sodium ion taurocholate cotransports polypeptide, and described microballoon is agarose microbeads.
4. the preparation method of hepatitis B virus surface proteins adsorbent according to claim 1, it is characterized in that: described step 4) in, add amino acid in adsorption microspheres after, the condition that amino acid is attached on adsorption microspheres is, in the environment of 25 ~ 40 DEG C, leave standstill containing adsorption microspheres and amino acid whose solution more than 30 minutes.
5. the preparation method of hepatitis B virus surface proteins adsorbent according to claim 1, is characterized in that: described step 4) in, washing away the cleaning solution that unnecessary amino acid adopts is PBS.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112717902A (en) * | 2020-12-31 | 2021-04-30 | 武汉瑞法医疗器械有限公司 | Hepatitis B virus adsorbent and preparation method and application thereof |
| CN113578288A (en) * | 2021-07-30 | 2021-11-02 | 北京中科太康科技有限公司 | Adsorbent for cell inflammatory factors and preparation method thereof |
-
2015
- 2015-03-29 CN CN201510142632.6A patent/CN104772121A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112717902A (en) * | 2020-12-31 | 2021-04-30 | 武汉瑞法医疗器械有限公司 | Hepatitis B virus adsorbent and preparation method and application thereof |
| CN112717902B (en) * | 2020-12-31 | 2022-05-17 | 武汉瑞法医疗器械有限公司 | Hepatitis B virus adsorbent and preparation method and application thereof |
| CN113578288A (en) * | 2021-07-30 | 2021-11-02 | 北京中科太康科技有限公司 | Adsorbent for cell inflammatory factors and preparation method thereof |
| CN113578288B (en) * | 2021-07-30 | 2023-11-14 | 北京中科太康科技有限公司 | Cell inflammatory factor adsorbent and preparation method thereof |
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