Luciferase complementary image detection method and carrier kit for detecting protein interaction

A luciferase and image detection technology, applied in the field of protein research, can solve problems such as gateways that have not yet been seen

Inactive Publication Date: 2014-06-11
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no report on the combination of gateway technology and LCI technology

Method used

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  • Luciferase complementary image detection method and carrier kit for detecting protein interaction
  • Luciferase complementary image detection method and carrier kit for detecting protein interaction
  • Luciferase complementary image detection method and carrier kit for detecting protein interaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Example 1. Construction of LCI technology Gateway vector: (Nluc is located at the C-terminus of the recombination site)

[0105] 1.1.pNG ( figure 1 ) build

[0106] Step 1. Digest pUC-Nluc (i.e. 35S::Nluc) with restriction enzyme KpnI-SalI ( image 3 ), excise the multiple cloning site sequence, and smooth the end of the obtained fragment to facilitate insertion of the RFC2.1 recombinant fragment.

[0107] Step 2. Use T4DNApolymerase (NEB) to simultaneously cut the 3'-overhanging end of the verified fragment in step 1 (KpnI forms the end), and fill in the 5'-overhanging end (SalI forms the end); then use alkaline Phosphatase dephosphorylates its terminus to prevent self-ligation in the ligation reaction to obtain pUC-Nluc-KS.

[0108] Step 3. Cloning of RFC2.1 recombination site into pUC-Nluc-KS: Ligate pUC-Nluc-KS with RFC2.1 fragment (see Invitrogen), and transform the ligation product into One ccdB Survival TM 2T1 R Competent cells (Invitrogen, resistant to cc...

Embodiment 2

[0121] Example 2. The LCI technology Gateway vector is used to detect the interaction between protein genes AtSGT1a and AtRAR1 to construct a fusion vector and introduce it into Agrobacterium GV3101 or GV2260 (both are acceptable).

[0122] Objective: To detect the interaction between AtSGT1a and AtRAR1, and use the interaction between AtSGT1a and AtR77 as a negative control. AtR77 comprises only the N-terminal 77 amino acids of AtRAR1.

[0123] The original LCI expression vectors pCAMBIA-Nluc and pCAMBIA-Cluc were used as controls to verify the applicability of pNG and pCG expression vectors in LCI technology.

[0124] Step 1. Construct the entry vector of the target gene (see invitrogen Catalog nos.12535-019 manual for details)

[0125] The construction process of the entry vector of AtSGT1a gene, AtRAR1 gene and AtR77 gene:

[0126] (1) PCR amplification of target gene with attB recombination site

[0127] By using primers with attB recombination sites to amplify the gen...

Embodiment 3

[0180] Example 3. Operational steps for high-throughput screening of protein interactions.

[0181] Step 1. cDNA library construction (see Invitrogen Ca.no.18248-013 "Super Plasmid System with Technology for cDNA Synthesis and Cloning"):

[0182] (In the pNG and pCG vectors, there are BamHI and SalI restriction sites adjacent to the attR1 and attR2 recombination sites, and there are no restriction sites outside attR1-attR2 on the vector, so the BamHI and SalI restriction sites are suitable as To construct the linker of cDNA library, see Figure 16 .

[0183] Specific steps are as follows:

[0184] Extract the mRNA of the sample; synthesize the first strand of cDNA; use poly(T)+BamHI digestion recognition site (ggatcc) as the reverse transcription primer;

[0185] Synthesize the second strand of cDNA; add SalI linker (gtcgac); obtain double-stranded cDNA with BamHI and SalI restriction sites at both ends.

[0186] BamHI-SalI digestion of double-stranded cDNA, and vector...

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Abstract

The invention discloses a luciferase complementary image detection method and a carrier kit for detecting protein interaction, belonging to a technology of protein research. The method is characterized in that a gateway technique is introduced into the luciferase complementary image detection method, thus a gateway expression carrier, a kit and an optimized protein interaction detection method for luciferase complementary image detection can be obtained, and the interaction of protein can be effectively and rapidly detected with high throughput.

Description

technical field [0001] The invention relates to protein research technology, in particular to a luciferase complementary image detection method and a carrier kit for detecting protein interaction. Background technique [0002] Currently, detection technologies for protein interaction include Yeast two hybrid (Y2H), fluorescence resonance energy transfer (FRET), chemiluminescence resonance energy transfer (BRET) and bimolecular fluorescence complementary technology (BiFC), etc. Wait. [0003] The yeast two-hybrid system (Y2H) uses hybrid genes to detect protein-protein interactions by activating the expression of reporter genes. Fluorescence resonance energy transfer (FRET) is an energy transfer phenomenon between two fluorescent molecules that are very close together. When the emission spectrum of the donor fluorescent molecule overlaps with the absorption spectrum of the acceptor fluorescent molecule, and the distance between the two molecules is within Within the range o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/84C12Q1/66C40B30/04
Inventor 陈华民周俭民何晨阳田芳
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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