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Replication-defective Ad29 adenovirus vector as well as construction method and application thereof

A replication-deficient type and construction method technology, applied in the field of replication-deficient Ad29 adenovirus vector and its construction, can solve the problems of complicated and time-consuming operation

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

[0005] Currently, the construction of replication-deficient adenovirus vectors usually adopts traditional molecular manipulation methods, including the following steps: (i) using cosmid to assemble the wild-type adenovirus genome (~35kb) into a plasmid vector (~3kb) to obtain an adenovirus vector (ii) using different dedicated shuttle plasmids to obtain replication-deficient adenoviral vectors by knocking out target genome segments at different sites through multi-step homologous recombination. long

Method used

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  • Replication-defective Ad29 adenovirus vector as well as construction method and application thereof
  • Replication-defective Ad29 adenovirus vector as well as construction method and application thereof
  • Replication-defective Ad29 adenovirus vector as well as construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0084] Example 1 Obtaining of human adenovirus type 29 genome

[0085] 1. Selection of human adenovirus type 29 adenovirus

[0086] The trans complement cell line of human adenovirus type 5 E1 deletion vector is HEK293. HEK293 cells are derived from human embryonic kidney cells. The E1A and E1B genes of human adenovirus type 5 are inserted into the cells, and the nucleotide sequence is 1 to 4344bp , the insertion site is the q13.2 region of chromosome 19.

[0087] A disadvantage of the HEK293 cell line is the possible contamination of the virus preparation with restorer mutations (RCA). This is because the 400th to 3500th base of the first-generation Ad5 vector is knocked out, but still maintains significant homology with the cellular DNA, and a double-crossover recombination event will occur to generate wild adenovirus.

[0088] In order to prevent the production of RCA, there are two ways, one is to construct a cell line containing only 505-4034 bases of human adenovirus t...

Embodiment 2

[0096] Example 2 Containing Fluorescent Labeled Adenovirus Shuttle Plasmid Construction

[0097] The human adenovirus type 29 genome is a linear DNA with a length of about 35,000 bp, and it is difficult to carry out effective molecular manipulation on it. Therefore, it is necessary to connect the elements related to replication in bacteria, the replicon (pACYC), and the resistance gene (kanamycin), to the left ~ 1400bp fragment of the Ad29 genome, and the right ~ 1400bp fragment, through homologous recombination in BJ5183 competent , constituting a circular plasmid, which can effectively replicate in bacteria; at the same time, in order to monitor the transfection efficiency of the plasmid in cells, a green fluorescent expression cassette (RSV promoter, EGFP, BGHpolyA) was added, and the TPO after transfection into cells The transfection efficiency of the large vector can be judged by the amount of fluorescence.

[0098] 1. Construction of the shuttle plasmid pAd29-shuttle fo...

Embodiment 3

[0121] Example 3 CRISPR / cas9 excision of Ad29 adenovirus E1 region

[0122] Ad29 replication-related genes are E1, E3, and E4 genes. E1 gene is adjacent to PIX. E1 gene is composed of E1A, E1B and their related promoter genes. Its upstream is the non-structural protein region, and its downstream position is PIX gene. It is a non-open reading frame and does not encode a protein. Use specific gRNA and Cas9 protein to cleave the double-stranded DNA of the E1 gene and the double-stranded DNA of the non-open reading frame downstream of the PIX gene, and realize the knockout of the E1 gene by double enzyme digestion, while using the human HPRT intron gene The excised sequences were ligated as nonsense sequences (stuff sequences) to obtain an Ad29 plasmid with E1 gene deletion, which was named pAd29ΔE1. The HPRT intron gene sequence is an intron of the human genome, which does not encode amino acids and does not express any genes, so it will not affect the packaging of adenovirus an...

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Abstract

The invention provides a replication-defective Ad29 adenovirus vector as well as a construction method and application thereof. The replication-defective Ad29 adenovirus vector is more efficiently and conveniently prepared through constructing a shuttle plasmid containing a replicon and a resistance gene, preparing an Ad29 adenovirus cyclized plasmid, finding the most suitable knockout site through a CRISPR / Cas9 technology, designing optimal specific targeting gRNA, and specifically knocking out E1 and E3 genes from the Ad29 adenovirus. The adenovirus is low in popularity and low in average neutralization titer in people, can be used for preparing novel coronavirus vaccines, is high in stability, and can be used for inducing very high humoral immunity and cellular immunity.

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: 202010945735.7, 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 field of genetic engineering and the field of immunology, in particular to a replication-deficient Ad29 adenoviral vector and its construction method and application. Background technique [0003] Adenovirus is a double-stranded DNA virus with a non-encapsulated spherical structure. Its virions are often arranged in a lattice in the infected cell nucleus. Each virion contains a 36kb linear double-stranded DNA with a 100 kb at each end. ~600bp inverted terminal repeat (inverted terminal re-peat, ITR). The adenovirus genome contains early expressed E1-E4 genes related to adenovirus replication...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/65C12N15/64C12N15/55C12N15/34C12N15/50A61K39/215A61P31/14
CPCC12N15/86C12N15/65C12N9/22C07K14/005A61K39/12A61P31/14C12N2710/10343C12N2710/10322C12N2710/10352C12N2800/50C12N2800/107C12N2770/20022C12N2770/20034A61K2039/5256A61K2039/53
Inventor 陈平张婷婷李娜钟鑫涛
Owner JIAXING ANYU BIOTECH CO LTD
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