CN102863535A - Fusion protein GPR45-Gilalpha, its coding gene and application - Google Patents

Abstract

The invention discloses a fusion protein GPR45-Gilalpha, its coding gene and an application. The provided fusion protein is show as the following (a) or (b):, (a) is the protein composed of an amino acid sequence showed as a sequence 1 in a sequence table; (b) is the protein derived from the sequence 1 with G protein antigen activity and formed by the amino acid sequence in (a), in which, one or more residues are replaced and/or deleted and/or added. The individual GPR45 or Gilalpha protein can not combine after disengaging the life state (living cell environment), a protein compound capable of being identified by the Gilalpha protein specific antibody can be combined with the help of cell inner environment. The provided fusion protein has the antigen activity of Gilalpha protein after disengaging the life state (living cell environment), and can be taken as an in vitro screening system for screening the candidate compound or micromolecule which is taken as a medicine(investigating whether the candidate compound or the micromolecule can combine with the fusion protein or not).

Description

Fusion rotein GPR45-Gi1 α and encoding gene thereof and application

Technical field

The present invention relates to a kind of fusion rotein GPR45-Gi1 α and encoding gene and application.

Background technology

G albumen (G-protein) is a kind of gtp binding protein that plays an important role in the Cellular Signaling Transduction Mediated approach, and by α, three different subunits of beta, gamma form.G albumen has the GTP enzymic activity, and α is arranged, other G albumen three classes of beta, gamma tripolymer G albumen, low-molecular-weight monomer small G-protein and high molecular.

G protein coupled receptor (G-protein coupled receptor) is a kind of and cell surface receptor tripolymer G albumen coupling, contains 7 transmembrane domains, is the receptor superfamily of the maximum found so far, and its member has more than 1000.By activating coupling G albumen, start different signal transduction pathways and cause various biological effects behind g protein coupled receptor and the ligand binding.The interactions such as g protein coupled receptor and its aglucon-hormone, neurotransmitter and cytohormone, and serve as the molecular switch that promotes or close a lot of functional biological processes.G protein coupled receptor is played the part of pivotal player in the various diseases such as blood pressure regulation, inflammation and mental illness.

The key of modern new drug research and exploitation at first is to seek, determine and preparation drug screening target-molecule medicine target.Drug target refers to medicine effect binding site in vivo, comprises the biomacromolecules such as gene locus, acceptor, enzyme, ionic channel, nucleic acid.Select to determine that novel active drug target is the top priority of new drug development.Found so far total about 500 as the medicine target spot, and the acceptor of G-albumen coupling (GPCR) target spot accounts for the wherein overwhelming majority of acceptor.High-flux medicaments sifting is one of most important instrument of developing drugs commitment.

Summary of the invention

The purpose of this invention is to provide a kind of fusion rotein GPR45-Gi1 α and encoding gene and application.

Protein provided by the invention is following (a) or (b):

(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of (a) through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the protein of being derived by sequence 1 with G proteantigen activity.

In order to make the protein in (a) be convenient to purifying, can connect upper label as shown in table 1 at N-terminal or the C-terminal of the protein of (a).

The sequence of table 1 label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned (b) but in the protein synthetic, also can synthesize first its encoding gene, carry out again biological expression and obtain.The encoding gene of the protein in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The gene of encoding said proteins also belongs to protection scope of the present invention.

Described gene specifically can be following 1) to 4) in arbitrary described dna molecular:

1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal 1 to 2178 Nucleotide;

2) dna molecular shown in the sequence 2 in the sequence table;

3) under stringent condition with 1) or 2) dna molecular with albumen of G proteantigen activity of the dna sequence dna hybridization that limits and coding;

4) with 1) or 2) dna sequence dna that limits has the dna molecular that 90% above homology and coding have the albumen of G proteantigen activity.

The recombinant expression vector, expression cassette, reconstitution cell (transgenic cell line) or the recombinant bacterium that contain described gene all belong to scope of the present invention.

Available existing expression vector establishment contains the recombinant expression vector of described gene.Described expression vector also can comprise 3 ' end untranslated zone of foreign gene, namely comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, can add any enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can use separately or be combined with other promotor; In addition, when using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.Carry out purifying for the ease of described albumen, can process used recombinant expression vector, as adding affinity tag etc.

Described recombinant expression vector specifically can be described gene is inserted the recombinant plasmid that the multiple clone site of pFASTBac1 plasmid obtains.

Described recombinant expression vector also can be described recombinant plasmid is imported intestinal bacteria DH10Bac bacterial strain, by the baculovirus plasmid that contains described gene of recombinating and obtaining in the bacterium.

The present invention also protects a kind of method for preparing described albumen, comprises the steps:

(1) with described baculovirus plasmid transfection insect cell, collect supernatant after cultivating, be P1 for virus liquid;

(2) with described P1 for the virus liquid infected insect cell, obtain described albumen after the cultivation.

In described step (1) and/or the described step (2), described insect cell specifically can be the sf9 cell.

In the described step (2), the infective dose of described infection specifically can be MOI=5, and the incubation time of described cultivation specifically can be 72h.

In the described step (1), the incubation time of described cultivation specifically can be 72 hours.

The present invention also protects a kind of test kit of expressing described albumen, comprises intestinal bacteria DH10Bac bacterial strain, insect cell and contains the recombinant expression vector of described gene.Described recombinant expression vector specifically can be described gene is inserted the recombinant plasmid that the multiple clone site of pFASTBac1 plasmid obtains.Described insect cell specifically can be the sf9 cell.

Described albumen can be used for drug screening.

Fusion rotein GPR45-Gi1 α provided by the invention can by the identification of the specific antibody of Gi1 α albumen, illustrate that fusion rotein has good antigenic activity and due higher structure.Independent GPR45 or Gi1 α albumen have broken away from life state (viable cell environment) and can't combine, and need to by intracellular environment, just can be combined into by the albumen composition of Gi1 α protein specific antibody identification.And fusion rotein provided by the invention, break away from life state (viable cell environment) and namely had the antigenic activity of Gi1 α albumen, can be used as the in-vitro screening system, candidate's compound or small molecules as medicine screened (can investigate it be combined with fusion rotein).

Description of drawings

Fig. 1 is the structural representation of pFASTBac1 plasmid.

Fig. 2 is the pcr amplification product electrophorogram of the encoding gene of the encoding gene of Gi1 α albumen and GPR45 albumen; 1:DNA Marker D2000 (2000,1000,750,500,250,100); The encoding gene of 2:Gi1 α albumen (about 1065bp); The encoding gene of 3:GPR45 albumen (about 1119bp).

Fig. 3 is the pcr amplification product electrophorogram of the encoding gene of fusion rotein GPR45-Gi1 α; 1:DNA Marker D2000 (2000,1000,750,500,250,100); 3: the encoding gene of fusion rotein GPR45-Gi1 α.

Fig. 4 is for containing the photo of the positive colony of restructuring rod granule pBacmi d-GPR45-Gi1 α by blue hickie screening on the LB flat board.

Fig. 5 identifies electrophorogram for the PCR of restructuring rod granule pBacmid-GPR45-Gi1 α; 1:DNAMarkerD15000 (15000,10000,7500,5000,2500,1000); 2: the pcr amplification product of restructuring rod granule pBacmid-GPR45-Gi1 α.

Fig. 6 is the form of sf9 cell transfecting P1 after for virus liquid; 1: normal sf9 cell (negative control); 2: the sf9 cell of transfection P1 after for virus liquid 72h.

Fig. 7 is the Western-blot detected result of GPR45-Gi1 alpha fusion protein among the embodiment 2.

Fig. 8 is film total protein under the different infective dose conditions (containing the GPR45-Gi1 alpha fusion protein) Western-blot detected result; 1:MOI=1; 2:MOI=2; 3:MOI=5; 4:MOI=10; 5: normal sf9 cell.

Fig. 9 is different infection time condition film total proteins (containing the GPR45-Gi1 alpha fusion protein) Western-blot detected results; 1:24h; 2:48h; 3:72h; 4:96h.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.

Total DNA extraction reagent TRIzol:Invitrogen company.RT-PCR test kit: TaKaRa company.Newborn calf serum: GIBCO-BRL company.TransFast TM transfection reagent: Promega company.IPTG: Beijing is glad through biotech company of section.The multifunctional dna purifying reclaims test kit: Bo Maide company.Sf900II nutrient solution: GIBCO-BRL company.Anti-Gi1 α protein polyclone antibody G α i-1/2/3 (N-20): SANTACRUZ company.The anti-goat of the rabbit of horseradish enzyme labelling two is anti-: Bioisystech Co., Ltd of middle China fir Golden Bridge.The super quick luminescent solution of Super ECL Plus: Puli's lema gene technology company limited.

PMD18-T carrier: TaKaRa company.

PFASTBac1 plasmid: Invitrogen company, catalog number: 10359-016.

HepG2 cell: ATCC product, numbering: HB8065.

Sf9 cell: ATCC product, numbering: CRL-1711.

Intestinal bacteria (E.coli) DH10Bac bacterial strain: intestinal bacteria (E.coli) DH10Bac bacterial strain is for being purchased bacterial strain, itself contains baculovirus shuttle back and forth rod granule (Bacmid) and helper plasmid; Invitrogen company, catalog number: 10361012.

The structure of embodiment 1, recombinant plasmid

One, the structure of recombinant plasmid pMD18T-Gi1 α

1, (GenBank Accession number:NM 002069 claims again Gi1 α albumen with reference to the GNAI1 albumen among the GenBank; The encoding gene of this albumen is present in the HepG2 cell, and do not have intron) encoding gene design pair of primers: Gi1 α-P1 (upstream primer, underscore marks EcoR I recognition sequence) and Gi1 α-P2 (downstream primer, underscore mark HindIII recognition sequence).

Gi1α-P1：5′-CG GAATTCATGGGCTGCACGCTGAGCG-3′；

Gi1α-P2：5′-CCC AAGCTTTTAAAAGAGACCACAATC-3′。

2, extract the genomic dna of HepG2 cell, genomic dna as template, is obtained pcr amplification product (electrophorogram of pcr amplification product is seen the swimming lane 2 of Fig. 2) with the primer of Gi1 α-P1 and Gi1 α-P2 composition to carrying out pcr amplification.

3, the pcr amplification product with step 2 is connected with the pMD18-T carrier, obtains recombinant plasmid pMD18-T-Gi1 α.

Recombinant plasmid pMD18-T-Gi1 α is checked order, sequencing result shows, in the pMD18-T carrier, inserted the sequence 2 of sequence table from the DNA (sequence 1 of code sequence tabulation is from the albumen shown in N-terminal the 373rd to the 726 amino acids residue, i.e. Gi1 α albumen) shown in 5 ' terminal the 1117th to 2181 Nucleotide.

Two, the structure of recombinant plasmid pMD18-T-GPR45

1, with reference to (the GenBank Accession number:NM_007227 of the GPR45 albumen among the GenBank; The encoding gene of this albumen is present in the HepG2 cell, and do not have intron) encoding gene design pair of primers: GPR45-P1 (upstream primer, underscore marks EcoR I recognition sequence) and GPR45-P2 (downstream primer, underscore mark HindIII recognition sequence).

GPR45-P1：5′-CG GAATTCATGGCCTGCAACAGCACGT-3′；

GPR45-P2：5′-CCC AAGCTTAACCGCAGACTGGTTTTC-3′。

2, extract the genomic dna of HepG2 cell, genomic dna as template, to carrying out pcr amplification, is obtained pcr amplification product (electrophorogram of pcr amplification product is seen the swimming lane 3 of Fig. 2) with the primer of GPR45-P1 and GPR45-P2 composition.

3, the pcr amplification product with step 2 is connected with the pMD18-T carrier, obtains recombinant plasmid pMD18-T-GPR45.PMD18-T-GPR45 checks order to recombinant plasmid, sequencing result shows, (sequence 1 of code sequence tabulation is from the albumen shown in N-terminal the 1st to the 372 amino acids residue, i.e. GPR45 albumen from the DNA shown in 5 ' terminal the 1st to 1116 Nucleotide for the sequence 2 of having inserted sequence table in the pMD18-T carrier.

Three, the structure of recombinant plasmid pFASTBac1-GPR45-Gi1 α

1, take recombinant plasmid pMD18-T-GPR45 as template, to carrying out pcr amplification, obtains pcr amplification product with the primer of GPR45-P1 and GPR45-P2 ' composition.

GPR45-P2′：5′- GCGTGCAGCCCATAACCGCAGACTGGTTTTC-3′。

Pcr amplification condition: 94 ℃ of 45s, 52 ℃ of 30s, 72 ℃ of 1.2min, 25 circulations.

2, take recombinant plasmid pMD18-T-Gi1 α as template, the primer that forms with Gi1 α (45)-P3 ' and Gi1 α-P2 obtains pcr amplification product to carrying out pcr amplification.

Gi1α(45)-P3′：5′- CCAGTCTGCGGTTATGGGCTGCACGCTGAGC-3′。

Pcr amplification condition: 94 ℃ of 45s, 55 ℃ of 30s, 72 ℃ of 1.2min, 25 circulations.

3, with after the pcr amplification product balanced mix of the pcr amplification product of step 1 and step 2 as template, primer with GPR45-P and Gi1 α-P2 composition obtains pcr amplification product (electrophorogram of pcr amplification product is seen the swimming lane 3 of Fig. 3) to carrying out pcr amplification.

94 ℃ of 45s of pcr amplification condition, 55 ℃ of 30s, 72 ℃ of 1.2min, 25 circulations.

4, with the pcr amplification product of restriction enzyme EcoR I and HindIII double digestion step 3, reclaim enzyme and cut product.

5, with restriction enzyme EcoR I and HindIII double digestion pFASTBac1 plasmid (structural representation is seen Fig. 1), reclaim carrier framework (about 4775bp).

6, the enzyme of step 4 is cut the carrier framework connection that product is connected with step, obtained recombinant plasmid pFASTBac1-GPR45-Gi1 α.It is as follows according to sequencing result recombinant plasmid pFASTBac1-GPR45-Gi1 α to be carried out structrual description: take the pFASTBac1 plasmid as skeleton carrier, between the EcoR of skeleton carrier I and HindIII restriction enzyme site, inserted the DNA shown in the sequence 2 of sequence table (DNA is the encoding gene of fusion rotein GPR45-Gi1 α shown in the sequence 2 of sequence table, the fusion rotein GPR45-Gi1 α shown in the sequence 1 of code sequence tabulation).

Expression and the evaluation of embodiment 2, fusion rotein GPR45-Gi1 α

Adopt the Bac-to-Bac baculovirus insect cell expression system to realize the efficient fast expression of fusion rotein GPR45-Gi1 α.Principle is as follows: after will containing the recombinant plasmid importing intestinal bacteria DH10Bac bacterial strain of fusion rotein encoding gene, the locus specificity swivel base occurs in recombinant plasmid under the helper plasmid effect, the fusion rotein encoding gene inserts baculovirus and shuttles back and forth in the rod granule, obtains the rod granule of recombinating; With the restructuring rod granule transfection insect sf9 cell behind the purifying, the cell after the transfection can be secreted recombinant baculovirus; With recombinant baculovirus transfection sf9 cell, can realize the expression of fusion rotein again.

One, the acquisition of restructuring rod granule

1, in 1.5ml Ep pipe, adds 100ul intestinal bacteria DH10Bac bacterial strain competent cell and 1ng (about 5 μ l) recombinant plasmid pFASTBac1-GPR45-Gi1 α, the tapped tube wall makes it mixing, place 30min in the mixture of ice and water, then transfer to heat-shocked 45s in 42 ℃ of water-baths, take out fast and be positioned over 2min in the mixture of ice and water.

2, in the Ep of step 1 pipe, add 900 μ l LB nutrient solutions, (37 ℃, 225rpm) shaking culture 5h in shaking table (4～6h all can).

3, the cell suspension of step 2 is coated on the LB culture medium flat plate (containing kantlex 50 μ g/ml, gentamicin 7 μ g/ml, tsiklomitsin 10 μ g/ml, X-gal 100 μ g/ml, IPTG 40 μ g/ml) after with the dilution of LB nutrient solution, placed 37 ℃ of incubators to cultivate 36h (24～48h all can).

4, show blue white two kinds of spots (seeing Fig. 4) on the LB flat board of step 3, choose a blue clone in contrast, 10 white clones of picking are scoring on the new LB culture medium flat plate (containing kantlex 50 μ g/ml, gentamicin 7 μ g/ml, tsiklomitsin 10 μ g/ml, X-gal 100 μ g/ml, IPTG 40 μ g/ml) simultaneously, are inverted overnight incubation in 37 ℃ of incubators; The single white of picking is cloned into 3ml LB nutrient solution (containing kantlex 50 μ g/ml, gentamicin 7 μ g/ml, tsiklomitsin 10 μ g/ml), 37 ℃ of shaking table 275rpm (250～300rpm all can) jolting spend the night (about 12 hours).

5, the extraction of restructuring rod granule pBacmid-GPR45-Gi1 α

1. get the culture of 1.5ml step 4 in 1.5ml Ep pipe, the centrifugal 1min of 14000g, vacuum take-off move and abandon supernatant, stand upside down on thieving paper.

2. add 0.3ml solution I (Tris-HCl 15mM, EDTA 10mM, RNaseA 100 μ g/ml; Filtration sterilization and 4 ℃ of preservations) blow and beat gently re-suspended cell with the rifle head.

3. add 0.3ml solution II (NaOH 0.2M, 1%SDS; Filtration sterilization and 4 ℃ of preservations), mixing gently.Incubated at room 5min.

4. slowly add 0.3ml solution III (potassium acetate 3M, adjust pH to 5.5; Filtration sterilization and 4 ℃ of preservations), limit edged light shaking when observing a large amount of white precipitate, places on ice 8min (5～10min all can) with sample.

5. the centrifugal 10min of 14000g room temperature transfers to supernatant in the Ep pipe that contains Virahol, and the Ep that reverses gently pipe is mixing for several times, places on ice 8min (5～10min all can).

6. the centrifugal 15min of 14000g room temperature moves and abandons supernatant, adds 70% ethanol 0.5ml in each pipe precipitation, and upset Ep pipe is washing and precipitating for several times.

7. the centrifugal 5min of 14000g room temperature moves and abandons supernatant, makes to be deposited in seasoning in the air.

8. add 40ul TE solution, rap the pipe end, make it dissolving ,-20 ℃ save backup.

6, the PCR of restructuring rod granule pBacmid-GPR45-Gi1 α identifies

Use for baculovirus shuttle back and forth in the rod granule in the complementary district of lacZ α little-primer at the two ends in attTn7 site carries out PCR to (PUCF and PUCR) to restructuring rod granule (solution that step 5 is preserved) to be identified, can cause pcr amplification product to increase that (the shuttle back and forth pcr amplification product of rod granule of original baculovirus is about 2.2kp, and the pcr amplification product of restructuring rod granule that inserts the encoding gene of fusion rotein GPR45-Gi1 α is contemplated to about 4.4kb if insert foreign gene.

PUC F (pUC/M13 upstream primer): 5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ';

PUC R (pUC/M13 downstream primer): 5 '-TCACACAGGAAACAGCTATGAC-3 '.

PCR condition: 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 5min, 35 circulations.

Pcr amplification product carries out 0.8% agarose gel electrophoresis, sees Fig. 5 (swimming lane 2 represents pcr amplification product).The PCR product is about 4.4kb, and is identical with theoretical value, illustrates that the encoding gene of fusion rotein GPR45-Gi1 α successfully has been cloned in the baculovirus shuttle vectors, and restructuring rod granule pBacmid-GPR45-Gi1 α successfully constructs.

Two, the preparation of recombinant baculovirus maternal plant

1, restructuring rod granule transfection sf9 cell

(1) the day before yesterday is pressed 9 * 10 with the sf9 cell in transfection ⁵Individual/hole is inoculated in 6 orifice plates, makes cell attachment.

(2) by the sf9 cell of TransFast TM transfection reagent (Promega company) with the restructuring rod granule pBacmid-GPR45-Gi1 α transfection step (1) of step 1 acquisition, by specification operates, and hatches for 27 ℃.

Transfection method is specific as follows: obtain solution A (5 μ l restructuring rod granule pBacmid-GPR45-Gi1 α is mixed with 100 μ lsf900II nutrient solutions) and solution B (5 μ l TransFast TM transfection reagents are mixed with 100 μ l sf900II nutrient solutions), solution A and solution B mixing, obtain the A-B mixed solution, incubated at room 30min (15-45min all can); Clean every porocyte once with 2ml sf900II nutrient solution; With A-B mixed solution and 800 μ l sf900II nutrient solutions mixing gently, add in the cell hole after cleaning; Hatch 5h for 27 ℃; Move the supernatant liquor of abandoning in the cell hole, add the 2mlsf900II nutrient solution, hatch for 27 ℃.

2, the separation of viral maternal plant and storage

When virus infection sign (about 72h after the transfection) appears in the cell of step 1, upper strata nutrient solution (about 2ml) is transferred in the aseptic Ep pipe, the centrifugal 5min of 500g (to remove cell and large fragment), collect supernatant liquor, be P1 for virus liquid, wherein contain the viral maternal plant (P1) of recombinant baculovirus.P1 is stored in aseptic EP pipe for virus liquid.

3, plaque measuring virus titer

Determine P1 for the titre of virus liquid by the plaque that fixing monolayer culture forms, concrete grammar is as follows:

1. every hole adds 1 * 10 in 6 orifice plates ⁶The sf9 cell of individual logarithmic phase places 27 ℃ of incubators to cultivate at least 1h, makes cell attachment, inhales and abandons supernatant.

2. the thawing of 40ml 4% agar gel microwave oven is placed on 70 ℃ of water-baths and prevents that it from condensing; A 100ml Erlenmeyer flask and one bottle of 1.3 * sf900II nutrient solution are placed 40 ℃ of heating in water bath.

3. in the sterilization centrifuge tube, P1 is carried out 10 times of gradient dilutions for virus liquid with the sf900II nutrient solution, obtain each diluent (virus inoculation liquid).

4. each cell hole in step 6 orifice plates is 1. inoculated respectively each virus inoculation liquid, every hole 2ml is hatched 1h for 27 ℃, inhales and abandons supernatant.

5. prepare the plaque covering liquid: rapidly 1.3 * sf900II nutrient solution of 30ml step in 2. and 10ml step 4% agar gel in is 2. added in the Erlenmeyer flask of step in 2., mixing places 40 ℃ of water-baths until use gently.

6. the every hole of each cell hole in step 6 orifice plates is 4. added 2ml step plaque covering liquid 5., places 27 ℃ of incubators to be inverted and cultivate, observe also counting plaque every day, until its continuous 2 days numbers constant till.

7. tire (pfu/ml) with the good counting region 3-9 of each Kongzui on 6 orifice plates Plaque assay virus inoculation liquid, calculation formula is as follows: virus titer (pfu/ml)=1/ extension rate * every hole plaque number * 1/ (every hole inoculation ml number).

The result shows, P1 for the titre of virus liquid between 1 * 10 ⁶～1 * 10 ⁷Between.

4, the amplification of viral maternal plant

Adopt following method to carry out the amplification of viral maternal plant: to adopt six orifice plates, every hole inoculation 2 * 10 ⁶Individual sf9 cell (vigor＞97%), in each hole, add 2ml P1 for virus liquid, (virus that nonspecific infection 48h collects can increase 100 times nearly to hatch 48h, the viral quality that surpasses the 48h collection is lower) the rear supernatant of collecting, the centrifugal 5min of 500g, collect supernatant liquor, be P2 for virus liquid.P2 is stored in aseptic EP pipe for virus liquid.

The P2 virus liquid is increased again as stated above, obtain P3 for virus liquid, detect P3 for the titre of virus liquid, method is with step 3, virus titer liquid P3 for the titre of virus liquid between 1 * 10 ⁷～1 * 10 ⁸Between.

Three, the preparation of contrast virus liquid

1, replaces recombinant plasmid pFASTBac1-GPR45-Gi1 α to carry out step 1 with pFASTBac 1 plasmid (being empty plasmid pFASTBac 1), obtain contrasting rod granule.

2, replace restructuring rod granule pBacmid-GPR45-Gi1 α to carry out step 2 with the contrast rod granule, obtain P1 for the contrast virus liquid.

Four, the acquisition of fusion rotein GPR45-Gi1 α and evaluation

1, at 25cm ²Inoculate 1 * 10 in the Tissue Culture Flask ⁷The sf9 cell of individual logarithmic phase places at least 1h (making cell attachment) of 27 ℃ of incubators, adds P1 for virus liquid, makes MOI equal 5 (definition of MOI is the quantity of average each cell infection virus); Place 27 ℃ of incubators to cultivate 72h.The photo of cultivating after 72 hours is seen Fig. 6, and the visible cell pathology is apparent in view, show as cell proliferation slowly, form swelling becomes circle and refractivity strengthens, cell is easier to be floating.

2, collect the sf9 cell of transfection P1 after for virus liquid 72h, adopt film/hydrophobin and hydrophilic albumen two-phase separation system's test kit (Puli's lema gene technology company limited) extraction cytolemma total protein, operate by the explanation of test kit.

3, the cytolemma total protein that step 2 is extracted carries out SDS-PAGE electrophoresis (12% separation gel, 5% concentrated glue; The 60V constant voltage is run concentrated glue, and the 90V constant voltage is run separation gel).

4, the running gel that step 3 is obtained carries out Western Blot, primary antibodie is anti-Gi1 α protein polyclone antibody G α i-1/2/3 (N-20) (working concentration is dilution in 1: 500), the anti-goats two of two anti-rabbits for the horseradish enzyme labelling anti-(working concentration is dilution in 1: 2000), in the darkroom, carry out chemoluminescence with the super quick luminescent solution of Super ECL Plus, detect the expression of fusion rotein.

The results are shown in Figure 7, fusion rotein GPR45-Gi1 α can be identified by the specific antibody of Gi1 α albumen.Independent GPR45 or Gi1 α albumen have broken away from life state (viable cell environment) and can't combine, and need to by intracellular environment, just can be combined into by the albumen composition of Gi1 α protein specific antibody identification.And fusion rotein provided by the invention, break away from life state (viable cell environment) and namely had the antigenic activity of Gi1 α albumen, can be used as the in-vitro screening system, candidate's compound or small molecules as medicine screened (can investigate it be combined with fusion rotein).

5, replace P1 to carry out step 1 to step 4 for virus liquid for the contrast virus liquid P1, do not demonstrate hybridization signal.

The optimization of embodiment 3, expressing fusion protein condition

One, the optimization of infective dose

1, at 25cm ²Inoculate 1 * 10 in the Tissue Culture Flask ⁷The sf9 cell of individual logarithmic phase places at least 1h (making cell attachment) of 27 ℃ of incubators, adds P1 for virus liquid, and 4 infective doses (MOI=1,2,5 or 10) are set respectively; Place 27 ℃ of incubators to cultivate 72h.

2, collect the sf9 cell of transfection P1 after for virus liquid 72h, adopt film/hydrophobin and hydrophilic albumen two-phase separation system's test kit (Puli's lema gene technology company limited) extraction cytolemma total protein.

3, the cytolemma total protein that step 2 is extracted carries out SDS-PAGE electrophoresis (12% separation gel, 5% concentrated glue; The 60V constant voltage is run concentrated glue, and the 90V constant voltage is run separation gel).

4, the running gel that step 3 is obtained carries out Western Blot, primary antibodie is anti-Gi1 α protein polyclone antibody G α i-1/2/3 (N-20) (working concentration is dilution in 1: 500), the anti-goats two of two anti-rabbits for the horseradish enzyme labelling anti-(working concentration is dilution in 1: 2000), in the darkroom, carry out chemoluminescence with the super quick luminescent solution of Super ECL Plus, detect the expression of fusion rotein.

The results are shown in Figure 8.When MOI=5, fusion protein expression is the highest.

Two, the optimization of infection time

1, at 25cm ²Inoculate 1 * 10 in the Tissue Culture Flask ⁷The sf9 cell of individual logarithmic phase places at least 1h (making cell attachment) of 27 ℃ of incubators, adds P1 for virus liquid, makes MOI equal 5; Place 27 ℃ of incubators to cultivate, sampling carry out step 2 after 24 hours, 48 hours, 72 hours and 96 hours respectively.

2, collecting cell adopts film/hydrophobin and hydrophilic albumen two-phase separation system's test kit (Puli's lema gene technology company limited) to extract the cytolemma total protein.

3, the cytolemma total protein that step 2 is extracted carries out SDS-PAGE electrophoresis (12% separation gel, 5% concentrated glue; The 60V constant voltage is run concentrated glue, and the 90V constant voltage is run separation gel).

4, the running gel that step 3 is obtained carries out Western Blot, primary antibodie is anti-Gil α protein polyclone antibody G α i-1/2/3 (N-20) (working concentration is dilution in 1: 500), the anti-goats two of two anti-rabbits for the horseradish enzyme labelling anti-(working concentration is dilution in 1: 2000), in the darkroom, carry out chemoluminescence with the super quick luminescent solution of Super ECL Plus, detect the expression of fusion rotein.

The results are shown in Figure 9.In the time of 72 hours, fusion protein expression is the highest.

Claims (10)

1. protein is following (a) or (b):

(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of (a) through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and the protein of being derived by sequence 1 with G proteantigen activity.

2. the gene of coding claim 1 described albumen.

3. gene as claimed in claim 2, it is characterized in that: described gene is following 1) to 4) in arbitrary described dna molecular:

1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal 1 to 2178 Nucleotide;

2) dna molecular shown in the sequence 2 in the sequence table;

3) under stringent condition with 1) or 2) dna molecular with albumen of G proteantigen activity of the dna sequence dna hybridization that limits and coding;

4) with 1) or 2) dna sequence dna that limits has the dna molecular that 90% above homology and coding have the albumen of G proteantigen activity.

4. the recombinant expression vector, expression cassette, reconstitution cell or the recombinant bacterium that contain claim 2 or 3 described genes.

5. recombinant expression vector as claimed in claim 4 is characterized in that: the recombinant plasmid that described recombinant expression vector obtains for the multiple clone site with claim 2 or 3 described genes insertion pFASTBac1 plasmids.

6. recombinant expression vector as claimed in claim 4, it is characterized in that: described recombinant expression vector is for the described recombinant plasmid of claim 5 is imported intestinal bacteria DH10Bac bacterial strain, by the baculovirus plasmid that contains claim 2 or 3 described genes of recombinating and obtaining in the bacterium.

7. a method for preparing the described albumen of claim 1 comprises the steps:

(1) with the described baculovirus plasmid transfection of claim 6 insect cell, collect supernatant after cultivating, be P1 for virus liquid;

(2) with described P1 for the virus liquid infected insect cell, obtain the described albumen of claim 1 after the cultivation.

8. method as claimed in claim 7, it is characterized in that: described insect cell is the sf9 cell; In the described step (1), the incubation time of described cultivation is 72 hours; In the described step (2), the infective dose of described infection is MOI=5, and the incubation time of described cultivation is 72h.

9. a test kit of expressing the described albumen of claim 1 comprises intestinal bacteria DH10Bac bacterial strain, insect cell and claim 4 or 5 described recombinant expression vectors.

10. the application of the described albumen of claim 1 in drug screening.

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