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shRNA inhibiting expression of rabbit Deptor gene, lentivirus expression vector and construction method and application of lentivirus expression vector

An expression vector, psicor-shrna-508 technology, applied in the direction of retroRNA virus, DNA/RNA fragment, virus/bacteriophage, etc., can solve the problem of reducing the activity of mTOR

Inactive Publication Date: 2019-04-16
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the absence of growth factors or in the presence of mTOR inhibitors, mTOR-Deptor binding is enhanced, thereby reducing mTOR activity

Method used

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  • shRNA inhibiting expression of rabbit Deptor gene, lentivirus expression vector and construction method and application of lentivirus expression vector
  • shRNA inhibiting expression of rabbit Deptor gene, lentivirus expression vector and construction method and application of lentivirus expression vector
  • shRNA inhibiting expression of rabbit Deptor gene, lentivirus expression vector and construction method and application of lentivirus expression vector

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] The construction of embodiment 1 rabbit Deptor gene expression vector

[0058] According to the predicted sequence of the rabbit (Oryctolagus cuniculus) Deptor gene (XM_002710752) published by NCBI GeneBank, primers F, R, and Rr for amplifying the full-length CDs of the Deptor gene were designed with OLigo7.0 software. Among them, the Deptor gene amplified from F and R is subsequently connected to the pLVX-IRES-ZsGreen1 vector. F and Rr amplify the Deptor gene, which is subsequently connected to the pEGFP-N1 vector used to detect the interference efficiency of the interference vector. pEGFP-N1 is a fusion expression vector. When constructing a recombinant plasmid, it must be ensured that the newly inserted sequence does not affect the expression of subsequent fluorescent proteins. Therefore, an additional downstream primer Rr (two bases more than the R primer) was designed. The primer sequence is shown in Table 1 below. , the underline in Table 1 is the enzyme cutting ...

Embodiment 2

[0066] Example 2 Cell Expression Detection of Recombinant Plasmid pEGFP-Deptor / pLVX-Deptor-IRES-ZsGreen1

[0067] Extract the constructed pEGFP-Deptor / pLVX-Deptor-IRES-ZsGreen1 recombinant plasmid with an endotoxin-free plasmid extraction kit. According to the instructions of the Viafect transfection reagent, take the amount of plasmid and transfection reagent corresponding to the size of the culture dish, and add In the corresponding amount of opti-MEM, the mass / volume ratio of the plasmid to the transfection reagent is 1:3 (that is, 3 μL of transfection reagent is needed to transfect 1 μg of the plasmid), mix well and let stand for 20 minutes. The transfection complex was evenly added dropwise to HEK 293T cell culture medium, and fresh culture medium was changed after 12 hours, and the expression of GFP in cells was observed under a fluorescent microscope after 72 hours.

[0068] The result is as Figure 6 and Figure 7 as shown, Figure 6 It was shown that a large number...

Embodiment 3

[0070] Example 3 Construction of shRNA lentiviral expression vector

[0071] According to THEROMO FISHER company's siRNA online design website (http: / / rnaidesigner.thermofisher.com / rnaiexpress / ), the CDs sequence of the amplified rabbit Deptor gene was screened for interfering sequences, because there is no rabbit gene bank on this website for sequence comparison Yes, then compared the human and mouse gene banks, and finally selected 3 target sequences with good conservation and high scores for shRNA design. BLAST comparison confirmed that the above sequences had no homology with other gene sequences, and the It is designed as an shRNA construct. The shRNA structure includes a 19nt siRNA sense strand, a 9nt loop loop, a 19nt siRNA antisense strand and a termination signal, and enzyme cutting sites Hpal and Xhol are introduced at both ends of the shRNA. Named as pSicoR-shRNA-477 or pSicoR-shRNA-508 or pSicoR-shRNA-585, respectively. See Table 3 below for details, F chain 5'→3...

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Abstract

The invention discloses shRNA inhibiting the expression of a rabbit Deptor gene, a lentivirus expression vector and a construction method and application of the lentivirus expression vector. The shRNAis pSicoR-shRNA-508 and is composed of a siRNA positive-sense strand, a loop, a siRNA antisense chain and a termination signal. shRNA can effectively and stably inhibit the expression of the rabbit Deptor gene, the influence and action mechanism of the Deptor gene on the exogenous gene expression efficiency can be studied by inhibiting the Deptor gene expression, the influence of the Deptor geneon rabbit embryonic stem cell multfunctionality maintenance and autophagy pathway related gene expression can be further verified, and a foundation is laid for stable rabbit embryonic stem cell establishment.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a shRNA for inhibiting the expression of rabbit Deptor gene, a lentiviral expression vector and a construction method and application thereof. Background technique [0002] Deptor is a 48kDa protein derived from the DEPDC6 gene (NCBI gene ID 64798). Peterson et al. identified this protein in the immunoprecipitation of mTOR, so two DEP domains were connected in series at the N-terminus of the protein and interacted with mTOR, so the protein was named Deptor. The C-terminus of Deptor contains a PDZ domain, which regulates the interaction between Deptor and the C-terminal region of mTOR protein. Deptor is a component of the mTOR complex mTORC1 / 2 and negatively regulates both complexes. Interfering with the expression of Deptor increases the phosphorylation of mTORC1 / 2 downstream signaling substrates. On the contrary, overexpression of Deptor inhibits the signaling downstream of mTORC1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N5/10
CPCC12N5/0606C12N15/1136C12N15/86C12N2310/14C12N2510/00C12N2740/15043C12N2800/107C12N2310/531
Inventor 邓彦飞农恬颖陈凤杨素芳石德顺
Owner GUANGXI UNIV
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