CN112717902B - Hepatitis B virus adsorbent and preparation method and application thereof - Google Patents
Hepatitis B virus adsorbent and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a hepatitis B virus adsorbent and a preparation method and application thereof. Mixing N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and dissolving the mixture in a buffer solution to prepare a crosslinking catalyst; and adding the obtained cross-linking catalyst into the carboxylated agarose gel microsphere suspension, fully and uniformly mixing, then adding the dissolved amino-modified nucleic acid aptamer, and utilizing amidation reaction to directly couple the carboxylated agarose gel microsphere and the amino-modified nucleic acid aptamer, thereby simply, conveniently and efficiently preparing the hepatitis B virus adsorbent. Through the mode, the nucleic acid aptamer can be used for replacing a traditional antibody to serve as a ligand, so that the prepared adsorbent has higher adsorption efficiency, and the effective adsorption of the hepatitis B surface antigen is realized, so that the content of the hepatitis B surface antigen in blood is effectively reduced, and the method has higher practical application value.
Description
Technical Field
The invention relates to the technical field of biomedical adsorption materials, in particular to a hepatitis B virus adsorbent and a preparation method and application thereof.
Background
Hepatitis b is a liver-like disease caused by infection with Hepatitis B Virus (HBV), including acute and chronic hepatitis. Positive serological detection of HBV surface antigen (HBsAg) is the main standard for definitive diagnosis, and in clinical practice, removal of HBsAg is considered to be the most critical therapeutic endpoint of chronic hepatitis b, associated with improved clinical outcome, prolonged survival, reduced incidence of cirrhosis and hepatocellular carcinoma. Currently, the treatment methods for hepatitis b mainly focus on drug screening, drug administration, targeted therapy and the like, but these treatment schemes have limited effects and poor long-term treatment effects.
Based on the problems with the existing treatment protocols, extracorporeal blood purification systems are receiving increasing attention from researchers and are being applied to the treatment of hepatitis b. The extracorporeal blood purifying system includes blood filtering, blood perfusion and blood adsorption, and has the operation principle of lowering the toxicity degree and time via lowering the concentration of pathogen related molecule in blood, wherein the blood adsorption is different from other purifying modes in that ligand capable of combining with target molecule specifically is introduced to produce corresponding adsorbent to eliminate specific target molecule effectively. Since the adsorption effect of the adsorbent is closely related to the adsorption material used as a carrier, the type of ligand and the binding mode thereof, how to select an appropriate carrier, ligand and binding mode thereof has become a major research point in the development process of the hepatitis b virus adsorbent.
The patent with publication number CN111659355A provides an alkylation modified hepatitis B virus immunoadsorbent and a preparation method thereof, the patent combines an activated adsorption carrier and a hepatitis B virus antibody in a coupling way, carries out alkylation modification on sulfydryl in the hepatitis B virus antibody, and can obtain the alkylation modified hepatitis B virus immunoadsorbent with high adsorption efficiency, good specificity and good reproducibility after reduction treatment, thereby increasing the operability and the reutilization rate in clinical use and further reducing the production and use costs. However, this patent uses an antibody as a ligand, and the production of the antibody requires a high cost and a long time, and the antibody undergoes irreversible denaturation at room temperature or higher for a certain period of time, and has high immunogenicity. Therefore, there is still a need to find alternatives to antibodies to improve the performance of adsorbents.
In view of the above, there is a need for an improved hepatitis b virus adsorbent and a method for preparing the same to solve the above problems.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a hepatitis b virus adsorbent and a preparation method and application thereof. The carboxylated agarose gel microspheres are directly coupled with the amino-modified nucleic acid aptamer by using the cross-linking agent, so that the hepatitis B virus adsorbent is simply, conveniently and efficiently prepared, and the effective adsorption of the hepatitis B surface antigen is realized, so that the content of the hepatitis B surface antigen in blood is reduced.
In order to achieve the above object, the present invention provides a method for preparing a hepatitis b virus adsorbent, comprising the steps of:
s1, washing the carboxylated agarose gel microspheres to obtain agarose gel microsphere suspension;
s2, placing the amino modified nucleic acid aptamer into an aptamer dissolving buffer solution, carrying out water bath treatment after oscillation and dissolution, placing the solution in a room temperature environment, and obtaining the dissolved aptamer after renaturation;
s3, mixing N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride according to a preset molar ratio, and dissolving the mixture in a 2- (N-morpholine) ethanesulfonic acid buffer solution to obtain a crosslinking catalyst;
s4, adding the crosslinking catalyst obtained in the step S3 into the agarose gel suspension obtained in the step S1, fully and uniformly mixing, adding the dissolved aptamer obtained in the step S2, and carrying out an amide reaction;
and S5, adding a trihydroxymethyl aminomethane hydrochloride buffer solution to stop the reaction after the amide reaction is finished in the step S4, and rinsing to obtain the hepatitis B virus adsorbent.
As a further improvement of the invention, in step S2, the temperature of the water bath treatment is 50-70 ℃, and the time is 5-15 min.
As a further improvement of the invention, in step S3, the preset molar ratio is 1 (1-4).
As a further improvement of the invention, in step S4, the reaction temperature of the amide reaction is 25-30 ℃, and the reaction time is 4-20 h.
As a further improvement of the invention, in step S2, the aptamer dissolution buffer solution contains Na2HPO4And MgCl2Said Na2HPO4And MgCl2The concentrations are respectively 150-250 mmol/L and 3-7 mmol/L; the pH value of the aptamer dissolving buffer solution is 7.5-8.5.
In a further improvement of the invention, in step S3, the concentration of the 2- (N-morpholine) ethanesulfonic acid buffer solution is 0.04-0.06 mol/L, and the pH value is 5.0-6.0.
In a further improvement of the present invention, in step S5, the pH value of the tris hydrochloride buffer is 8.5 to 9.5; the rinsing process uses 0.01mol/L phosphate buffer solution for rinsing.
As a further improvement of the present invention, in step S2, the sequence of the aptamer is 5'-TGAGC CTCTG GATAC TTTTT ACCCA CAGCG AACAG CGGCG GACAT AATAG TGCTT ACTAC GACGC-3'.
In order to achieve the purpose, the invention also provides a hepatitis B virus adsorbent which is prepared according to any one of the technical schemes and comprises carboxylated agarose microspheres and an aptamer coupled with the carboxylated agarose microspheres.
The invention also provides application of the hepatitis B virus adsorbent in the field of blood adsorption.
The invention has the beneficial effects that:
(1) the invention uses N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride as cross-linking agent, controls reaction conditions, and can make the carboxylated agarose gel microspheres and amino-modified nucleic acid aptamers generate amide reaction, thereby successfully coupling the agarose gel and the nucleic acid aptamers, simply and efficiently preparing the hepatitis B virus adsorbent, realizing effective adsorption of hepatitis B surface antigen, and reducing the content of hepatitis B surface antigen in blood.
(2) According to the invention, the nucleic acid aptamer is selected to replace the traditional antibody as the ligand, so that the defects of high cost, high changeability, high immunogenicity and the like of the antibody can be avoided, and the prepared adsorbent has higher adsorption efficiency, more specific affinity, lower cost and longer storage period, and has higher practical application value.
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FIG. 1 is a schematic diagram of the preparation principle of the hepatitis B virus adsorbent provided by the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
It should be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and/or processing steps closely related to the aspects of the present invention are shown in the drawings, and other details not closely related to the present invention are omitted.
In addition, it is also to be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention provides a preparation method of a hepatitis B virus adsorbent, which comprises the following steps:
s1, washing the carboxylated agarose gel microspheres to obtain agarose gel microsphere suspension;
s2, placing the amino modified nucleic acid aptamer into an aptamer dissolution buffer solution, oscillating for dissolution, carrying out water bath treatment, placing in a room temperature environment, and obtaining a dissolved aptamer after renaturation;
s3, mixing N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride according to a preset molar ratio, and dissolving the mixture in a 2- (N-morpholine) ethanesulfonic acid buffer solution to obtain a crosslinking catalyst;
s4, adding the crosslinking catalyst obtained in the step S3 into the agarose gel suspension obtained in the step S1, fully and uniformly mixing, adding the dissolved aptamer obtained in the step S2, and carrying out an amide reaction;
and S5, adding a trihydroxymethyl aminomethane hydrochloride buffer solution to stop the reaction after the amide reaction is finished in the step S4, and rinsing to obtain the hepatitis B virus adsorbent.
In step S2, the sequence of the aptamer is 5'-TGAGC CTCTG GATAC TTTTT ACCCA CAGCG AACAG CGGCG GACAT AATAG TGCTT ACTAC GACGC-3'; the aptamer dissolving buffer solution contains Na2HPO4And MgCl2Said Na2HPO4And MgCl2The concentrations are respectively 150-250 mmol/L and 3-7 mmol/L; the pH value of the aptamer dissolving buffer solution is 7.5-8.5; the temperature of the water bath treatment is 50-70 ℃, and the time is 5-15 min.
In step S3, the preset molar ratio is 1 (1-4); the concentration of the 2- (N-morpholine) ethanesulfonic acid buffer solution is 0.04-0.06 mol/L, and the pH value is 5.0-6.0.
In step S4, the reaction temperature of the amide reaction is 25-30 ℃ and the reaction time is 4-20 h.
In step S5, the pH value of the tris hydrochloride buffer is 8.5-9.5; the rinsing process uses 0.01mol/L phosphate buffer solution for rinsing.
The invention also provides a hepatitis B virus adsorbent which is prepared according to the technical scheme and comprises carboxylated agarose microspheres and an aptamer coupled with the carboxylated agarose microspheres.
The invention also provides application of the hepatitis B virus adsorbent in the field of blood adsorption.
The following will explain the hepatitis b virus adsorbent provided by the present invention, its preparation method and application by referring to specific examples.
Example 1
The embodiment provides a preparation method of a hepatitis b virus adsorbent, the reaction principle of which is shown in fig. 1, and the preparation method specifically comprises the following steps:
s1, uniformly shaking the carboxylated agarose gel microspheres, extracting 10mL of the uniformly mixed microspheres by using a pipette gun, transferring the microspheres into a 100mL conical flask, standing for 5min at room temperature, slowly sucking and removing the upper layer of protection buffer solution, adding 30mL of distilled water for re-suspension, transferring the microspheres into a filter flask, washing three times by using 200mL of distilled water to completely remove part of the protection buffer solution, quickly transferring the washed microspheres into the 100mL conical flask, and keeping the microspheres moist in the whole process to obtain the agarose gel microsphere suspension.
S2, taking out the ammonia with the content of 250 mu g from a refrigerator at the temperature of 20 ℃ below zeroStanding the modified nucleic acid aptamer-based EP tube at room temperature for 5min, and centrifuging at the rotation speed of 5000r/min at room temperature for 2min to make the aptamer attached to the EP tube wall gather at the tube bottom. Then taking aptamer dissolving buffer (containing 200mmol/L Na)2HPO4And 5mmol/L MgCl2pH 8.0), slightly shaking, standing at room temperature for 10min, treating in a water bath at 60 deg.C for 10min, and standing at room temperature for 20min to obtain the soluble aptamer.
S3, weighing 0.23g N-hydroxysuccinimide (NHS) and 0.78g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) respectively to ensure that the molar ratio of the NHS to the EDC is 1: 2.5; then, the solution was dissolved in a buffer solution of 2- (N-morpholine) ethanesulfonic acid (MES) at a pH of 5.5 and a concentration of 0.05mol/L to obtain a crosslinking catalyst.
S4, adding the crosslinking catalyst obtained in the step S3 into the agarose gel suspension obtained in the step S1, fully and uniformly mixing, adding the dissolved aptamer obtained in the step S2, placing in a thermostat at 27 ℃ and reacting for 12 hours;
s5, after the amide reaction in step S4 is completed, Tris-HCl (Tris-HCl) buffer solution with pH 9 is added to terminate the reaction, and the remaining active ester is blocked, and the solution is rinsed twice with Phosphate Buffered Saline (PBS) having a concentration of 0.01mol/L to obtain the hepatitis b virus adsorbent.
Examples 2 to 10
Examples 2 to 10 each provide a method for preparing a hepatitis b virus adsorbent, which is different from example 1 in that the molar ratio of NHS to EDC in step S3 or the reaction time in step S4 is changed, and the molar ratio and the reaction time in each example are shown in table 1.
TABLE 1 reaction parameters corresponding to examples 2 to 10
Static blood samples from hepatitis B patients were taken, and the hepatitis B virus adsorbents prepared in examples 1 to 10 were used, respectively, according to the following: the blood sample is adsorbed according to the proportion of 1:5, and the content of hepatitis B surface antigen in the sample before and after adsorption is detected. Wherein the content of hepatitis B surface antigen in the sample before adsorption was 66.29IU/mL, and the content of hepatitis B surface antigen obtained after adsorption with the hepatitis B virus adsorbents provided in examples 1 to 10, respectively, is shown in Table 2.
TABLE 2 hepatitis B surface antigen content after adsorption with the adsorbents provided in examples 1-10
| Examples | Hepatitis B surface antigen content after adsorption (IU/mL) |
| Example 1 | 8.61 |
| Example 2 | 18.56 |
| Example 3 | 15.24 |
| Example 4 | 11.26 |
| Example 5 | 12.59 |
| Example 6 | 13.92 |
| Example 7 | 13.25 |
| Example 8 | 11.93 |
| Example 9 | 9.24 |
| Example 10 | 10.60 |
As can be seen from Table 2, with the increase of EDC content or the extension of the amide reaction time, the content of hepatitis B surface antigen after the adsorption of the prepared hepatitis B virus adsorbent shows a tendency of first decreasing and then increasing, i.e. the adsorption efficiency of the adsorbent is first increasing and then decreasing, which indicates that the coupling of agarose gel and nucleic acid aptamer is not facilitated when the EDC content is too small or too large, and the reaction time is too short or too long, thereby affecting the adsorption effect of the prepared adsorbent. Therefore, the optimal molar ratio of NHS to EDC is 1 (1-4), the reaction time is 4-20 h, and the hepatitis B virus adsorbent prepared in the range can achieve a good adsorption effect, so that the requirement of practical application is met.
Comparative example 1
Comparative example 1 provides a method for preparing a hepatitis b virus adsorbent, comprising the steps of:
s1, taking 10mL of the agarose gel microspheres which are evenly mixed, washing the agarose gel microspheres, and adding 10mL of NaIO with the concentration of 0.2mol/L into the drained microspheres4The aqueous solution of (1) was mixed with gentle stirring. Then the beaker is placed on a magnetic stirrer, the stirring speed is kept slow, and the reaction is carried out for 90min at room temperature. Standing the beaker at room temperature for 5min, removing supernatant with a pipette after microspheres in the beaker are completely precipitated, adding 200mL of distilled water,and (4) resuspending the microspheres, transferring the microspheres into a filter flask, washing the microspheres with 200mL of distilled water for three times under a wet state, and terminating the oxidation reaction to obtain aldehyde group activated microspheres.
And S2, adding the aldehyde group activated agar gel microspheres into a beaker in ventilation, and fully stirring to suspend the agar gel microspheres to obtain aldehyde group activated gel microsphere suspension.
S3, accurately weighing 80mg NaCNBH by using a balance3And slowly adding the aldehyde group-activated gel microsphere suspension obtained in the step S2 into the aldehyde group-activated gel microsphere suspension, keeping the mixture stirred slowly, and reacting for 2 hours at room temperature.
S4, after the reaction in the step S3 is finished, the beaker is kept stand for 5min, after microspheres in the beaker are completely precipitated, supernatant is removed by a pipette gun, 200mL of distilled water is added, the microspheres are resuspended, the mixture is transferred to a filter flask, the filter flask is washed by 200mL of distilled water for three times under the condition of keeping the humidity, then the filter flask is washed by 200mL of NaCl with the concentration of 1mol/L, and finally the filter flask is washed by 200mL of distilled water to remove unreacted NaCNBH3To obtain the hepatitis B virus adsorbent.
The method provided in comparative example 1 is to activate agarose microspheres with sodium periodate, so that the activated agarose microspheres are coupled with antibodies to form the hepatitis b virus adsorbent.
To compare the adsorption effects of the hepatitis B virus adsorbents prepared in example 1 and comparative example 1, 15 HBV samples from different patients were divided into three portions each for measuring the content of hepatitis B surface antigen in the samples before adsorption, after adsorption by the adsorbent provided in comparative example 1, and after adsorption by the adsorbent provided in example 1, respectively, and the results are shown in Table 3.
Table 3 adsorption results of the adsorbents prepared in comparative example 1 and example 1 on different samples
As can be seen from table 3, compared with the hepatitis b virus adsorbent provided in comparative example 1, the content of hepatitis b surface antigen in blood after adsorption by the hepatitis b virus adsorbent provided in example 1 is significantly lower, and the higher the content of hepatitis b surface antigen in a sample is, the more significant the adsorption advantage of the adsorbent provided in example 1 is. The invention shows that the nucleic acid aptamer is selected to replace the traditional antibody as the ligand, so that the adsorption efficiency of the adsorbent can be effectively improved, a better adsorption effect is achieved, the requirement of practical application is met,
in conclusion, the invention provides a hepatitis B virus adsorbent and a preparation method and application thereof. Mixing N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and dissolving the mixture in a buffer solution to prepare a crosslinking catalyst; and adding the obtained cross-linking catalyst into the carboxylated agarose gel microsphere suspension, fully and uniformly mixing, then adding the dissolved amino-modified nucleic acid aptamer, and utilizing amidation reaction to directly couple the carboxylated agarose gel microsphere and the amino-modified nucleic acid aptamer, thereby simply, conveniently and efficiently preparing the hepatitis B virus adsorbent. Through the mode, the nucleic acid aptamer can be used for replacing a traditional antibody to serve as a ligand, so that the prepared adsorbent has higher adsorption efficiency, and the effective adsorption of the hepatitis B surface antigen is realized, so that the content of the hepatitis B surface antigen in blood is effectively reduced, and the method has higher practical application value.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the present invention.
Claims (10)
1. The preparation method of the hepatitis B virus adsorbent is characterized by comprising the following steps:
s1, washing the carboxylated agarose gel microspheres to obtain agarose gel microsphere suspension;
s2, placing the amino modified nucleic acid aptamer into an aptamer dissolving buffer solution, carrying out water bath treatment after oscillation and dissolution, placing the solution in a room temperature environment, and obtaining the dissolved aptamer after renaturation;
s3, mixing N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride according to a preset molar ratio, and dissolving the mixture in a 2- (N-morpholine) ethanesulfonic acid buffer solution to obtain a crosslinking catalyst;
s4, adding the crosslinking catalyst obtained in the step S3 into the agarose gel suspension obtained in the step S1, fully and uniformly mixing, adding the dissolved aptamer obtained in the step S2, and carrying out an amide reaction;
and S5, adding a trihydroxymethyl aminomethane hydrochloride buffer solution to stop the reaction after the amide reaction is finished in the step S4, and rinsing to obtain the hepatitis B virus adsorbent.
2. The method for preparing an adsorbent for hepatitis B virus according to claim 1, wherein: in step S2, the temperature of the water bath treatment is 20-80 ℃ and the time is 5-30 min.
3. The method for preparing the hepatitis B virus adsorbent according to claim 1, characterized in that: in step S3, the preset molar ratio is 1 (1-4).
4. The method for preparing the hepatitis B virus adsorbent according to claim 1, characterized in that: in step S4, the reaction temperature of the amide reaction is 25-30 ℃ and the reaction time is 4-20 h.
5. The method for producing a hepatitis B virus adsorbent according to claim 1 or 2, characterized in that: in step S2, the aptamer dissolution buffer contains Na2HPO4And MgCl2Said Na2HPO4And MgCl2The concentrations are respectively 150-250 mmol/L and 3-7 mmol/L; the pH value of the aptamer dissolving buffer solution is 7.5-8.5.
6. The method for preparing an adsorbent for hepatitis B virus according to claim 1, wherein: in step S3, the concentration of the 2- (N-morpholine) ethanesulfonic acid buffer solution is 0.04-0.06 mol/L, and the pH value is 5.0-6.0.
7. The method for preparing the hepatitis B virus adsorbent according to claim 1, characterized in that: in step S5, the pH value of the tris hydrochloride buffer is 8.5-9.5; the rinsing process uses 0.01mol/L phosphate buffer solution for rinsing.
8. The method for preparing a hepatitis B virus adsorbent according to claim 1 or 5, characterized in that: in step S2, the sequence of the aptamer is 5'-TGAGC CTCTG GATAC TTTTT ACCCA CAGCG AACAG CGGCG GACAT AATAG TGCTT ACTAC GACGC-3'.
9. A hepatitis B virus adsorbent, characterized in that: the hepatitis B virus adsorbent is prepared by the preparation method of any one of claims 1 to 8, and comprises carboxylated agarose microspheres and aptamers coupled with the carboxylated agarose microspheres.
10. The use of the hepatitis b virus adsorbent produced by the production method according to any one of claims 1 to 8 or the hepatitis b virus adsorbent according to claim 9, characterized in that: the hepatitis B virus adsorbent is used in the field of blood adsorption.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4710378A (en) * | 1984-03-13 | 1987-12-01 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Lyophilized hepatitis B vaccine |
| CN103111270A (en) * | 2013-02-26 | 2013-05-22 | 武汉真福医药科技发展有限公司 | Adsorbing material of hepatitis B antigen protein and preparation method of material |
| CN104772121A (en) * | 2015-03-29 | 2015-07-15 | 武汉瑞法医疗器械有限公司 | Preparation method of hepatitis B virus surface protein adsorbent |
| CN111659355A (en) * | 2020-06-23 | 2020-09-15 | 武汉瑞法医疗器械有限公司 | Alkylation modified hepatitis B virus immunoadsorbent and preparation method thereof |
-
2020
- 2020-12-31 CN CN202011641413.XA patent/CN112717902B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4710378A (en) * | 1984-03-13 | 1987-12-01 | Juridical Foundation The Chemo-Sero-Therapeutic Research Institute | Lyophilized hepatitis B vaccine |
| CN103111270A (en) * | 2013-02-26 | 2013-05-22 | 武汉真福医药科技发展有限公司 | Adsorbing material of hepatitis B antigen protein and preparation method of material |
| CN104772121A (en) * | 2015-03-29 | 2015-07-15 | 武汉瑞法医疗器械有限公司 | Preparation method of hepatitis B virus surface protein adsorbent |
| CN111659355A (en) * | 2020-06-23 | 2020-09-15 | 武汉瑞法医疗器械有限公司 | Alkylation modified hepatitis B virus immunoadsorbent and preparation method thereof |
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