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Hepatitis c virus adsorbent as well as preparation method and application thereof

A technology of hepatitis C virus and adsorbent, which is applied in the field of medical biomaterials to achieve the effect of stable ligand binding, short safe process route and high adsorption efficiency

Active Publication Date: 2013-11-20
WUHAN RUIFA MEDICAL DEVICES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the adsorption of HCV in plasma by aptamers screened by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) has not been reported yet.

Method used

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  • Hepatitis c virus adsorbent as well as preparation method and application thereof
  • Hepatitis c virus adsorbent as well as preparation method and application thereof
  • Hepatitis c virus adsorbent as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 in situ PCR method prepares hepatitis C virus adsorbent

[0037] (1) Synthesize a single-stranded deoxyribonucleic acid ligand that binds to the envelope protein of hepatitis C virus. The sequence is as follows: 5'-gggatccccgCGCCCTAGGATACTGCGTAACTGGGAGTCGCTTCCTCGATCTAATAAGGAG TAAGCCGAGCCTGACAAGGGATATCACTCAGCATAATCTTAAGGCGaacgcgtctg-3'.

[0038] (2) Design primers to amplify the single-stranded deoxyribonucleic acid ligand binding to hepatitis C virus envelope protein, primer A: 5'-gggatccccgCGCCCTA-3', primer B: 5'-cagacgcgttCGCCTTA-3'.

[0039] (3) Entrust General Electric Company to connect primer A to high-flow agarose microsphere 6FF by chemical cross-linking method, use ligand as template, support carrier as one end primer, and primer B as opposite primer for in situ PCR reaction , so that the primer A immobilized on the carrier grows to the sequence described in step (1).

[0040] The system composition of the in situ PCR reaction is basically the sa...

Embodiment 2

[0043] Embodiment 2 Asymmetric PCR method prepares hepatitis C virus adsorbent

[0044] (1) and (2) are the same as embodiment 1;

[0045] (3) Convert the single-stranded deoxyribonucleic acid ligand bound to the hepatitis C virus envelope protein into double-stranded DNA by PCR reaction, and insert the double-stranded DNA into the cloning vector pUC19 for preservation; use the cloning vector or double-stranded DNA as The template was subjected to an asymmetric PCR reaction to obtain the target single-stranded DNA; the asymmetric PCR reaction conditions were as follows: the content of primer A was more than 100 times that of primer B; PCR reaction system: TaKaRa Ex Taq (5U / μL) 0.5μL, 10× Ex Taq Buffer 5 μL, dNTP Mixture (2.5 mM each) 4 μL, template DNA 1 μL, primer A (25 μmol / L) 2 μL, primer B (0.25 μmol / L) 2 μL, ddH 2 O 35.5 μL. PCR reaction program: 94°C for 3min; 94°C for 30s, 62°C for 90s, 32 cycles; 72°C for 20min to obtain single-stranded DNA.

[0046] (4) After purif...

Embodiment 3

[0051] Example 3 The Hepatitis C Virus Adsorbent Measures the Maximum Adsorption Capacity of Hepatitis C Antigen E2

[0052] (1) Determination of standard curve of hepatitis C E2 concentration

[0053] Accurately weigh 25mg of HCV E2 standard substance, and prepare 25mL of 1.0mg / mL HCV E2 original solution with 0.01M PBS buffer solution with pH=7.4. Diluted with PBS to 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg / mL of HCV E2 solution, 4 mL each. Using PBS as a blank control, the absorbance at 280 nm of each solution was detected. Draw the standard curve with the mass concentration as the abscissa and the absorbance as the ordinate, and the relationship between the two is y=2.31x through linear fitting; R 2 =0.9973.

[0054] (2) Hepatitis C E2 static adsorption

[0055] Use PBS (0.01M pH7.4) buffer to prepare HCV E2 solutions with a certain mass concentration gradient, the mass concentrations are 0.5, 0.81, 1.0, 1.5, 1.87, 2.0, 4.62, 5.78 mg / mL, and take 10 mL of HCV E2 ...

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Abstract

The invention discloses a hepatitis c virus adsorbent as well as a preparation method and an application thereof, and belongs to the field of medical and biological materials. The hepatitis c virus adsorbent comprises a carrier and a single strand desoxyribonucleic acid ligand combined with a hepatitis c virus envelope protein and connected to the carrier. The sequence of the ligand is as shown in SEQINNO.2. The hepatitis c virus adsorbent is obtained by obtaining a sensitive carrier surface through a chemical activating reaction and performing covalent linkage of the carrier surface with the artificially synthesized single strand desoxyribonucleic acid. The adsorbent is simple and convenient in synthetic method, short in process line and safe to prepare. The covalent bond is firm to connect and the ligand is stable to combine. The product has the characteristics of strong specificity, high absorption efficiency to hepatitis c virus particles, good regenerability and no toxicity and heat source reaction, and can be used for absorptive treatment of clinically and specifically eliminating hepatitis c virus particles in plasma and can be used for preparing anti-hepatitis c virus medicines.

Description

technical field [0001] The invention belongs to the field of medical biological materials, and in particular relates to a hepatitis C virus adsorbent and its preparation method and application. Background technique [0002] Hepatitis C is caused by hepatitis C virus (HCV), which is widely distributed and easily evolves into chronic hepatitis, liver cirrhosis and liver cancer. Except for a very small number of acute hepatitis C that can be cured, the vast majority of patients will live with the disease for life once they are infected. At present, there is no completely effective treatment plan, and there is no clinical vaccine; the only recognized effective treatment is interferon, and it must be implemented in the early stage of infection, otherwise there will be no obvious effect. [0003] Viruses in the blood are the causative agent of hepatitis C. Although there have been early explorations, what is still inconclusive is how to specifically and effectively remove these ...

Claims

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Application Information

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IPC IPC(8): B01J20/24B01J20/30A61K47/42A61K31/7088A61P31/14
Inventor 王业富森林韩振伟张磊严玲
Owner WUHAN RUIFA MEDICAL DEVICES CO LTD
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