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 intra-batch and inter-batch differences, increased material costs and labor, and easy misjudgment. , to achieve the effect of reducing the cost of experiments, improving repeatability and reliability, and having a wide range of applications

Inactive Publication Date: 2013-03-20
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, which is planned to be cloned between the Renilla luciferase gene and the ampicillin resistance gene on the reporter vector pRL-TK. The chimera primer sequence used is:

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

Embodiment 2

[0090] Example 2 One-step binary bridge coupling 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 article number is KOD-201, and stored at -20°C. Reporting vectors pGL3-promoter and pRL-TK were purchased from Promega, USA, and were extracted and purified according to the instructions of the small extraction medium-volume ultrapure plasmid extraction kit provided by Beijing Tiangen Biotechnology Co., Ltd., and frozen at -20°C.

[0093] ii) The system composition is shown in Table 1.

[0094] Table 1 PCR components and concentration

[0095] Ingredients

The initial concentration

Dosage

Final concentration

PCR buffer

10×

5μl

1×

dNTP

2mmol / L

5μl

200μmol / L

Magnesium sulfate MgSO4...

Embodiment 3

[0104] Example 3 DpnI digests PCR products

[0105] The restriction endonuclease DpnI specifically recognizes and cleaves the methylated GATC sequence, purchased from Fermentas, Lithuania, catalog number ER1702. The composition of the reaction system is shown in Table 2.

[0106] Table 2 Dpn I digestion system

[0107] Ingredients

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

[0108] Add the above ingredients one by one on ice and mix well.

[0109] Incubate in a 37°C water bath for 2h, and place on ice after completion 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 suitable for 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 field of molecular biology and cell biology, and relates to a report carrier for microRNA (microRNA) target molecule identification and activity analysis. Background technique [0002] MicroRNA (microRNA) is a type of small single-stranded non-coding RNA (CHIANG, HR; SCHOENFELD, LW; RUBY, JG; AUYEUNG, VC; SPIES) that is about 19-25 nt in length and can negatively regulate the expression of target mRNA after transcription. , N.; BAEK, D.; JOHNSTON, WK; RUSS, C.; LUO, S.; BABIARZ, JE; BLELLOCH, R.; SCHROTH, GP; NUSBAUM, C. andBARTEL, DP(2010). Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. Genes Dev, vol.no.p.doi: gad.1884710[pii]10.1101 / gad.1884710.). They are widely present 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.; ZHAN...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/65C12N15/52C12Q1/68
Inventor 毕延震郑新民邵长伟潘雯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苹华再东魏庆信
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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