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XRCC2 promoter, carrier containing XRCC2 promoter, construction and application of carrier

A promoter and carrier technology, applied in the field of genetic engineering, can solve the problems of insufficient activity difference, limitation, and low tumor targeting

Inactive Publication Date: 2015-10-21
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the insufficient difference in activity between tumor cells and normal cells, the tumor targeting is not high, or the promoter fragment is too long, which makes it difficult to use traditional methods to transport it into tumor cells at a high level, which greatly limits the discovery. The possibility of applying the promoter with high activity in tumor cells to tumor gene therapy

Method used

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  • XRCC2 promoter, carrier containing XRCC2 promoter, construction and application of carrier
  • XRCC2 promoter, carrier containing XRCC2 promoter, construction and application of carrier
  • XRCC2 promoter, carrier containing XRCC2 promoter, construction and application of carrier

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Real Time RT-PCR technology to detect the expression level of XRCC2mRNA in different cells

[0047] (1) Extraction of total cellular RNA

[0048] After the cells were collected by centrifugation, 1ml Trizol reagent was added to fully lyse them. Add 200 μl of chloroform and shake vigorously for 30 seconds, place it in an ice bath for 2-3 minutes, centrifuge at 12000 rpm at 4°C for 5 minutes. The solution is layered, carefully pipette the quantitative upper layer liquid into another RNase-free centrifuge tube, add an equal volume of isopropanol, shake slightly to mix, and place it in an ice bath for 10 minutes at 4°C. Centrifuge at 12000rpm for 5 minutes. The supernatant was discarded, and the pellet was washed twice with 75% ethanol. After washing, discard the ethanol solution and evaporate to dryness. Dissolve the RNA with an appropriate amount of DEPC water, and dissolve it in a water bath at 65°C for 15 minutes. The purity and concentration of the extracted RNA w...

Embodiment 2

[0057] vector cloning

[0058] (1) Construction of pXRCC2-GFP plasmid

[0059] Genomic DNA of MJT cells was used as a template, XRCC2 5' and XRCC2 3' were upstream and downstream primers respectively, and the XRCC2 promoter (pXRCC2) sequence containing EcoRI at the 5' end and AgeI restriction site at the 3' end was amplified by PCR. The pRad51C-GFP plasmid and the amplified pXRCC2 fragment were digested with restriction endonucleases EcoRI and AgeI, the gel was run to identify and the corresponding fragments were recovered, and then the ligation reaction was carried out to construct the target plasmid.

[0060] (2) Construction of pXRCC2-Luciferase plasmid

[0061] Using the pXRCC2-GFP plasmid as a template, the upstream and downstream primers XRCC2-Luciferase 5' and XRCC2-Luciferase 3' were used to PCR amplify pXRCC2 containing NheI and HindIII restriction sites at both ends, respectively. The pXRCC2 fragment and pGL3-basic plasmid were digested with NheI and HindIII. Afte...

Embodiment 3

[0071] plasmid transfection

[0072] (1) Transfection of pXRCC2-GFP plasmid

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Abstract

The invention relates to an XRCC2 promoter, a carrier containing the XRCC2 promoter, the construction and application of the carrier. A sequence of the XRCC2 promoter is shown in SEQ ID NO.1, and the carrier containing the XRCC2 promoter comprises pXRCC2-GFP, pXRCC2-Luciferase, pXRCC2-DTA, pXRCC2-other toxalbumin, AAV-pXRCC2-Luciferase and Fugw-pXRCC2-Luciferase. The carrier containing the XRCC2 promoter is applied to the preparation of tumor therapeutic drugs or tumor diagnostic reagents. Compared with other existing tumor specific promoters, the XRCC2 promoter has the obvious advantages of large activity difference, small fragment length, easiness in construction of virus vectors and the like in normal tumor cells, and therefore, the XRCC2 promoter has enormous potential in the application to oncogene treatment and diagnosis.

Description

technical field [0001] The invention relates to an XRCC2 promoter, a carrier containing the XRCC2 promoter, construction and application of the carrier, and belongs to the technical field of genetic engineering. Background technique [0002] Tumor is one of the diseases that seriously endanger human health and life, and its mortality rate ranks first among all kinds of diseases. Therefore, in addition to applying traditional surgery, radiotherapy, and chemotherapy to treat tumors, it is imperative to continuously develop new tumor treatment methods to alleviate the huge threat that tumors pose to human health. With the continuous development of pathology and molecular biology and other disciplines, people gradually realize that tumor is a "genetic disease" involving multiple factors, multiple steps and multiple genes. Inactivation is considered to be an important factor in tumorigenesis. Therefore, gene therapy based on changing human genetic material is an essential treat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/66A61K48/00A61P35/00C12Q1/68C12Q1/66C12Q1/02
Inventor 张文俊李珍张蕾郭文煊蒋颖毛志勇
Owner TONGJI UNIV