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an adeasy based tm Preparation method and application of systematic high-efficiency recombinant adenovirus vector

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

Active Publication Date: 2022-07-01
WUHAN KEQIAN BIOLOGY CO LTD
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  • 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

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  • an adeasy based <sup>tm</sup> Preparation method and application of systematic high-efficiency recombinant adenovirus vector
  • an adeasy based <sup>tm</sup> Preparation method and application of systematic high-efficiency recombinant adenovirus vector
  • an adeasy based <sup>tm</sup> Preparation method and application of systematic high-efficiency recombinant adenovirus vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Mutant screening of prokaryotic constitutive expression promoter 16S RNA promoter prrn

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

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

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

[0038] 2.1 Primer design and PCR

[0039] First, primers (Table 1) were designed to obtain the prrn sequence and the shuttle vector sequence by PCR (Table 2), and the prrn gene sequence was inserted into AdEasy by homologous recombination. TM A new shuttle vector pShuttle-IRES-prrn-hrGFP-1 is constructed between the IRES and hrGFP of the system shuttle vector pShuttle-IRES-hrGFP-1. The sequence and position of the insert were verified by sequencing. And the tra...

Embodiment 2

[0052] Example 2: The screened prrn mutants were inserted into the shuttle vector pShuttle-IRES-hrGFP-1, and the vector had hrGFP green fluorescence indicator function 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 normally expresses hrGFP and emits light in eukaryotic cells 293T, we carried out a cell verification test. The steps are as follows:

[0055] 2.1 Extraction of recombinant adenovirus

[0056] (1) Pick a single colony that glows green and does not grow on ampicillin into 5 ml of LB medium (Kana: 50 μg / ml), grows at 250 rpm and 37° C. for 12 h.

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

[0058] (3) Remove the residual liq...

Embodiment 3

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

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

[0073] The primers (Table 3) were designed to obtain the E2 protein gene sequence and the shuttle vector sequence by PCR, and the E2 gene sequence (SEQ ID NO: 3) was inserted 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 sequences and shuttle vector primer sequences

[0075]

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

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Abstract

The present invention relates to an AdEasy-based TM Preparation method and application of a systematic high-efficiency recombinant adenovirus vector, the recombinant adenovirus vector is a shuttle vector pShuttle-IRES-hrGFP-1 containing a prrn gene, and the prrn gene includes a nucleoside as shown in SEQ ID NO.1 acid sequence. In the present invention, the prokaryotic 16S RNA promoter Prrn whose sequence has been optimized is integrated into the shuttle vector pShuttle-IRES-hrGFP-1, so that the colony successfully recombined between pAdEasy-1 and the shuttle vector will lose the ampicillin resistance and emit green light, which can easily Screening of successfully recombined adenovirus recombinants. When the recombinant adenovirus vector provided by the present invention is applied to the expression of recombinant protein, the screening efficiency of the adenovirus recombinant can be greatly improved, and the screening time can be shortened from 2-3 days to immediate realization.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a kind of 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 influence or inhibit the growth of cancer cells. It enables immunization against tumor-specific antigens, which can be achieved by adenovirus-mediated tumor-associated antigens (TAAs) or immunomodulatory molecules. [0003] Recombinant adenoviral vectors have been widely used to deliver foreign genes in vitro and in vivo in various cells and tissues. They can easily grow...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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
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