Dual-luciferase reporter gene plasmid of 3-UTR region of BCL2 gene as well as construction method and application thereof

A 3-UTR, dual luciferase technology, applied in the fields of biochemical equipment and methods, introduction of foreign genetic material using vectors, determination/inspection of microorganisms, etc.

Inactive Publication Date: 2013-04-24
SHANGHAI UNIV OF TRADITIONAL CHINESE MEDICINE PUTUO DISTRICT CENT HOSPITAL +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The prior art discloses that the dysregulation of apoptosis by the BCL2 oncogene family has been an important pathway in the process of tumorigenesis

Method used

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  • Dual-luciferase reporter gene plasmid of 3-UTR region of BCL2 gene as well as construction method and application thereof
  • Dual-luciferase reporter gene plasmid of 3-UTR region of BCL2 gene as well as construction method and application thereof
  • Dual-luciferase reporter gene plasmid of 3-UTR region of BCL2 gene as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] 2) According to sequence information (NCIBI: NM_000633 ), using the primer 5 software to design the primer sequence of the 3-UTR region of BCL2, and adding the sequence of the restriction site Xba I. Using the total human genome DNA sequence as a template, the amplified size is 2270bp. Cycle conditions: 95°C for 30s, 59.1°C for 30s, 72°C for 120s, a total of 35 cycles, and finally 72°C for 10 minutes.

[0017] Sense: GCTCTAGA TTGGAAATCCGACCACTA

[0018] Antisense: GCTCTAGA TGGCAGGATAGCAGCAC

[0019]

[0020] Obtain the full length of the sequence: (sequence 4)

[0021] 1 GCTCTAGATT GGAAATCCGA CCACTAATTG CCAAGCACCG CTTCGTGTGGCTCCACCTGG

[0022] 61 ATGTTCTGTG CCTGTAAACA TAGATTCGCTTTCCATGTTGTTGGCCGGATCACCATCTGA

[0023] 121 AGAGCAGACG GATGGAAAAA GGACCTGATC ATTGGGGAAG CTGGCTTTCTGGCTGCTGGA

[0024] 181GGCTGGGGAG AAGGTGTTCA TTCACTTGCA TTTCTTTGCC CTGGGGGCTGTGATATTAAC

[0025] 241 AGAGGGAGGG TTCCTGTGGG GGGAAGTCCA TGCCTCCCTG GCCTGAAGAA GAGACTCTTT

[0026] 301 GCATATG...

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Abstract

The invention belongs to the field of biotechnology, and relates to a dual-luciferase reporter gene plasmid of a 3-UTR region of a BCL2 gene as well as a construction method and an application thereof. According to the invention, an Xba I locus of a dual-luciferase report plasmid pGL3-promoter is inserted into a 3-UTR region of an inhibitor of apoptosis gene BCL2, and the dual-luciferase reporter gene plasmid pGL3-p-BCL2-3-UTR containing the BCL2 gene in the 3-UTR region is used for detecting a regulating effect of hsa-mir-1915 to the BCL2 gene, so that the intracellular microRNA regulation is detected. The expression of the intracellular hsa-mir-1915 is detected to mark the specific intracellular microRNA expression level, so that a new research method is provided for the intracellular microRNA detection; the built dual-luciferase reporter gene plasmid in the 3-UTR region of the BCL2 gene can be used for detecting the regulating effect of the intracellular hsa-mir-1915 to the BCL2 gene; and furthermore, the dual-luciferase reporter gene plasmid in the 3-UTR of the BCL2 gene is used for the anticancer research, thereby having an excellent application foreground.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a BCL2 gene 3-UTR dual-luciferase reporter gene plasmid and a construction method thereof. The plasmid is used to detect the regulatory effect of hsa-mir-1915 on the BCL2 gene, and is convenient for the detection of intracellular microRNA regulation , the present invention is particularly applicable to the identification of tumors and the study of microRNA anti-tumor mechanisms. Background technique [0002] According to the "World Cancer Report" of the World Health Organization (WHO), by 2020, the global cancer incidence rate will increase by 50%. At present, there are 10 million new cancer patients every year. Compared with 1990, the incidence of cancer patients in the world has increased by 19%, and the mortality rate has increased by 18%. The prior art discloses that the dysregulation of cell apoptosis by the BCL2 oncogene family has always been an important pathway in the...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/66C12Q1/68
Inventor 范忠泽肖海娟许建华刘建文孙珏崔大苓徐可梁欣李琦殷佩浩高虹赵成根陆海金泉克王国娟余文燕朱晏伟张勇
Owner SHANGHAI UNIV OF TRADITIONAL CHINESE MEDICINE PUTUO DISTRICT CENT HOSPITAL
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