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Process for preparing target ribonuclease for curing hepatitis B virus infection

A technology of ribonuclease and hepatitis B virus, which is applied in the field of preparation technology of targeted ribonuclease, can solve the problem of reducing the inhibitory effect of hepatitis B virus replication, affecting the correct folding of effector molecules and target molecules, and affecting the inhibition of fusion proteins on hepatitis B virus replication Effect and other issues

Inactive Publication Date: 2002-08-21
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current research of Beterams and Nassal is aimed at the DNA formed after the reverse transcription of pgRNA, which inhibits the replication of hepatitis B virus through the degradation of DNA outside the cell; they (Beterams and Nassal are the same research, that is, the two authors of the same paper ,) all adopt staphylococcal nuclease, staphylococcal nuclease can induce human immune response, may cause allergic reaction, the antibody that immune response produces has neutralizing effect to effector molecule, can reduce the inhibitory effect to the replication of hepatitis B virus; The target molecule is the core protein; the effector molecule is directly connected to the target molecule, which may affect the correct folding of the effector molecule and the target molecule, and then affect the inhibitory effect of the fusion protein on the replication of hepatitis B virus

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1) Amplification and sequencing of hEDN coding gene

[0046] 1.1 Primer design based on hEDN gene sequence

[0047] Forward primer: 5′-GCG CGG ATC CAC CAT GAA ACC TCC ACA GTT TAC-3′;

[0048] Reverse primer: 5′-GCG CGA GCT CGA TGA TTC TAT CCA GGT G-3′;

[0049] The 5' ends of the forward primer and reverse primer have BamH I and Sac I restriction sites respectively;

[0050] 1.2 Amplify the target gene

[0051] at 5×10 6 Add 1ml of Trizol to the HL-60 cells  , repeated pipetting, placed at 15°C for 5 minutes, then added 0.2ml of chloroform, shaken vigorously for 15 seconds, placed at 15°C for 2 minutes, centrifuged at 2°C, 12000g relative centrifugal force for 15 minutes, and absorbed the upper aqueous phase;

[0052] Add 0.5ml of isopropanol to the upper aqueous phase, place it at 15°C for 10 minutes, then centrifuge at 2°C under a relative centrifugal force of 12000g for 10 minutes, remove the supernatant, and obtain the total RNA of HL-60 cells; 60% total RNA ...

Embodiment 2

[0083] 1) Amplification and sequencing of hEDN coding gene

[0084] 1.1 Primer design based on hEDN gene sequence

[0085] Forward primer: 5′-GCG CGG ATC CAC CAT GAA ACC TCC ACA GTT TAC-3′;

[0086] Reverse primer: 5′-GCG CGA GCT CGA TGA TTC TAT CCA GGT G-3′;

[0087] The 5' ends of the forward primer and reverse primer have BamH I and Sac I restriction sites respectively;

[0088] 1.2 Amplify the target gene

[0089] at 10 x 10 6 Add 1ml of Trizol to the HL-60 cells  , repeated pipetting, placed at 30°C for 5 minutes, then added 0.2ml of chloroform, shaken vigorously for 15 seconds, placed at 30°C for 3 minutes, centrifuged at 8°C, 12000g relative centrifugal force for 15 minutes, and absorbed the upper aqueous phase;

[0090] Add 0.5ml of isopropanol to the upper aqueous phase, place it at 30°C for 10 minutes, then centrifuge at 8°C and a relative centrifugal force of 12000g for 10 minutes, remove the supernatant, and obtain the total RNA of HL-60 cells; 60% total RNA...

Embodiment 3

[0121] 1) Amplification and sequencing of hEDN coding gene

[0122] 1.1 Primer design based on hEDN gene sequence

[0123] Forward primer: 5′-GCG CGG ATC CAC CAT GAA ACC TCC ACA GTT TAC-3′;

[0124] Reverse primer: 5′-GCG CGA GCT CGA TGA TTC TAT CCA GGT G-3′;

[0125] The 5' ends of the forward primer and reverse primer have BamH I and Sac I restriction sites respectively;

[0126] 1.2 Amplify the target gene

[0127] in 8×10 6 Add 1ml of Trizol to the HL-60 cells  , repeated blowing, placed at 20°C for 5 minutes, then added 0.2ml of chloroform, shaken vigorously for 15 seconds, placed at 25°C for 2.5 minutes, centrifuged at 6°C, 12000g relative centrifugal force for 15 minutes, and absorbed the upper aqueous phase;

[0128] Add 0.5ml of isopropanol to the upper aqueous phase, place it at 18°C ​​for 10 minutes, then centrifuge at 5°C and a relative centrifugal force of 12000g for 10 minutes, remove the supernatant, and obtain the total RNA of HL-60 cells; 60% total RNA ...

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Abstract

The present invention relates to the process of preparing target ribonuclease for curing hepatitis B virus infection. By means of molecular biological measures, it is constituted fusion protein of neurotoxin hEDN and HBV polymerase end protein structural domain DNAPTP or core protein originated from human eosinophil. The fusino protein, that is the said target ribonuclease, may be used in treating huma hepatitis B virus infection via gene therapy or protein medicine mode. Experiment shows that the target ribonuclease can reduce in vitro HBSAg for 46-59 % and inhibit obviously the duplicatino of hepatitis B virus. Available gene therapy or protein medicine mode may be refered to for its specific administratino way or mode.

Description

1. Technical field [0001] The invention belongs to the field of biotechnology, in particular to a preparation process of targeted ribonuclease for treating hepatitis B virus infection. 2. Background technology [0002] HBV infection is a worldwide health problem. A considerable number of HBV-infected patients, especially those infected with HBV in childhood, will develop into chronic hepatitis, liver cirrhosis or liver cancer with delay. Although hepatitis B vaccine can prevent its infection, there is an urgent need for effective treatment in the face of a large number of infected people, about 5% of those who do not respond to the vaccine, and possible mutant strains of hepatitis B virus that escape vaccine-induced immunity. For the treatment of patients with chronic hepatitis B infection, alpha interferon is currently the most commonly used drug, but its effective rate is not high (about 30%-40%), and its tolerance is poor. Nucleoside analogues including lamivudine, famc...

Claims

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Application Information

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IPC IPC(8): A61K38/46A61P31/14C12N9/22C12N15/63C12N15/70C12N15/79C12Q1/68
Inventor 刘军薛采芳
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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