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Novel chimpanzee adenovirus vector as well as construction method and application thereof

A construction method and adenovirus technology, applied in the field of genetic engineering technology and immunology, can solve the problems of complex vaccine carrier process, low titer of chimpanzee adenovirus, and difficulty in industrialization

Pending Publication Date: 2022-01-07
JIAXING ANYU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The titer of the existing chimpanzee adenovirus is generally low, and the process of constructing a vaccine vector is complicated and difficult to industrialize

Method used

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  • Novel chimpanzee adenovirus vector as well as construction method and application thereof
  • Novel chimpanzee adenovirus vector as well as construction method and application thereof
  • Novel chimpanzee adenovirus vector as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] The isolation of embodiment 1 novel chimpanzee virus strains

[0118] Chimpanzee anal swabs were collected, and chimpanzee adenoviruses were cultured in HEK293 cells (purchased from ATCC, lot number 70009859).

[0119] First, thaw the collected anal swab on ice until room temperature, put it into a 5ml cryovial, add 3ml DMEM, vortex for 30s, 4°C, 2000rpm, centrifuge for 5min, filter the above supernatant through a 0.2μm pinhole Membrane filter about 200 μl into a sterile 48-well plate for later use, and then inoculate into 48-well HEK293 cells prepared in advance, and observe the lesion every day. If the cells are full in 48 hours, collect the cell suspension and freeze-thaw twice After freezing, store at -20°C. If there is no overgrowth, collect the cell suspension on the 5th day, continue to inoculate HEK293 cells until the lesion occurs, and extract the virus genome for PCR amplification and identification. The identified correct strain was further expanded, and the...

Embodiment 2

[0120] Example 2 Classification of novel chimpanzee adenoviruses

[0121] The chimpanzee adenovirus genomic DNA obtained in Example 1 was subjected to whole-genome sequencing, and Sangon Bioengineering (Shanghai) Co., Ltd. was responsible for determining the final viral genome sequence. The chimpanzee adenovirus is preserved in the China Center for Type Culture Collection, named as: chimpanzee adenovirus sAd-AY01, and the preservation number is: CCTCC NO: V202120. The full-length genome of chimpanzee adenovirus sAd-AY01 is 36687bp.

[0122] Human adenoviruses belong to the family Adenoviridae and the genus Mammalian Adenoviruses. So far, 52 serotypes of human adenoviruses have been isolated, belonging to subgenus A to G7. At present, the diagnosis and typing of adenovirus infection mainly use virus isolation technology, antibody detection technology, and molecular biology technology (such as polymerase chain reaction, restriction fragment length polymorphism analysis technolo...

Embodiment 3

[0124] Example 3 Construction of a novel chimpanzee adenovirus (psAd) vector containing a fluorescent marker

[0125] The chimpanzee adenovirus (sAd) genome is a linear DNA longer than 36kb, and it is difficult to edit it efficiently. Therefore, it is necessary to link the elements related to replication in bacteria, the replicon (pACYC), and the resistance gene (kanamycin), to the chimpanzee genome to form a circular plasmid.

[0126] Therefore, the replicon (pACYC), resistance gene (kanamycin) and the left arm (~1400bp) and right arm (~1400bp) of the chimpanzee genome need to be constructed into a circular plasmid with a size of about 6000bp, and then linearized Co-transformed with the chimpanzee adenovirus genome into BJ5183 competent, and performed homologous recombination to form a chimpanzee circular plasmid, which allows it to replicate effectively in bacteria; at the same time, in order to monitor the transfection efficiency of the plasmid in cells, a green fluorescent...

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Abstract

The invention provides a novel chimpanzee adenovirus vector as well as a construction method and application thereof. The novel chimpanzee adenovirus vector has higher virus titer. The invention also provides a method for measuring a virus titer of the chimpanzee adenovirus vector. The chimpanzee adenovirus annular vector is creatively constructed through a shuttle plasmid, E1 and E3 are knocked out through CRISPR / Cas9, and a replication-defective chimpanzee adenovirus vector is constructed. The chimpanzee adenovirus vector does not have a preexisting antibody in people, knockout of the E1 and the E3 is safe, and meanwhile, the chimpanzee adenovirus vector is remarkably different from E1 of human adenovirus type 5 in 293 cells, so that recovery mutation (RCA) can be greatly avoided, and the chimpanzee adenovirus vector is safer. After passage of dozens of generations, the stability of a constructed novel coronavirus vaccine is very high, mutation is not caused, and very strong humoral immunity and cellular response can be induced in a mouse model.

Description

[0001] This application claims the priority of the earlier Chinese application, application number: 2020106427453, and the filing date of July 6, 2020; the priority of the earlier Chinese application, application number: 202010945335.6, and the filing date of September 10, 2020; all content as part of the present invention. technical field [0002] The invention relates to the technical fields of genetic engineering and immunology, in particular to a novel chimpanzee adenovirus vector and its construction method and application. Background technique [0003] Adenoviruses are common viruses that cause upper respiratory tract and lung diseases, and the human immune system will mount a strong immune response to adenoviruses. Therefore, it is necessary to transform the adenovirus into a harmless vaccine carrier. After decades of research and development, the human adenovirus type 5 vaccine carrier is widely used in the market. Adenovirus is very popular as a vaccine virus. The ...

Claims

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Application Information

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IPC IPC(8): C12N15/861C12N15/50C12N15/64A61K39/215A61P31/14
CPCC12N15/86C07K14/005C12N9/22A61K39/12A61P31/14C12N2710/10343C12N2770/20022C12N2770/20034A61K2039/5256A61K2039/575A61K2039/57A61P35/00C12N2710/10321C12N7/00
Inventor 陈平李娜张婷婷钟鑫涛李楠
Owner JIAXING ANYU BIOTECH CO LTD
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