Anti-HIV-1 (human immunodeficiency virus 1) gene, application thereof, DC vaccine containing gene and preparation method of vaccine

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

Active Publication Date: 2018-04-24
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 (human immunodeficiency virus 1) gene, application thereof, DC vaccine containing gene and preparation method of vaccine
  • Anti-HIV-1 (human immunodeficiency virus 1) gene, application thereof, DC vaccine containing gene and preparation method of vaccine
  • Anti-HIV-1 (human immunodeficiency virus 1) gene, application thereof, DC vaccine containing gene and preparation method of vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Anti-HIV-1 gene, the gene is composed of GM-CSF nucleic acid artificial sequence and gp120-CD4 module in series, 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 concatenated.

[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); can also be GM- CSF-(2)-(1), GM-CSF-(3)-(1), GM-CSF-(3)-(2),...

Embodiment 2

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

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

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

[0059] Use agar gel electrophoresis to cut off the agar gel part containing the HIV-gp120-CD4 DNA fragment, and put it in two centrifuge tubes. DNA was extracted from the agar gel using a DNA extraction kit (purchased from ThermoFisher) and concentrated. First, 500 μl DF buffer was added to the above-mentioned centrifuge tube for 10 minutes at 55° C. and shaken every 2-3 minutes until the agar gel was completely dissolved. Inhale...

Embodiment 3

[0061] Preparation of plasmids containing the 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, it is prepared as follows:

[0064] Simultaneously carry out Fast Digest AsiSI (purchased from ThermoFisher Company) and Fast Digest NotI (purchased from ThermoFisher Company) double enzyme digestion of pUC-HIV-gp120-CD4 (prepared in the first step of Example 2) and pLent-C-GFP vector , 37°C, enzyme digestion for 20min. 100μl enzyme digestion system: 10×buffer: 10μl; DNA 6μg; AsiSI enzyme: 3μl; NotI enzyme: 3μl; deionized water to make up the volume. Use agar gel electrophoresis to cut out the agar gel parts containing the HIV-gp120-CD4 DNA fragment and the linearized pLent-C-GFP DNA fragment respectively, and place them in two centrifuge tubes. Use DNA extraction kit (purchased from ThermoFisher Company) to dissolve and conc...

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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 anti-HIV-1 gene, application of the gene, DC vaccine with the gene and 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 type 1 (human immunodeficiency virus 1, HIV-1), and has become one of the most serious viral diseases threatening human beings. one. According to the estimates of the World Health Organization, there are currently about 40 million people infected with HIV in the world, of which 2 million are children, and the death toll has reached 35 million. At present, more than 40 kinds of HIV vaccines have been used to prevent or treat AIDS, including inactivated vaccines, live attenuated vaccines, recombinant subunit vaccines, recombinant live vector vaccines, and DNA vaccines. Although the HIV vaccine has entered cli...

Claims

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Application Information

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Patent Type & Authority Applications(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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