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Construction and application of E3 region-deleted complete-copy-type recombinant adenovirus 4 carrier system

A recombinant adenovirus and vector system technology is applied in the field of construction of a complete recombinant adenovirus type 4 vector system that lacks the E3 region to replicate, and can solve the problems of reducing the application effect of the adenovirus type 5 vector, insufficient antibodies, and short-lived.

Active Publication Date: 2016-05-11
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because adenovirus type 5 is harmful to the human body, it cannot be directly used as a live carrier vaccine, and can only be prepared as a replication-deficient (missing E1 region) vaccine, so only short-lived and limited antigens can be produced in clinical use. expression, and the final antibody produced is also correspondingly insufficient
[0003] Studies in recent years have also found that the pre-existing immunity against adenovirus type 5 in the body greatly reduces the application effect of adenovirus type 5 vectors. Epidemiological surveys show that human infection with adenovirus type 5 is very common. In developed countries, the seropositive rate of adenovirus type 5 is about 60-70%; in Africa and Southwest Asia, the seropositive rate of adenovirus type 5 is as high as 98%; in China, the seropositive rate of adenovirus type 5 has also reached 72%. %, the above situation further hampers the use of adenovirus type 5 viral vectors

Method used

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  • Construction and application of E3 region-deleted complete-copy-type recombinant adenovirus 4 carrier system
  • Construction and application of E3 region-deleted complete-copy-type recombinant adenovirus 4 carrier system
  • Construction and application of E3 region-deleted complete-copy-type recombinant adenovirus 4 carrier system

Examples

Experimental program
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Effect test

Embodiment 1

[0034] 1. Construction of backbone plasmid pAd4FAST and preparation of electroporation competent cells

[0035] (1) Construction of backbone plasmid pAd4FAST:

[0036] ① Using the pAdeasy-1 vector (stratagene company of the United States, GenBank accession number AY370909) as a template, use primers P1: CCTTAATTAA (PacI) CATGC (SEQ ID NO: 1) and P2: GGACTAGA (SpeI) TCTAGTTTCG (SEQ ID NO: 2) to amplify its position The fragment at 27246-31123bp (the fragment size is 3878bp), which contains the basic components of the vector such as pBR322 replication origin, ampicillin resistance gene, etc., the product is digested with PacI and SpeI, purified and recovered by DNA gel recovery kit to obtain the product 1;

[0037] Amplification was carried out in a standard polymerase chain reaction (PCR) system, the reaction system was 50 μl system: 5×PCRBuffer 10uL, template 10ng, 10mM deoxyribonucleoside triphosphate (dNTP) 1μl, upstream and downstream primers (25μM) 1μl each , high-fideli...

Embodiment 2

[0058] This example is the preparation process of a recombinant AdV4 virus carrying a gene of interest, specifically taking the preparation of "recombinant adenovirus AdV4-EGFP carrying an EGFP reporter gene" as an example:

[0059] The recombinant AdV4 adenovirus carrying the target gene can be prepared by using the vector system, and the target gene can be cloned into the cloning site of the shuttle plasmid pAd4FAST-Shuttle. After the shuttle plasmid carrying the target gene was linearized by restriction endonuclease SwaI, it was transformed into Escherichia coli BJ5183 strain (Stratagenen, USA) carrying the backbone plasmid pAd4FAST, and the target gene was obtained through homologous recombination in BJ5183 cells. The adenovirus plasmid, which is digested with the restriction endonuclease PacI, releases the recombinant adenovirus genome, and uses the method of DNA transfection to transfect the recombinant adenovirus genome into the packaging cell line HEK-293, and rescues t...

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Abstract

The invention relates to construction and application of an E3 region-deleted complete-copy-type recombinant adenovirus 4 carrier system. The system comprises a framework plasmid, a shuttle plasmid and a packaging cell line; the framework plasmid contains 1-26569bp segments on the left side of an adenovirus (AdV) 4 genome, and the adenovirus (AdV) 4 genome has E3 region genes serving as deletion genes; the shuttle plasmid contains cloning sites used for being inserted into a target gene and an inverted repeat sequence on the right side of the adenovirus (AdV) 4 genome; an HEK-293 cell line is adopted as the packaging cell line. Due to the fact that the anthropogenic adenovirus (AdV) 4 genome is safe and reliable, the recombinant adenovirus (AdV) 4 is expected to be applied to gene therapy and a recombinant vaccine in a host which can be infected by the recombinant adenovirus (AdV) 4, that is, the produced recombinant adenovirus (AdV) 4 has safe and wide application.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the construction and application of a recombinant adenovirus type 4 (AdV4) vector system with deletion of E3 region and complete replication in the field of gene therapy and recombinant vaccines. technical background [0002] The adenovirus type 5 recombinant system is currently the most widely used adenovirus system. Since the system was successfully constructed in 1998, it has been widely used in vitro due to the advantages of the adenovirus type 5 itself and the ease of use of the system. Various fields such as gene delivery, in vivo vaccination and gene therapy. However, because adenovirus type 5 is harmful to the human body, it cannot be directly used as a live carrier vaccine, and can only be prepared as a replication-deficient (missing E1 region) vaccine, so only short-lived and limited antigens can be produced in clinical use. Expression, the fina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N7/01
CPCC12N7/00C12N15/86C12N2710/10321C12N2710/10343C12N2800/107C12N2800/40C12N2820/55
Inventor 赵军王川庆高冬生李永涛杨霞刘红英常洪涛陈陆王新卫王永生郑鹿平姚惠霞黄慧敏
Owner HENAN AGRICULTURAL UNIVERSITY
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