RNA interference virus vector

A technology of RNA interference and virus vector, which is applied in the field of RNA interference, can solve the problems of not being able to observe the interference of multiple genes in any combination at the same time, and not be able to observe the effect of individual interference of multiple genes at the same time, and achieve the effect of overcoming cytotoxicity

Pending Publication Date: 2022-04-05
BINZHOU MEDICAL COLLEGE
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, traditional multiple RNA interference can only interfere with multiple genes at the same time, but it cannot observe the individual int

Method used

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  • RNA interference virus vector
  • RNA interference virus vector
  • RNA interference virus vector

Examples

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Embodiment 1

[0072] Prepare a lentiviral vector with fluorescent protein as a reporter molecule (such as Figure 4 shown).

[0073] S11. Using the PLVX-Puro lentiviral vector as the backbone, through the AfeI and KpnI restriction sites, an artificially synthesized sequence (SEQ ID NO.1) including the miR-AB sequence and the Venus fluorescent protein controlled by the PGK promoter The gene sequence and the WPRE sequence are inserted into the vector to prepare the LCAPVW vector;

[0074] S12. Using the LCAPVW vector as the backbone, using the ClaI and AfeI restriction sites, replace the hCMV promoter with mCMV (SEQ ID NO.2), CBH (SEQ ID NO.3), hEF1a (SEQ ID NO.4), EH (SEQ ID NO. 5) or AH (SEQ ID NO. 6) promoters to make LMAPVW, LBAPVW, LEAPVW, LHAPVW and LAAPVW. These promoters have different transcriptional activities in eukaryotic cells, thus ensuring that the miR-AB-based RNA interference process can be carried out smoothly in a variety of eukaryotic cells;

[0075] Since these promote...

Embodiment 2

[0080] Prepare a retroviral vector using fluorescent protein as a reporter molecule for RNA interference based on the miR-AB sequence (such as Figure 4 shown).

[0081]S21. Using the MIGR1 retroviral vector as the backbone, through the BglII and ClaI restriction sites, an artificially synthesized sequence (SEQ ID NO.1) including the miR-AB sequence and the Venus fluorescent protein gene sequence controlled by the PGK promoter and the WPRE sequence, inserted into the vector, so as to prepare the MAPVW vector;

[0082] Using the MAPVW vector as the backbone, using the PacI and PmeI restriction sites at both ends of the Venus gene sequence, the Venus sequence was replaced with Azurite (SEQ ID NO.7), mTagBFP2 (SEQ ID NO.8), EGFP (SEQ ID NO.9 ), Ametrine (SEQ ID NO.10), mOrange (SEQ ID NO.11), mCherry2 (SEQ ID NO.12) or E2-Crimson (SEQ ID NO.13) sequence, thereby preparing MAPZW, MAPTW, MAPGW, MAPAW , MAPOW, MAPCW, MAPEW;

[0083] The present invention optimizes the coding sequ...

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Abstract

The invention relates to the technical field of RNA (Ribonucleic Acid) interference, in particular to an RNA interference virus vector which interferes the expression of a target gene by expressing shRNAmir carried by a miR-AB framework based on human miR-30pril-miRNA. The miR-AB sequence is regulated and controlled by a plurality of eukaryotic promoters; the RNA interference virus vector traces cells subjected to RNA interference through fluorescent protein reporter molecules, or screens the cells subjected to RNA interference through antibiotics. According to the present invention, the miR-AB sequence in the RNA interference virus vector is regulated and controlled by a variety of eukaryotic promoters so as to easily achieve the application of the RNA interference virus vector in a variety of eukaryotic cells, and the virus vector can screen the cells subjected to RNA interference through antibiotics by depending on the Puromycin resistance, and can also trace the cells subjected to RNA interference by depending on the fluorescent protein reporter molecule; therefore, a plurality of target genes in the eukaryotic cell can be subjected to single-gene interference and multi-gene arbitrary combination interference at the same time in a multi-virus co-infection mode, so that the functions of the plurality of target genes in the eukaryotic cell are analyzed at the same time.

Description

technical field [0001] The invention relates to the technical field of RNA interference, in particular to an RNA interference virus vector. Background technique [0002] Target gene deletion research has always been a commonly used method in life sciences to study the function of target genes. [0003] RNA interference (RNA interference) is a means of deleting target genes, which refers to the phenomenon that small molecule double-stranded RNA can specifically degrade or inhibit the expression of homologous mRNA, thereby inhibiting or shutting down the expression of specific genes. For example, as long as people know the causative gene of a certain disease, they can design small interfering RNA (Smallinterfering RNA, siRNA) targeting the mRNA of the gene to inhibit or block the expression of the causative gene, so as to achieve the purpose of treating the disease Specifically, siRNA can specifically bind to the target gene mRNA, and then degrade the mRNA through the RISC co...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/65C12N15/113C12Q1/02G01N21/64
Inventor 王大鹏
Owner BINZHOU MEDICAL COLLEGE
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