CN105018564B - A kind of g protein coupled receptor vivo tracking 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 vivo tracking method and application

Technical field

The present invention relates to molecular imaging field, specifically Novel G protein-coupled receptor (G Protein-Coupled Receptor, GPCR) vivo tracking method, taking people's rhodopsin as a example, illustrating in its nonfunctional area is first intracellular In ring (Cytoplasmic Loop 1, CL1), insertion one molecule eGFP, successfully builds a kind of new rhodopsin^CL1- eGFP melts Hop protein, is distributed in the cell by fluoroscopic examination rhodopsin.

Background technology

G-protein-coupled receptor (G Protein-Coupled Receptor, GPCR) is the participation being present on cell membrane The important albumen of cell signalling, participates in multiple important physiological process, such as synapse transmission, the propagation of cell and differentiation and Perception of the sense of taste and tactile etc..GPCR is constituted by seven transbilayer helixs, by three intracellular rings and three extracellular rings between spiral Connect, its N-terminal is typically closely related with the slotting film of albumen, and its C-terminal is usually phosphorylation site and participates in swashing of downstream G-protein Live, therefore there is important effect.Due to the important function of GPCR, GPCR mutation can lead to the generation of multiple diseases.Due to GPCR is eukaryotic protein, and it typically can experience glycosylation processing and the folding process of complexity, only finally folds correct albumen The complete procedure being finally successively inserted into cell membrane from endoplasmic reticulum to Golgi body could be experienced.After GPCR is mutated, the lighter's albumen Conformation changes, and severe one can cause the false folding of albumen, leads to albumen correctly can not insert film, or even occurs in the cell Assemble, and cause the serious consequences such as cell death.Therefore, the molecular imaging of GPCR has important value, and it can react GPCR Whether correctly insert film, expression height and the abundant information such as whether assemble, this is for the related disease tool of research GPCR There is important meaning.

Traditional molecular imaging method dyes in a organized way or the method such as immunostaining, immunofluorescence.Pass through specific Dyestuff or fluorescent labeling specific antibody combining target molecule, finally carry out object observing molecule by chromogenic reaction or fluoroscopic examination Distribution.But said method be only applicable to fixing after tissue and cell it is impossible to be applied to active somatic cell or tissue.Fluorescence egg White appearance has greatly driven the development of molecules in vivo spike, and people carry out amalgamation and expression fluorescin and target protein, Distribution finally by fluorescence direct object observing albumen in active somatic cell or tissue.Green fluorescent protein (Green Fluorescent Protein, GFP) have that fluorescence intensity is big, interference factor is few, the advantages of stable luminescence, is extensively should at present A kind of fluorescin.Therefore, GPCR-GFP fusion protein is the first-selected strategy of GPCR molecules in vivo spike.

Rhodopsin (rhodopsin) is the light receptor being widely present on vertebrate retina rod cell cell membrane Albumen, it is made up of a molecule ligand retinal (retinal) and a molecule opsin (opsin), belongs to GPCR Class A Family.After light stimulation, there is isomery in retinal, promotes rhodopsin albumen that a series of conformation change occurs, and swashs The signal transduction in downstream alive, then produces a signal being transmitted to optic nerve, ultimately results in the generation of vision.Rhodopsin has altogether There are 348 residues, but wherein have being mutated of more than 100 site closely related with the generation of congenital oculopathy, such as retinal pigment Degeneration and nyctalopia etc., in addition to minority mutant rhodopsin has been furtherd investigate, most rhodopsin mutant Research also in blank out.The generation therefore exploring rhodopsin mutation with oculopathy still has huge development space.

Research rhodopsin distribution in the cell, transhipment and expression etc. are for exploring rhodopsin mutation and oculopathy There is important value.As led to modal rhodopsin P23H of North America human retinal pigment's degeneration, due to mutation Albumen afterwards there occurs that grave error folds, and is therefore trapped in endoplasmic reticulum it is impossible to by normal transport to cell membrane, institute So that normal light receptor protein function cannot be executed.The pathogenesis of P23H are obtained for extensively in cellular level and animal level Checking, this mainly has benefited from the foundation of rhodopsin-GFP fusion protein, people can by GFP in cell or retina Directly observe the distribution of rhodopsin.Prior art is typically connected to GFP the C-terminal (CT) of rhodopsin, its objective is to keep away Exempt from the interference to its N-terminal, thus reach not affecting rhodopsin^CTThe purpose of the slotting film of-GFP.But the C due to rhodopsin End is interfered, compared with wild type rhodopsin, rhodopsin^CTThe G-protein transduction function of-GFP and phosphorylation efficiency are big Big reduction, has huge gap with wild type rhodopsin.Obviously, rhodopsin^CT- GFP is not one and preferably merges Expression strategy.

The invention provides a kind of improved rhodopsin-eGFP amalgamation and expression strategy, that is, first rhodopsin's Nonfunctional area-the first intracellular ring (Cytoplasmic Loop 1, CL1) place inserts a molecules enhancing green fluorescent protein (enhanced Green Fluorescent Protein, eGFP), constructs a kind of new fusion protein rhodopsin^CL1- eGFP, thus be prevented effectively from the interference of the N-terminal to rhodopsin and C-terminal.The experiment proved that, rhodopsin^CL1- eGFP assumes similar glycosylation processing and expression to wild type rhodopsin, and can correctly transport To on cell membrane, it is therefore that a kind of preferable rhodopsin vivo tracking indicates probe.Further, since the fluorescence intensity of eGFP For GFP more than 6 times, therefore rhodopsin^CL1The fluorescence intensity of-eGFP is greatly enhanced, thus being more favorable for fluorescence inspection Survey the increase of tissue depth.In a word, the present invention is that research rhodopsin associated ophthalmopathy provides strong instrument, and is other The research of GPCR provides beneficial reference and new inspiration.

Content of the invention

The invention aims to providing a kind of new GPCR vivo tracking method, it is exactly specifically to pass through structure one Kind of new GPCR-eGFP fusion protein directly to observe distributing position in active somatic cell or tissue for the GPCR, expression with And state etc., thus providing valuable reference, and the structure for fusion protein for the research of the pathogenesis of GPCR relevant disease Build and new thinking is provided.It is characteristic of the invention that eGFP is inserted in the nonfunctional area of GPCR, thus avoiding the function to GPCR to produce Raw interference.

The concrete technical scheme realizing the object of the invention is：

A kind of Novel G protein-coupled receptor (G Protein-Coupled Receptor, GPCR) vivo tracking method, Feature is that the method is：Insert a molecules enhancing green fluorescent protein in the nonfunctional area of G-protein-coupled receptor (enhanced Green Fluorescent Protein, eGFP), realizes GPCR-eGFP amalgamation and expression, by fluoroscopic examination Distribution in active somatic cell for the GPCR.

A molecules enhancing green fluorescent protein (eGFP) is inserted in described nonfunctional area in G-protein-coupled receptor, be On the premise of not disturbing GPCR function, fluorescent labeling is realized to GPCR molecule, realize the live body real-time monitored of GPCR molecule.

A kind of new people's rhodopsin (rhodopsin) and enhanced green fluorescence protein (eGFP) fusion expression method, It is characterized in that the method is：Insert one in first intracellular ring region (Cytoplasmic Loop 1, CL1) of rhodopsin Molecule eGFP, builds rhodopsin^CL1-eGFP；

Specifically include following steps：

1) segmented-PCR method is utilized to introduce a simple point mutation K66M in first intracellular ring region of rhodopsin, that is, the The residue lysine of 66 sports methionine, introduces a unique Nde I restriction enzyme site in rhodopsin (CATATG)；

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 rhodopsin, so that it is determined that K66 is non-pass Key residue, 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 the 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, use BamH I Connect experiment with the experiment of Hind III double digestion and T4 ligase and rhodopsinK66M is transferred to pBAD24 from pCEP4 plasmid PBAD24-rhodopsinK66M plasmid is obtained on plasmid；

5) after Nde I single endonuclease digestion being carried out to pBAD24-rhodopsinK66M plasmid, then use calf intestinal alkaline phosphatase (Calf Intestine Alkaline Phosphatase, CIAP) is processed, and prevents pBAD24-rhodopsinK66M plasmid from existing Connect certainly connecting in experiment；

6) T4 ligase Connection Step 3) in Nde I enzyme action after eGFP gene and step 5) in through Nde I enzyme action PBAD24-rhodopsinK66M plasmid after processing with calf intestinal alkaline phosphatase, connection product is converted escherichia coli Top10 bacterial strain, then filters out the clone of the positive insertion of eGFP by bacterium colony PCR method, builds into an identification finally by being sequenced PBAD24-rhodopsinK66M-eGFP plasmid be pBAD24-rhodopsin^CL1- eGFP plasmid；

7), after being sequenced correctly, 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 experiments identify rhodopsin^CL1The expression of-eGFP.

Described rhodopsin^CL1- eGFP, it is to avoid the interference of the critical function area C-terminal to rhodopsin for the eGFP, is one Plant the rhodopsin fluorescent reporter protein after improving, be more suitable for the viviperception of rhodopsin associated ophthalmopathy.

The present invention compared with prior art, its advantage：

Fluorescin, as common label protein, can carry out amalgamation and expression with destination protein, thus by fluorescence to mesh Albumen be observed.General convergence strategy is N-terminal or C section fluorescin being inserted in destination protein, such as people Construct the fusion protein of multiple rhodopsin and fluorescin, such as rhodopsin-GFP albumen (Invest Ophthalmol Vis Sci 52(13)：9728-9736.), rhodopsin-eGFP or rhodopsin-mCherry fusion protein (PLoS One 7(1)：E30101.), they all achieve the fluorescent labeling to rhodopsin, therefore can observe in cell and tissue Rhodopsin wild type and the expression and distribution of mutant.But in above-mentioned fusion protein, fluorescin is both connected to The C-terminal of rhodopsin.But because the C-terminal of rhodopsin is phosphorylation site, and participate in downstream G-protein activation process, because This has important function.After fluorescin is connected on the C-terminal of rhodopsin, impact can be produced on the function of the latter, such as significantly drop The phosphorylation of low albumen and G-protein activation efficiency (J Biol Chem 276 (30)：28242-28251.).For solving above-mentioned difficulty Topic, present invention firstly provides the nonfunctional area position of rhodopsin insert eGFP it is therefore an objective to reduce to greatest extent right The interference of rhodopsin functional areas.First intracellular ring (Cytoplasmic Loop 1, CL1) of Rhodopsin is generally acknowledged that There is no important function, and through it is demonstrated experimentally that the mutant rhodopsinK66M of CL1 also presents and wild type The property that rhodopsinWT is similar to, therefore CL1 is a preferable eGFP on position.The present invention successfully constructs rhodopsin^CL1This new fusion protein of-eGFP, thus effectively prevent the interference to rhodopsin functional areas, be The fluorescin amalgamation and expression of rhodopsin and other GPCR provides new thinking.

Brief description

Fig. 1 is rhodopsin^CL1- eGFP structural representation；

Fig. 2 is pCEP4-rhodopsin^CL1- eGFP eukaryon expression plasmid schematic diagram；

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

Fig. 4 is clone's schematic diagram that bacterium colony PCR method screens eGFP positive insertion rhodopsin；

Fig. 5 identifies rhodopsin for Western Blot^CL1Expression schematic diagram in the HEK293S cell for-eGFP；

Fig. 6 is rhodopsin^CL1Fluoroscopic examination schematic diagram in the HEK293S cell for-eGFP.

Specific embodiment

More detailed experimental technique refers to embodiment.The present embodiment is for more fully understanding present disclosure, and It is not to limit the present invention with method in any form.

Embodiment 1：Build rhodopsin K66M mutant using segmented-PCR method

The purpose building rhodopsinK66M mutant has two, and one is to detect that whether the K66 of rhodopsin be Key residues, if eGFP can be inserted；Two is to introduce a unique Nde I restriction enzyme site in rhodopsin, with profit Insertion in eGFP.The present invention utilizes segmented-PCR method to build rhodopsinK66M mutant, and key step is as follows：

1) with laboratory existing pcDNA4/TO-rhodopsin plasmid as template, using the general upstream of pcDNA4/TO plasmid Primer CMV and downstream primer Rho (CL1) 2 (comprising mutational site) amplifies fragment 1, and its size is about 390bp.The primer As follows：

CMV：5’-CGCAAATGGGCGGTAGGCGTG-3’

Rho(CL1)2：5’CGTGCGCAGCTTCATATGCTGGACGGT-3’(Nde I)

2) with laboratory existing pcDNA4/TO-rhodopsin plasmid as template, using the general downstream of pcDNA4/TO plasmid Primer BGH and forward primer Rho (CL1) 1 (comprising mutational site) amplifies fragment 2, and its size is about 990bp.The primer As follows：

Rho(CL1)1：5’ACCGTCCAGCATATGAAGCTGCGCACG-3’(Nde I)

BGH：5’-ACTAGAAGGCACAGTCGAGGCT-3’

3) glue reclaim fragment 1 and fragment 2, then with this two fragments as template, is expanded using this two primers of CMV and BGH Increase the complete fragment (from CMV to BGH) rhodopsinK66M, size is about 1380bp (with reference to Fig. 3), and condition used is as follows：

The first step：

PCR condition used by the first step is：

After the first EOS, concentration is added to be each 0.5 μ l of CMV and BGH primer of 10pM in PCR pipe

Used by second step, PCR condition is：

4) glue reclaim 3) in PCR primer, then carry out BamH I and Hind III double digestion and process.In kind locate Reason pCEP4 plasmid.The above-mentioned digestion products of glue reclaim.Enzyme action system used is as follows：

37 DEG C, it is incubated 2h

5) utilize T4 ligase connect 4) in through double digestion process after pCEP4 plasmid and PCR primer, obtain pCEP4- Rhodopsin K66M plasmid.Enzyme action system used is as follows：

16 DEG C, it is incubated 16h

6) by 5) in connection product Transformed E .Coli Top10 bacterial strain, on the solid LB flat board containing ammonia benzyl cultivate.

7) picking single bacterium colony being inoculated in the LB liquid medium containing ammonia benzyl, is sequenced after extracting plasmid, is determined RhodopsinK66M is successfully prepared.

Embodiment 2：rhodopsin^CL1- eGFP is gene constructed

rhodopsin^CL1The gene constructed thinking of-eGFP is insertion eGFP gene in pCEP4-rhodopsinK66M, Obtain pCEP4-rhodopsin^CL1- eGFP (see Fig. 2).

Comprise the following steps that：

1) rhodopsinK66M genetic fragment is replaced pBAD24 plasmid from pCEP4 plasmid：First use BamH I and Hind III double digestion processes pCEP4-rhodopsinK66M and pBAD24 plasmid, then uses T4 ligase handle PBAD24 plasmid after rhodopsinK66M fragment and double digestion couples together, and obtains pBAD24-rhodopsinK66M plasmid.

2) with primer eGFP1 and eGFP2, two ends are amplified from pcDNA3.1-3 '-eGFP plasmid and carry NdeI enzyme action position The eGFP fragment (720bp about) of point, glue reclaim PCR primer.

Forward primer eGFP1：5’-GGAATTCCATATGGTGAGCAAGGGCGAGGAG-3’(Nde I)

Downstream primer eGFP2：5’-CCCTTAAGCATATGCTTGTACAGCTCGTCCAT-3’(Nde I)

3) Nde I enzyme processes the eGFP fragment amplifying and pBAD24-rhodopsinK66M plasmid respectively, then distinguishes Glue reclaim digestion products.Nde I enzyme action system is as follows：

37 DEG C, it is incubated 2h

4) calf intestinal alkaline phosphatase (Calf Intestine Alkaline Phosphatase, CIAP) is used to process Nde PBAD24-rhodopsinK66M plasmid after I enzyme action, glue reclaim genetic fragment.This step purpose is to prevent pBAD24- Certainly the connecting of rhodopsinK66M plasmid, thus improve eGFP plasmid insertion success rate.

CIAP processing procedure is as follows：

First it is incubated 15min at 37 DEG C, then 57 DEG C of incubation 15min

5) T4 ligase connect Nde I enzyme action after eGFP fragment and 4) in after Nde I enzyme action and CIAP ferment treatment PBAD24-rhodopsinK66M plasmid, obtaining pBAD24-rhodopsinK66M-eGFP is pBAD24-rhodopsin^CL1- EGFP plasmid.

6) bacterium colony PCR method identification direction of insertion in pBAD24-rhodopsinK66M plasmid for the eGFP, filters out forward direction The pBAD24-rhodopsin of insertion^CL1- eGFP plasmid.

Choose 12 single bacterium colonies to be tested, experimental result is shown in Fig. 4.Due to being that single endonuclease digestion connects experiment, eGFP can be positive Or in reverse insertion rhodopsin, swimming lane reference numerals represent single bacterium colony clone's numbering.Left half figure swimming lane 1-10 represents use The forward primer Op1 of rhodopsin and eGFP downstream of gene primer eGFP2 bacterium colony PCR result.Rho1 is about 200bp, and eGFP is about For 720bp, the therefore amplified fragments of Op1 and eGFP2 are about 920bp.Cloning for positive insertion of band can be amplified, result shows Show that bacterium colony 2,4,8 is positive insertion.Right half figure swimming lane 1-10 (italic) represents forward primer Op1 and eGFP with rhodopsin Upstream region of gene primer eGFP1 bacterium colony PCR result (be similarly 920bp about), can amplify band for reverse insertion clone, knot Fruit display bacterium colony 1,3,7,10 is reverse insertion.Twice PCR does not all occur band for inserting loser, that is, unloaded Body, is followed successively by bacterium colony 5,6,9.

7) the pBAD24-rhodopsin of positive insertion^CL1Through sequencing identification ,-eGFP plasmid proves that eGFP is properly inserted into The CL1 position of rhodopsin.

rhodopsin^CL1- eGFP gene order is shown in sequence table.This gene order total length is 1767bp, wherein comprises people Rhodopsin full-length gene (containing start codon and termination codon) 1047bp, and eliminate start codon and termination The eGFP gene 714bp of codon, finally also includes 6 bases that the Nde I restriction enzyme site at eGFP gene two ends introduces.

8) BamH I and Hind III double digestion and T4 ligase is used to test rhodopsin^CL1- eGFP genetic fragment from PBAD24 replaces on pCEP4 plasmid, obtains pCEP4-rhodopsin^CL1- eGFP (see Fig. 2).

Embodiment 3：pCEP4-rhodopsin^CL1Detection of expression in the HEK293S cell for-eGFP

pCEP4-rhodopsin^CL1Detection of expression in the HEK293S cell for-eGFP mainly comprises two aspects, and one is Rhodopsin is checked by Western Blot result^CL1The expression of-eGFP and glycosylation processing situation (with reference to Fig. 5), two It is that rhodopsin is detected by fluorescence microscope^CL1Whether whether-eGFP can normally send green fluorescence and gather in the cell Collection etc. (with reference to Fig. 6).Concrete operation step is as follows：

1) plasmid prepares：Prepare plasmid with plasmid extraction test kit, choose plasmid concentration and be not less than 200ng/ μ l, A₂₆₀/ A₂₈₀High-purity pCEP4-rhodopsin between 1.9～2.0^CL1- eGFP plasmid.Same method prepares pCEP4- RhodopsinWT and pCEP4-rhodopsinK66M plasmid is as comparison.

2) cell prepares：HEK293S twice is at least passed on as host cell, with DMEM (10% after choosing recovery FBS) culture medium carrys out cultured cells.When cell state is good, with 2 × 10⁵The density of cell/ml inoculates the hole 1 of one or six orifice plates To hole 4, it is possible to carry out plasmid transfection when cell confluency rate is 80%～90%.Before transfection, 24h changes liquid, and culture medium is still It is so DMEM (10%FBS).

3) plasmid transfection：Specifically transfection process is：1. 2 μ g DNA are diluted respectively using Opti-MEM I, that is, every hole adds 2 μ g DNA (only transfection hole 1, hole 2 and hole 3, hole 4 is as negative control), cumulative volume is 100 μ l.Gently mix；2. using Opti- The homemade transfection reagent (abbreviation PEI) based on Polyetherimide in MEM I dilution experiment room, needs altogether 18 μ g PEI, that is, Every hole need to add 6 μ g PEI (PEI: DNA=3: 1).Cumulative volume is 300 μ l.Gently mix；3. the PEI after dilution is added respectively Enter (DNA after 100 μ l dilutions and the PEI after 100 μ l dilution) in the DNA after dilution, gently mix；4. incubated at room 20- To form PEI-DNA complex in 30min；5. hole 1 adds the PEI-pCEP4-rhodopsin that 200 μ l are incubated^CL1- eGFP matter Grain complex, hole 2 adds the PEI-pCEP4-rhodopsinWT plasmid composite that 200 μ l are incubated, and hole 3 adds 200 μ l incubations Good PEI-pCEP4-rhodopsinK66M plasmid composite, hole 4 adds 200 μ l Opti-MEM I, as negative control.Gently Jog shakes flat board so as to be evenly distributed；6. cell is placed in 37 DEG C, 5%CO₂Damp and hot constant incubator culture.

4), after plasmid transfection 48h, with fluorescence microscope, fluoroscopic examination is carried out to six orifice plates.Blue light excites down, and hole 1 can send Substantially green fluorescence, and hole 2, the equal unstressed configuration in hole 3 and hole 4.

Specifically refer to Fig. 6.Wherein, left figure is rhodopsin^CL1- eGFP, right figure is that HEK293S is blank for control Cell.Result shows rhodopsin^CL1- eGFP sends obvious fluorescence, and is uniformly distributed in cell, does not form obvious egg White gathering bright spot.

5), after fluorescence observation terminates, harvest each hole cell, in case Western Blot detection.

6) Western Blot detection

Carry out sample treatment first：Every solencyte adds 100 μ l lysates and 1 μ l PMSF (100mM), and piping and druming is uniformly.So Afterwards using liquid nitrogen multigelation method cell lysis.Then 12,000g centrifugation 10min, takes supernatant.

Then carry out SDS-PAGE and run glue.Then by half-dried transferring film method, the protein delivery on SDS-PAGE glue is made to arrive On pvdf membrane, transferring film condition is 10V, 40min.

Then carry out antibody incubation, detailed process is：1. TBST wash film once after, with 5% defatted milk powder room temperature close 2h； 2. after TBST washes film, anti-(Mus source rhodopsin monoclonal antibody) incubated at room 1h；3., after TBST washes film three times, carry out HRP Labelling two resists (sheep anti mouse) incubated at room 1h；4., after two anti-incubations terminate, TBST washes film 6 times, each 5min.5. finally carry out ECL exposure colour developing.

If carrying out β-actin detection, film 30s need to be washed with 0.8M NaOH solution, to go rhodopsin's in membrane removal One is anti-anti- with two.Then carry out antibody incubation, step, such as the detection of rhodopsin, only resists one and changes anti-Mus source into β-actin monoclonal antibody.

Concrete outcome is with reference to Fig. 5.The size of rhodopsin is 36kD, due to mammalian cell expression eukaryotic protein, meeting Albumen is carried out with different degrees of glycosylation processing, therefore albumen assumes dispersivity band, can be according to western blot band The glycosylation machining status to judge rhodopsin for the distribution.Albumen use respectively monoclonal antibody 1D4 of rhodopsin and β- The monoclonal antibody of actin is identified, the content that swimming lane represents is as shown in the figure.Result shows, the expression of rhodopsinK66M Similar with rhodopsinWT, illustrate that K66 is not Key residues, be preferable eGFP insertion point.Secondly, rhodopsin^CL1- EGFP also can correctly express, and assumes the expression similar with rhodopsinWT and glycosylation processing situation, but due to rhodopsin^CL1- eGFP molecular weight is more much bigger than rhodopsin, migration rate relative reduction, therefore western blot band The overall movement at macromolecule.Wherein Control represents HEK293S blanc cell, the therefore monoclonal antibody of rhodopsin Band cannot be detected.

In a word, from experimental result as can be seen that rhodopsin^CL1- eGFP can be used as a preferable new rhodopsin Vivo tracking fusion protein.

<110>East China Normal University

<120>A kind of G-protein-coupled receptor vivo tracking method and application

<160> 1

<210> 1

<211> 1767

<212> DNA

<213>Artificial sequence

<220>

<223> rhodopsin^CL1- eGFP gene order

<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 ctctctgcat 1020

ggatacttcg tcttcgggcc cacaggatgc aatttggagg gcttctttgc caccctgggc 1080

ggtgaaattg ccctgtggtc cttggtggtc ctggccatcg agcggtacgt ggtggtgtgt 1140

aagcccatga gcaacttccg cttcggggag aaccatgcca tcatgggcgt tgccttcacc 1200

tgggtcatgg cgctggcctg cgccgcaccc ccactcgccg gctggtccag gtacatcccc 1260

gagggcctgc agtgctcgtg tggaatcgac tactacacgc tcaagccgga ggtcaacaac 1320

gagtcttttg tcatctacat gttcgtggtc cacttcacca tccccatgat tatcatcttt 1380

ttctgctatg ggcagctcgt cttcaccgtc aaggaggccg ctgcccagca gcaggagtca 1440

gccaccacac agaaggcaga gaaggaggtc acccgcatgg tcatcatcat ggtcatcgct 1500

ttcctgatct gctgggtgcc ctacgccagc gtggcattct acatcttcac ccaccagggc 1560

tccaacttcg gtcccatctt catgaccatc ccagcgttct ttgccaagag cgccgccatc 1620

tacaaccctg tcatctatat catgatgaac aagcagttcc ggaactgcat gctcaccacc 1680

atctgctgcg gcaagaaccc actgggtgac gatgaggcct ctgctaccgt gtccaagacg 1740

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.