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Proliferation and tumor cell specific gene operating system for gene therapy of malignant tumor

A system-manipulating and specific technology, applied in the field of highly proliferative and specific recombinant eukaryotic cell promoters, can solve the problems of adverse effects on normal stem cells and the inability to guarantee the absolute safety of gene therapy, and achieve the effect of improving transcription efficiency and increasing expression efficiency

Inactive Publication Date: 2010-03-17
GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As mentioned above, although the PCNA gene promoter cannot initiate the expression of exogenous therapeutic genes in normal cells in a non-proliferative state, since all normal stem cells are also in an active state of proliferation, the promoter can be used to initiate the expression of exogenous therapeutic genes. There is still the possibility of adverse effects on these normal stem cells
Therefore, simply solving the proliferation specificity of exogenous therapeutic genes expressed in malignant tumor cells cannot guarantee the absolute safety of gene therapy.

Method used

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  • Proliferation and tumor cell specific gene operating system for gene therapy of malignant tumor

Examples

Experimental program
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Effect test

Embodiment 1

[0036] The method for obtaining the highly proliferative specific eukaryotic promoter PCNATAI: using human genomic DNA as a template, using polymerase chain reaction (PCR) technology to amplify the -778 ~ +61 region of the human wild-type PCNA promoter, and subcloning To the commercial T vector pBS-T of Tiangen Biochemical Technology Co., Ltd., a recombinant plasmid pBS-PCNAP containing the -778~+61 region of the wild-type PCNA promoter was constructed. Digest pBS-PCNAP with restriction endonucleases AatII and KpnI to release the -57~+61 region of the wild-type PCNA promoter. The -57~+61 region of the PCNATAI promoter was synthesized by DNA synthesis technology, the -33~-21 region contained the TATA box structure of the adenovirus E1B gene promoter, the -2~+5 region contained the typical Inr structure, and the rest of the sequence was It is the same as the corresponding sequence of the wild-type PCNA promoter, and at the same time, the recognition sequences of AatII and KpnI a...

Embodiment 2

[0038] The method for obtaining the operating system PCNATAI-LoxP-stop-LoxP: use DNA synthesis technology to synthesize the LoxP-stop-LoxP nucleic acid sequence in the commercialized plasmid pBS302 of Invitrogen Company of the United States, and introduce restriction internals at its 5' end and 3' end respectively Recognition sites of Dicer SpeI and NotI. The recombinant plasmid pBS-PCNATAI containing the highly proliferative specific eukaryotic promoter PCNATAI was linearized with SpeI and NotI, and the LoxP-stop-LoxP nucleic acid sequence digested by SpeI and NotI was inserted into the linearized plasmid to construct a pBS-PCNATAI containing PCNATAI-LoxP The recombinant plasmid pBS-PCNATAI-loxP-stop-loxP of -stop-LoxP was sequenced to confirm the correctness of its sequence.

Embodiment 3

[0040] Qualitative detection of PCNATAI transcription efficiency in different in vitro cultured cells and cell lines

[0041] Using the enhanced green fluorescent protein EGFP gene as a reporter gene, the -300~+61 region (P300), -600~+61 region (P600) and PCNATAI transformed the prototype PCNA promoter -788 ~ +61 region transcription efficiency was observed qualitatively. The PCNA promoter -300~+61 region, -600~+61 region, -788~+61 region and PCNATAI promoter were respectively inserted into the plasmid vector pEGFP-1 (Clontech Company, USA) to construct recombinant plasmids p300-EGFP, p600- EGFP, pPCNA-EGFP and pPCNATAI-EGFP. Four recombinant plasmids were used to transfect primary cultured rat astrocytes, human embryonic kidney immortalized HEK293 cell line, malignant glioma cell line TJ905 and U251 respectively, and observed under a fluorescent inverted microscope after continuing to culture for 72 hours The expression of EGFP. The results showed that the transcription ef...

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Abstract

The invention relates to the field of biotechnology and discloses a high-proliferation specific recombination eucaryotic cell promoter PCNATAI; an eucaryotic cell operating system PCNATAI-LoxP-stop-LoxP taking a PCNATAI as a core transcription initiator and a LoxP-stop-LoxP as a transcriptional repression element; and a set of general scheme for eliminating the repression effect of the LoxP-stop-LoxP to the transcription process of the PCNATAI promoter through a Cre recombinase provided by a tumor cell specific dependent model trans form and enabling the downstream exogenous tumor suppressor gene or the suicide gene to be effectively and specifically expressed in the malignant tumor cell in a tumor cell specific pattern and a proliferation specific pattern. The scheme not only can remarkably improve the tumor inhibition rate during the gene therapy of the malignant tumor or the expression efficiency of the suicide gene in the malignant tumor cell, but also can ensure the security of the gene therapy.

Description

【Technical field】 [0001] The invention belongs to the field of biotechnology, and in particular relates to a highly proliferative specific recombinant eukaryotic cell promoter (PCNATAI); a set of PCNATAI as a promoter for gene transcription is activated by Cre recombinase expressed by a tumor cell specific promoter , a eukaryotic cell manipulation system that activates the high-efficiency and specific expression of exogenous therapeutic genes in malignant tumor cells in a dual-dependent mode of high proliferation specificity and tumor cell specificity; and the corresponding eukaryotic cells constructed with each component of the manipulation system Cell expression plasmid vector, recombinant adenovirus shuttle plasmid vector and recombinant adenovirus expression vector. 【Background technique】 [0002] Malignant tumor is a major disease that seriously endangers human health. It ranks third among the causes of death in the world. Since 2006, malignant tumor has become the lead...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12P19/34C12N15/85A61K48/00A61P35/00
Inventor 于士柱王虔孙翠云安同岭
Owner GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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