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Anti-HIV-1 drugs and preparation and application thereof

An HIV-1, drug technology, applied in antiviral agents, pharmaceutical formulations, chemical instruments and methods, etc., can solve the problems of low efficiency, adverse reactions, drug resistance, huge medical costs, drug side effects, etc., and achieve the effect of avoiding mutation

Active Publication Date: 2014-11-05
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although there are many anti-HIV-1 drugs on the market and HARRT is widely used, the resulting drug resistance, huge medical expenses, and side effects of drugs should not be underestimated.
Although HAART can reduce the plasma viral load of a considerable number of patients below the level that can be detected, the rebound after drug withdrawal and serious side effects have not been resolved; although gene therapy has shown its antiviral potential, and some studies The results have entered the stage of clinical trials, but its low efficiency and possible adverse reactions caused by exogenous vectors are still a major obstacle in research and development

Method used

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  • Anti-HIV-1 drugs and preparation and application thereof
  • Anti-HIV-1 drugs and preparation and application thereof
  • Anti-HIV-1 drugs and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction model of chimeric vector Rev-Vif-C

[0031] The two oligomerization binding domains (amino acids 9-26 and amino acids 51-65) on the HIV-1 Rev gene were respectively cloned into the N-terminus of the Vif gene, and replaced with the binding site of APOBEC3G (1- 79 amino acids), and then connected to the pcDNA3.1 vector to form three different chimeric chimeras, which were named ROL1-Vif-C, ROL2-Vif-C, and ROL12-Vif-C. Among them, ROL1 represents the oligomerization binding domain of the N-terminal part of Rev, that is, amino acids 9-26; ROL2 represents the oligomerization binding domain of the C-terminal part of Rev, that is, amino acids 51-65; ROL12 represents the tandem Two oligomerization binding domains at the N-terminal and C-terminal of Rev, namely amino acids 9-26 and amino acids 51-65.

[0032] Specific steps are as follows:

[0033] (1) There are two oligomerization domains in the protein structure of Rev, and these two oligomerization do...

Embodiment 2

[0036] Example 2 The chimeric vector Rev-Vif-C degrades Rev protein through the ubiquitination pathway

[0037] Fusion expression of HIV-1 Rev gene and RFP and cloned into the vector of pcDNA3.1, so that the expression of Rev can be reflected by observing the expression of RFP; at the same time, the fusion expression of Rev gene and HA tag is convenient for western blot to further Verify the expression of Rev.

[0038](1) In 293t cells in a 24-well plate, co-transfect Rev-RFP and ROL1-Vif-C (or ROL2-Vif-C or ROL12-Vif-C) vectors, the amount of plasmids was 1:0, 1 : 1, 1: 2, 1: 3, observe the expression of RFP 48 hours after transfection, and co-transfect Rev-HA and ROL1-Vif-C vectors in 293t cells in a 6-well plate, and the amount of plasmids is 1 :0, 1:1, 1:2, 1:3, 48 hours after transfection, the cells were harvested and lysed for Western Blot to detect the expression of Rev

[0039] This experiment shows that all three chimeric vectors can inhibit the expression of Rev pr...

Embodiment 3

[0044] Example 3 The chimeric vector Rev-Vif-C inhibits the replication of various wild-type HIV-1 strains by inhibiting the nuclear export function of Rev-RRE

[0045] There are SD and SA cleavage sites on PDM628. When the Rev protein does not exist, the luciferase gene carried on PDM 628 is clipped, resulting in a small amount of luciferase expression; when the two plasmids are co-transfected, Rev and RRE combine to convert luciferase The gene fragments are taken out of the nucleus, avoiding being edited by SD and SA, so that luciferase can be expressed in large quantities. Therefore, when Rev protein expression is inhibited, the Rev-RRE-related nuclear export system will be inhibited, and the expression of luciferase will decrease at this time. Based on this principle, it can be judged whether the function of the Rev protein will be affected.

[0046] (1) In 293t cells in a 96-well plate, 10 ng of pDM628 plasmid and 10 ng of Rev plasmid were co-transfected, and then co-tra...

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Abstract

The invention provides anti-HIV-1 drugs (also known as chimeric vectors Rev-Vif-C) prepared by splicing connection of Rev protein binding sites and a Vif protein C end, wherein the connection comprises substituting a Vif N end with Rev multimerization structural domains. In the study, the Rev-Vif-C vectors are designed and constructed, various experiments prove that the Rev-Vif-C vectors have a good anti-virus effect, and a new anti-virus technology aiming at an HIV-1 Rev protein is provided. The Vif-C vectors are successfully applied in degradation of the Rev protein, and are expected to become a novel gene knockout means. The Vif-C vectors can specifically degrade targeted proteins and are especially suitable for drug development of virus target proteins easy to produce high-frequency mutation, as long as the protein binding sites are not mutated, the chimeric vectors can be effective for a long term, and thus the drug resistance of the anti-virus drugs can be reduced.

Description

technical field [0001] [0002] The present invention relates to the field of antiviral drugs, more specifically, relates to an anti-HIV-1 virus drug and its preparation and application. Background technique [0003] In the late 1970s and early 1980s, a disease characterized by dysfunction of the immune system emerged in Europe and the United States. Subsequently, scientists from various countries began to explore the cause of the disease and its treatment options. Until 1983, after the French Pasteur research team first successfully isolated this new retrovirus, there were more and more theoretical research and medical treatment of HIV-1. At present, drugs against HIV-1 mainly act on different links in the virus life cycle, especially some necessary enzymes such as reverse transcriptase and protease. [0004] Although there are many anti-HIV-1 drugs on the market and HARRT is widely used, the resulting drug resistance, huge medical expenses, and side effects of drugs sho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K38/16A61P31/18C12N15/85C12N15/62
Inventor 张辉潘婷
Owner SUN YAT SEN UNIV
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