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Double-promoter and double-expression specific shRNA (Short Hairpin Ribonucleic Acid) lentiviral vector and application thereof

A lentiviral vector and double promoter technology, applied in the field of biomedicine, can solve the problems of affecting the interference effect of siRNA and the easy mutation of viral genes, and achieve the effect of avoiding loss of exchange.

Inactive Publication Date: 2013-08-21
中国疾病预防控制中心病毒病预防控制所 +4
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the targeted gene is an RNA virus gene, since the viral gene is prone to mutation, RNA interference is highly sequence-specific. Even if there is a mutation in the targeted gene, it will seriously affect the interference effect of siRNA

Method used

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  • Double-promoter and double-expression specific shRNA (Short Hairpin Ribonucleic Acid) lentiviral vector and application thereof
  • Double-promoter and double-expression specific shRNA (Short Hairpin Ribonucleic Acid) lentiviral vector and application thereof
  • Double-promoter and double-expression specific shRNA (Short Hairpin Ribonucleic Acid) lentiviral vector and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Construction of double-promoter double-expression shRNA lentiviral vector

[0034] 1. Linker design and synthesis

[0035]The linker sequence linker, positive strand linker(+) and negative strand linker(-) were designed and synthesized. The positive chain linker (+) and the negative chain linker (-) were dissolved in double-distilled water to a concentration of 100 mM, and 2 μL of each was heated to 90 ° C in a 50 μL reaction system, and naturally cooled and annealed at room temperature. The double-stranded oligonucleotide oligos contains four restriction sites of BamHI, EcoRI, XbaI and XhoI in the sequence, and the sequence is as follows BamHI-EcoRI-XbaI-XhoI such as ( figure 1 )

[0036] 2. Analysis and processing of lentiviral vector plvx-shRNA2

[0037] The recombinant lentiviral vector involved in the present invention is plvx-shRNA2 ( figure 2 ). Sequence analysis showed that there were a large number of enzyme cutting sites in the vector, includin...

Embodiment 2

[0042] Example 2 Construction of double shRNA lentiviral vectors targeting HIV-1vpr and tat genes and research on inhibition of targeted genes

[0043] 1. Construction of plvx-vpr-tatshRNA plasmid

[0044] For HIV-1NL4-3, our laboratory screened the siRNA sequences for vpr and tat, and designed the corresponding shRNA, vprshRNA and tatshRNA. The recombinant vectors plvx-vprshRNA and plvx-tatshRNA capable of expressing a single shRNA are respectively connected with the linearized plvx-shRNA2. The vprshRNA was inserted into the BamHI and EcoRI sites in plvx-DS, and the tatshRNA was inserted between the XbaI and XhoI sites to form plvx-vpr-tatshRNA ( image 3 ).

[0045] 2. Co-transfection experiment to detect shRNA inhibition efficiency

[0046] 293T cells by 3×10 5 Each cell / well was inserted into a 6-well cell culture plate, and when the cell density reached 80-85%, the plvx-vprshRNA, plvx-vpr-tatshRNA (1 μg) and the recombinant plasmid pEGFP-vpr (1 ​​μg) containing the ta...

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Abstract

The invention provides a double-promoter and double-expression specific shRNA (Short Hairpin Ribonucleic Acid) lentiviral vector and application thereof. The double-promoter and double-expression specific shRNA lentiviral vector comprises an U6 promoter with a nucleotide sequence shown as SEQ ID NO: 1, and an H1 promoter with the nucleotide sequence shown as SEQ ID NO: 2. The experiment results show that the lentiviral vector can express two types of shRNA at the same time, can inhibit the expression of a vpr (Viral Protein Regulatory) gene and / or reduce the expression level of a tat (Transactivator) gene, and can be used for developing and preparing a medicine for preventing HIV (Human Immunodeficiency Virus) infection from occurring.

Description

Technical field: [0001] The invention belongs to the field of biomedicine, and in particular relates to a recombinant lentiviral vector and a shuttle plasmid that use dual promoters to simultaneously express two shRNAs. The invention also relates to the application of the carrier and the shuttle plasmid in the preparation of medicines for preventing and treating HIV infection. Background technique: [0002] RNA interference (RNA interference, RNAi) refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), which is highly conserved during evolution. Since the use of RNAi technology can specifically knock out or shut down the expression of specific genes, this technology has been widely used in the field of gene therapy for exploring gene functions and infectious diseases and malignant tumors. [0003] RNA interference can be used to transiently transfect cells with chemically synthesized mature small interfering...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867A61K48/00A61P31/18C12N15/66
Inventor 曾毅滕智平郝彦哲杨怡姝
Owner 中国疾病预防控制中心病毒病预防控制所
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