Fusion gene building method for effectively screening GPCR (G Protein-Coupled Receptor) expression

A technology of coupled receptors and fusion genes, applied in the fields of molecular imaging and cell biology, can solve the problems of inconvenient protein research, high cost, and experimental failure, and achieve the effect of increasing protein expression, increasing ratio, and increasing yield

Inactive Publication Date: 2015-12-16
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Western Blot detection is the most commonly used protein expression detection method, but it needs to go through complex operation steps such as cell transfection, harvesting, protein extraction, electrophoresis, membrane transfer, blocking, primary antibody and secondary antibody incubation, and color development. The experimental period is long and unstable. There are many factors, any error in any link will lead to the failure of the experiment, and due to the need for specific antibodies, the cost is high
In addition, because many GPCRs lack specific monoclonal antibodies or antibodies with low specificity, Western Blot that relies on antibody detection is not universal
Therefore, the traditional expression detection method cannot meet the requirements of rapid detection of GPCR expression, causing inconvenience to further protein research.

Method used

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  • Fusion gene building method for effectively screening GPCR (G Protein-Coupled Receptor) expression
  • Fusion gene building method for effectively screening GPCR (G Protein-Coupled Receptor) expression
  • Fusion gene building method for effectively screening GPCR (G Protein-Coupled Receptor) expression

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Experimental program
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Effect test

Embodiment 1

[0048] The fusion gene Rho33-protease-CX3CR1-1D4-protease-eGFP was constructed by overlapping extension PCR method and expressed in HEK293 cells. The steps are as follows:

[0049] 1) Using the existing pCDNA4 / TO-rhodopsin plasmid in the laboratory as a template, use the pcDNA4 / TO plasmid general upstream primer CMV and the synthetic downstream primer Rho(33aa)1 to amplify fragment 1, whose size is about 300bp. The primers used are as follows:

[0050] CMV: 5'-CGCAAATGGGCGGTAGGCGTG-3'

[0051] Rho(33aa) 1:5'- GGGGTTCTCTCAT CTCAGCCAGGTAG-3' ("_" means CX3CR1 fragment)

[0052] 2) Using the existing pcDNA4 / TO-CX3CR1 plasmid in the laboratory as a template, the synthetic upstream primers Rho(33aa)2 and eGFP1 were used to amplify fragment 2 with a size of about 1100bp. The primers used are as follows:

[0053] Rho(33aa)2: 5'-CTACCTGGCTGAG ATGAGAGAACCCC -3' ("_" indicates CX3CR1 fragment)

[0054] eGFP1: 5'- CTTGCTCACCAT GAGAAGGAGC-3' ("_" indicates eGFP fragment)

[005...

Embodiment 2

[0084] The fusion gene Rho33-protease-ADRB3-1D4-protease-eGFP was constructed by one-step gene cloning and expressed in HEK293 cells. The steps are as follows:

[0085] 1) Using the existing pCDNA4 / TO-rhodopsin plasmid in the laboratory as a template, the synthetic upstream primer FP and downstream primer Rho(33aa) 1' were used to amplify fragment 1 with a size of about 150 bp. The primers used are as follows:

[0086] FP: 5'-CTAGCGTTTAAACTTAAGCTT ATGAATGGCACAG -3, ("_" indicates a Rhodopsin fragment)

[0087] Rho(33aa) 1':5'- CCACGGAGCCAT CTCAGCCAGGTAG-3' ("_" indicates ADRB3 fragment)

[0088] 2) Using the existing pcDNA4 / TO-ADRB3 plasmid in the laboratory as a template, the synthetic upstream primer Rho(33aa) 2' and downstream primer RP were used to amplify fragment 2 with a size of about 1200bp. The primers used are as follows:

[0089] Rho(33aa) 2':5'-CTACCTGGCTGAG ATGGCTCCGTGG -3' ("_" indicates ADRB3 fragment)

[0090] RP: 5'- CTTGCTCACCAT AGAAACTCCCAAG-3' (...

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Abstract

The invention relates to the field of molecular imaging and cytobiology, and discloses a fusion gene building method for screening GPCR (G Protein-Coupled Receptor) expression. According to the method, 33 residues at the starting end of human Rhodopsin and corresponding protein endonuclease sites are inserted into the front section of a GPCR; His (5 residues) or ID4 (9 residues) epitopes, corresponding endonuclease sites and eGFP (Enhanced Green Fluorescent Protein) are inserted into the tail end of the GPCR; and Rho33-protease-GPCR-1D4-protease-eGFP is built, wherein the Rho33 can effectively guide GPCR insertion into a membrane, can increase the functional molecule proportion and can improve the protein yield; the 1D4 epitope is used for Western Blot detection and protein purification; the eGFP is a main index for screen authentication; and the endonuclease sites can be used for cutting parts beyond target proteins and maintaining the protein function. The method has the advantages that the function protein proportion can be effectively improved; the protein yield is improved; the influence of fusion protein can be completely eliminated through protein digestion in a later period; and high efficiency, convenience and high speed are realized.

Description

technical field [0001] The present invention relates to the fields of molecular imaging and cell biology, and specifically includes the construction of a novel G protein-coupled receptor, the fusion gene of GProtein-CoupledReceptor (GPCR), which is beneficial to improve the expression of GPCR under the condition of expression of the fusion gene. yield and rapid screening identification. Background technique [0002] GPCRs are a class of membrane proteins with seven transmembrane structures, and they are also the largest family of membrane proteins and cell surface receptors. GPCRs are widely distributed in eukaryotic cells, ranging from yeast to human cells. There are more than 800 GPCRs in the human body, and the response range includes: light, ions, odor molecules, hormones, neurotransmitters and peptides. GPCR is almost involved in various physiological regulation of the human body, and its mutation can cause various diseases. It has long been a hot field and frontier di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/62
Inventor 赵欣孙超
Owner EAST CHINA NORMAL UNIV
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