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Anti-hiv-1 gene, application of the gene, dc vaccine with the gene, and vaccine preparation method

An HIV-1 and vaccine technology, applied in the field of genes, can solve the problems of difficult exposure of antigenic epitopes, immune evasion of virus mutation, weak binding site affinity, etc. Effect

Active Publication Date: 2019-06-11
SHANDONG XINRUI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the experiment, it was found that the antigenic epitope is relatively difficult to expose, resulting in weak binding site affinity, and the possibility of virus mutation immune escape is greatly increased

Method used

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  • Anti-hiv-1 gene, application of the gene, dc vaccine with the gene, and vaccine preparation method
  • Anti-hiv-1 gene, application of the gene, dc vaccine with the gene, and vaccine preparation method
  • Anti-hiv-1 gene, application of the gene, dc vaccine with the gene, and vaccine preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] An anti-HIV-1 gene, which is composed of a GM-CSF nucleic acid artificial sequence in series with a gp120-CD4 module, and the gp120-CD4 module contains at least one of the following gene fragments;

[0048] (1) gp120(B) nucleic acid artificial sequence, G4S nucleic acid artificial sequence, CD4 nucleic acid artificial sequence;

[0049] (2) gp120(C) nucleic acid artificial sequence, G4S nucleic acid artificial sequence, CD4 nucleic acid artificial sequence;

[0050] (3) gp120(AE) nucleic acid artificial sequence, G4S nucleic acid artificial sequence, CD4 nucleic acid artificial sequence;

[0051] When the gp120-CD4 module contains two or more gene fragments, the contained gene fragments are connected in series.

[0052] In more detail, the anti-HIV-1 gene of the present invention can be the following gene sequence: GM-CSF-(1), GM-CSF-(2), GM-CSF-(3), GM-CSF-(1) -(2), GM-CSF-(1)-(3), GM-CSF-(2)-(3), GM-CSF-(1)-(2)-(3); it can also be GM- CSF-(2)-(1), GM-CSF-(3)-(1), GM-CSF-(3)-(...

Embodiment 2

[0056] Purification of HIV-gp120-CD4 gene.

[0057] In more detail, the above steps are:

[0058] The synthesized HIV-gp120-CD4 gene was inserted into the standard vector pUC, so it was named pUC-HIV-gp120-CD4, and pUC-HIV-gp120-CD4 was subjected to Fast Digest AsiSI (purchased from ThermoFisher) and FastDigest NotI (purchased from ThermoFisher) double digestion, 37°C, digestion for 20 min. The 100μl digestion system is: 10×buffer: 10μl; DNA 6μg; AsiSI enzyme: 3μl; NotI enzyme: 3μl; deionized water to make up the volume.

[0059] Use agar electrophoresis to cut off the agar containing HIV-gp120-CD4 DNA fragments and place them in two centrifuge tubes. A DNA extraction kit (purchased from ThermoFisher) was used to dissolve the DNA from the agar and concentrate it. First, add 500 μl DF buffer to the above centrifuge tube, incubate at 55°C for 10 minutes, and shake it every 2-3 minutes until the agar is completely dissolved. Then suck all the agar solution into the DF Column and put ...

Embodiment 3

[0061] Preparation of plasmid containing HIV-gp120-CD4 gene.

[0062] Insert the HIV-gp120-CD4 gene synthesized in Example 2 into the lentiviral expression vector pLent-C-GFP (Invitrogen) NotI-AsiSI site (see figure 2 ).

[0063] In more detail, use the following method to prepare:

[0064] At the same time, pUC-HIV-gp120-CD4 (prepared in the first step of Example 2) and pLent-C-GFP vector were digested with Fast Digest AsiSI (purchased from ThermoFisher) and Fast Digest NotI (purchased from ThermoFisher) , 37℃, digestion for 20min. The 100μl digestion system is: 10×buffer: 10μl; DNA 6μg; AsiSI enzyme: 3μl; NotI enzyme: 3μl; deionized water to make up the volume. Use agar electrophoresis to cut off the agar containing HIV-gp120-CD4 DNA fragment and linearized pLent-C-GFP DNA fragment, and place them in two centrifuge tubes. DNA extraction kit (purchased from ThermoFisher) was used to dissolve the DNA from the agar and concentrate. First, add 500 μl DFbuffer to the above centrifug...

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Abstract

The invention discloses an anti-HIV-1 (human immunodeficiency virus 1) gene, an application thereof, a DC vaccine containing the gene and a preparation method of the vaccine. The gene is formed by a GM-CSF nucleic acid artificial sequence and a gp120-CD4 module through series connection, wherein the gp120-CD4 module contains at least one of the following gene fragments: (1) a gp120 (B) nucleic acid artificial sequence, a G4S nucleic acid artificial sequence and a CD4 nucleic acid artificial sequence; (2) a gp120 (C) nucleic acid artificial sequence, the G4S nucleic acid artificial sequence andthe CD4 nucleic acid artificial sequence; (3) a gp120 (AE) nucleic acid artificial sequence, the G4S nucleic acid artificial sequence and the CD4 nucleic acid artificial sequence. Gp120 is linked with CD4 analogues or CD4 'mini-proteins', conformation of gp120 is changed and epitope is exposed on a CD4 binding site, so that an antibody identifies and binds with the exposed antigen, and the organism is stimulated to produce cellular immunity and humoral immunity responses.

Description

Technical field [0001] The invention relates to the field of gene technology, in particular to the anti-HIV-1 gene, the application of the gene, a DC vaccine with the gene, and a vaccine preparation method. Background technique [0002] AIDS (acquired immunodeficiency syndrome, AIDS) is a chronic infectious disease with extremely high mortality caused by human immunodeficiency virus 1 (HIV-1) infection, and has become one of the most serious viral diseases threatening humans. One. According to estimates by the World Health Organization, about 40 million people are currently infected with HIV, of which 2 million are children, and 35 million have died. At present, there are more than 40 kinds of HIV vaccines used to prevent or treat AIDS, including inactivated vaccines, live attenuated vaccines, recombinant subunit vaccines, recombinant live vector vaccines, and DNA vaccines. The HIV vaccine has many types and attacks its own immune system. Although the HIV vaccine has entered cl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/867A61K39/21A61P31/18
CPCA61K39/12A61K2039/53A61K2039/572A61K2039/575C07K14/005C07K14/53C07K14/70514C07K2319/00C12N15/86C12N2740/15043C12N2740/16022C12N2740/16034
Inventor 刘明录王立新冯建海金海锋卢永灿韩国英马洪华万磊张传鹏强邦明刘敏韩庆梅王亮
Owner SHANDONG XINRUI BIOTECH CO LTD
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