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Human tumour specific Ki67 gene promotor

A tumor-specific, promoter technology, applied in the field of medical genetic engineering, can solve the problems of unclear biological function and little research, and achieve the effect of good application value, high tumor specificity, and high promoter activity

Inactive Publication Date: 2010-12-22
郑骏年
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In 1991, the Ki67 gene was located on chromosome 10 [18] , but due to its unique primary structure, no homologous sequence has been found in biological databases [19] , so that its biological function is still unclear, and there are few studies on the function of Ki67 gene and the mechanism of gene expression regulation.

Method used

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  • Human tumour specific Ki67 gene promotor
  • Human tumour specific Ki67 gene promotor
  • Human tumour specific Ki67 gene promotor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 Contains the construction of the eukaryotic expression plasmid of human Ki67 gene promoter

[0027] 1. Cloning and sequence identification of the 5' flanking sequence (-820~+771) of the human Ki67 gene

[0028] The 5' flanking sequence of the transcription start point of Ki67 gene was searched in Genbank, and a pair of primers were designed and synthesized to amplify the 1591 fragment upstream of the start codon of Ki67 gene. Design upstream primers in the -820~-805 region, and introduce the Xho I restriction site, design downstream primers in the +758~+771 region, and introduce the Hind III restriction site. The upstream primer is named KU800, the sequence is: 5′-ttgt ctcgagtttttctgtgcttttg-3′, the downstream primer is named KD1, and the sequence is: 5′-tgtg aagctttcgaccccgctcct-3′.

[0029] Using this pair of primers, using the genomic DNA of human kidney cancer Ketr-3 cells as a template, a DNA fragment of about 1.6kb was amplified by PCR, and the targe...

Embodiment 2

[0040] Example 2 Cell culture, plasmid transfection and human Ki67 gene promoter activity analysis

[0041] After each experimental plasmid (eukaryotic expression plasmid of the Ki67 promoter fragment) was successfully constructed, the plasmid was extracted with the PureYieldPlasmid Midiprep System kit, and the positive control plasmid pGL3-Control (containing SV40 promoter / enhancer) and the negative control were extracted at the same time. Plasmid pGL3-Basic (without SV40 promoter / enhancer) and internal reference plasmid pRL-TK (expressing renilla luciferase under the control of HSV-TK promoter). The experimental plasmid and control plasmid were diluted to 100ng / μl, and the internal reference plasmid pRL-TK was diluted to 20ng / μl.

[0042] In the present invention, each experimental plasmid and control plasmid were respectively transfected into human cervical cancer cells (Hela), bladder cancer cells (BIU-87), renal cancer cells (Ketr-3), lung cancer cells (A549) and human um...

Embodiment 3

[0057] Embodiment 3 Ki67-promoter, hTERT promoter, Survivin promoter activity comparison

[0058] In order to further verify the magnitude of Ki67-promoter transcriptional activity, we compared it with the two tumor-specific promoters hTERT and Survivin promoters that have been studied more. Experimental method and steps are the same as in Example 2. The results showed that in all tumor cells, the activity of Ki67-promoter was stronger than that of hTERT and Survivin promoters, and in normal cells, all three promoters were inactive (see Table 6, Figure 12 , 13 ). Ki67-promoter showed better promoter activity.

[0059] Table 6 The activity comparison of Ki67 core promoter, hTERT promoter and Survivin promoter in each cell

[0060]

[0061] b Ratio of relative luciferase activity to pGL3-Control plasmid

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Abstract

The invention discloses a promoter for a human tumor cell proliferation antigen Ki67 protein encoding gene. The invention concretely comprises the cloning, the sequence, the functions and an application of the promoter. The invention discloses a 1591bp sequence which contains the Ki67 gene promoter and is positioned at the positions from -820 to +771 on the 5' flank of the Ki67 gene. A deletion analysis method is used to respectively delete fragment by fragment from the 5' end and the 3' end of the 5' flank of the Ki67 gene to obtain DNA chopped fragments with different lengths. The fragmentsare inserted into the upper stream of a firefly luciferase reporter gene to construct a series of expression plasmids. The plasmids are transfected to four tumor cells and normal cells for transient expression, thereby determining that the Ki67 gene core promoter is positioned at the positions from -223 to +30 of the Ki67 gene. Due to high transcription activity and tumor specificity in the tumorcells, the Ki67 gene core promoter is a very useful tumor specificity promoter. The promoter can be used for adjusting and controlling therapeutic gene expression or for constructing conditional proliferative viruses, thereby realizing the selective killing of the tumor cells. The promoter is widely applied to the tumor targeted therapy.

Description

technical field [0001] The invention belongs to the technical field of medical genetic engineering and relates to cell biology, molecular biology, cancer biology and the like. Specifically, it is the cloning and functional identification of the human tumor-specific Ki67 promoter, which has high transcriptional activity in tumor cells or tissues, so that foreign genes can be specifically expressed in tumor cells or tissues, so it can be widely used Targeted gene therapy for tumors. Background technique [0002] Tumors seriously endanger human health. Although the improvement of chemotherapy, radiotherapy and surgical techniques has greatly improved the effect of tumor treatment, the effect is still unsatisfactory, and the tumor often eventually becomes resistant to chemotherapy and radiotherapy. In recent years, great progress has been made in tumor gene therapy research, which brings hope to human beings to overcome tumors. Clinical gene therapy includes many kinds, such ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C12N15/63A61K48/00A61P35/00
Inventor 郑骏年裴冬生刘晓昀孙方浩韩东李圆李望
Owner 郑骏年
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