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Therapeutic DC composite vaccine against herpes simplex viruses and preparation method thereof

A herpes simplex virus, therapeutic technology, applied in the field of vaccines, to increase transcription and expression, reduce lesions, and enhance immune effects

Active Publication Date: 2019-05-03
上海兴瑞一达生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

HSV-2 vaccines with envelope proteins such as gD, gB, gH, and gL as antigens can induce HSV-specific neutralizing antibodies, among which gD has the strongest ability to induce neutralizing antibodies, but for effective HSV-2 vaccines, Stimulation with gD is not sufficient and immunization with envelope is not sufficient for effective HSV-2 vaccine

Method used

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  • Therapeutic DC composite vaccine against herpes simplex viruses and preparation method thereof
  • Therapeutic DC composite vaccine against herpes simplex viruses and preparation method thereof
  • Therapeutic DC composite vaccine against herpes simplex viruses and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The antigen gene fragments GM-CSF-gC+US10, GM-CSF-gD+ICP4, and GM-CSF-UL47 were respectively inserted into the lentiviral expression vector pLent-C-GFP, and three corresponding plasmids were prepared, and the gC+US10 Nucleic acid artificial sequence, gD+ICP4 nucleic acid artificial sequence, and UL47 nucleic acid artificial sequence are respectively added with NotI and AsiSI site restriction sites at both ends to synthesize a complete expression frame, which is inserted into the lentiviral pLent-C-GFP vector after restriction digestion (Invitrogen) NotI-AsiSI site, transformed into E.coli (Top10), after the correct identification by nucleic acid sequencing, use the endotoxin-free plasmid purification kit to extract and purify the plasmid to obtain the recombinant expression vector plasmid

[0042] In more detail, this embodiment adopts the following steps:

[0043] The antigen gene fragments GM-CSF-gC+US10, GM-CSF-gD+ICP4, and GM-CSF-UL47 were respectively inserted into...

Embodiment 2

[0047] Inoculate the lentiviral packaging cell line 293T in a 10cm culture dish containing DMEM+10% FBS; prepare the reaction system, mix well, and place it at room temperature for 20 minutes, then evenly add it dropwise to the culture dish containing 293T cells, and then place it in a CO2 incubator Medium culture; culture after transfection, to obtain lentivirus virus liquid carrying GM-CSF-gC+US10, GM-CSF-gD+ICP4, GM-CSF-UL47 encoding genes.

[0048] In more detail, this embodiment adopts the following specific steps:

[0049] Use the Lentiviral Packaging Kit lentiviral packaging kit, the specific method is as follows: inoculate the lentiviral packaging cell line 293T in a 10cm culture dish containing DMEM+10%FBS, culture at 37°C, 5% CO2, and the adhesion rate is 70% -80% ready for transfection. Take a sterile 1.5ml EP tube or 15ml centrifuge tube, and prepare the reaction system according to the following components: serum-free DMEM: 4ml; pLent-gC+US10, pLent-gC+US10, pLen...

Embodiment 3

[0051] Collect peripheral venous blood from patients with herpes simplex virus, isolate peripheral blood mononuclear cells, retain adherent cells, add cytokines to induce mononuclear cells to differentiate into DC, and culture imDC.

[0052] In more detail, this embodiment adopts the following specific steps:

[0053] Collect 50 ml of peripheral venous blood from patients with herpes simplex virus, use TBD sample density separation medium (purchased from Tianjin Haoyang Huake Biology), separate peripheral blood mononuclear cells, and culture them in 10 ml of culture medium (purchased from TaKaRa Company, GT-T551), After culturing at 37°C and 5% CO2 for 3-4 hours, discard the suspended T cells, keep the adherent cells, and add cytokines rhIL-4 (final concentration: 50ng / ml), rhGM-GSF (final concentration 100ng / ml) to induce mononuclear cells to differentiate into DCs, the medium was replaced every 48 hours, and imDCs were obtained on the 5th day of culture.

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Abstract

The present invention discloses a therapeutic DC composite vaccine against herpes simplex viruses and a preparation method thereof. The therapeutic DC composite vaccine comprises dendritic cells modified with three loaded HSV-2 virus-specific immune antigen gene fragments. The three loaded HSV-2 virus-specific immune antigen gene fragments are gC+US10, DC-UL47 and gD+ICP4, respectively. The therapeutic DC composite vaccine uses granulocyte-macrophage cluster factor receptor signal peptides to guide expressions of antigens, can improve expression efficiency of the antigens, enhances immune effects, and generates a stronger anti-HSV-specific immune response.

Description

technical field [0001] The invention relates to the technical field of vaccines, in particular to a therapeutic DC compound vaccine against herpes simplex virus and a preparation method thereof. Background technique [0002] Herpes simplex virus (HSV) infection is a common infectious disease in humans. The virus is divided into two serotypes, HSV-1 and HSV-2. Among them, HSV-2 infection is one of the most common sexually transmitted diseases, and is the main cause of genital herpes and ulcers. It has infected more than 500 million people worldwide, and it is estimated that 23 million people are newly infected every year. The current clinical treatment of genital herpes mainly relieves symptoms through the use of antiviral drugs, but it cannot completely eliminate the HSV-2 virus and effectively control the recurrence of the disease. [0003] The genome of HSV contains approximately 85 open reading frames, enabling HSV to produce at least 85 unique proteins. It mainly inclu...

Claims

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Application Information

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IPC IPC(8): A61K39/245A61P31/22
Inventor 刘明录王立新刘敏金海锋强邦明张传鹏王亮马洪华冯建海
Owner 上海兴瑞一达生物科技有限公司
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