Method for replicating adenovirus by way of selection and mutual compensation

A technology of adenovirus and virus, applied in the field of biomedicine, can solve problems such as the influence of human hematopoiesis, and achieve the effect of flexible and diverse treatment and high safety

Inactive Publication Date: 2011-09-21
THE WEST CHINA SECOND UNIV HOSPITAL OF SICHUAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This replication-type adenovirus may replicate in cells such as hematopoietic stem cells, which may affect important functions such as human hematopoiesis

Method used

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  • Method for replicating adenovirus by way of selection and mutual compensation
  • Method for replicating adenovirus by way of selection and mutual compensation
  • Method for replicating adenovirus by way of selection and mutual compensation

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Embodiment 1

[0021] The tumor-specific TERT promoter and E1a gene were isolated by molecular cloning technology.

[0022] The present invention uses PCR technology to amplify the TERT promoter and the E1a gene sequence respectively using human cervical cancer Hela cells and wild-type adenovirus Ad5DNA as templates, inserting them into the shuttle plasmid pAdtrack, and constructing a gene sequence controlled by the TERT promoter to express E1a. pAdtrack-TERTp-E1a plasmid.

[0023] Primer sequences for amplifying the TERT promoter:

[0024] Upstream primer 5'-CAATGATATCTTCCCAGGGCTCCACATCATG-3'

[0025] Downstream primer 5'-ATAGTTTAGCGGCCGCACGCAGCGCTGCCTGAAACTC-3'

[0026] Primer sequences for amplifying the E1a gene:

[0027] Upstream primer 5'-ACCGGGACTGAAAATGAGAC-3'

[0028] Downstream primer 5'-TTAAGCATAATCTGGAACATCATATGGATATGGCCTGGGGCGTTTAC-3'

Embodiment 2

[0030] A replication-defective virus Ad-TERTp-E1aΔE4 with deletion of E4 gene and expression of E1a gene controlled by TERT promoter was constructed.

[0031] The present invention utilizes the AdEasy adenovirus vector system to construct a replication-deficient adenovirus, and the implementation steps are as follows:

[0032] 1. The backbone plasmid pAdEasy-2 (without E4 gene) was electrotransformed into BJ5183 bacteria, screened on an antibiotic plate, the clone was picked out, amplified, the plasmid was extracted, the integrity of pAdEasy-2 was checked by enzyme digestion, and the correct clone was selected as BJ5183- Ad-2 bacteria were prepared as electrocompetent cells.

[0033] 2. The recombinant plasmid pAdtrack-TERT p-E1a was constructed by using the shuttle plasmid pAdtrack (carrying the green fluorescent protein gene), TERT promoter and E1a fragment.

[0034] 3. BJ5183-Ad-2 was electrotransformed with pAdtrack-TERTp-E1a, screened with antibiotic plates, clones were pi...

Embodiment 3

[0039] Infection and Replication of Selective Complementary Replication Adenovirus in Cultured Cells in Vitro (1)

[0040] Currently, the replication-deficient adenoviruses commonly used in gene therapy generally have E1 gene deletion and a therapeutic gene inserted at this site, and this virus carries the E4 gene. Taking the replication-defective adenovirus Ad-GFP with the green fluorescent protein GFP gene as an example, we used our constructed virus Ad-TERTp-E1aΔE4 to co-infect the cultured cells in vitro.

[0041] Inoculate human cervical cancer Hela cells and normal human embryonic lung fibroblasts MRC-5 into six-well cell culture plates, 5×10 per well 5 cells. The viruses were added into Hela and MRC-5 cells according to the following three groups: the group with Ad-TERTp-E1aΔE4 alone; the group with Ad-GFP alone; the group with double viruses Ad-TERTp-E1aΔE4 and Ad-GFP. The amount of added virus was calculated according to 10 MOI of each cell infection, and the amount...

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Abstract

The invention provides a method for replicating adenovirus by means of selection and mutual compensation, belonging to the field of biomedicine and relating to a method for replicating replication-defective adenovirus by means of selection and compensation. The method is characterized in that by mutual compensation, two replication-defective adenoviruses are provided with one early gene which is not contained in the other one, wherein the early gene of one replication-defective adenovirus is controlled by a tumor specific promoter, the other replication-defective adenovirus is provided with the early gene and the tumor therapy gene which are not contained in the former replication-defective adenovirus, and the adenoviruses can be selectively compensated and replicated in tumour cells by simultaneously adopting the two replication-defective adenoviruses. The method can selectively kill the tumour cells, without affecting normal cells. The invention can improve the safety, the targetingand the effectiveness of the operation in which adenoviruses carry target genes to carry out gene therapy on tumors.

Description

technical field [0001] The invention belongs to the field of biomedicine. It involves the establishment of a method for selective and complementary replication of two replication-deficient adenoviruses in tumor cells. Background technique [0002] Malignant tumors are one of the most serious diseases threatening human health and the leading cause of death worldwide. The World Health Organization reported that in 2007, the number of cancer deaths in the world reached 7.9 million (accounting for about 13% of all deaths), and the number of cancer deaths is expected to continue to increase. In 2030, 12 million people will die of cancer. According to the results of the third sample survey on the causes of death of residents released by the Ministry of Health in April 2008, malignant tumors are the first cause of death in my country's urban population (accounting for 25.0% of the total number of deaths), and the second cause of death in rural areas (accounting for 21.0% of the to...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N7/04A61K48/00A61P35/00
Inventor 王和刘珊玲陈新莲谢晓砚
Owner THE WEST CHINA SECOND UNIV HOSPITAL OF SICHUAN
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