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Combined deletion 19 Kda, 55KDA, E1B code sequence recombination adenovirus construction body and its use

A technology of recombinant viruses and constructs, applied in the field of medical genetic engineering, can solve problems such as hindering the maximum therapeutic effect of tumor cells

Inactive Publication Date: 2005-01-12
深圳市天达康基因工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The 19kDa E1B protein is equivalent to the human anti-apoptotic gene bcl-2, which has a significant anti-apoptotic effect and hinders the maximum therapeutic effect of killing tumor cells, which has become a deficiency of existing methods

Method used

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  • Combined deletion 19 Kda, 55KDA, E1B code sequence recombination adenovirus construction body and its use
  • Combined deletion 19 Kda, 55KDA, E1B code sequence recombination adenovirus construction body and its use
  • Combined deletion 19 Kda, 55KDA, E1B code sequence recombination adenovirus construction body and its use

Examples

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example 2

[0029] Example 2: Construction of recombinant adenovirus constructs with deletion of the entire coding sequence of 19kDa E1B (1713-2243nt) and E1B 55kDa (2018-3328nt) coding sequence.

[0030] pBHGE3 was purchased from Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number: PD-01-12). This plasmid contains the entire genome sequence of human type 5 adenovirus except the packaging signal (194-358nt). See the attached Figure 3. The 293 cell line is a human embryonic kidney cell transformed with sheared human adenovirus type 5 (AdV5) DNA to obtain a permanent passage cell line, purchased from ATCC (American typical culture collection, ATCC, USA, catalog number: ATCC CRL- 1573, published in 1972). At the time of acquisition, the number of passages of cells was 32. 293 cells were adherent cells, low triploid, 30% of the cells had 64 chromosomes, and 4.2% of the cells had higher ploidy. Co-transfect 293 cells with pBHGE3 and pCX1-E1B, and homologous recombination occur...

example 3

[0031] Example 3: In vivo and in vitro experiments of recombinant adenovirus constructs selectively killing tumor cells.

[0032] The following cell lines were selected as experimental subjects:

[0033] U-2OS (ATCC, HTB96) P53+, RB-human osteoblastoma cell line;

[0034] HS700T (ATCC, HTB147) P53-, RB-, human metastatic adenoma cell line;

[0035] DLD-1 (ATCC, CCL221) P53-, RB-, human colon cancer cell line;

[0036] IMR90 (ATCC, CCL186) P653+, RB+, human lung fibroblast cell line;

[0037] 293 (ATCC, CRL1573) P53+, RB+, a human embryonic kidney cell line, is a transgenic cell of adenovirus.

[0038] Cells were incubated in 10% FBS DMEM, 37°C, 5% CO 2 cultured in a 6-well plate, 5×10 per well 5 cell.

[0039] Adenoviruses used in the experiment: wild-type human adenovirus 5; recombinant ADV / dE1B adenovirus; recombinant E1 and E3-deleted recombinant adenoviruses were used as controls.

[0040] The adenovirus infection index (MOI) used for adenovirus transfection is 10. ...

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Abstract

A recombinant adenovirus configurator deleted 19kDa E1B (1713-2243nt) and E1B 55KDa (2018-3328 nt) sequence in human adenovirus type 5 genom and its application to diagnosing and treating tumor are disclosed. The PCR amplification, enzyme severing, linking, subcloning, transfection, and single-cloning purification of recombinant adenovirus technologies, and the deleted 19kDa E1B coded area (1713-2243 nt) and E1B 55kDa (2018-3328 nt) sequence are used to screen the said comfigurator. It can selectively kill tumor cells, but not normal cells.

Description

1. Technical field [0001] The present invention relates to a construction scheme for point-directed deletion (delete) of 19kDa E1B and 55kDa E1B coding sequences in the genome of human adenovirus type 5 (Human adenovirus type 5), as well as the adenovirus with tumor treatment and diagnosis functions obtained through the scheme. The virus construct belongs to the technical field of medical genetic engineering. 2. Background technology [0002] The mortality rate of malignant tumors has become the second leading cause of death after cardiovascular and cerebrovascular diseases in China, and the whole society has a great demand for the diagnosis and treatment of malignant tumors. At present, tumor chemotherapy, radiotherapy, and surgery, which are dominant in clinical treatment, have formed a relatively mature treatment system after years of development and improvement, and have achieved good clinical efficacy. However, these classic treatment methods were established and matur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61P35/00C12N7/01C12N15/31C12N15/86C12N15/861C12Q1/70G01N33/574
Inventor 周剑锋马丁卢运萍王世宣徐钢王世安李震中
Owner 深圳市天达康基因工程有限公司
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