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shRNA lentiviral expression vector for specifically inhibiting hepatic cell CYP2E1 gene expression, constructing method and application thereof

A CYP2E1 and expression vector technology, applied in the field of genetic engineering, can solve the problems of inability to achieve long-term stable interference, insufficient interference effect, and high cytotoxicity, and achieve good inhibition effect, good operation effect, and less dosage

Inactive Publication Date: 2012-10-03
SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, Xu Yun et al. connected siRNA to the carrier pmU6 to form a recombinant vector, transfected E47 cells, and constructed an siRNA expression vector that inhibits the transcription site of the CYP2E1 gene, although the siRNA expression vector can silence CYP2E1 in a short time Gene expression, but its interference effect is not obvious enough, the transfection efficiency is low, the cytotoxicity is high, and the shortcoming of long-term stable interference cannot be achieved
The realization of RNA interference gene expression function depends on the selection of siRNA sequence and the construction of high-efficiency expression vector, but there is no shRNA expression vector with high transfection efficiency, long-term stable expression, and specific inhibition of CYP2E1 gene expression in the prior art

Method used

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  • shRNA lentiviral expression vector for specifically inhibiting hepatic cell CYP2E1 gene expression, constructing method and application thereof
  • shRNA lentiviral expression vector for specifically inhibiting hepatic cell CYP2E1 gene expression, constructing method and application thereof
  • shRNA lentiviral expression vector for specifically inhibiting hepatic cell CYP2E1 gene expression, constructing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Design of the shRNA oligonucleotide sequence targeting the CYP2E1 gene of hepatocytes.

[0039] The complete mRNA sequence of CYP2E1 (NM_000773.3) was found in GenBank, and the specificity was confirmed by BLAST homology comparison, and the secondary structure of the target mRNA sequence was evaluated by using RNA structure 4.4 software, and finally three 19nt target nuclei were screened out Nucleotide sequence, the specific sequence is shown in Table 1.

[0040]

[0041] According to the target nucleotide sequence, the DNA template single strands of two shRNAs are designed and synthesized. The design is as follows: Age I restriction site + 19 nt target nucleotide sequence + stem-loop structure (TTCAAGAGA) + target sequence complementary sequence + RNA Poly III polymerase transcription termination site (TTTTTT) + EcoRI restriction site six regions, They were named shRNA1, shRNA2, and shRNA3, respectively, and a pair of control sequences siRNAc was design...

Embodiment 2

[0043] Example 2 Construction of shRNA lentiviral expression vector.

[0044] Mix the synthetic shRNA oligonucleotide single strands in equal amounts, anneal to form double-stranded shRNA, and extract the plasmid PLKO.1-puro (map as figure 1 shown), double-digested with restriction endonucleases AgeⅠ and EcoRI, recovered the vector by electrophoresis and gel cutting, and then ligated the double-stranded shRNA1, shRNA2, shRNA3, and shRNAc into the vector PLKO.1-puro with T4 DNA ligase, respectively. The recombinant plasmids pLKO-2E1shRNA1, pLKO-2E1shRNA2, pLKO-2E1shRNA3, pLKO-2E1shRNAc were formed. Transform the ligation product into competent Escherichia coli JM107, smear it on a plate containing ampicillin LB medium, and incubate at 37°C for 14 hours, and set a negative control group 1 (uniformly spread competent cells on a plate without ampicillin). plate), negative control group 2 (uniformly spread competent cells on a plate containing 100 μg / ml ampicillin), positive con...

Embodiment 3

[0047] Example 3 Lentiviral packaging.

[0048] 293FT cells were cultured, and the cells in good growth state were inoculated into six-well plates, 10 per well 6 2 μg of the extracted recombinant plasmids pLKO-2E1shRNA1, pLKO-2E1shRNA2, pLKO-2E1shRNA3, and pLKO-2E1shRNAc were respectively mixed with 1 μg of lentiviral packaging plasmids pCMV-VSV-G and 2 μg of pCMV-dR8.91 by using Lipofectamine® 2000. Transfect into 293FT cells, collect virus-containing supernatant medium at 48h and 72h respectively, filter the virus liquid with a 0.45 μm sieve, and use it to transduce L-02 liver cells. The titer of the virus detected by the Lenti-X GoStix kit is 5×10 6 ~5×10 7 IFU.

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Abstract

The invention provides a shRNA lentiviral expression vector for specifically inhibiting hepatic cell CYP2E1 gene expression, comprising basic sequences, resistance gene sequences, multiple cloning site sequences, promoter sequences of PLKO.1-puro expression vector, and shRNA oligonucleotides sequences of target CYP2E1 genes; the shRNA oligonucleotides sequences are inserted into multiple clone sites forwards. shRNA lentiviral expression vector provided by the invention has the advantages of high transfection efficiency, little dosage, sustainability, high efficiency, stability and specificityfor inhibiting hepatic cell CYP2E1 gene expression, and can be used for preparing medicines for treating diseases related to abnormal CYP2E1 gene expression as powerful tools.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an shRNA lentiviral expression vector for specifically inhibiting hepatocyte CYP2E1 gene expression, a construction method and application thereof. Background technique [0002] CYP2E1 is one of the important members of the cytochrome oxidase (cytochrome, CYP) superfamily, accounting for 7% of the total CYP. CYP2E1 has a wide range of metabolic substrates, and there are more than 70 known metabolic substrates, mainly including environmental pollutants such as trichlorethylene, tetrachloroethylene, ethanol, aniline, and nitrous acid compounds. Most of these environmental pollutants are former Carcinogens are transformed into final carcinogenic active substances through CYP2E1 catalyzed metabolism. Many studies have shown that CYP2E1 mediates the liver injury caused by various compounds, such as trichlorethylene, ethanol, chloroform and so on. There are tw...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66A61K48/00C12N5/10
Inventor 徐新云毛吉炎程锦泉彭朝琼
Owner SHENZHEN CENT FOR DISEASE CONTROL & PREVENTION
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