CN101045925A - Mutual action of CAL and mGluR5 and its effect on mGluR5 expression - Google Patents

Abstract

The present invention relates to the discovery of the interaction between two proteins, mGluR ₅ /CAL, and the discovery that the cellular expression of the CAL protein can promote the expression of mGluR ₅ and that the CAL protein does not increase the transcription of the mGluR ₅ gene. The important point of the present invention is to discover and confirm the specific binding between CAL and mGluR ₅ , and the expression of mGluR ₅ in CAL cells. This provides a new theoretical basis for exploring the physiological and pathological effects of mGluR ₅ in the central nervous system, and provides new potential drugs for the treatment of related diseases (Parkinson's disease, spasticity, epilepsy, depression, anxiety and pain) target of action.

Description

Interaction between CAL and mGluR5 and its effect on mGluR5 expression

Technical field:

The present invention discovers and confirms CAL and metabotropic glutamate receptor- 5 (mGluR ₅ ) interaction. And through transfection, Western Blot, RT-PCR and other techniques, it was confirmed that the expression of CAL protein can promote the expression of mGluR ₅ , and the possible mechanism of CAL promoting the expression of mGluR ₅ was speculated. It provides a new molecular mechanism for elucidating the role of mGluR ₅ in the central nervous system, and provides a new potential drug target for the treatment of related nervous system diseases. The invention relates to the field of development and application of new methods and new proteins in biomedical high technology.

Background of the invention:

mGluR ₅ is abundantly expressed in the striatum, hippocampus and cerebral cortex. It exerts important biological effects through G protein, 1, 4, 5-triphosphate inositol (IP ₃ ) and Ca ²⁺ cell signal transduction pathways, such as participating in the regulation of the plasticity of central nervous system development, strengthening the cerebral cortex γ -Inhibitory synaptic transmission in aminobutyric acid (GABA)ergic neurons. In the striatum, it can strengthen the neurotoxic effect of NMDA receptors, and connect with the subthalamic nucleus and the dopamine (DA) system, may participate in the regulation of the movement of the basal ganglia, and may be associated with Parkinson's disease (PD) animal models Associated with extrapyramidal symptoms. In the hippocampus, mGluR ₅ can form long-term potentiation (LTP), which is related to learning and memory. In addition, convulsions, epilepsy, depression, anxiety and pain are all closely related to mGluR ₅ , but the molecular regulation mechanism of mGluR ₅ is still not very clear.

The carboxyl terminus of many GPCRs contains a region that can bind to a PDZ domain. mGluR ₅ is a member of the GPCRs family, and its carboxy-terminus also has a PDZ binding motif (PDZ binding motif): ST/SL sequence. In 2002, Kitano J et al. reported that mGluR ₅ can specifically bind to PDZ protein Tamalin to promote the expression of mGluR ₅ on the plasma membrane.

In the research field of GPCRs, the regulation of receptors by non-G proteins is one of the hotspots. The research results of our laboratory show that: adrenergic receptor (β ₁ AR) can specifically bind to some PDZ proteins such as MAGI-2, MAGI-3 and CAL (cystic fibrosis transmembrane conductance regulator a ssociated ligand ). Among them, MAGI-2 can promote the desensitization of β ₁ AR, MAGI-3 can inhibit the activation of MAPK caused by β ₁ AR, and CAL can regulate the transport of newly synthesized β ₁ AR in cells. Based on our research experience on β ₁ AR and the report of Kitano J, we speculate that other members of the PDZ family may also combine with mGluR ₅ to regulate its physiological properties.

Since mGluR ₅ is abundantly expressed in the striatum, hippocampus and cerebral cortex, we used the GST fusion protein at the carboxy-terminal of mGluR ₅ to sediment the brain tissue protein lysate. The results showed that compared with GST negative control, GST-mGluR ₅ -CT could precipitate several proteins at the position between 75KD-58KD and above 75KD, suggesting that the carboxy terminus of mGluR ₅ may bind to several rabbit brain proteins. The samples were analyzed by two-dimensional electrophoresis. According to the isoelectric point and molecular weight of the difference points, and on the basis of the previous work in the laboratory, we speculated that CAL may exist in these several proteins. The sedimentation complex was analyzed by immunoblotting with a specific CAL antibody, and the results showed that the carboxyl terminus of mGluR ₅ could bind to CAL.

Purpose of the invention:

The PDZ protein combined with mGluR ₅ was screened by GST fusion protein sedimentation in tissues: CAL. The interaction between mGluR ₅ and CAL was fully studied by combining in vitro experiments (GST-Pull down) with intracellular experiments (co-immunoprecipitation), biochemical and molecular biology methods and morphological methods (cellular immunofluorescence). After establishing the CAL/mGluR ₅ interaction, the effect of cellular expression of CAL on mGluR ₅ expression was continued, and the CAL protein did not increase the transcription of the mGluR ₅ gene.

The interaction between CAL and mGluR ₅ and the promotion of its expression provide a new theoretical basis for the elucidation of the molecular mechanism of mGluR ₅ in the central nervous system, and provide new potential drug targets for the treatment of related nervous system diseases. Content and requirements:

CAL and mGluR ₅ interact, and the cellular expression of CAL protein promotes the expression of mGluR ₅ , while CAL protein does not increase the transcription of mGluR ₅ gene. This invention involves the following experimental results:

1. GST-mGluR ₅ on rabbit brain tissue homogenate Pull-down Coomassie Brilliant Blue staining and Western blot analysis results,

2. Pull-down of GST-mGluR ₅ on the PDZ domain of CAL,

3. Pull-down of GST-mGluR ₅ on the complete protein molecule of CAL,

4. Co-immunoprecipitation of intact protein molecules of mGluR ₅ and CAL in COS-7 cells,

5. Confocal subcellular colocalization of mGluR ₅ and CAL intact protein molecules in BHK cells,

6. Western blot analysis of CAL promoting mGluR ₅ expression in COS-7,

7. The effect of the cellular expression of CAL protein on the transcription of mGluR ₅ gene.

The experimental content and results mentioned above are also the technical indicators of the present invention.

Description of drawings:

figure 1.

Organization Pull-down

A. Coomassie Brilliant Blue staining results, the results show that compared with GST negative control, GST-mGluR ₅ -CT can precipitate several proteins at positions between 75KD-58KD and above 75KD. It suggested that the carboxy terminus of mGluR ₅ may bind to several rabbit brain proteins.

B. Western blot analysis with specific antibody CAL, the first lane is the supernatant of rabbit brain tissue lysate containing CAL protein, after the GST fusion protein sedimentation experiment, it can be detected in the sedimentation complex of GST-mGluR ₅ -CT Presence of CAL protein (lane 3), CAL was not detectable in the sedimented complex of GST protein alone (lane 2). It shows that the carboxyl terminus of mGluR ₅ interacts with rabbit brain protein CAL, and the arrow points to the protein CAL.

figure 2.

Prokaryotic Pull-down analysis results, the first lane is the bacterial lysate supernatant containing His-CAL-PDZ fusion protein, after the GST fusion protein sedimentation experiment, it can be detected in the sedimentation complex of GST-mGluR ₅ -CT-wt In the presence of His-CAL-PDZ protein (lane 3), His-CAL-PDZ was not detected in the sedimentation complex of GST protein alone (lane 2), when mGluR ₅ carboxy-terminal -1 and -3 positions respectively When mutated to alanine, the presence of His-CAL-PDZ protein was not detected in the sedimentation complex of the corresponding GST-mGluR ₅ -CT fusion protein (lanes 4 and 6), when mGluR ₅ carboxy-terminal When positions 0 and -2 were mutated to alanine, respectively, the presence of His-CAL-PDZ protein was detected in the sedimentation complex of the corresponding GST-mGluR ₅ -CT fusion protein (lanes 5 and 7). It shows that mGluR ₅ carboxy-terminal -1 and -3 mutations can significantly inhibit its binding to CAL. The arrow points to His-CAL.

image 3.

Eukaryotic Pull-down analysis results, the first lane is the cell lysate transfected with HA-CAL, after the same amount of HA-CAL transfected cell lysate was subjected to the GST fusion protein sedimentation experiment, the GST-mGluR ₅ -CT The presence of HA-CAL protein could be detected in the sedimentation complex of -wt (lane 3), HA-CAL was not detected in the sedimentation complex of GST protein alone (lane 2), when the mGluR ₅ carboxy-terminal last When the amino acid Ser was mutated to Ala, the presence of CAL protein was not detectable in the sedimentation complex of the corresponding GST-mGluR ₅ -CT fusion protein (lane 4). It shows that the mGluR ₅ carboxy terminus can interact with the complete protein molecule of CAL protein, and the mutation of mGluR ₅ carboxyl terminus can obviously inhibit its combination with CAL. The arrow points to HA-CAL.

Figure 4.

Co-immunoprecipitation analysis of the interaction of the intact protein molecule mGluR ₅ and CAL.

Lane 2 in Figure B is the cell lysate transfected with HA-CAL alone, and Lane 3 in Figures B and C is the cell lysate co-transfected with HA-CAL and Flag-mGluR ₅ . In cells transfected with HA-CAL alone, HA-CAL protein was not detected in the immunoprecipitation complex (A, lane 2); in the blank cell COS-7, HA-CAL protein was not detected in the immunoprecipitation complex (A lane 1); while in cells co-transfected with HA-CAL and Flag-mGluR ₅ , HA-CAL could be detected in its immunoprecipitated complex (lane 3). This indicates that the intact CAL protein can interact with mGluR ₅ in cells.

Figure 5.

Immunofluorescence confocal confocal localization

a.mGluR ₅ alone transfected in BHK subcellular localization b.CAL alone transfected in BHK subcellular localization

c. mGluR ₅ subcellular localization in BHK in the presence of CAL d. subcellular localization of CAL in BHK in the presence of mGluR ₅

e. CAL and mGluR ₅ co-localize in BHK subcells

Figure 6.

As intracellular CAL increases, so does _mGluR5 . After statistical analysis (9B), it showed that: compared with the control group, the difference between the 0.60ug group and the control group was statistically significant (**P<0.01, n=4); 0.15ug group, 0.30ug, 1.20ug Compared with the control group, the difference between the two groups was statistically significant (*P<0.05, n=4). It shows that CAL can increase the expression level of mGluR ₅ protein.

Figure 7.

Panel A: total RNA extracted from cells, the first lane is the total RNA of -/pCDNA ₃ -Flag-mGluR ₅ group, the second lane is the total RNA of pBK-CMV-HA-CAL/pCDNA ₃ -Flag-mGluR ₅ group.

Panel B: the first and third lanes are respectively -/pCDNA ₃ -Flag-mGluR ₅ , pBK-CMV-HA-CAL/pCDNA ₃ -Flag-mGluR ₅ groups of β-actin PCR products, the second and fourth lanes are respectively - /pCDNA ₃ -Flag-mGluR ₅ , pBK-CMV-HA-CAL/pCDNA ₃ -Flag-mGluR ₅ group mGluR ₅ PCR products.

Panel C: the RNA level of mGluR ₅ in the pBK-CMV-HA-CAL/pCDNA ₃ -Flag-mGluR ₅ group was lower than that in the -/pCDNA ₃ -Flag-mGluR ₅ group. Statistical analysis showed that the difference between the two groups was statistically significant ( ^* P<0.05, n=3).

Example:

1. GST-mGluR ₅ -CT fusion protein screening of PDZ protein bound to it in rabbit brain tissue

1.1 Brain protein preparation:

1) Take a healthy female New Zealand rabbit, cut off the head with surgical scissors, and peel off the rabbit brain with bone forceps. Put the rabbit brain on ice, wash the brain tissue with PBS, cut it into pieces, and weigh the wet weight.

2) Homogenization: Put the tissue into a homogenizer and add tissue lysate at a ratio of 100 mg/ml. Homogenize manually. Pay attention to keep the temperature as low as possible and homogenate quickly.

3) Centrifuge at 12000rpm, 4°C, 20min.

4) Take a small amount of supernatant for quantification.

Preparation method of tissue lysate:

HEPES 10mM

NaCl 50mM

EDTA 5mM

Triton X-100 1.0%

Benzamidine 1mM

1.2 GST-fusion protein induction and purification:

1.2.1 Induced expression of GST-fusion protein

1) Transform the constructed GST fusion protein expression plasmid into Escherichia coli BL-21, pick a single clone strain and inoculate it into 5-10ml LB culture medium (add corresponding antibiotics according to the strain resistance), and inoculate in a shaker at 37°C at 250rpm /min Shake the bacteria overnight.

2) Inoculate 3ml of bacterial solution into 1L of LB culture medium (add corresponding antibiotics according to the resistance of the strain), shake the bacteria at 250rpm/min in a shaker at 37°C for 2-3 hours, and measure the OD ₆₀₀ absorbance of the bacterial solution intermittently The best value is between 0.6-0.7.

3) Add IPTG (final concentration reaches 500 μmol/L) to induce bacterial growth for 2 hours in a shaker at 37° C. at 250 rpm/min.

4) Collect the culture medium: centrifuge at 3000 rpm for 30 min at 4°C, discard the supernatant, and keep the precipitate.

5) Add 25ml pre-prepared pre-cooled Harvest buffer to resuspend the bacteria, and ultrasonically break the bacteria (output power 10w, total 10-15 minutes, pulse 1 time/1.5s). Centrifuge at 15,000 rpm for 20 min at 4°C, and transfer the supernatant to a new high-speed centrifuge tube. Take a part of the supernatant and add an equal amount of 2× protein loading buffer to mix well. After 5 minutes at 95°C, take out an appropriate amount of samples for SDS-PAGE electrophoresis and Coomassie brilliant blue staining.

1.2.2 Identification of GST-fusion protein (Coomassie brilliant blue staining)

(1) Protein samples were subjected to SDS-PAGE.

(2) Put the PAGE gel into the staining solution and shake gently at room temperature for 1 hour.

(3) Then put the glue into the decolorization solution and shake until the background of the glue turns white.

(4) Dry the glue with a glue dryer and save it.

Preparation of reagents:

(1) Coomassie brilliant blue staining solution: (1L/1×)

Coomassie Blue R250 1g

Deionized water 500ml

Acetic acid 100ml

Methanol 400ml

(2) Decolorizing solution: (1L/1×)

Deionized water 500ml

Acetic acid 100ml

Methanol 400ml

1.2.3 GST-fusion protein purification

1) After identifying the successful expression of the protein, perform protein purification, add streptomycin sulfate (10mg/L) and mix well, centrifuge at 15000rpm at 4°C for 20min, and transfer the supernatant to another new low-speed centrifuge tube.

2) Add Glutathione-Sepharose 4B 60mg, and incubate upside down at 4°C for 3h.

3) Centrifuge at 3000rpm/min at 4°C for 1min, discard the supernatant, add 30ml of ice-cold Harvest buffer, mix well and then centrifuge (4°C, 3000rpm/min, 1min at 3000rpm).

4) Repeat the above step (3) twice.

5) Finally, resuspend Glutathione-Sepharose 4B with 1ml Harvest buffer.

6) Take 50 μl of the above sample and add 50 μl of 2× protein loading buffer to mix, place at 95°C for 5 minutes, take out an appropriate amount of sample for protein purification, SDS-PAGE electrophoresis and Coomassie brilliant blue staining to identify the effect of protein purification.

Harvest buffer preparation method: Total volume 25ml

HEPES solution (1mol/l pH7.4) 250μl

Nacl solution (1mol/l) 1.25ml

Benzamidine 0.004g

EDTA solution (0.5mol/l PH8.0) 250μl

H ₂ O 23.25ml

1.3 GST fusion protein sedimentation experiment:

1) Take an equal amount of GST and GST-mGluR5-CT fusion proteins bound to Glutathione-Sepharose 4B, add 1ml of cerebroprotein supernatant, and incubate at 4°C for 3h with rotation.

2) Collect Glutathione-Sepharose 4B by centrifugation at 3000rpm at 4°C for 2min, carefully discard the supernatant and keep the precipitate.

3) Add 1ml Wash Buffer I to the precipitate, mix gently at 3000rpm 4°C for 2min, carefully discard the supernatant, and keep the precipitate.

4) Repeat step 3) three times.

5) Add 1ml Wash Buffer II to the precipitate, mix gently at 3000rpm 4°C for 2min, carefully discard the supernatant, and keep the precipitate.

6) Add an appropriate amount of protein electrophoresis loading buffer to the sedimentation complex, centrifuge at 95° C. for 5 minutes, and take the supernatant. After the samples were subjected to SDS-PAGE, Coomassie brilliant blue staining and western blot analysis were performed.

Wash Buffer I:

HEPES 10mM

NaCl 50-100mM

EDTA 5mM

Tween20 0.1%

BSA 3g

Benzamidine 1mM (temporarily added before use)

Wash Buffer II:

HEPES 10mM

NaCl 50-100mM

EDTA 5mM

Tween20 0.1%

Benzamidine 1mM (temporarily added before use)

1.3.1 Coomassie Brilliant Blue staining sedimentation complex samples (see 1.2.2 for specific methods)

1.3.2 Western blot analysis of sedimentation complex samples:

(1) The sample was transferred to a nitrocellulose membrane by SDS-PAGE.

(2) Block with blocking solution for 1 h at room temperature, wash the membrane three times with TBST, 10 min each time.

(3) Add the primary antibody (anti-PIST, 1:1000) diluted with antibody diluent, shake gently at room temperature for 1 h, and wash the membrane three times with TBST, 10 min each time.

(4) Add HRP-labeled secondary antibody (1:3000) diluted with antibody diluent, shake gently at room temperature for 1 h, and wash the membrane three times with TBST, 10 min each time.

(5) ECL kit develops color for 3 minutes, and X-ray film develops.

2. GST fusion protein sedimentation experiment in prokaryotic cells

2.1 Induced expression and identification of His fusion protein:

1) Transform the recombinant expression plasmid His-CAL-PDZ into Escherichia coli BL-21(DE3), inoculate the single clone strain into 5-10ml LB culture medium (add corresponding antibiotics according to the strain resistance), shake at 37°C The bacteria were shaken overnight at 250rpm/min in the bed.

2) Inoculate 3ml of bacterial solution into 1L of LB culture medium (add corresponding antibiotics according to the resistance of the strain), shake the bacteria at 250rpm/min in a shaker at 37°C for 2-3 hours, and measure the OD ₆₀₀ absorbance of the bacterial solution intermittently The value is best between 0.6-0.7.

3) Add IPTG (final concentration reaches 500 μmol/L), and induce bacterial growth in a shaker at 37° C. at 250 rpm/min for 2 hours.

4) Harvest the bacteria by centrifuging at 3000 rpm for 30 minutes at 4°C, discard the supernatant and keep the precipitate.

5) Add 25ml pre-prepared pre-cooled Harvest buffer to resuspend the bacteria, and ultrasonically break the bacteria (output power 10w, total 10-15 minutes, pulse 1 time/1.5s). Centrifuge at 15,000 rpm for 20 min at 4°C, and transfer the supernatant to a new high-speed centrifuge tube. Take a part of the supernatant and add an equal amount of 2× protein loading buffer to mix well. After 5 minutes at 95°C, take an appropriate amount of samples for SDS-PAGE electrophoresis and Coomassie brilliant blue staining for identification.

2.2 GST fusion protein sedimentation experiment:

(1) Take an equal amount of bacterial lysate supernatant containing His-CAL-PDZ induced by IPTG.

(2) Equal amounts of GST bound to Glutathione-Sepharose 4B, GST-mGluR ₅ -CT and fusion proteins of four mutants thereof were added, and incubated at 4° C. for 2 h with rotation.

(3) Collect Glutathione-Sepharose 4B by centrifugation, and wash thoroughly according to the method in 1.3.

(4) Add an appropriate amount of protein electrophoresis loading buffer to the sedimentation complex, heat at 95° C. for 5 min, and then centrifuge to obtain the supernatant to obtain a sample for western blot analysis.

(5) The samples were subjected to Western blot analysis, see 1.3.2 for specific methods, and the primary antibody was anti-His (1:1000).

3. GST fusion protein sedimentation experiment in eukaryotic cells

3.1 Cell preparation:

COS-7 cells were cultured in a complete medium (high glucose DMEM, 10% fetal calf serum, 1% penicillin, 1% streptomycin sulfate) in a constant temperature incubator at 37°C and 5% CO ₂ .

3.2 Transfection:

Before transfection, the cells were inoculated into a plastic plate, observed under a light microscope, and transfected when the cell density accounted for 70%-80% of the growth surface of the culture plate. Depending on the purpose of the experiment, the amount of DNA required varies.

Specific steps:

(1) Dilution of DNA: Dilute with 250ul 0.9% NaCl if transfected into a 90mm plate; dilute with 100ul 0.9% NaCl if transfected into a 6-Well plate; shake gently to mix, and let stand for 5min.

(2) Dilute Hifectin II: Dilute Hifectin II with 0.9% NaCl according to the ratio of 1ugDNA: 2ul Hifectin II; shake gently to mix and let stand for 5min.

(3) Add the diluted Hifectin II to the diluted DNA, vortex gently to mix, and let stand for 20 minutes.

(4) At the same time, replace the medium in the culture dish with fresh serum-free medium.

(5) Culture in a constant temperature incubator at 37°C and 5% CO ₂ , and replace with complete medium after 4 hours.

(6) Continue culturing in a CO ₂ constant temperature incubator at 37°C for 24-48 hours to express the gene.

3.3 GST fusion protein sedimentation experiment:

3.3.1 Extraction of cell protein:

1) After removing the culture medium, rinse with warm PBS 2 to 3 times (cold PBS may cause the cells to fall off).

2) Add 1ml of ice-precooled lysate to a 90mm petri dish and put it on ice for 2-5min.

3) Scrape the cells with a cell scraper, and mix them upside down for 30 minutes at 4°C.

4) Centrifuge at 12000rpm at 4°C for 15min, and transfer the supernatant to 1.5ml EP sterilized by fresh autoclaving.

Preparation method of cell lysate:

HEPES 10mM

NaCl 50mM

EDTA 5mM

Triton X-100 1.0%

Benzamidine 1mM (temporarily added before use)

3.3.2 GST fusion protein sedimentation experiment:

1) Take an equal amount of supernatant of cell lysate expressing HA-CAL through transient transfection.

2) Equal amounts of GST bound to Glutathione-Sepharose 4B, fusion protein of GST-mGluR ₅ -CT and its mutant SSSA were added respectively, and incubated at 4° C. for 2 hours with rotation.

3) Collect Glutathione-Sepharose 4B by centrifugation, and wash thoroughly according to the method in 1.3.

4) Add an appropriate amount of protein electrophoresis loading buffer to the sedimentation complex, heat at 95° C. for 5 minutes, and then centrifuge to obtain the supernatant to obtain a sample for western blot analysis.

5) The samples were subjected to Western blot analysis, see 1.3.2 for specific methods, and the primary antibody was anti-HA (1:1000).

4. Co-immunoprecipitation

4.1. Cell culture and transfection

COS-7 cells were cultured in DMEM (high glucose) medium containing 10% fetal bovine serum and 1% penicillin and streptomycin at 37°C in an incubator with 5% CO ₂ . COS-7 cells with appropriate density were transfected with -/PBK-CMV-HA-CAL and PBK-CMV-HA-CAL/pCDNA ₃ -Flag-mGluR ₅ , and the transfection reagent was Hifectin II. For specific methods, see 3.2.

4.2. Extraction of cell protein: see 3.3.1 for specific methods

4.3 Co-immunoprecipitation

1) Take the supernatant, keep a small amount for immunoblotting control, add 30μl anti-Flag M2 affinitygel to 1ml protein supernatant, and incubate at 4°C for 2h with rotation.

2) Centrifuge at 3000rpm at 4°C for 1 min to collect the immunoprecipitated complex and wash thoroughly according to the method in 1.3.

3) Add an appropriate amount of protein electrophoresis loading buffer to the sedimentation complex, heat at 95° C. for 5 minutes, and then centrifuge to collect the supernatant to obtain samples for western blot analysis.

4) Perform Western blot analysis on the samples, see 1.3.2 for specific methods, the primary antibody is anti-HA (1:1000)

5. Cell Immunofluorescence

5.1 Treatment of glass climbing pieces:

The coverslips were soaked in concentrated acid for 2 hours, washed with deionized water and dried at room temperature. Put the acid-treated coverslip into ten-fold diluted poly-lysine solution, leave it at room temperature for 5 minutes, take it out and dry it at 60°C for 1 hour or overnight at room temperature. Soak coverslips in 75% ethanol for at least 30 min before use.

5.2 Cell culture and transfection

BHK cells were inoculated onto glass slides and cultured in DMEM (high glucose) medium containing 10% fetal bovine serum and 1% penicillin and streptomycin in an incubator at 37°C and 5% CO ₂ . When the cells reach an appropriate density, transfect -/PBK-CMV-HA-CAL, PBK-CMV-HA-CAL/pCDNA ₃ -Flag-mGluR ₅ into BHK cells, and the transfection reagent is Hifectin II. For details, see 3.2 .

5.3 Immunofluorescent staining of cells

(1) Cell fixation: 36 hours after transfection, the cells were washed 3 times with D-PBS, 5 minutes each time, the coverslip was taken out, the residual liquid was blotted dry with filter paper, and fixed with 4% paraformaldehyde at room temperature for 30 minutes.

(2) Rinse the cells with 2% BSA/D-PBS 3 times, 5 minutes each time.

(3) Cell permeabilization: at room temperature, incubate with Saponin buffer at room temperature for 30 minutes.

(4) Primary antibody incubation: place the cell side of the coverslip facing up, dilute the primary antibody with Saponin buffer (anti-HA 1:100anti-Flag1:250) and spot on the coverslip (for double immunization as before) For fluorescence analysis, add two kinds of primary antibodies at the same time), at room temperature for 1 hour or at 4°C overnight.

(5) Wash the cells 3 times with Saponin buffer, 5 minutes each time.

(6) Incubate the secondary antibody: place the cell side of the coverslip upward, dilute the secondary antibody with Saponin buffer (FITC-labeled goat anti-rabbit IgG 1:100, rhodamine-labeled goat anti-rat IgG 1:100) and Spot on the coverslip (if performing double immunofluorescence analysis, add two secondary antibodies at the same time), room temperature for 30 minutes.

(7) Wash the cells with D-PBS 3 times, 5 minutes each time.

(8) Mounting: Take 15ul of mounting medium and drop it on the glass slide, and seal the cover slip with the cell side facing down. Regarding buffer preparation:

(1) concentrated acid: concentrated nitric acid: concentrated hydrochloric acid = 2:1 (V/V)

(2) D-PBS (PH: 7.4 1L/1×): NaCl: 8.0g

KCl: 0.2g

MgCl ₂ 6H ₂ O: 0.1g

Na ₂ HPO ₄ ·12H ₂ O: 2.90g

_CaCl2 : 0.1g

_KH2HPO4 : _0.2g

(3) Blocking solution: 2% BSA/D-PBS

(4) Permeabilization solution: Saponin Buffer (2% BSA/D-PBS/0.04% Saponin)

(5) 4% paraformaldehyde fixative solution: add 4g paraformaldehyde to 60ml D-PBS, add 1mol/ml NaOH dropwise in a water bath at 60-80°C until dissolved and transparent. When the solution was cooled to 15°C, the pH was adjusted to 7.0 with HCl. Finally, dilute to 100ml with D-PBS.

(6) Mounting agent: prepare 70% glycerol with D-PBS

6. The experiment of CAL promoting the expression of mGluR ₅ protein

6.1. Cell culture and transfection:

(1) COS-7 cells were cultured in a DMEM (high glucose) medium containing 10% fetal bovine serum and 1% penicillin and streptomycin in an incubator at 37°C and 5% CO ₂ .

(2) Inoculate COS-7 cells into a 35ml cell culture dish, and when the cell density reaches about 80%, different doses of pBK-CMV-HA-CAL plasmid (0, 0.15, 0.30, 0.60, 1.20ug) COS-7 cells were co-transfected with the same dose of pCDNA ₃ -Flag-mGluR ₅ (0.5ug), respectively, and the transfection reagent was HifectinII. For specific methods, see 3.2.

6.2 Collection of cell samples:

1) 36 hours after transfection, cell samples were collected.

2) Discard the old cell culture medium and wash the cells twice with warm PBS. Invert the Petri dish onto the filter paper to remove residual PBS.

3) Add 200ul of 1.5× protein loading buffer, and scrape the cells with a cell scraper into a 1.5ml EP tube.

4) Ultrasound (100-200w) for 3s, twice.

6.3 Western blot analysis of cell samples

For details, see 1.3.2. The primary antibodies were anti-HA (1:1000), anti-mGluR ₅ (1:2000) and anti-β-actin (1:500).

7. Extraction of total cellular RNA and RT-PCR experiment:

7.1 Transfection:

COS-7 cells were transfected with pBK-CMV-HA-CAL/pCDNA ₃ -Flag-mGluR ₅ and -/pCDNA ₃ -Flag-mGluR ₅ , respectively.

7.2 Extraction of total RNA: 36 hours after transfection, extract the total RNA of cells in each dish. (All pipette tips and centrifuge tubes are treated with DEPC):

1) After the cells transfected with the corresponding plasmids, suck out the medium, wash twice with DEPC-treated PBS, add Trizol (1ml Trizol/plate with a diameter of 35mm) to blow off the sample and transfer it to a 1.5ml centrifuge tube, and place it on ice 10min.

2) Add 200μl chloroform/1ml Trizol lysate, mix thoroughly by inversion, and place on ice for 5min.

3) Centrifuge at 12000 rpm at 4°C for 15 minutes.

4) Carefully pipette the upper aqueous phase into a new centrifuge tube, add an equal volume of isopropanol (generally 500 μl/1ml Trizol lysate), and place on ice for 10 minutes.

5) Centrifuge at 12000 rpm at 4°C for 10 min.

6) Discard the supernatant, add 1 ml RNase-free 75% ethanol, and suspend the RNA precipitated at the bottom of the tube.

7) Centrifuge at 12000 rpm at 4°C for 5 minutes.

8) Discard the ethanol and suck up the liquid on the tube wall as much as possible, and open the tube mouth to dry at room temperature for 10 minutes.

9) Add 20μl DEPC-treated water/1ml Trizol lysate to dissolve the RNA precipitate. 55 ℃ -60 ℃ water bath to promote its dissolution.

10) Take 1 μl of RNA for quantification with a UV spectrophotometer, and store the rest in a -80°C refrigerator.

11) Identify the extracted total RNA by 1% agarose gel electrophoresis.

7.3 RNA reverse transcription into cDNA:

Reverse transcription reaction: add various components in the following table into a PCR tube, the total volume is 20 μl. Element Volume (μl) Final concentration Template RNA5×bufferdNTP mixtureRnase inhibitor AMVOligdTNuclease-free Water 1.04.08.00.52.01.0 updated to 20.0 1.0μg/20μl reaction system 1.0mmol/l1.0U/μl0.5U/μl2.5pmol/l

Mix well, place at room temperature for 10 minutes, keep warm at 42°C for 1 hour, and place on ice for 2 minutes. The obtained cDNA was stored at -20°C until use.

7.4 RT-PCR

1) Primer design:

Carboxy-terminal primer for mGluR ₅ :

5′-AAA CCA GAT CTG GAG GAG C-3′

5′-CGA TGA AGA ACT CTG CGT G-3′

β-actin primer

5′-CCC ATC TAC GAG GGC TAC GC-3′

5′-AGG AAG GAG GGC TGG AAC A-3′

2) The reaction system is as follows: Element Volume (μl) Final concentration 10×bufferdNTP mixture 5' primer 3' primer cDNA template Taq enzyme deionized water 2.5002.0001.0001.0001.0000.125 up to 25.000 0.500mmol/l0.400mmol/l0.400mmol/l0.025U/μl

3) The optimal reaction procedure for PCR is as follows:

94°C for 5 minutes → 94°C for 40 seconds → 55°C for 40 seconds → 72°C for 40 seconds → 72°C for 7 minutes → 30 cycles.

4) Identification and analysis of PCR products:

Take 5 μl of PCR product, add 1 μl of 6× loading buffer, electrophoresis in 2% agarose gel, observe the results under ultraviolet light, scan and save the results with an ultraviolet gel imager, and use ImageJ software for quantitative analysis.

7.5 Statistical method:

The obtained data were processed by SPSS11.5 statistical software package. Data are presented as mean ± standard deviation ( X±SD) indicates that the two groups of data were subjected to independent sample t test, and P<0.05 was considered statistically significant.

Claims (5)

1. the present invention relates to two albumen mGluR ₅Interaction between the/CAL and the cell expressing of CAL can promote mGluR ₅Express, and CAL albumen does not increase mGluR ₅Gene transcription.

2. two albumen: the mGluRs according to claim 1 ₅Interaction between the/CAL is characterized in that mGluR ₅Four amino acid of C-terminal, the Ser of 0 ,-1 ,-No. 2 position is changed to Ala, and the Leu of-No. 3 positions is changed to Ala, external protokaryon GST-Pulldown result: mGluR ₅-1 and-3 sudden changes can thoroughly destroy mutual do effect with CAL.

3. two albumen: the mGluRs according to claim 1 ₅Interaction between the/CAL is characterized in that two whole protein interactions of molecules in cell, at mGluR ₅Influence down CAL is distributed in endochylema by disperse and transfers to and being distributed on the cytolemma.

4. two albumen: the mGluRs according to claim 1 ₅Interaction between the/CAL is characterized in that the cell expressing of CAL can promote mGluR ₅Expression.

5. promote mGluR according to the described CAL of claim 4 ₅The mechanism of expressing, CAL albumen does not increase mGluR ₅Gene transcription.

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