CN105018564B - A kind of G protein-coupled receptor living body tracing method and application - Google Patents

Abstract

The present invention relates to molecular imaging field, specifically a kind of g protein coupled receptor GPCR vivo tracking method, insert a molecules enhancing green fluorescent protein eGFP in the nonfunctional area of GPCR, construct a kind of new GPCR eGFP fusion protein, the imaging for GPCR molecule and tracking.Taking people's rhodopsin rhodopsin as a example, it is the light receptor albumen being widely present on vertebrate retina rod cell film to the present invention, and its mutation can cause albumen can not insert film, false folding is even assembled etc., causes serious ocular disease.Therefore, build rhodopsin eGFP fusion protein and detect that by fluorescence rhodopsin distribution situation in the cell has important value for oculopathy research.The present invention is that first intracellular ring CL1 inserts a molecule eGFP in the nonfunctional area of rhodopsin first, it is demonstrated experimentally that the new rhodopsin that the present invention builds^CL1EGFP fusion protein has the expression similar to wild-type protein and function, is the powerful of rhodopsin associated ophthalmopathy research.

Description

A kind of G protein-coupled receptor living body tracing method and application

technical field

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, namely the first A molecule of eGFP was inserted into Cytoplasmic Loop 1 (CL1), and a new type of rhodopsin ^CL1 -eGFP fusion protein was successfully constructed, and the intracellular distribution of rhodopsin was detected by fluorescence.

Background technique

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 intracellular loops and three extracellular loops. The N-terminal is generally closely related to the insertion of proteins, while the C-terminal is generally a phosphorylation site and Participates in the activation of downstream G proteins and therefore plays an important role. Due to the important role of GPCRs, GPCR mutations can lead to the occurrence of various diseases. Since GPCR is a eukaryotic protein, it generally undergoes complex glycosylation processing and folding processes. Only proteins that are finally correctly folded can undergo a complete process from the endoplasmic reticulum to the Golgi apparatus, and finally successfully inserted into the cell membrane. When the GPCR is mutated, the conformation of the protein changes in mild cases, and in severe cases, it can cause misfolding of the protein, resulting in the incorrect insertion of the protein into the membrane, and even aggregation in the cell, resulting in serious consequences such as cell death. Therefore, the molecular imaging of GPCR is of great value. It can reflect rich information such as whether GPCR is correctly inserted into the membrane, the level of expression, and whether it is aggregated, which is of great significance for the study of GPCR-related diseases.

Traditional molecular imaging methods include tissue staining or immunostaining, immunofluorescence and other methods. That is, specific dyes or fluorescently labeled specific antibodies are used to bind target molecules, and finally the distribution of target molecules is observed by means of color reaction or fluorescence detection. However, the above method is only applicable to fixed tissues and cells, and cannot be applied to living cells or tissues. The emergence of fluorescent proteins has greatly promoted the development of molecular in vivo tracers. People express fluorescent proteins and target proteins in fusion, and finally directly observe the distribution of target proteins in living cells or tissues through fluorescence. Green Fluorescent Protein (GFP) has the advantages of high fluorescence intensity, few interfering factors, and stable luminescence, and is currently a widely used fluorescent protein. Therefore, GPCR-GFP fusion protein is the preferred strategy for in vivo tracking of GPCR molecules.

Rhodopsin (rhodopsin) is a photoreceptor protein widely present on the cell membrane of vertebrate retinal rod cells. It is composed of a molecule of ligand retinal (retinal) and a molecule of opsin (opsin), which belongs to GPCR Class A family. After being stimulated by light, retinal isomerizes, prompting a series of conformational changes of rhodopsin protein, and activating downstream signal transduction, which then generates a signal that is transmitted to the optic nerve, eventually leading to the occurrence of vision. Rhodopsin has a total of 348 residues, but mutations in more than 100 sites are closely related to the occurrence of congenital eye diseases, such as retinitis pigmentosa and night blindness. Except for a few mutant rhodopsins that have been deeply studied, Most of the rhodopsin mutants are still blank. Therefore, there is still a huge room for development to explore the relationship between rhodopsin mutations and eye diseases.

It is of great value to study the distribution, transport and expression level of rhodopsin in cells to explore the relationship between rhodopsin mutation and eye diseases. For example, rhodopsin P23H, which is the most common cause of retinitis pigmentosa in North Americans, is trapped in the endoplasmic reticulum due to severe misfolding of the mutated protein and cannot be normally transported to the cell membrane, so it cannot perform normal light. Receptor protein function. The pathogenic principle of P23H has been extensively verified at the cellular level and animal level, mainly due to the establishment of rhodopsin-GFP fusion protein. People can directly observe the distribution of rhodopsin in cells or retina through GFP. In the prior art, GFP is generally connected to the C-terminal (CT) of rhodopsin, the purpose of which is to avoid interference with its N-terminal, so as to achieve the purpose of not affecting the membrane insertion of rhodopsin ^CT -GFP. However, because the C-terminus of rhodopsin is disturbed, compared with wild-type rhodopsin, the G protein transduction function and phosphorylation efficiency of rhodopsin ^CT -GFP are greatly reduced, and there is a huge gap with wild-type rhodopsin. Obviously, rhodopsin ^CT -GFP is not an ideal fusion expression strategy.

The present invention provides an improved rhodopsin-eGFP fusion expression strategy, that is, inserting a molecule of enhanced green fluorescent protein (enhanced Green Fluorescent) at the non-functional region of rhodopsin - the first intracellular loop (Cytoplasmic Loop 1, CL1) for the first time Protein, eGFP), a new type of fusion protein rhodopsin ^CL1 -eGFP was constructed, thereby effectively avoiding interference to the N-terminal and C-terminal of rhodopsin. Experiments have proved that rhodopsin ^CL1 -eGFP exhibits similar glycosylation processing and expression levels to wild-type rhodopsin, and can be correctly transported to the cell membrane, so it is an ideal rhodopsin in vivo tracer indicator probe. In addition, since the fluorescence intensity of eGFP is more than 6 times that of GFP, the fluorescence intensity of rhodopsin ^CL1 -eGFP is greatly enhanced, which is more conducive to the increase of fluorescence detection tissue depth. In conclusion, the present invention provides a powerful tool for studying rhodopsin-related eye diseases, and provides useful reference and new inspiration for the research of other GPCRs.

Contents of the invention

The purpose of the present invention is to provide a novel GPCR tracer method in vivo, specifically by constructing a novel GPCR-eGFP fusion protein to directly observe the distribution position, expression level and state of GPCR in living cells or tissues, etc. Therefore, it provides a valuable reference for the study of the pathogenesis of GPCR-related diseases, and provides new ideas for the construction of fusion proteins. The feature of the present invention is that eGFP is inserted in the non-functional region of GPCR, so as to avoid interference to the function of GPCR.

The concrete technical scheme that realizes the object of the invention is:

A novel G protein-coupled receptor (G Protein-Coupled Receptor, GPCR) in vivo tracing method, characterized in that the method is: inserting a molecule of enhanced green fluorescent protein into the non-functional region of the G protein-coupled receptor (enhanced Green Fluorescent Protein, eGFP), realize the fusion expression of GPCR-eGFP, and detect the distribution of GPCR in living cells by fluorescence.

The insertion of a molecule of enhanced green fluorescent protein (eGFP) in the non-functional region of the G protein-coupled receptor is to realize fluorescent labeling of GPCR molecules without interfering with GPCR functions, and realize real-time observation of GPCR molecules in vivo .

A novel human rhodopsin (rhodopsin) and enhanced green fluorescent protein (eGFP) fusion expression method is characterized in that the method is: inserting a molecule in the first intracellular loop region (Cytoplasmic Loop 1, CL1) of rhodopsin eGFP, construct rhodopsin ^CL1 -eGFP;

Specifically include the following steps:

1) Introduce a single point mutation K66M in the first intracellular loop region of rhodopsin by segmented PCR, that is, mutate the 66th residue lysine to methionine, and introduce a unique Nde in rhodopsin I enzyme cutting site (CATATG);

2) HEK293S cells were transfected with the pCEP4-rhodopsinK66M plasmid, and the expression level and glycosylation processing of rhodopsinK66M and wild-type rhodopsin were determined to be similar by Western Blot bands, thus confirming that K66 was a non-key residue, and this position was suitable for the insertion of eGFP;

3) Amplify the eGFP gene with Nde I restriction sites at both ends from pcDNA3.1-3'-eGFP by PCR method, and then perform single digestion with Nde I, and recover the eGFP after Nde I digestion by gel Gene;

4) Since the pCEP4 plasmid contains multiple Nde I restriction sites, it is not suitable for single restriction experiments. Use BamH I and Hind III double restriction experiments and T4 ligase ligation experiments to transfer rhodopsinK66M from the pCEP4 plasmid to the pBAD24 plasmid Get the pBAD24-rhodopsinK66M plasmid;

5) After the pBAD24-rhodopsinK66M plasmid was digested with Nde I, it was treated with Calf Intestine Alkaline Phosphatase (CIAP) to prevent the self-ligation of the pBAD24-rhodopsinK66M plasmid in the ligation experiment;

6) T4 ligase ligated the eGFP gene after Nde I digestion in step 3) and the pBAD24-rhodopsinK66M plasmid after Nde I digestion and bovine intestinal alkaline phosphatase treatment in step 5), and transformed the ligated product into Escherichia coli For the Top10 strains, the colony PCR method was used to screen out the clones with forward insertion of eGFP, and finally the constructed pBAD24-rhodopsinK66M-eGFP plasmid was identified by sequencing, that is, the pBAD24-rhodopsin ^CL1 -eGFP plasmid;

7) After the sequencing is correct, the rhodopsin ^CL1 -eGFP is transferred from the pBAD24 plasmid to the pCEP4 plasmid through the BamH I and Hind III double enzyme digestion experiment and the T4 ligase ligation experiment again to obtain the pCEP4-rhodopsin ^CL1 -eGFP plasmid;

8) The pCEP4-rhodopsin ^CL1 -eGFP plasmid was transfected into HEK293S cells, and the expression of rhodopsin ^CL1 -eGFP was identified by Western Blot and fluorescence experiments.

The rhodopsin ^CL1 -eGFP avoids the interference of eGFP on the C-terminus of the important functional region of rhodopsin, is an improved fluorescent reporter protein of rhodopsin, and is more suitable for in vivo research of rhodopsin-related eye diseases.

The present invention compares with prior art, its beneficial effect:

As a common tag protein, fluorescent protein can be fused and expressed with the target protein, so that the target protein can be observed through fluorescence. The general fusion strategy is to insert the fluorescent protein at the N-terminal or C segment of the target protein. For example, people have constructed fusion proteins of various rhodopsin and fluorescent proteins, such as rhodopsin-GFP protein (Invest OphthalmolVis Sci 52 (13): 9728- 9736.), rhodopsin-eGFP or rhodopsin-mCherry fusion protein (PLoS One7(1): e30101.), they all realize the fluorescent labeling of rhodopsin, so the expression of rhodopsin wild type and mutant can be observed in cells and tissues distributed. However, in the above fusion proteins, the fluorescent proteins are all connected to the C-terminus of rhodopsin. However, since the C-terminus of rhodopsin is a phosphorylation site and participates in the downstream G protein activation process, it has an important function. When the fluorescent protein is attached to the C-terminus of rhodopsin, it will affect the function of the latter, such as greatly reducing the phosphorylation of the protein and the activation efficiency of the G protein (J Biol Chem 276(30): 28242-28251.). In order to solve the above problems, the present invention proposes to insert eGFP at the non-functional region of rhodopsin for the first time, so as to minimize the interference to the rhodopsin functional region. The first intracellular loop of Rhodopsin (Cytoplasmic Loop 1, CL1) is generally considered to have no important functions, and experiments have proved that the mutant rhodopsinK66M of CL1 also exhibits similar properties to wild-type rhodopsinWT, so CL1 is an ideal eGFP insert position. The invention successfully constructs a novel fusion protein of rhodopsin ^CL1 -eGFP, thereby effectively avoiding the interference to the rhodopsin functional region, and providing a new idea for the fusion expression of rhodopsin and other GPCR fluorescent proteins.

Description of drawings

Figure 1 is a schematic diagram of the structure of rhodopsin ^CL1 -eGFP;

Figure 2 is a schematic diagram of pCEP4-rhodopsin ^CL1 -eGFP eukaryotic expression plasmid;

Fig. 3 is the electropherogram of the PCR result of rhodopsinK66M gene;

Figure 4 is a schematic diagram of colony PCR screening of eGFP positively inserted into rhodopsin clones;

Figure 5 is a schematic diagram of Western Blot identification of the expression of rhodopsin ^CL1 -eGFP in HEK293S cells;

Fig. 6 is a schematic diagram of fluorescence detection of rhodopsin ^CL1 -eGFP in HEK293S cells.

detailed description

More detailed experimental method can refer to embodiment. This embodiment is used to better understand the content of the present invention, but not to limit the present invention in any form or method.

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

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:

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:

CMV: 5'-CGCAAATGGGCGGTAGGCGTG-3'

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

2) Using the existing pcDNA4/TO-rhodopsin plasmid in the laboratory as a template, use the pcDNA4/TO plasmid general downstream primer BGH and upstream primer Rho(CL1)1 (including the mutation site) to amplify fragment 2, with a size of about 990bp . The primers used are as follows:

Rho(CL1)1: 5'ACCGTCCAG CATATG AAGCTGCGCACG-3'(Nde I)

BGH: 5'-ACTAGAAGGCACAGTCGAGGCT-3'

3) Gel recovery fragment 1 and fragment 2, and then using these two fragments as templates, use the two primers CMV and BGH to amplify the complete fragment of rhodopsinK66M (from CMV to BGH), the size is about 1380bp (refer to Figure 3) , using the following conditions:

first step:

The PCR conditions used in the first step are:

After the first step is completed, add 0.5 μl each of CMV and BGH primers at a concentration of 10 pM to the PCR tube

The PCR conditions used in the second step are:

4) Recover the PCR product in 3) by gel, and then perform double digestion treatment with BamH I and Hind III. Treat pCEP4 plasmid in the same way. Gel recovery of the above digestion products. The enzyme digestion system used is as follows:

37°C, incubate for 2h

5) Using T4 ligase to connect the pCEP4 plasmid and the PCR product after the double digestion treatment in 4), to obtain the pCEP4-rhodopsin K66M plasmid. The enzyme digestion system used is as follows:

16°C, incubate for 16h

6) The ligation product in 5) was transformed into E. Coli Top10 strain, and cultured on a solid LB plate containing ampicillin.

7) Pick a single colony and inoculate it in a liquid LB medium containing ampicillin, extract the plasmid and perform sequencing to confirm that rhodopsinK66M has been successfully prepared.

Embodiment 2: rhodopsin ^CL1 -eGFP gene construction

The idea of rhodopsin ^CL1 -eGFP gene construction is to insert the eGFP gene into pCEP4-rhodopsinK66M to obtain pCEP4-rhodopsin ^CL1 -eGFP (see Figure 2).

Specific steps are as follows:

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.

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.

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

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

3) The amplified eGFP fragment and pBAD24-rhodopsinK66M plasmid were treated with Nde I enzyme, and then the digested products were recovered by gel respectively. The Nde I digestion system is as follows:

37°C, incubate for 2h

4) The NdeI digested pBAD24-rhodopsinK66M plasmid was treated with calf intestinal alkaline phosphatase (Calf Intestine Alkaline Phosphatase, CIAP), and the gene fragment was recovered from the gel. The purpose of this step is to prevent the self-connection of the pBAD24-rhodopsinK66M plasmid, thereby increasing the success rate of eGFP plasmid insertion.

The CIAP process is as follows:

Incubate at 37°C for 15 minutes, then at 57°C for 15 minutes

5) T4 ligase ligated the eGFP fragment digested with Nde I and the pBAD24-rhodopsinK66M plasmid after Nde I digestion and CIAP enzyme treatment in 4) to obtain pBAD24-rhodopsinK66M-eGFP, namely pBAD24-rhodopsin ^CL1 -eGFP plasmid.

6) Colony PCR was used to identify the insertion direction of eGFP in the pBAD24-rhodopsinK66M plasmid, and the pBAD24-rhodopsin ^CL1 -eGFP plasmid inserted in the forward direction was screened out.

12 single colonies were selected for testing, and the experimental results are shown in Figure 4. Since it is a single enzyme cut ligation experiment, eGFP can be inserted into rhodopsin in the forward or reverse direction, and the number of the lane mark indicates the number of the single colony clone. Lanes 1-10 in the left half of the figure represent the results of colony PCR using the upstream primer Op1 of rhodopsin and the downstream primer eGFP2 of eGFP gene. Rho1 is about 200bp, and eGFP is about 720bp, so the amplified fragment of Op1 and eGFP2 is about 920bp. The clones that can amplify the bands are positive insertion clones, and the results show that colonies 2, 4, and 8 are positive insertions. Lanes 1-10 (italics) in the right half of the figure represent the colony PCR results of rhodopsin upstream primer Op1 and eGFP gene upstream primer eGFP1 (also about 920bp), and the bands that can be amplified are reverse insertion clones, and the results show that colony 1 , 3, 7, 10 are reverse insertion. The ones with no bands in the two PCRs were insertion failures, that is, empty vectors, colonies 5, 6, and 9 in turn.

7) The positively inserted pBAD24-rhodopsin ^CL1 -eGFP plasmid was sequenced and identified to prove that eGFP was correctly inserted into the CL1 position of rhodopsin.

The rhodopsin ^CL1 -eGFP gene sequence is shown in the sequence listing. The full length of this gene sequence is 1767bp, which includes 1047bp of the full-length human rhodopsin gene (including start codon and stop codon), and 714bp of the eGFP gene without the start codon and stop codon, and finally includes two eGFP gene sequences. The 6 bases introduced by the Nde I restriction site at the end.

8) Replace the rhodopsin ^CL1 -eGFP gene fragment from pBAD24 to the pCEP4 plasmid using BamH I and Hind III double enzyme digestion and T4 ligase experiments to obtain pCEP4-rhodopsin ^CL1 -eGFP (see Figure 2).

Example 3: Expression detection of pCEP4-rhodopsin ^CL1 -eGFP in HEK293S cells

The expression detection of pCEP4-rhodopsin ^CL1 -eGFP in HEK293S cells mainly includes two aspects, one is to check the expression level and glycosylation of rhodopsin ^CL1 -eGFP through Western Blot results (refer to Figure 5), and the other is to use fluorescence microscopy Check whether rhodopsin ^CL1 -eGFP can normally emit green fluorescence and whether it accumulates in the cells (refer to FIG. 6 ). The specific operation steps are as follows:

1) Plasmid preparation: Plasmids were prepared with a plasmid extraction kit, and high-purity pCEP4-rhodopsin ^CL1 -eGFP plasmids with a plasmid concentration of not less than 200ng/μl and an A ₂₆₀ /A ₂₈₀ between 1.9 and 2.0 were selected. The same method was used to prepare pCEP4-rhodopsinWT and pCEP4-rhodopsinK66M plasmids as controls.

2) Cell preparation: select HEK293S that has been passaged at least twice after thawing as host cells, and use DMEM (10% FBS) medium to culture the cells. When the cells are in good condition, inoculate wells 1 to 4 of a six-well plate at a density of 2×10 ⁵ cells/ml. When the cell confluency is 80% to 90%, plasmid transfection can be carried out. The medium was changed 24 hours before transfection, and the medium was still DMEM (10% FBS).

3) Plasmid transfection: The specific transfection process is as follows: ① Use Opti-MEM I to dilute 2 μg DNA respectively, that is, add 2 μg DNA to each well (only transfect well 1, well 2 and well 3, and well 4 is used as a negative control). The volume is 100 μl. Mix gently; ② Use Opti-MEM I to dilute the laboratory-made polyetherimide-based transfection reagent (referred to as PEI), a total of 18 μg PEI is required, that is, 6 μg PEI is required to be added to each well (PEI: DNA = 3:1). The total volume is 300 μl. Mix gently; ③ Add the diluted PEI to the diluted DNA (100 μl diluted DNA and 100 μl diluted PEI), and mix gently; ④ Incubate at room temperature for 20-30 minutes to form a PEI-DNA complex ⑤ Add 200 μl of incubated PEI-pCEP4-rhodopsin ^CL1 -eGFP plasmid complex to well 1, add 200 μl of incubated PEI-pCEP4-rhodopsinWT plasmid complex to well 2, and add 200 μl of incubated PEI-pCEP4-rhodopsinK66M to well 3 For the plasmid complex, 200 μl Opti-MEM I was added to well 4 as a negative control. Gently shake the plate to make it evenly distributed; ⑥The cells are cultured in a 37°C, 5% CO ₂ humid heat incubator.

4) After 48 hours of plasmid transfection, fluorescence detection was performed on the six-well plate with a fluorescence microscope. Under blue light excitation, well 1 can emit obvious green fluorescence, while well 2, well 3 and well 4 have no fluorescence.

Please refer to Figure 6 for details. Among them, the left picture is rhodopsin ^CL1 -eGFP, and the right picture is control HEK293S blank cells. The results showed that rhodopsin ^CL1 -eGFP emitted obvious fluorescence and distributed uniformly in the cells without forming obvious protein aggregation spots.

5) After the fluorescence observation, the cells in each well were harvested for Western Blot detection.

6) Western Blot detection

Firstly, sample processing: add 100 μl lysate and 1 μl PMSF (100 mM) to each tube of cells, and pipette evenly. Cells were then lysed using liquid nitrogen repeated freeze-thaw cycles. Then centrifuge at 12,000 g for 10 min, and take the supernatant.

Then run SDS-PAGE gel. Then, the protein on the SDS-PAGE gel was transferred to the PVDF membrane by the semi-dry transfer method, and the transfer condition was 10V, 40min.

Then carry out antibody incubation, the specific process is: ①After washing the membrane once with TBST, seal it with 5% skimmed milk powder at room temperature for 2h; HRP-labeled secondary antibody (goat anti-mouse) was incubated at room temperature for 1 hour; ④ After secondary antibody incubation, the membrane was washed 6 times with TBST, 5 minutes each time. ⑤Finally, ECL exposure and color development are carried out.

If β-actin detection is performed, wash the membrane with 0.8M NaOH solution for 30s to remove the primary and secondary antibodies to rhodopsin on the membrane. Then carry out antibody incubation, the steps are similar to the detection of rhodopsin, except that the primary antibody is replaced by anti-mouse β-actin monoclonal antibody.

Refer to Figure 5 for specific results. The size of rhodopsin is 36kD. Since mammalian cells express eukaryotic proteins, they will undergo different degrees of glycosylation processing on the protein, so the protein presents diffuse bands. The glycosylation of rhodopsin can be judged according to the distribution of western blot bands Processing status. The proteins were identified with the monoclonal antibody 1D4 of rhodopsin and the monoclonal antibody of β-actin respectively, and the contents represented by the swimming lanes are shown in the figure. The results showed that the expression of rhodopsinK66M was similar to that of rhodopsinWT, indicating that K66 is not a key residue and is an ideal eGFP insertion site. Secondly, rhodopsin ^CL1 -eGFP can also be correctly expressed, showing similar expression levels and glycosylation processing conditions as rhodopsinWT, but because the molecular weight of rhodopsin ^CL1 -eGFP is much larger than that of rhodopsin, the migration rate is relatively lower, so the overall Western blot bands are towards Move at large molecular weight. Among them, Control represents HEK293S blank cells, so the monoclonal antibody to rhodopsin cannot detect the band.

In conclusion, it can be seen from the experimental results that rhodopsin ^CL1 -eGFP can be used as an ideal fusion protein for tracking rhodopsin in vivo.

<110> East China Normal University

<120> A G protein-coupled receptor tracing method in vivo and its application

<160> 1

<210> 1

<211> 1767

<212>DNA

<213> Artificial sequence

<220>

<223> rhodopsin ^CL1 -eGFP gene sequence

<400> 1

atgaatggca cagaaggccc taacttctac gtgcccttct ccaatgcgac gggtgtggta 60

cgcagcccct tcgagtaccc acagtactac ctggctgagc catggcagtt ctccatgctg 120

gccgcctaca tgtttctgct gatcgtgctg ggcttcccca tcaacttcct cacgctctac 180

gtcaccgtcc agcatatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc 240

ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag 300

ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc 360

gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac 420

cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag 480

gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc 540

gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc 600

aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc 660

gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc 720

agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 780

ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag 840

cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 900

gagctgtaca agcatatgaa gctgcgcacg cctctcaact acatcctgct caacctagcc 960

gtggctgacc tcttcatggt cctaggtggc ttcaccagca ccctctacac ctctctgcat1020

ggatacttcg tcttcgggcc cacaggatgc aatttggagg gcttctttgc caccctgggc1080

ggtgaaattg ccctgtggtc cttggtggtc ctggccatcg agcggtacgt ggtggtgtgt1140

aagcccatga gcaacttccg cttcggggag aaccatgcca tcatgggcgt tgccttcacc1200

tgggtcatgg cgctggcctg cgccgcaccc ccactcgccg gctggtccag gtacatcccc1260

gagggcctgc agtgctcgtg tggaatcgac tactacacgc tcaagccgga ggtcaacaac1320

gagtcttttg tcatctacat gttcgtggtc cacttcacca tccccatgat tatcatcttt1380

ttctgctatg ggcagctcgt cttcaccgtc aaggaggccg ctgcccagca gcaggagtca1440

gccaccacac agaaggcaga gaaggaggtc acccgcatgg tcatcatcat ggtcatcgct1500

ttcctgatct gctgggtgcc ctacgccagc gtggcattct acatcttcac ccaccagggc1560

tccaacttcg gtcccatctt catgaccatc ccagcgttct ttgccaagag cgccgccatc1620

tacaaccctg tcatctatat catgatgaac aagcagttcc ggaactgcat gctcaccacc1680

atctgctgcg gcaagaaccc actgggtgac gatgaggcct ctgctaccgt gtccaagacg1740

gagacgagcc aggtggcccc ggcctaa 1767

Claims (2)

1. a kind of G-protein-coupled receptor Cellular tracking method is it is characterised in that the method is：In G-protein-coupled receptor GPCR Nonfunctional area, that is, first intracellular ring region CL1 insert a molecules enhancing green fluorescent protein eGFP, do not disturbing GPCR work( The amalgamation and expression of GPCR-eGFP is realized, by the fluorescent labeling of GPCR molecule, real-time detection GPCR is in cell on the premise of energy Distribution, avoid the interference to GPCR critical function area's C-terminal and downstream signal transduction for the eGFP simultaneously.

2. a kind of people's rhodopsin is rhodopsin and enhanced green fluorescence protein eGFP fusion expression method, and its feature exists In the method it is：Insert a molecule eGFP in first intracellular ring region CL1 of people's rhodopsin, build rhodopsin^CL1- eGFP；Specifically include following steps：

1）Introduce a simple point mutation K66M using segmented-PCR method in first intracellular ring region of people's rhodopsin, that is, the 66th The residue lysine of position sports methionine, introduces a unique Nde I restriction enzyme site CATATG in people's rhodopsin；

2）PCEP4-rhodopsinK66M plasmid transfection HEK293S cell, is determined by Western Blot band RhodopsinK66M is similar in terms of expression and glycosylation processing to wild type rhodopsinWT, so that it is determined that K66 right and wrong Key residues, this position is suitable for the insertion of eGFP；

3）The eGFP gene that two ends carry Nde I restriction enzyme site is amplified from pcDNA3.1-3 '-eGFP by PCR method, then EGFP gene after Nde I single endonuclease digestion, after glue reclaim Nde I enzyme action；

4）Due to containing multiple Nde I restriction enzyme sites on pCEP4 plasmid, be not suitable for carrying out single endonuclease digestion experiment, with BamH I and The experiment of Hind III double digestion and T4 ligase connect experiment and rhodopsinK66M are transferred to pBAD24 matter from pCEP4 plasmid PBAD24- rhodopsinK66M plasmid is obtained on grain；

5）After pBAD24-rhodopsinK66M plasmid is carried out with Nde I single endonuclease digestion, then with calf intestinal alkaline phosphatase CIAP Reason, prevents pBAD24-rhodopsinK66M plasmid certainly connecting in connecting experiment；

6）T4 ligase Connection Step 3）In Nde I enzyme action after eGFP gene and step 5）In through Nde I enzyme action and cattle PBAD24-rhodopsinK66M plasmid after intestinal alkaline phosphatase process, connection product is converted escherichia coli Top10 bacterium Plant, then filter out the clone of the positive insertion of eGFP by bacterium colony PCR method, identify structure further finally by sequencing PBAD24-rhodopsinK66M-eGFP is pBAD24-rhodopsin^CL1- eGFP plasmid；

7）After sequencing is correct, connect experiment handle again by BamH I and the experiment of Hind III double digestion and T4 ligase rhodopsin^CL1- eGFP is transferred to pCEP4 plasmid from pBAD24 plasmid, obtains pCEP4-rhodopsin^CL1- eGFP plasmid；

8）By pCEP4-rhodopsin^CL1- eGFP plasmid transfection in HEK293S cell, by Western Blot and fluorescence Experimental identification rhodopsin^CL1The expression of-eGFP.