Eukaryotic expression vector for expressing shRNA (short hairpin Ribonucleic Acid) in manner of targeting in cancer cells

A technology for eukaryotic expression vectors and tumor cells, applied in the field of eukaryotic expression vectors, can solve problems such as reducing siRNA production, shRNA expression and shearing effects, and achieve the effects of increasing production, solving non-specific interference problems, and increasing expression.

Inactive Publication Date: 2011-03-30
HUNAN NENGRUN MEDICAL DIAGNOSIS TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this kind of shRNA expression structure can specifically express shRNA in tumor cells, because it adopts the structure of polII type promoter + shRNA, it cannot be terminated precisely like U6 and other polIII type promoters, so the expression of shRNA and Cutting will be affected to a certain extent, thereby reducing the yield of siRNA

Method used

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  • Eukaryotic expression vector for expressing shRNA (short hairpin Ribonucleic Acid) in manner of targeting in cancer cells
  • Eukaryotic expression vector for expressing shRNA (short hairpin Ribonucleic Acid) in manner of targeting in cancer cells
  • Eukaryotic expression vector for expressing shRNA (short hairpin Ribonucleic Acid) in manner of targeting in cancer cells

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Experimental program
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Embodiment 1

[0033]Construction of vector pYrbio-hTERT-Mir30shRNA targeting expression of shRNA in tumor cells

[0034] Methods: Using the commercial vector pYrbio-Mir30-shRNA (produced by Changsha Yingrun Biotechnology Co., Ltd., as shown in SEQID2) as the backbone, through a series of transformations, the vector pYrbio-hTERT- Mir30shRNA.

[0035] (1) Synthesize hTERT promoter (about 385bp), and load it on pMD19-T Simple Vector. In the upstream of hTERT promoter fragment, add BglII, EcoRI, PstI restriction site; in the downstream of hTERT promoter fragment, add SpeI restriction site. The constructed vector was named TS-hTERTp.

[0036] (2) PCR amplify the SV40 Enhancer fragment from pCDNA3.1(+), add EcoRI and PstI restriction sites at both ends. Using EcoRI and PstI as subcloning sites, the SV40 Enhancer fragment was subcloned into TS-hTERTp. The constructed vector was named TS-SV40E-hTERTp.

[0037] (3) PCR amplify the CMV Enhancer fragment from pCDNA3.1(+), and add BglII and EcoRI ...

Embodiment 2

[0043] Specific Interfering with the Expression of CD146 Gene in Hepatocellular Carcinoma Cells

[0044] Methods: The shRNA targeting human CD146 gene was inserted into the shRNA Insertion Site of the pYrbio-hTERT-Mir30shRNA vector to construct the pYrbio-hTERT-Mir30-CD146 vector.

[0045] The pYrbio-hTERT-Mir30-CD146 vector was transfected into the liver cancer cell line HepG2, the cells were collected 48 hours later, RNA and protein were extracted, and the target gene CD146 was detected by RT-PCR and Western-blot. HepG2 cells not transfected with any plasmid served as the control group.

[0046] Results: After the constructed shRNA expression vector pYrbio-hTERT-Mir30-CD146 was transfected into HepG2 cells, obvious green fluorescence could be observed under the fluorescence microscope 24 hours later, indicating that the entire plasmid expression cassette was working normally, and the expression level of shRNA was relatively low. high. However, no green fluorescence was see...

Embodiment 3

[0050] Study on the specificity of hTERT promoter

[0051] Methods: The pYrbio-hTERT-Mir30shRNA vector was transfected into liver cancer cell line HepG2 and human normal fibroblast cell line HELF, respectively, and the fluorescence was observed under a fluorescence microscope 24 hours later.

[0052] Results: After the pYrbio-hTERT-Mir30shRNA vector was transfected into the liver cancer cell line HepG2 cells, obvious green fluorescence could be observed under the fluorescence microscope 24 hours later, indicating that the entire plasmid expression cassette was working normally. However, after pYrbio-hTERT-Mir30shRNA was transfected into human normal fibroblast cell line HELF, no green fluorescence was seen. The fluorescence observation photos of HepG2 cells and HELF cells are shown in Figure 4 .

[0053] Conclusion: The pYrbio-hTERT-Mir30shRNA vector constructed by the method provided by the present invention, its hTERTpromoter-EGFP-Mir30shRNA-SV40 PolyA expression cassette...

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Abstract

The invention provides a eukaryotic expression vector for expressing shRNA (short hairpin Ribonucleic Acid) in manner of targeting in cancer cells, comprising the structure as follows: (1) an expression cassette structure which is driven by a polII-type promoter and connects a fluorescent protein gene and a mirshRNA structure in series together; (2) a hTERT (human telomerase reverse transcriptase) promoter enhanced by a CMV (cytomegalovirus) enhancer and a SV40 (simian virus 40) enhancer; (3) mirshRNA structures based on mir30: mir30 left arm-shRNA-mir30 right arm, and multiple mirshRNA structures can be connected in series; (4) a Kan or Amp resistance selection marker; and (5) LR homologous recombination arms. By utilizing the method to construct the shRNA eukaryotic expression vector, one or more than one shRNA can be specifically expressed in the cancer cells in manner of targeting; normal cells are not influenced while the RNA interference and the gene therapy are carried out on the cancer cells, thereby solving the problem of non-specific interference during carrying out the gene therapy by utilizing the RNA interference and being beneficial to the research and the application of the RNA interference in the cancer gene therapy aspect.

Description

technical field [0001] The invention relates to a eukaryotic expression vector capable of expressing single or multiple shRNAs in tumor cells and its construction. Background technique [0002] RNA interference (RNA interference) is a kind of gene silencing induced by double-stranded RNA. During this process, messenger RNA (mRNA), which has a homologous sequence to the double-stranded RNA, is degraded, thereby inhibiting the expression of the gene. RNA interference technology has broad application prospects in probing gene functions and treating human diseases. It is widely believed in the scientific community that by using RNAi, the manipulation of the cell's ribonucleic acid (genetic messenger), to interfere or suppress target genes to prevent the formation of those proteins that cause disease, it is possible to produce promising therapeutic drugs for the treatment of cancer, including , blindness and AIDS, among other diseases. [0003] At present, people have successf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85A61K48/00A61P35/00C12N7/01C12R1/93
Inventor 易银沙吕媛孙永林袁炳秋
Owner HUNAN NENGRUN MEDICAL DIAGNOSIS TECH
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