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SiRNA expression vector and application thereof

A vector and recombinant vector technology, which is applied in recombinant DNA technology, the use of vectors to introduce foreign genetic material, gene therapy, etc., can solve the problems of weak expression and inability to stably knock down target genes, so as to avoid competitive inhibition and improve knockdown. Reduced efficiency, high efficiency and stable transfection effect

Inactive Publication Date: 2015-07-01
重庆沃康生物科技有限公司
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is that the existing siRNA overexpression technology mainly uses shRNA plasmids initiated by RNA mimics and RNA Pol III promoters. The former cannot stably knock down the target gene, while the latter exists The disadvantage of weak expression

Method used

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  • SiRNA expression vector and application thereof
  • SiRNA expression vector and application thereof
  • SiRNA expression vector and application thereof

Examples

Experimental program
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Embodiment 1

[0058] Experimental reagents and equipment:

[0059] The main instruments are: 5417R desktop refrigerated high-speed centrifuge, thermo company, product number cat#5417R;

[0060] PCR instrument, cat#2500;

[0061] ThermoForma310 series direct heating CO 2 Incubator, thermo company, cat#3111;

[0062] Microplate reader Biotek, VE-186;

[0063] qPCR instrument, BioRad Company, CFX Connect, product number: BR001139.

[0064] The main reagents are: Reverse transcriptase, Takara company, article number: cat#U1240;

[0065] E.coli Poly(A) polymerase, NEB company, article number: #M0276S;

[0066] pCDH-CMV-MCS-EF1-copGFP plasmid (see plasmid map figure 2 ), purchased from SystemBiosciences (SBI), product number: CD511B-1;

[0067] Endonuclease: EcoRI, NEB Company, cat#R0101V; SalI, NEB Company, cat#R0138V; BsmBI, NEB Company, cat#R0580L; BamHI, NEB Company, cat#0136V;

[0068] Genomic DNA extracted from human liver was purchased from Hangzhou Zandao Technology Co., Ltd., ...

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Abstract

The invention discloses an empty carrier for construction of a siRNA expression vector, and a recombinant vector for siRNA and application thereof. The empty carrier, from terminal 5' to terminal 3', comprises: 1) a CMV-derived RNA polymerase II dependent promoter sequence; 2) a pri-MiR21 front fragment sequence as represented by SEQ ID No. 1; 3) the sequence of a cleavage site of restriction endonuclease; 4) a pri-MiR21 rear fragment sequence as represented by SEQ ID No. 2; and 5) a terminator sequence. The recombinant vector for siRNA is obtained by inserting the sequence of siRNA sense sequence-MiR21 ring structure sequence-siRNA antisense sequence to the cleavage site of the restriction endonuclease. According to the invention, since an RNA Pol II promoter is employed, shRNA can be conveniently and effectively connected to the skeleton of pri-MiR21 and be stably expressed through the RNA Pol II promoter, high efficiency expression is realized, and efficiency of knockout and deletion of RNAi is improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an siRNA expression vector and its application, in particular to an empty vector for constructing an siRNA expression vector, its recombinant vector and its application. Background technique [0002] siRNA formed by cleavage of dsRNA. siRNAs are 21-25nt long with a 3' overhang. The 2-nt overhang at the 3' end of the siRNA plays a role in the specificity of target recognition, limiting it to the position of the first base pair adjacent to the unpaired base. Then the siRNA binds to the RNA-induced silencing complex (RISC) and unwinds into a single strand. The activated RISC is guided by the single-stranded siRNA, sequence-specifically binds to the target mRNA and cuts it off. , triggering specific breakdown of target mRNAs, thereby blocking post-transcriptional gene silencing of corresponding gene expression. In recent years, RNAi libraries have been created targeting thousands of h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85A61K48/00
Inventor 姚远颋王海峰费倩岚谈竹君劳昕元
Owner 重庆沃康生物科技有限公司
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