Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation method and application of efficient recombinant adenovirus vector based on AdEasy<TM> system

A recombinant adenovirus, adenovirus technology, applied in the field of gene editing, can solve problems such as inability to work

Active Publication Date: 2020-12-29
WUHAN KEQIAN BIOLOGY CO LTD
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The prokaryotic 16S RNA promoter Prrn is a constitutive strong promoter, which can only be expressed in prokaryotic cells, but cannot be recognized by the specific promoter recognition factor σ factor in the transcription complex in the nucleus of higher eukaryotic cells, and cannot kick in

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Preparation method and application of efficient recombinant adenovirus vector based on AdEasy&lt;TM&gt; system
  • Preparation method and application of efficient recombinant adenovirus vector based on AdEasy&lt;TM&gt; system
  • Preparation method and application of efficient recombinant adenovirus vector based on AdEasy&lt;TM&gt; system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: the mutant screening of prokaryotic constitutive expression promoter 16S RNA promoter prrn

[0035] 1. Acquisition of prokaryotic constitutive expression promoter 16S RNA promoter prrn gene sequence

[0036] The Prrn promoter gene sequence (SEQ ID NO: 2) was artificially synthesized with reference to the Prrn promoter sequence in the literature (Daniell H., Lee S B., Panchal T., Wiebe P O. (2001). The gene was provided by Biotech Synthesized by Engineering (Wuhan) Co., Ltd.

[0037] 2. Screening of 16S RNA promoter prrn mutants.

[0038] 2.1 Primer design and PCR

[0039] First, design primers (Table 1) to obtain the prrn sequence and the shuttle vector sequence respectively by means of PCR (Table 2), and insert the prrn gene sequence into AdEasy through homologous recombination TM The new shuttle vector pShuttle-IRES-prrn-hrGFP-1 is constructed between IRES and hrGFP of the system shuttle vector pShuttle-IRES-hrGFP-1. After sequencing verification, th...

Embodiment 2

[0052] Example 2: The screened prrn mutant is inserted into the shuttle vector pShuttle-IRES-hrGFP-1, and the vector has the green fluorescent indicator function of hrGFP in both prokaryotic and eukaryotic cells.

[0053] 1. It can be seen from Example 1 that the screened prrn mutant still has the function of initiating transcription in prokaryotic cells, and can transcribe and express hrGFP green fluorescent protein.

[0054] 2. In order to verify whether the new shuttle vector pShuttle-IRES-prrn-hrGFP-1 can normally express hrGFP and emit light in eukaryotic cell 293T, we conducted a cell verification experiment, the steps are as follows:

[0055] 2.1 Extraction of recombinant adenovirus

[0056] (1) Pick a single colony that emits green light and does not grow on the ampicillin antibiotic, and put it in 5 ml of LB medium (Kana: 50 μg / ml), grow at 250 rpm, 37°C for 12 hours.

[0057] (2) Add 1.5-3ml of the culture to the EP tube, and centrifuge at 14000g for 1min at room te...

Embodiment 3

[0071] Embodiment 3: Screening of recombinant classical swine fever virus E2 protein adenovirus

[0072] 1. Construction of pShuttle-E2-IRES-prrn-hrGFP-1 shuttle vector and transformation of Escherichia coli BJ5183

[0073] Design primers (Table 3) to obtain the E2 protein gene sequence and the shuttle vector sequence by PCR, and insert the E2 gene sequence (SEQ ID NO: 3) into AdEasy through homologous recombination TM In the system shuttle vector pShuttle-IRES-prrn-hrGFP-1, the recombinant vector pShuttle-E2-IRES-prrn-hrGFP-1 was constructed.

[0074] Table 3 E2 protein primer sequence and shuttle vector primer sequence

[0075]

[0076] The recombinant plasmid pShuttle-E2-IRES-prrn-hrGFP-1 and its mutants were transformed into Escherichia coli BJ5183, placed in the EP tube containing the transformed plasmid on ice for 5 minutes, and amplified with 50 μL of BJ5183 competent cells, mixed gently, and placed in an ice bath 30min. Heat shock at 42°C for 45s, and quickly ice...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a preparation method and application of an efficient recombinant adenovirus vector based on an AdEasy<TM> system. The recombinant adenovirus vector is a shuttle vector pShuttle-IRES-hrGFP-1 containing a prrn gene, and the prrn gene comprises a nucleotide sequence as shown in SEQ ID NO.1. A prokaryote 16S RNA promoter Prrn with an optimized sequence is integrated to the shuttle vector pShuttle-IRES-hrGFP-1, so that a bacterial colony in which pAdEasy-1 and the shuttle vector are successfully recombined loses ampicillin resistance and emits green light, and thereby the screening of successfully recombined adenovirus recombinants can be easily realized. When the recombinant adenovirus vector provided by the invention is applied to the expression of recombinant protein, the screening efficiency of adenovirus recombinants can be improved to a greater extent, and the screening time is shortened from 2-3 days to immediate realization.

Description

technical field [0001] The present invention relates to the technical field of gene editing, in particular to an AdEasy-based TM The preparation method and application of a systematic high-efficiency recombinant adenovirus vector. Background technique [0002] Adenoviral vectors have been widely used as vaccine delivery systems for the treatment of various infectious diseases such as rabies, human immunodeficiency virus type 1 (HIV-1), malaria, hepatitis C virus (HCV), and influenza. Adenoviral vectors are also used as therapeutic cancer vaccines, which enable the immune system to recognize cancer cells and affect their growth or inhibit their growth. It enables immunization against tumor-specific antigens, which can be achieved through adenovirus-mediated tumor-associated antigens (TAAs) or immunomodulatory molecules. [0003] Recombinant adenoviral vectors have been widely used in the in vitro and in vivo delivery of foreign genes in various cells and tissues. They can ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12N15/861C12Q1/70C12Q1/686C12N15/11C12N15/64C12N15/65C12N1/02G01N21/64
CPCC12N15/86C12Q1/701C12Q1/686C12N15/64C12N15/65C07K14/005C12N1/02G01N21/6486C12N2710/10343C12N2710/12022C12Q2565/125
Inventor 周明光邵伟汤细彪徐高原曾小燕郝根喜金建云陈章表
Owner WUHAN KEQIAN BIOLOGY CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com