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G protein-coupled receptor (GPCR) in-vivo tracking method and application

A technology for coupling receptors and G proteins, applied in chemical instruments and methods, biochemical equipment and methods, preparations for in vivo experiments, etc., can solve the problems of G protein transduction function and phosphorylation efficiency reduction, gap, etc.

Active Publication Date: 2015-11-04
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the disturbance of the C-terminus of rhodopsin, compared with wild-type rhodopsin, rhodopsin CT - The G protein transduction function and phosphorylation efficiency of GFP are greatly reduced, and there is a huge gap with wild-type rhodopsin
Apparently, rhodopsin CT -GFP is not an ideal fusion expression strategy

Method used

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  • G protein-coupled receptor (GPCR) in-vivo tracking method and application
  • G protein-coupled receptor (GPCR) in-vivo tracking method and application
  • G protein-coupled receptor (GPCR) in-vivo tracking method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of rhodopsin K66M mutant by segmented PCR method

[0032] The purpose of constructing the rhodopsinK66M mutant is twofold, one is to detect whether K66 of rhodopsin is a key residue and whether it can be inserted into eGFP; the other is to introduce a unique Nde I restriction site in rhodopsin to facilitate the insertion of eGFP . The present invention utilizes segmental PCR method to construct rhodopsinK66M mutant, main steps are as follows:

[0033] 1) Using the existing pcDNA4 / TO-rhodopsin plasmid in the laboratory as a template, use the pcDNA4 / TO plasmid general upstream primer CMV and downstream primer Rho(CL1)2 (including the mutation site) to amplify Fragment 1 with a size of about 390bp . The primers used are as follows:

[0034] CMV: 5'-CGCAAATGGGCGGTAGGCGTG-3'

[0035] Rho(CL1)2: 5'CGTGCGCAGCTT CATATG CTGGACGGT-3' (Nde I)

[0036] 2) Using the existing pcDNA4 / TO-rhodopsin plasmid in the laboratory as a template, use the pcDNA4 / TO...

Embodiment 2

[0055] Example 2: rhodopsin CL1 -eGFP gene construct

[0056] rhodopsin CL1 - The idea of ​​eGFP gene construction is to insert the eGFP gene into pCEP4-rhodopsinK66M to obtain pCEP4-rhodopsin CL1 -eGFP (see figure 2 ).

[0057] Specific steps are as follows:

[0058] 1) Replace the rhodopsinK66M gene fragment from the pCEP4 plasmid to the pBAD24 plasmid: first, pCEP4-rhodopsinK66M and pBAD24 plasmids were digested with BamH I and Hind III, and then the rhodopsinK66M fragment was ligated with the double digested pBAD24 plasmid with T4 ligase Up, the pBAD24-rhodopsinK66M plasmid was obtained.

[0059] 2) Use primers eGFP1 and eGFP2 to amplify the eGFP fragment (about 720bp) with NdeI restriction sites at both ends from the pcDNA3.1-3'-eGFP plasmid, and recover the PCR product from the gel.

[0060] Upstream primer eGFP1: 5'-GGAATTC CATATG GTGAGCAAGGGCGAGGAG-3' (Nde I)

[0061] Downstream primer eGFP2: 5'-CCCTTAAG CATATG CTTGTACAGCTCGTCCAT-3' (Nde I)

[0062] 3) The...

Embodiment 3

[0076] Example 3: pCEP4-rhodopsin CL1 -Expression detection of eGFP in HEK293S cells

[0077] pCEP4-rhodopsin CL1 - The detection of eGFP expression in HEK293S cells mainly includes two aspects, one is to check rhodopsin through Western Blot results CL1 -Expression level and glycosylation processing of eGFP (reference Figure 5 ), the second is to detect rhodopsin by fluorescence microscopy CL1 -Whether eGFP can normally emit green fluorescence and whether it accumulates in cells, etc. (refer to Figure 6 ). The specific operation steps are as follows:

[0078] 1) Plasmid preparation: Prepare the plasmid with a plasmid extraction kit, and select a plasmid concentration of not less than 200ng / μl, A 260 / A 280 High-purity pCEP4-rhodopsin between 1.9 and 2.0 CL1 - eGFP plasmid. The same method was used to prepare pCEP4-rhodopsinWT and pCEP4-rhodopsinK66M plasmids as controls.

[0079] 2) Cell preparation: select HEK293S that has been passaged at least twice after thawin...

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Abstract

The invention relates to the field of molecular Imaging, in particular to a G protein-coupled receptor (GPCR) in-vivo tracking method. The GPCR in-vivo tracking method is characterized by inserting a molecular enhanced green fluorescent protein (eGFP) to a non-functional region of the GPCR to construct a novel GPCR-eGFP fusion protein for imaging and tracking of GPCR molecules. In the GPCR in-vivo tracking method, once mutating, rhodopsin which is a photoreceptor protein existing on retinal rod cell membrane of vertebrate widely may cause failure of protein membrane insertion, misfolding and even aggregation and the like to further cause severe eye diseases; therefore, construction of the rhodopsin-eGFP fusion protein and detection distribution of rhodopsin in cells by means of fluorescence are great significant in research on eye diseases. As the molecular eGFP is inserted to the non-functional region, namely cytoplasmic loop 1 (CL1), of rhodopsin originally, the novel rhodopsin CL1-eGFP fusion protein, which is constructed by the method, is of expression level and function similar to those of wild-type proteins, and is an effective tool for research on eye diseases related to rhodopsin.

Description

technical field [0001] The present invention relates to the field of molecular imaging, specifically a novel G protein-coupled receptor (G Protein-Coupled Receptor, GPCR) tracing method in vivo. Taking human rhodopsin as an example, it is specifically described in its non-functional region, that is, the first A molecule of eGFP was inserted into an intracellular loop (Cytoplasmic Loop 1, CL1), and a new type of rhodopsin was successfully constructed CL1 -eGFP fusion protein, the intracellular distribution of rhodopsin is detected by fluorescence. Background technique [0002] G Protein-Coupled Receptor (GPCR) is an important protein that exists on the cell membrane and participates in cell signal transduction, and participates in a variety of important physiological processes, such as synaptic transmission, cell proliferation and differentiation, and Taste and touch perception etc. GPCRs are composed of seven transmembrane helices, which are connected by three intracellula...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02C07K19/00C12N15/85C12N15/65A61K49/00
Inventor 赵欣姜彩虹
Owner EAST CHINA NORMAL UNIV
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