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Construction and application of multi-cistron double-label expression lentivirus vector

A technology of lentiviral vectors and recombinant lentiviruses, which is applied in the direction of viruses/bacteriophages, applications, and the use of vectors to introduce foreign genetic materials, etc. It can solve the problems of tandem promoters that are easy to interfere with each other, abnormal protein folding or transportation, and upstream and downstream expression imbalances. and other issues, to achieve the effect of being beneficial to application, good practical value and application prospect, and widely applicable

Active Publication Date: 2014-11-26
思路迪科技(上海)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the commonly used strategies for constructing lentiviral polycistronic vectors are: (1) Internal ribosome entry site (IRES) connection, about 1000bp, but this method has its own large structure and unbalanced upstream and downstream expression (2) Gene fusion, which has caused abnormal protein folding or transport and affects its expression; (3) Using multiple promoters, each of which promotes an independent coding frame
The disadvantage is that the tandem promoters are easy to interfere with each other

Method used

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  • Construction and application of multi-cistron double-label expression lentivirus vector
  • Construction and application of multi-cistron double-label expression lentivirus vector
  • Construction and application of multi-cistron double-label expression lentivirus vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1U6-E

[0041] Example 1 Construction of U6-EF1α-MCS-FLAG-P2A-Tomato-T2A-Puro (abbreviated as pUETP) lentiviral vector

[0042] (1) Amplify U6, EF1α-MCS-FLAG, Tomato and Puromycin respectively

[0043] 1. Amplification of the U6 promoter

[0044] 1) Whole gene synthesis U6 sequence

[0045] The U6 fragment was artificially synthesized from the whole gene, and its sequence is shown in SEQ ID NO.1.

[0046] 2) Using the whole gene synthesis U6 fragment as a template, the Asc I and Nhe I restriction sites in the U6 fragment were removed by overlap extension PCR, and the EcoR I and BamH I restriction sites were introduced into the sequence at the same time. Wherein, in the first round of PCR, the U6-1 fragment is amplified by U6-F1 and U6-R1 primers, and the U6-2 fragment is amplified by U6-F2 and U6-R2 primers. In the second round of PCR, U6-1 and U6-2 fragments were used as templates, and U6-F1 and U6-R2 were used as primers for PCR amplification to obtain U6 fragments with EcoR Ⅰ an...

Embodiment 2U6-E

[0109] Example 2 Construction of U6-EF1α-MCS-FLAG-P2A-copGFP-T2A-Hygromycin (pUEGH for short) lentiviral vector

[0110] (1) Amplify U6, EF1α-MCS-FLAG, P2A-copGFP and T2A-Hygromycin respectively

[0111] 1. Amplify U6, EF1α-MCS-FLAG

[0112] The method is the same as pUETP.

[0113] 2. Amplification of T2A-Hygromycin

[0114] Using the linear hygro linearized fragment (purchased from Clontech) as a template, the EcoRI cleavage site in Hygromycin was removed by overlapping extension PCR point mutation. Among them, in the first round of PCR, Hygro-1 was amplified using linear hygro as a template, Hygro-F and Hygro-R were used as primers, and Hygro-2 was amplified using Hygro-MF and Hygro-MR as primers. In the second round of PCR, Hygro-1 and Hygro-2 were used as templates, and Hygro-F and Hygro-MR were used as primers for PCR amplification to obtain Hygromycin without an EcoRI restriction site, and T2A was introduced upstream of Hygromycin. Wherein the primer sequence is as ...

Embodiment 3

[0140] Example 3 Application examples of pUETP and pUEGH lentiviral vectors

[0141] (1) Application of pUETP in the construction of CDH1 gene RNAi stable strain

[0142] 1. CDH1 gene shRNA primer design

[0143] According to the principle of shRNA primer design, a pair of shRNA primers were designed for CDH1 gene (NCBI number: NM_004360.3).

[0144] shCDH1-F:

[0145] 5'-GATCCGGCGATTCAAAGTGGGCACAGATGGTACCATCTGTGCC CACTTTGAATCGTTTTTG-3';

[0146] shCDH1-R:

[0147] 5'-AATTCAAAAACGATTCAAAGTGGGCACAGATGGTACCATCTGT GCCCACTTTGAATCGCCG-3'.

[0148] 2. Construction of pUETP-shCDH1 lentiviral vector

[0149] Primer F (10 μM) 10 μl, primer R (10 μM) 10 μl, 10× annealing buffer 5 μl, ddH 2 O25μl reaction system was placed in a 100°C constant temperature water bath for 5 minutes, and then cooled naturally to room temperature for annealing. After the CDH1 shRNA double strand is obtained, it is connected to the EcoRI and BamHI sites of the modified lentiviral vector U6 of the presen...

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Abstract

The invention discloses a lentiviral vector, wherein the original hUbc promoter in FUGW is replaced by a U6 promoter or a strong promoter EF1alpha, the original EGFP gene is replaced by a strong fluorescent protein copGFP or a Tomato gene, a eukaryon resistance label gene, multiple clone sites and tag protein Flag are linked to the same cistron of the gene, and splicing protein T2A and P2A are adopted to link a plurality of genes on the same cistron. Compared with the conventional lentivirus vector, the lentiviral vector of the present invention has the following advantages that: a plurality of proteins can be concurrently and efficiently expressed with the same cistron, fluorescence intensity is high, induced function recovery is performed on the interfered gene, the vector is suitable for carrying large fragment genes, and the like. In addition, the lentivirus vector has characteristics of wide application, high efficiency, benefits for applications of the lentiviral vector in clinical and science researches, good practical value and good application prospects.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to the construction and application of a polycistronic double-marked expression lentiviral vector. Background technique [0002] Lentiviral vector refers to a viral vector derived from human immunodeficiency virus-1 (HIV-1). The lentiviral vector contains the genetic information required for packaging, transfection, and stable integration, and is the main component of the lentiviral vector system. part. With the assistance of lentiviral packaging plasmids and cell lines, the lentiviral vectors carrying foreign genes are packaged into infectious virus particles, and the exogenous genes are expressed in cells or living tissues by infecting cells or living tissues . Lentiviral particles have a wide infection spectrum, and can not only efficiently infect dividing cells, but also infect non-dividing cells, and can also efficiently infect primary cells and embryonic stem cells that are...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N15/65C12N7/00C12N7/01
Inventor 熊磊谢正华李风庆
Owner 思路迪科技(上海)有限公司
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