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shRNA (short hairpin ribonucleic acid) sequence for silencing RhoB in targeting manner, primer, application, shRNA lentiviral vector and construction method

A lentiviral vector and targeting technology, applied in the field of molecular biology and biomedicine, can solve the problem of poor selectivity of different tissue cells with Rho kinase specificity

Inactive Publication Date: 2016-01-20
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a shRNA sequence, primer, application, shRNA lentiviral vector and construction method for targeted silencing of RhoB, aiming to solve the problem of existing Rho kinase inhibitors on Rho kinase. Specificity and selectivity to different tissue cells are not good enough

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  • shRNA (short hairpin ribonucleic acid) sequence for silencing RhoB in targeting manner, primer, application, shRNA lentiviral vector and construction method
  • shRNA (short hairpin ribonucleic acid) sequence for silencing RhoB in targeting manner, primer, application, shRNA lentiviral vector and construction method
  • shRNA (short hairpin ribonucleic acid) sequence for silencing RhoB in targeting manner, primer, application, shRNA lentiviral vector and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1: Construction of a lentiviral backbone vector for expressing shRNA

[0057] (1) Construct vector backbone pLVX-EF1-copGFP-Puro:

[0058] Using pCDF1-MCS2-EF1-copGFP (SBI) vector as a template, PCR amplifies the EF1-copGFP fragment (see Table 1 for the primer sequence), digests the PCR product with MluI and AfeI, and inserts it into the MluI and AfeI. Enzyme digestion of the lentiviral vector pLVX-Puro (Clontech company) to obtain the vector backbone pLVX-EF1-copGFP-PGK-Puro.

[0059] (2) Insert promoter U6:

[0060] The vector pLVX-EF1-copGFP-PGK-Puro obtained by double digestion with ClaI and XhoI in step (1) was recovered by electrophoresis after dephosphorylation; after the PCR product of U6 promoter was digested with ClaI and XhoI (for primer sequences, see Table 2 ), connected to the vector pLVX-EF1-copGFP-Puro, transferred to E. coli competent cells, coated on LB-Amp plates, cultured overnight, and the vector obtained after identification was pLVX-U6-EF1-copGFP...

Embodiment 2

[0063] Example 2: Designing siRNA and shRNA sequences against RhoB gene

[0064] In this example, siRNA sequences were first designed for the gene RhoB (GeneID: 338), and corresponding shRNA oligonucleotide primers were synthesized. The primer sequences are shown in Table 4.

[0065] Table 4 Oligonucleotide primers for annealing synthesis of shRhoB sequence

[0066]

[0067] Add samples and mix according to the system in Table 5. After evenly mixing, place them in boiling water, and let stand until it drops to room temperature (4-6h).

[0068] Table 5 The annealing system of shRhoB primers forming double strands

[0069] Reagent

Embodiment 3

[0070] Example 3: Construction of shRNA lentiviral vector for targeted silencing of RhoB gene

[0071] The shRNA lentiviral vector pLVX-U6-ccdB-EF1-copGFP-PGK-Puro constructed in Example 1 was digested with BsmBI. After the gel was cut and recovered, the shRhoB double strand annealed and synthesized in Example 1 was combined with pLVX-U6- The EF1-copGFP-PGK-Puro vector backbone is connected through the corresponding restriction sites, transferred into E. coli competent cell stable3, coated with LB-Amp plate and cultured overnight at 37°C, a single clone is picked and sequenced to construct targeted silencing RhoB Gene shRhoB lentiviral vector pLVX-U6-shRhoB-EF1-copGFP-PGK-Puro.

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Abstract

The invention discloses an shRNA (short hairpin ribonucleic acid) sequence for silencing RhoB in a targeting manner, a primer, an application, an shRNA lentiviral vector and a construction method. The shRNA sequence is designed aiming at the key factor RhoB of an Rho kinase signal pathway and the corresponding shRNA lentiviral silencing vector is constructed. The shRNA sequence comprises a sequence 5'-CGTGTTCAGTAAGGACGAGTT-3'. Experimental results prove that shRNA can obviously silence expression of RhoB. Seeing that RhoB serves as a potential pulmonary hypertension treatment target, shRNA of RhoB and related biological reagents, which are provided by the invention, can be used for studying and preparing medicines for treating pulmonary hypertension related diseases.

Description

Technical field [0001] The invention relates to the fields of molecular biology and biomedicine, and mainly relates to a shRNA sequence, primer, application, shRNA lentiviral vector and construction method for targeted silencing of RhoB. Background technique [0002] RNA interference (RNAinterference, RNAi) is an evolutionarily conserved gene defense mechanism, that is, a sequence-specific post-transcriptional gene silencing (PTGS). In recent years, the development of RNAi technology has not only greatly promoted the human post-genome project, but can also be applied to high-throughput screening of drug target genes, promote gene therapy and new drug development, and open up new ways to treat cancer, genetic diseases and other diseases. In the past few years, RNAi technology, as a new gene therapy, has been widely used in the research field of gene therapy of diseases, such as age-related macular degeneration, amyotrophic lateral sclerosis, rheumatoid arthritis, etc. In addition...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N15/867C12N15/66A61K31/7088A61P9/00
CPCY02A50/30
Inventor 苟德明康康李洁璇黄炼张利敏牛燕琴姚丽君吴伊可
Owner SHENZHEN UNIV
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