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A kind of human type 3 adenovirus replication defective recombinant virus, construction method and application

A technology for replication-deficient recombinant viruses, applied in the field of construction of human type 3 adenovirus replication-defective recombinant viruses, can solve the problems of only symptomatic treatment and non-specific drugs, and achieve good application prospects and simple preparation methods , to ensure the effect of immunogenicity

Active Publication Date: 2020-11-24
广东龙帆生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The treatment after adenovirus infection is the same as other diseases caused by viruses. At present, there are no specific drugs, and only symptomatic treatment is available. The antiviral neutralizing antibodies produced by the body's own immune system mainly rely on the antiviral neutralizing antibodies produced by the body's own immune system to eliminate the virus and cure the disease.

Method used

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  • A kind of human type 3 adenovirus replication defective recombinant virus, construction method and application
  • A kind of human type 3 adenovirus replication defective recombinant virus, construction method and application
  • A kind of human type 3 adenovirus replication defective recombinant virus, construction method and application

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Embodiment 1

[0038] Example 1 Construction of type 3 replication-deficient adenovirus and expression of target protein

[0039] 1. The construction process of the plasmid pSAd3H containing the hexon gene of human adenovirus type 3

[0040] The hexon gene of human type 3 adenovirus involved in the present invention is a complete human type 3 adenovirus hexon antigen gene, the nucleotide sequence is shown in SEQ ID NO.3, which comes from the wild type human adenovirus type 3 Strain GZ01 (GenBank: DQ099432). A pair of primers were designed according to the nucleotide sequence of the hexon protein of human type 3 adenovirus (HAdV-3GZ01 strain) and the restriction site in the multiple cloning site of the shuttle plasmid pShuttle-CMV-GFP:

[0041] The upstream outer primer sequence is the sequence shown in SEQ ID No.1 (Ad3-GZ01-EcoRV-HexF: TGAGATATCGCCACCATGGCCACCCCATC);

[0042] The downstream outer primer sequence is the sequence shown in SEQ ID No. 2 (Ad3-GZ01-HexR-XhoI: AGACTCGAGTTATGTGGTG...

Embodiment 2

[0101] Example 2 Immunogenicity of type 3 replication-deficient adenovirus

[0102] A large amount of replication-deficient adenovirus type 3 was amplified, and the virus was purified by the cesium chloride density gradient method. The operation was as follows:

[0103] (1) Harvesting cells: Repeated freezing and thawing 3 times of AD293 cells with 90% CPE in culture, centrifuging at 1000g for 10min, collecting the supernatant, and freezing at -80°C; (2) discontinuous density gradient centrifugation of the virus (all operations below are Take a 30mL centrifuge tube as an example): a. Pre-cool the rotor of the ultracentrifuge to 4°C. b. Slowly add 8mL 1.4g / mL cesium chloride to the centrifuge tube of the ultra-high speed centrifuge, and then add 6mL 1.2g / mL cesium chloride very gently; c. Add at the top of the discontinuous gradient cesium chloride solution 20mL virus collection solution, if the volume of virus solution is less than 20mL, use pH 7.9 10mmol / L Tris-HCl to balanc...

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Abstract

The invention belongs to the technical field of biomedicines and in particular relates to a human type-3 adenovirus replication defective recombinant virus, a construction method and application. A wild human type-3 adenovirus capsid protein hexon gene is adopted as a protective antigen gene, a human type-5 replication defective adenovirus with gene deletion in regions E1 and E3 is adopted as a vector, and AD293 cells for integrating genes of the region E1 are adopted as a packaging cell line. After mice are immunized by using a replication defective vaccine, high humoral immunity of a body can be induced, a specific neutral antibody for a wild human type-3 adenovirus is generated, the immunization way is intramuscular injection or nasal inhalation, and the vaccine candidate strain has theadvantages of being simple in preparation method, simple and convenient in immunization operation, safe and efficient, and the like, and has good application prospects.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a human type 3 adenovirus replication-deficient recombinant virus, a construction method and an application. Background technique [0002] Human adenovirus (HAdV) is a relatively common respiratory system infection pathogen, highly contagious, can cause multi-system multi-organ infection, the susceptible population is mainly infants and young children, can cause acute respiratory disease (Acute Respiratory Disease) , ARD) and fatal pneumonia in infants and young children. Adenovirus pneumonia is one of the viral pneumonias with the highest incidence in my country. There are 51 serotypes of human adenovirus, among which human adenovirus type 3 is endemic, epidemic and sporadic infection, and has been reported in Europe, Asia, America and Oceania. It is highly infectious, highly toxic and can cause severe disease. Clinical symptoms such as severe pneumonia and even ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/01A61K39/235A61P31/20
CPCA61K39/12A61P31/20C07K14/005C12N7/00C12N2710/10021C12N2710/10022C12N2710/10334
Inventor 张其威严雨茜吴建国谭秋萍
Owner 广东龙帆生物科技有限公司
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