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Lentiviral expression vector pLOX-CMV-E/P and construction and application thereof

A technology of lentiviral vector and expression vector, applied in the field of lentiviral expression vector pLOX-CMV-E/P, can solve problems such as safety risks and achieve the effect of stable expression

Inactive Publication Date: 2015-02-25
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the exogenous gene can be effectively integrated into the genome of the target cell for stable expression through the lentiviral vector, the integration of the exogenous gene and the backbone sequence of the lentiviral vector still poses a potential safety risk to the target cell, and the exogenous gene cannot be integrated as needed. Timely deletion of genes requires the development of a safer and more effective chronic disease system that can effectively screen and identify transfected positive cells, as well as effectively delete foreign genes and vector backbones as needed

Method used

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  • Lentiviral expression vector pLOX-CMV-E/P and construction and application thereof
  • Lentiviral expression vector pLOX-CMV-E/P and construction and application thereof
  • Lentiviral expression vector pLOX-CMV-E/P and construction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Design and synthesis of artificial multiple cloning site MCS

[0033] (1) Using the DNA of the pLOX-TERT-iresTK plasmid (Addgene, USA) as a template, use the NEBcutter V2.0 online analysis software (http: / / tools.neb.com / NEBcutter2 / ) to analyze the restriction enzyme site, in Select the SpeI single restriction site between the CMV promoter and the TERT coding region, select the restriction site KpnI between the TK gene coding region and the 3′LTR for double restriction, and remove the TRET-iresTK fragment (such as figure 1 shown in A).

[0034] (2) Select the commonly used restriction sites BclI, EcoRI, PacI, XhoI, SalI, BamHI, SbfI and MluI that are not in the remaining plasmid fragments to form a multiple cloning site sequence, and add SpeI enzyme at its 5' end The sticky end of the cleavage site is added to the 3' end of the sticky end of the KpnI restriction site for connecting the multiple cloning site into the vector.

[0035] (3) Design and synthesize ...

Embodiment 2

[0039] Example 2 Construction of pLOX-MCS vector

[0040] (1) The pLOX-TERT-iresTK plasmid is double digested with Spe I and Kpn I, and the digested products are separated by electrophoresis, and the gel recovery kit (Qiagen Company) is used to perform gel cutting to recover 8195bp size fragments, as in Example 1 The MCS fragments formed by the annealing in step (3) were ligated with T4 DNA ligase (ThermoFisher Company) at 16° C. overnight. Wherein, the connection reaction system is shown in Table 2.

[0041] Table 2 The ligation reaction system between multiple cloning sites and pLOX-TERT-riesTK restriction site

[0042] pLOX-TERT-iresTK vector fragment recovered from gel cutting

50ng

MCS fragment

0.1μL

T4 DNA ligase

1μL

10×T4Ligase Buffer

2μL

DDW (Ultrapure Water)

to 20μL

Total Volume

20 μL

[0043] (2) Transform Escherichia coli DH5α competent cells (Beijing Tiangen Biochemical Technology Co., Ltd....

Embodiment 3

[0045] Example 3 Construction of pLOX-CMV-E / P vector

[0046] (1) Using the pB513 plasmid (System Biosciences) as a template, design the forward primer EF1α-GFP / Pur-F: 5′-CG G GAT CC G AAG GAT CTG CGA TCG CTC C-3' (the underline is the BamHI restriction site, and the CG at the 5' end is the protection base of the restriction site), and the reverse primer EF1α-GFP / Pur-R: 5'-GG G GTA CC T CAG GCA CCG GGC TTG CGG GT-3' (the underline is the KpnI restriction site, and the 5' end GG is the protection base of the restriction site), using KOD-Plus-Neo High Fidelity Enzyme PCR Kit (Toyobo Company) Amplify the EF1α-EGFP-Puro fragment (1986bp in size), and separate by electrophoresis (see Figure 4 After A), use the gel recovery kit (Qiagen company) to recover the target size fragments. Wherein, the PCR reaction system is shown in Table 3.

[0047] Table 3 PCR reaction system used to amplify EF1α-EGFP-Puro fragment

[0048] pB513 plasmid (170ng / μL)

0.3μL

EF1α-G...

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Abstract

The invention discloses a lentiviral expression vector pLOX-CMV-E / P and construction and application thereof. Multiple cloning site which is inserted into the downstream part of a CMV promoter of the vector can be recognized by 7 restriction endonucleases, including SpeI, BclI, EcoRI, Paci, XhoI, SalI and BamHI. The application of the vector comprises the following steps: inserting a target gene to the multiple cloning site to realize the stable expression of the target gene; using Puromycin resistance gene and EGFP which is driven by EF1alpha promoter to perform fluorescence tracing and drug resistance screening; based on LoxP site inserted in 3'LTR region and the transcription and reverse transcription characteristic of lentivirus, realizing effective deleting of an exogenous gene and a reporter gene under the condition of Cre recombinase expression. The vector disclosed by the invention can realize the package of the lentiviral particles by commonly transfecting 293T cell together with pCMVR8.74 and PMD2.G vector.

Description

technical field [0001] The present invention relates to a lentiviral expression vector pLOX-CMV-E / P, in particular to a kind not only suitable for Cre-LoxP system, but also carrying EGFP and Puro dual reporter genes driven by EF1α promoter, and simultaneously carrying CMV promoter driven Artificially synthesized lentiviral expression vector with multiple cloning sites and its construction and application. Background technique [0002] The lentiviral vector is modified on the basis of human immunodeficiency virus (human immune-deficiency virus, HIV). It has a wide range of hosts, can infect dividing cells and non-dividing cells, and has high transfection efficiency. It can carry up to 8-10kb It is one of the important ways in the field of gene function research and gene therapy. In recent years, with the in-depth development of molecular biology techniques, lentiviral vectors have been divided into several different plasmids according to their physical characteristics, furth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/66
Inventor 齐绪峰夏景波陈卓莹蔡冬青吴彩红王健欢毛承舟刘光辉
Owner JINAN UNIVERSITY
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