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Reference internal type dual-luciferase reporter vector and application thereof

A dual-luciferase and luciferase technology, applied in the fields of molecular biology and cell biology, can solve the problems of large differences within batches and between batches, increased material costs and labor, and easy to cause misjudgment. , to reduce the experimental cost, improve the repeatability and reliability, and achieve the effect of a wide range of applications

Inactive Publication Date: 2011-06-15
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, this reporter system needs to be prepared and transfected with two vectors, and its disadvantages are obvious: (1) The steps are more complicated, which virtually increases material costs and labor; (2) Poor repeatability, intra-batch and inter-batch differences Larger, the result is unstable, and it is easy to cause misjudgment

Method used

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  • Reference internal type dual-luciferase reporter vector and application thereof
  • Reference internal type dual-luciferase reporter vector and application thereof
  • Reference internal type dual-luciferase reporter vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Design and synthesis of chimeric primers for cloning firefly luciferase gene

[0082] The sequence of the firefly luciferase gene comes from the reporter vector pGL3-promoter, and it is planned to be cloned between the Renilla luciferase gene and the ampicillin resistance gene on the reporter vector pRL-TK. The chimeric primer sequences used were:

[0083] pF-Fluc (SEQ ID No: 1)

[0084] 5' AAGGATCCAGGTGGCACTTTTCG

[0085] pR-Fluc (SEQ ID No: 2)

[0086] 5' GAAAAATAAACAAATAGGGGTTCCGCGCAC

[0087]

[0088] P1R (SEQ ID No: 3) 5'CGAAAAGTGCCACCTGGATCCTT3'

[0089] All primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd., packaged, transported, and stored in the form of dry powder. The primers were diluted with TE buffer (ph=8.0) to a solution of 10 μmol / L, and stored at -20°C for use.

Embodiment 2

[0090] Example 2 One-step binary bridging coupled long-distance PCR amplification of the linear fusion fragment of the firefly luciferase gene and the reporter vector pRL-TK

[0091] i) Sources of reagents and materials

[0092] High-fidelity DNA polymerase KOD plus and its matching buffer (10×buffer), dNTP, magnesium sulfate MgSO 4 All were purchased from Toyobo Biotechnology Co., Ltd., the product number is KOD-201, and stored at -20°C. The reporter vectors pGL3-promoter and pRL-TK were purchased from Promega Company in the United States, extracted and purified according to the instructions of the small-scale ultra-pure plasmid extraction kit provided by Beijing Tiangen Biotechnology Co., Ltd., and stored at -20°C.

[0093] ii) See Table 1 for system composition.

[0094] Table 1 PCR components and concentration

[0095] Element

The initial concentration

Dosage

Final concentration

PCR buffer

10×

5μl

1×

dNTP

2mmol / L

...

Embodiment 3

[0103] Example 3 DpnI digests PCR product

[0104] Restriction endonuclease DpnI specifically recognizes and cuts the methylated GATC sequence, which was purchased from Fermentas Company in Lithuania, Cat. No. ER1702. The composition of the reaction system is shown in Table 2.

[0105] Table 2 Dpn I enzyme digestion system

[0106] Element

The initial concentration

Dosage

Final concentration

Buffer Tango TM

10×

3μl

1×

Dpn I

10units / μl

1μl

0.33unit / μl

PCR product

26μl

total capacity

30μl

[0107] Add the above ingredients one by one on ice and mix thoroughly.

[0108] Incubate in a 37°C water bath for 2 hours, and place on ice for transformation.

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Abstract

The invention discloses a reference internal type dual-luciferase reporter vector and an application thereof. Firefly luciferase gene is fused between the renilla luciferase gene of the pRL-TK vector and the ampicillin resistance gene, the two luciferases are on the same vector, and the firefly luciferase gene is used as reference. The one-step binary bridging coupling long-distance polymerase chain reaction (PCR) and the Escherichia coli vivo homologous recombination method are utilized to place the firefly luciferase gene and the renilla luciferase gene on the same vector; and monoclonal sites are introduced in the 3' untranslated region of the renilla luciferase gene for conveniently cloning the target segment, thus the reference internal type dual-luciferase reporter vector can be constructed. The vector of the invention has the advantages of high repeatability, little multiple-pore variation, convenient operation and precise quantification. The vector of the invention is suitablefor the screening, identification and confirmation of the miRNA target molecule and also suitable for the quantitative analysis of the activity change of miRNA in cellular level.

Description

technical field [0001] The invention belongs to the fields of molecular biology and cell biology, and relates to a reporter carrier for microRNA (microRNA) target molecule identification and activity analysis. Background technique [0002] MicroRNA (microRNA) is a kind of small molecular single-stranded non-coding RNA (CHIANG, H.R.; SCHOENFELD, L.W.; RUBY, J.G.; AUYEUNG, V.C.; , N.; BAEK, D.; JOHNSTON, W.K.; RUSS, C.; LUO, S.; BABIARZ, J.E.; BLELLOCH, R.; experimental evaluation of novelland previously annotated genes. Genes Dev, vol. no. p. doi: gad.1884710 [pii] 10.1101 / gad.1884710.). They widely exist in eukaryotic organisms and participate in the regulation of various physiological and pathological pathways such as cell differentiation, proliferation, and apoptosis during the development of organisms (SONG, G.; ZHANG, Y. and WANG, L. (2009). MICRORNA- 206 targets NOTCH3, activates apoptosis, inhibits tumor cell migration and foci formation. J Biol Chem, vol. no. p. doi...

Claims

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Application Information

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IPC IPC(8): C12N15/65C12N15/52C12Q1/68
Inventor 毕延震郑新民邵长伟潘文姜黎欧阳辉伍乔宪凤刘西梅周荆荣华文君李莉肖红卫张立萍华再东魏庆信
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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