Recombinant replication-defective adenovirus for expressing hTERT (human telomerase reverse transcriptase) gene and application thereof

A recombinant adenovirus, adenovirus technology, applied in the field of recombinant adenovirus, can solve problems such as unclear, malignant diseases that cannot be completely cured, etc.

Inactive Publication Date: 2014-07-30
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main problem facing this kind of malignant disease is that it cannot be completely cured
At present, the impact of changes in hTERT gene expression on intracellular signaling pathways and gene expression is not clear

Method used

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  • Recombinant replication-defective adenovirus for expressing hTERT (human telomerase reverse transcriptase) gene and application thereof
  • Recombinant replication-defective adenovirus for expressing hTERT (human telomerase reverse transcriptase) gene and application thereof
  • Recombinant replication-defective adenovirus for expressing hTERT (human telomerase reverse transcriptase) gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 The recombinant plasmid pDC318-hTERT carrying hTERT gene was constructed.

[0024] Take the plasmid pIRES2-EGFP-hTERT previously constructed in our laboratory, cut it with the endonuclease EcoR I, and recover the 4015bp band (hTERT gene). Take the pSG218 vector, cut it with endonuclease EcoR I, and recover the band at 3926bp. The products recovered from the above two enzyme digestions were ligated at 12°C for 12 hours. Then the ligation product was transformed into DH5α Escherichia coli competent cells, spread on an agar plate (containing 100ug / ml AMP) and cultured for 12 hours, picked clones, and amplified on a shaker at 37°C for 12 hours. The plasmid DNA of positive clones was extracted, and identified by restriction enzyme digestion with BamH I, Kpn I + Sac I, Pvu II, and Pst I respectively, and the results were as follows: figure 1As shown (M1 is ΛDNA / EcoRI+HindIII marker, M2 is 100bp DNALadder, lane 1 is the 1519bp + 6422bp fragment obtained by BamH ...

Embodiment 2

[0025] Example 2 Liposome-mediated recombinant plasmid pDC318-hTERT and adenovirus backbone vector pPE3-F11B were co-transfected into human embryonic kidney 293 cells for virus packaging, identification, amplification and titer determination of the recombinant virus.

[0026] 1. Liposome-mediated recombinant plasmid pDC318-hTERT and adenovirus backbone vector pPE3-F11B were co-transfected into human embryonic kidney 293 cells for virus packaging

[0027] The day before transfection, inoculate 7.5 × 10 5 Personal embryonic kidney 293 cells, placed at 37°C, 5% CO 2 Cultured in a cell culture incubator. Change the medium 3-4 hours before transfection, and add 5ml of serum-free medium to ensure the exponential growth of cells. Prepare to transfect the recombinant plasmid pDC318-hTERT obtained in Example 1, the viral backbone vector plasmid pPE3-F11B and liposome Lipofectamine 2000 (purchased from Invitrogen) to transfect 293 cells (specific steps omitted), and the virus appear...

Embodiment 3

[0035] Example 3 The transfection efficiency and expression of the recombinant adenovirus VDC318-hTERT transfected into the target cells in vitro were detected.

[0036] Taking the human multiple myeloma cell line RPMI-8226 as an example, the expression and identification of hTERT gene after the recombinant adenovirus VDC318-hTERT of the present invention was transfected into target cells in vitro was detected, and the specific detection method was as follows:

[0037] 1. Culture of human multiple myeloma cell line RPMI-8226

[0038] Take out the frozen RPMI-8226 cell line from the liquid nitrogen tank, melt it in a 37°C water bath, add RPMI-1640 medium containing 10% FBS, and place it at 37°C, 5% CO 2 Cultured in an incubator for 6 hours, centrifuged at 1000rpm for 5 minutes, and replaced with RPMI-1640 medium containing 10% FBS to remove antifreeze DMSO. Placed at 37°C, 5% CO 2 Cell culture incubator, subculture every 1-2 days or change the medium once in half. Cells in...

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Abstract

The invention provides a recombinant replication-defective adenovirus for expressing an hTERT (human telomerase reverse transcriptase) gene. A target gene hTERT is obtained by virtue of enzyme digestion from a plasmid pIRES2-EGFP(enhanced green fluorescent protein)-hTERT, the target gene hTERT is inserted to downstream of a cytomegalovirus promoter in a pSG218 shuttle vector to obtain a recombinant shuttle vector with the hTERT gene; and the recombinant shuttle vector and a virus framework vector pPE3-F11B are co-transferred to a human embryo kidney 293 cell to obtain replication-defective adenovirus with hTERT. The recombinant adenovirus infects a human multiple myeloma cell, so that the cell excessively expresses the hTERT gene, and therefore, drug resistance of the cell on pharmorubicin is strengthened. The recombinant replication-defective adenovirus disclosed by the invention is mainly used for establishing a multiple myeloma cell strain model for excessively expressing hTERT gene, and further researching a mechanism that the hTERT gene participates in drug resistance of the multiple myeloma cell based on the model.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant adenovirus expressing hTERT gene and application thereof. Background technique [0002] Telomere (telomere) is a special cap-shaped structure at the end of eukaryotic chromosomes. It shortens with each chromosome division. When its length reaches a critical state, it induces cell aging and death. The main function of human telomerase is to use its own RNA subunit (TERC) as a template, use its reverse transcriptase subunit (hTERT) to reverse transcribe and synthesize a telomere repeat sequence (TTAGGG) and add it to the end of the chromosome, thereby maintaining Stabilization of telomere length. hTERT is the main rate-limiting enzyme of telomerase activity, which is activated in most malignant tumor cells, making the cells immortal; in addition, the activation of hTERT can also regulate the proliferation and apoptosis of tumor cells through various ways , E...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/861
Inventor 张越峰金洁
Owner ZHEJIANG UNIV
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