CN1990859B - Method of nucleus receptor ligand binding domain protein fusion expression on bacteriophage surface - Google Patents
Method of nucleus receptor ligand binding domain protein fusion expression on bacteriophage surface Download PDFInfo
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- CN1990859B CN1990859B CN2005101124835A CN200510112483A CN1990859B CN 1990859 B CN1990859 B CN 1990859B CN 2005101124835 A CN2005101124835 A CN 2005101124835A CN 200510112483 A CN200510112483 A CN 200510112483A CN 1990859 B CN1990859 B CN 1990859B
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Abstract
The invention discloses a bacteriophage, comprising capsid and bacteriophag nucleic acid inside capsid. The surface of said capsid is fused with out nuclear receptor or receptor ligand domain in nuclear receptor. The bacteriophage can be used to screen the substance combined with nuclear receptor or receptor ligand domain in nuclear receptor, and is characterized by high screen sensitivity and efficiency, and low amount of needed protein for screen.
Description
Technical field
The present invention relates to biological technical field, relate to that nuclear receptor that external source is arranged or the proteic phage of nuclear receptor LBD are merged in a kind of capsid surface and described nuclear receptor or nuclear receptor LBD protein fusion expression method at phage surface.
Background technology
Phage display technique is widely used in protein structure and functional study after it is set up.Foreign protein, peptide section, cDNA or genomic fragment be by being illustrated in phage surface with the phage ghost gene fusion expression, constitutive protein storehouse or peptide library at random.In theory, by biological " elutriation " (biopanning) process can be used for screening and target protein, acceptor, cell surface receptor and chemical small molecules (medicine) interactional albumen or polypeptide are arranged.This technology is mainly used in following each side: 1. screen the antibody of specific combination, perhaps by in conjunction with the triage techniques engineered antibody, improve the avidity and the bonded specificity of antibody; 2. study the interaction of protein-protein, be used for determining the conjugated protein or polypeptide of acceptor or part; 3. determine function determinant or paratope; 4. enzyme or proteic orthogenesis etc.But phage display expressed protein in the past all belongs to the protein of solubility class, does not relate to the foreign protein that is difficult to solubility expression as yet.So far, still there be not the relevant report of insoluble albumen presenting and expressing on phage capsid surface.
Nuclear receptor (nuclear receptor) is a class transcription factor superfamily, by the family member cDNA that has been found that and coded amino acid whose sequence are relatively found, structural similitude between them, quite conservative on evolving, nuclear receptor be activated after part combines, act on the particular responses element HRE (hormone respond sequence) of its target gene, thereby the mediation special genes is transcribed.
Nuclear receptor structurally has the common feature, generally comprise the transcription activating district that 6 functional zone of A～F: A/B contains in the district a non-dependence of part and be called AF-1 (Activation Function-1), by regulating and control goal gene with the interaction of other transcription factors such as auxilliary activation factor, auxilliary repressor; The C district be the most conservative DNA land (DNA binding domain, DBD), the homology between the various nuclear receptor DBD reaches more than 90%, is made of two zinc fingerses, the mediation acceptor combines with target gene; The D district is a hinge area, connects DNA land and ligand binding domain; The E district is that (Ligand Binding Domain LBD), is the long hydrophobic region that is made of about 250 amino acid to ligand binding domain, contains a nuclear positioning area and the transcription activating district (AF-2) that acceptor relies on, and is relatively large.Homology between the different nuclear receptor LBD is about 15%, though homology is less than the DNA land, but according to the LBD space structure data of having resolved, the three-dimensional structure of all nuclear receptor LBD is identical substantially, all form by 12 α spirals, and all have binding partner, receptor dimerization and in conjunction with the function of cofactor.The F district is positioned at the carboxyl terminal of nuclear receptor in addition.(Peroxisome Proliferators-Activated Receptors PPARs) belongs to the nuclear receptor family member to peroxisome proliferation-activated receptors, plays an important role in energy metabolism, cell proliferation and the differentiation of cell, inflammatory reaction.PPARs has three kinds of hypotype: α, β, γ in Mammals.The same with other nuclear receptor superfamilies member, PPAR is made of six structural areas (A-F) too.PPAR is with after its aglucon combines, with another activated nuclear receptor retinoid receptor (retinoid X receptor, RXR) form heterodimer, be incorporated into specific PPAR response element PPRE on the target gene (Peroxisome Proliferator Response Elements), thereby start transcribing of target gene.
Troglitazone (troglitazone) is by activating PPAR γ acceptor in the nuclear receptor family, suppress the TNF-a Role in Plant Signal Transduction, can strengthen insulin sensitivity effectively, be first thiazolidine dione compounds of being ratified by FDA as the type ii diabetes curative, but this medicine is withdrawn from market because of untoward reactions such as hepatotoxicities.Pioglitazone (pioglitazone) and rosiglitazone (rosiglitazone) are such medicines that is used for the treatment of type ii diabetes of present approved listing, they also can reduce insulin resistance effectively, but, equally also there is serious adverse effects in this type of medicine, as cause weight increase, may increase the weight of the state of an illness of heart failure patient etc.
Therefore, research and screening nuclear receptor ligands have crucial meaning for the medicine of metabolic diseases such as the better treatment of exploitation diabetes, obesity.Traditional based on acceptor and aglucon affinity analyzing and the technology and the method for the micromolecular compound in-vitro screening molecular model of setting up all need to obtain the soluble purifying protein of q.s, and need isolation identification bonded aglucon or albumen.But for the protein as nuclear receptor or this poorly soluble of nucleus receptor ligand binding domain (LBD), these two problems become the huge obstacle of development drug screening molecular model.
Although people attempt to adopt the whole bag of tricks to screen the small molecules part of nuclear receptor at present, but, still be difficult to set up a very effective screening molecular model because the ligand binding domain (LBD) of nuclear receptor is a kind of protein of insoluble and the soluble purifying protein that is difficult to obtain q.s.Therefore, press for a kind of method of exploitation in the prior art, acquisition can be used to screen the recombinant protein of nuclear receptor or its part, to satisfy ligand screening to lipidated protein and deliquescent requirement, so that find the medicine of metabolic diseases such as better treatment diabetes, obesity early.
Summary of the invention
The phage that the object of the present invention is to provide a kind of capsid surface to merge nuclear receptor or nucleus receptor ligand binding domain that external source is arranged.
The purposes of the nuclear receptor that the present invention also aims to provide described capsid surface to merge external source is arranged or the phage of nucleus receptor ligand binding domain.
The present invention also aims to provide and utilize the fusion of described surface to have the nuclear receptor of external source or the phage of nucleus receptor ligand binding domain to carry out the nuclear receptor ligands method for screening.
In a first aspect of the present invention, a kind of phage is provided, constitute by capsid and the bacteriophage nucleic acid that is positioned at capsid, merge on the capsid surface of described phage the nuclear receptor of external source or the ligand binding domain of nuclear receptor.
In a preference of the present invention, the capsid protein pD of described phage goes up to merge the nuclear receptor of external source or the ligand binding domain of nuclear receptor.
In another preference, described nuclear receptor or nucleus receptor ligand binding domain are the transcription factors that a class is distributed in the ligand dependent in endochylema or the nuclear, influence genetic transcription by the response element that is incorporated into target gene; Described nuclear receptor or nucleus receptor ligand binding domain are made of the transcription activating district of the non-dependence of part of N end, DNA land, hinge region, the conservative relatively ligand binding domain that contains nuclear positioning area and ligand-dependent transcription activating district that conservative zinc fingers constitutes usually; The ligand binding domain of the transcription activating district of N end and DNA land and C end is relatively independent on space structure.
In a preference of the present invention, described nuclear receptor is selected from down group: 1. steroid hormone receptor; 2. pth receptor; 3. orphan nuclear receptor.
In another preference, the nuclear receptor of steroid hormone receptor family includes but not limited to: glucocorticosteroid (GR), mineralocorticoid (MR), androgen receptor (AR), estrogen receptor (ER) and progesterone receptor (PR).
In another preference, the nuclear receptor of pth receptor family includes but not limited to: pth receptor (TR), retinoid receptor (RAR) and Vitamin D Receptor (VDR).
In another preference, described orphan nuclear receptor is meant the nuclear receptor of not finding its corresponding physiology part, includes but not limited to: subfamilies such as RXRs, LXRs, FXRs, PPARs, COUP-TFs, ERRs, HNFs.
In another preference, described PPARs includes but not limited to: PPAR α, PPAR β, PPAR γ, preferred, described PPARs is PPAR γ.
In another preference, described phage capsid is made of head and afterbody, and the ligand binding domain of described nuclear receptor or nuclear receptor is positioned at the capsid head.
In another preference, described nuclear receptor land is the ligand binding domain (LBD) of PPAR γ.
In a preference of the present invention, described phage is selected from down group: lambda particles phage, T7 phage, T4 phage, filobactivirus M13, fd and f1.
In another preference, described phage is a virulent phage, as λ, and T7, T4 phage.
In another preference, described phage is lambda particles phage, and is preferred, and described phage is the lambda particles phage that contains capsid protein pD.
In a preference of the present invention, described bacteriophage nucleic acid coding phage capsid protein and the nuclear receptor of external source or the ligand binding domain of nuclear receptor.
In a second aspect of the present invention, the purposes of described phage is provided, be used to screen ligand binding domain bonded material with nuclear receptor or nuclear receptor.
In another preference, described ligand binding domain bonded material with nuclear receptor or nuclear receptor is selected from down group: part, antibody, polypeptide or micromolecular compound.
In a third aspect of the present invention, the method for the ligand binding domain bonded material of a kind of screening and nuclear receptor or nuclear receptor is provided, comprise step:
(a) candidate substances is contacted with second phage with first phage respectively, wherein said first phage is the phage of merging the ligand binding domain that nuclear receptor or nuclear receptor are arranged on the foregoing capsid, and described second phage is ligand binding domain and the phage same kind of described phage that does not have the external source nuclear receptor or the nuclear receptor of fusion on the capsid surface;
(b) observe the situation that combines of candidate substances and first phage and second phage,
Wherein, be incorporated into first phage but debond is exactly a ligand binding domain bonded material with nuclear receptor or nuclear receptor in the candidate substances of second phage.
In a preference of the present invention, described ligand binding domain bonded material with nuclear receptor or nuclear receptor is selected from part, antibody, polypeptide or micromolecular compound.
In another preference, observe combining of candidate substances and first phage and second phage in the step (b) by the following method: phage and candidate substances in conjunction with after, reclaim phage or measure the bonded phage titre by the competition wash-out by directly infecting the host bacterium.
In a fourth aspect of the present invention, a kind of nucleic acid molecule is provided, it contains following element: promotor, operon, replication site, selection markers site, bacteriophage coat protein coding region, phage foreign protein coding region; And, the nuclear receptor of described phage foreign protein coding region encoding exogenous or the ligand binding domain of nuclear receptor.
In another preference, there are promotor (tac promoter), operon (lac operator) in phage foreign protein coding region and bacteriophage coat protein upstream of coding region, and the required replication site of plasmid amplification and antibiotic-screening mark (Ampicillin) etc.
In another preference, the capsid protein of described bacteriophage coat protein coding region coding phage.
In another preference, described phage foreign protein coding region links to each other with the proteic coding region of pD.
In a preference of the present invention, a kind of fusion rotein also is provided, comprise first part and second section, wherein first part is the capsid protein pD of phage; Second section is the ligand binding domain of described nuclear receptor or nuclear receptor.
In another preference, the ligand binding domain of described nuclear receptor or nuclear receptor is the ligand binding domain (PPAR γ LBD) of PPAR γ or PPAR γ.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown pCGMT-LBD, p171-LBD, pET-hPPGLBD plasmid construction mode chart.
Fig. 2 has shown the pulsating pcr amplification product electrophoretogram of PPAR γ LBD that is cloned on the p171Bio3, and wherein, control represents blank.
Fig. 3 has shown the EcoRI restriction enzyme mapping of plasmid p171Bio3 and the new plasmid p171-LBD that makes up.
Fig. 4 has shown that IPTG induces the protein electrophoresis collection of illustrative plates of PPAR γ LBD in host bacterium BL21 (DE3).S represents the lysate supernatant, and P represents lysate centrifugation.Albumen mainly exists with the inclusion body form, and accounts for thalline ultrasonication postprecipitation thing more than 90%.
Fig. 5 has shown that the Western blot that fusion rotein LBD-gp3 and pD-LBD express analyzes.
Fig. 6 has shown that Western blot display part pD-LBD fusion rotein is with in the soluble form expressive host mycetocyte matter.
Fig. 7 A has shown that anti-PPAR LBD antibody analysis shows that PPAR LBD is assembled in the lambda particles phage; Fig. 7 B has shown that anti-pD antibody analysis shows that PPAR LBD is assembled in the lambda particles phage.
Fig. 8 has shown that λ p171-LBD phagocytosis physical efficiency is incorporated into the enzyme plate hole of anti-PPAR γ LBD antibody sandwich, illustrates that PPAR γ LBD presenting and expressing is on the lambda particles phage surface.
Embodiment
Research and test that inventor's process is extensive and deep, the capsid surface that discovery can be illustrated in phage with nuclear receptor or its ligand binding domain of insoluble, thus taken the lead in obtaining merging on the capsid phage that nucleus receptor ligand binding domain is arranged.Described phage can be used for screening the material that combines with nuclear receptor or its ligand binding domain.
The inventor is at the beginning of research, be faced with nuclear receptor protein be difficult to soluble-expression, phage display express indissoluble albumen still do not have any prior art report as a means of use for reference and phage display to the foreign protein assembling size difficulty of restriction to some extent, through long term studies and test repeatedly, the position that has solved presenting and expressing is determined, the foreign protein local expression is assembled and problem such as solvability enhancing.In process of the test, the inventor selects nucleus receptor ligand binding domain as preferential presenting and expressing, and preferably be fit to the filamentous phage coat protein gp3 of high molecular weight protein presenting and expressing and lambda particles phage coat protein pD as carrier proteins, solvability by the external source recombinant protein of relatively expressing, lytic phages such as discovery λ are fit to the presenting and expressing of insoluble nuclear receptor more, thereby the capsid surface with nuclear receptor or its ligand binding domain of insoluble is illustrated in phage has obtained nuclear receptor or the nucleus receptor ligand binding domain recombinant protein of amalgamation and expression at phage ghost.Described amalgamation and expression can be used for screening the material that combines with nuclear receptor or its ligand binding domain at the recombinant protein of phage surface.
As used herein, described " nuclear receptor " albumen are meant that a class has the regulatory factor albumen common constructional feature, that homology is higher.The mechanism of causing a disease of metabolic diseases such as described nuclear receptor protein and diabetes, obesity is closely related, is a kind of useful drug target therefore.
As used herein, described " ligand binding domain of nuclear receptor " is meant a structural region that is present in nuclear receptor, has the ability of the part of syncaryon acceptor.The three-dimensional structure of all nuclear receptor LBD is identical substantially, all form by 12 α spirals, and all have binding partner, receptor dimerization and in conjunction with the function of cofactor.In preference of the present invention, (the particularly ligand binding domain of PPAR γ) of having selected to belong to nuclear receptor family is as demonstration example, because structural similarity, the member of other nuclear receptor family (or their ligand binding domain) also can be demonstrated the surface that is expressed in described phage.
As used herein, described " with the ligand binding domain bonded material of nuclear receptor or nuclear receptor " comprise ligand binding domain bonded material any and nuclear receptor or nuclear receptor, include but not limited to: part, antibody, polypeptide or micromolecular compound etc.
Should be understood that numerals such as composition consumption shown in embodiment or experiment material and method, reaction conditions or the employed numeral of present specification content is about numerical value.Therefore, unless dated especially in the literary composition, the above-mentioned digital parameters of this specification sheets is approximation, and it can be changed according to the result of the present invention who desires to try to achieve.And these parameters are not the principle that is used for limiting with the present patent application claim equalization, but use resulting preferable data under the normal running technology.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.Preferable implementation method described in the literary composition and material are only done the usefulness of demonstration.
Major advantage of the present invention is:
(1) with nuclear receptor or its ligand binding domain presenting and expressing on the capsid surface of phage, merge the phage that nuclear receptor or its ligand binding domain are arranged on the capsid thereby made up.
(2) nuclear receptor or its ligand binding domain can be demonstrated and be expressed in phage surface, and the host bacterium ability that infects that can directly utilize phage combines the detection index of situation with other material as the screening nuclear receptor.And, adopt phage of the present invention to carry out the screening substances that combines with nuclear receptor or its ligand binding domain, have the high and high characteristics of screening efficiency of screening sensitivity, only need very low expressing quantity to screen.
(3) with the capsid protein amalgamation and expression of nuclear receptor or its ligand binding domain and phage, can promote the solubility of the fusion rotein of expression, overcome since nuclear receptor or its ligand binding domain because of belonging to the problem that insoluble albumen is difficult to screen its binding substance.
I. material and experimental technique
Use following material and method in the specific embodiment of the invention, below the material and the method that are provided be not to be used for limitation of the present invention.
Bacterial strain and plasmid
DH5α：F
-，φ80d?lacZΔM15，Δ(lacZYA-argF)U169，deoR，recA1，endA1，hsdR17(rK
-mK
+)，phoA，supE44，λ
-，thi-1，gyrA96，relA1
BB4：supF58，supE44，hsdR514，galK2，galT22，trpR55，metB1，tonA，ΔlacU169/F’[proAB+，lacI
q，lacZΔM15Tn10(tet
r)]
BL21(DE3)：F-，ompT，hsdSB(rB-mB-)，gal(λcI857，ind1，Sam7，nin5，lacUV5-T7gene1)，dcm(DE3)
People PPAR γ 2 gene clones (Gnebank number: NM_015869) from liver cDNA library; PGEX-2T is an Amersham Pharmacia company product; Plasmid pET-21a (+) derives from Navogen company; Plasmid p171 Bio3 makes up referring to document (Santi E, Capone S, Mennuni C, Lahm A, Tramontano A, Luzzago A, Nicosia A:Bacteriophage lambda display of complex cDNA libraries:a newapproach to functional genomics.J.Mol.Biol.2000,296:497-508.); The pCGMT phagemid makes up referring to document (Gao C, Lin CH, Lo CH, Mao S, Wirsching P, Lerner RA, Janda KD:Making chemistry selectable by linking it to infectivity.Proc.Natl.Acad.Sci.U.S.A 1997,94:11777-11782); Lysogenic phage λ D1180 (λ Dam15 b538 cIts857 nin5 Sam100) preparation reference literature (Eguchi A, Akuta T, Okuyama H, Senda T, Yokoi H, Inokuchi H, Fujita S, Hayakawa T, Takeda K, Hasegawa M, Nakanishi M:Protein transduction domain ofHIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells.J.Biol.Chem.2001,276:26204-26210).
Enzyme and chemical reagent
Required chemical reagent and biochemical reagents all available from Shanghai China Shun biotechnology company limited, are the Amresco product in the experimentation.The required adjuvant of animal immune is the Sigma product; The pvdf membrane of Western blot (Immobilon-P) is a Millipore company product; The HRP-sheep anti-mouse igg is a Calbiochem company product.
Various restriction enzymes, T4 dna ligase, dna molecular amount standard are available from TaKaRa biotechnology company limited; The dNTP, pfu archaeal dna polymerase that is used for PCR can lottery industry biotech company available from the Shen, Shanghai, and the synthetic and sequencing of PCR primer can lottery industry biotech company be finished by the Shen, Shanghai.
Key instrument equipment
PTC-150 PCR instrument (U.S. MJ company); Low-temperature and high-speed whizzer TL-100 (U.S. Beckman company); Shaking culture shaking table (the special Analytical Instrument Co., Ltd of Shanghai fine jade); Electro-heating standing-temperature cultivator (Nereid restrains experimental installation company limited on the Shanghai); JY-882 ultrasonic cell disruptor (the new sesame biotechnology research in Shanghai institute); DY-A electrophoresis apparatus (west, Shanghai Bath biotechnology development corporation, Ltd.); The small-sized electrophoresis chamber of DY-24D; DY-1 electrophoretic blotting groove (Jiangsu Province Xinghua City analytical instrument factory).
Substratum and buffering solution
Microbial culture and phage titre are measured required substratum configuration with reference to molecular cloning experiment guide (SambrookJ., Russell DW: molecular cloning: lab guide, Cold Spring Harbor, NY.:Cold Spring HarborLaboratory Press; 2001.); Required TE, PBS buffered soln in the experimentation, SDS-PAGE electrophoresis related solution, Western blot detects related solution, and the configuration of other required solution is with reference to the molecular cloning experiment guide.
Main experimental methods
Conventional PCR, enzyme are cut, are connected, operations such as the preparation of competent cell, plasmid conversion and extraction are with reference to molecular cloning experiment guide (Sambrook J., Russell DW: molecular cloning: lab guide, Cold SpringHarbor, NY.:Cold Spring Harbor Laboratory Press; 2001.).
One. construction of recombinant plasmid and checking
1.PPAR the pulsating pcr amplification of γ LBD
CDNA (GenBank accession number NM_015869) with PPAR γ LBD is a template, utilize two primers of PPLBD_Fwd1 and PPLBD_Rev1 to carry out pcr amplification, amplification PPAR γ LBD segment is connected gained recombinant plasmid called after pET-hPPGLBD with plasmid pET-21a (+).Utilize PPLBD_Fwd2 and PPLBD Rev2 to carry out pcr amplification for primer, the PCR segment of gained PPAR γ LBD is connected with plasmid p171Bio3, gained recombinant plasmid called after p171-LBD.
Wherein, primer PPLBD_Fwd1:5 '-AG
GGATCCGTGGGGATGTCTCATAATGC-3 ' (BamHI); Primer PPLBD_Rev1:5 '-ACGC
GTCGACGTACAAGTCC TTGTAGAT-3 ' (SalI); Primer PPLBD_Fwd2:5 '-AG
AC TAGTGTGGGGATGTCTCATAATGC-3 ' (SpeI); Primer PPLBD_Rev2:5 '-TGTT
GCGGCCGCTACAAGTCCTTGTAGATC-3 ' (NotI).And the underscore indication is a restriction enzyme site shown in the bracket in the above-mentioned primer sequence.
2.PCR the amplification segment is connected, transforms and verify with phage vector
Introduced different restriction enzyme sites by the PCR primer at the pulsating two ends of PCR, the vector plasmid with same double digestion behind the PCR product double digestion is connected, and transforms host's bacterium competence cell, and concrete grammar is with reference to the molecular cloning experiment guide.Select the clone who grows on the plate, be inoculated in the 3ml LB substratum, after 37 ℃ of overnight incubation, extract plasmid with test kit, enzyme is cut checking.Cut checking through enzyme and connect correct recon in-70 ℃ of preservations.
Two. expression and the analysis of exogenous protein in intestinal bacteria
Conversion contains the host bacterium of foreign protein plasmid, and in 50ml LB (penbritin 60 μ g/ml, 0.1% glucose), 37 ℃ vibrate to OD
600Be about 0.4～0.5, adding IPTG is that 1mM induces exogenous protein expression to final concentration, 30 ℃ of shaking culture 8 hours.
8000rpm collected the thalline after IPTG induces in centrifugal 10 minutes, was suspended in (50mMTris pH8.0,150mM NaCl in the ultrasonic buffered soln, 1mM EDTA), ultrasonication cell under the ice bath, centrifugal 15 minutes of 4 ℃ of 10000rpm separate soluble part and throw out in the ultrasonic disruption thing.
Each component after centrifugal is separated through the 10%SDS-PAGE denaturing polyacrylamide gel electrophoresis.The tetrabromophenol sulfonphthalein electrophoresis when separating than the bottom, taking-up gel electrophoresis is finished after, 500mA constant current transferase 12 hour under the cooling conditions is transferred to albumen on the PVDF blotting membrane.
After electrophoretic transfer is finished, take out pvdf membrane, and confining liquid (2%BSA, TBS) sealing is spent the night, and rinsing is 3 times under TBST damping fluid (TBS, the 0.05%Tween 20) room temperature, each 10 minutes; Anti-as one with anti--LBD (preparation method as follows) or anti--pD serum (preparation method as follows) (confining liquid dilution in 1: 1000), 37 ℃ of jog temperature were bathed 1 hour; Repeat rinsing three times, each 10 minutes; Add HRP-sheep anti-mouse igg two anti-(confining liquid dilution in 1: 5000), 37 ℃ of jog temperature were bathed 1 hour; After repeating rinse step, add under the HRP substrate reactions liquid chamber temperature and develop the color, big water gaging flushing color development stopping when waiting to develop the color appropriateness, ddH
2The preservation of drying in the shade behind the O water rinse is preserved in 4 ℃.
Three. polyclonal antibody is anti--preparation of LBD and anti--pD
Conversion has the e. coli bl21 (DE3) of plasmid pET-hPPGLBD to utilize IPTG to induce PPAR γ LBD to express, ultrasonic treatment, and PPAR γ LBD albumen mainly exists with insoluble inclusion body, can go out PPAR γ LBD albumen by centrifugation.The intestinal bacteria BB4 that transforms plasmid p171Bio3 induces the pD protein expression with IPTG, and thalline ultrasonic treatment thing utilizes SDS-PAGE glue to separate, and cutting pD protein band place gel was isolated pD albumen in-70 ℃ of room temperature multigelations after electrophoresis was finished.
Utilize the PPAR γ LBD albumen and the pD albumen difference immune mouse of purifying.Immunity is five times altogether, gets blood from the mouse canthus, collects serum.After mensuration is tired, after the packing in-70 ℃ of preservations.Antibody mediated immunity method and titration are with reference to the molecular cloning experiment guide.
Four. foreign protein is in the presenting and expressing and the analysis of phage surface
In preference of the present invention, nuclear receptor or its ligand binding domain are blended on the capsid of lambda particles phage, concrete presenting and expressing and analytical procedure are as follows:
1. the preparation of lysogen BB4 (λ D1180)
Prophage λ D1180 (λ Dam15 b538 cIts857 nin5 Sam100) makes up and prepares aforesaid reference (Eguchi A, Akuta T, Okuyama H, Senda T, Yokoi H, Inokuchi H, Fujita S, Hayakawa T, Takeda K, Hasegawa M, Nakanishi M:Protein transduction domain ofHIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells.J.Biol.Chem.2001,276:26204-26210).The λ D1180 phage dilution 10 that obtains
4Doubly, get the fresh logarithmic phase intestinal bacteria BB4 of 1 μ l and 200 μ l and mix, 30 ℃ leave standstill 30 minutes after, on flat board, rule 32 ℃ of cultivations with the inoculating needle bacterium liquid that takes a morsel.Inferior daily toothpick picking mono-clonal is inoculated in respectively on two flat boards, and corresponding one by one.A flat board places 32 ℃, and another flat board places 42 ℃ to cultivate 12 hours, selects 42 ℃ of bacterium colonies of not growing of 32 ℃ of growths, is the BB4 lysogen that is integrated with prophage λ D1180, is called BB4 (λ D1180).
2. lysogen BB4 (λ D1180) competent preparation and conversion
The competent preparation of BB4 (λ D1180) is similar to other bacterial strain, but thalli growth, culture temperature are 32 ℃.Plasmid p171Bio3, recombinant plasmid p171-LBD transform BB4 (λ D1180) competence, and conversion process and other bacterium conversion process are similar, but need not 42 ℃ of heat shocks behind the ice bath, directly add the LB liquid culture based on 32 ℃ of recoveries.Behind the coated plate in 32 ℃ of cultivations.
3. lambda particles phage increases and titer determination
The cultivation of lambda particles phage, amplification and titre (plaque-forming unit pfu, plaque forming unit) are measured reference molecule cloning experimentation guide.
4. lambda particles phage binding analysis
Coated elisa plate hole after mouse-anti PPAR γ LBD resists (anti--LBD serum, on the preparation method sees) to dilute 100 times with coating buffer more, every hole 100 μ l, totally 6 holes.Aphylactic mice serum dilutes 100 times in contrast with coating buffer too simultaneously, adds in 3 enzyme plate holes, and 4 ℃ of bags are spent the night.Next day every hole add 200 μ l confining liquid PBST (2%BSA, 0.05%Tween-20) 37 ℃ the sealing 2 hours.In three holes of anti--LBD bag quilt, add 1 * 10 then
8λ p171Bio3 phage, and the λ pD-LBD phage of the same quantity of adding (merging the lambda particles phage that LBD is arranged on the pD albumen) in other three holes also adds 1 * 10 simultaneously in the hole of no antibody mice serum bag quilt
8λ pD-LBD phage, 37 ℃ of temperature were bathed 1 hour.Remove phage solution, the every hole of enzyme plate is with 200 μ l scavenging solution (PBS, 0.05%Tween-20,10mM MgSO
4) clean three times, clean once (10mM MgSO with PBS solution at last
4).Add logarithmic phase bacterium BB4 then, 37 ℃ of temperature were bathed 30 minutes.Utilize above-mentioned lambda particles phage titer determination method to determine to be combined in phage number in the enzyme plate hole.The gained result carries out statistical analysis, utilizes t check (student ' s paired t test) to determine to have or not significant otherness between two groups of data, thinks to have the significance difference opposite sex on the statistics between the two when p＜0.05.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
II. specific embodiment
The structure of foreign protein PPAR γ LBD expression plasmid
The synoptic diagram of plasmid construction is seen Fig. 1.Plasmid p171Bio3 includes the gene of coding lambda particles phage head capsid protein pD, and the pcr amplification segment of PPAR γ LBD is connected 3 ' end of pD protein gene.Utilize primer PPLBD_Fwd2 and PPLBD_Rev2, amplify PPAR γ LBD segment, gene segment is about 900bp.Agarose electrophoresis shows that segmental size of PCR product and expection match (Fig. 2), and the plasmid of structure is p171-LBD.The p171-LBD plasmid is cut checking with the EcoRI enzyme, and enzyme is cut into slices disconnected and size matches (Fig. 3) with expection, illustrates that the connection of foreign protein PPAR γ LBD gene segment has entered among the plasmid p171Bio3
Utilize the PPAR γ LBD of primer PPLBD_Fwd1 and PPLBD_Rev1 amplification, be connected with plasmid pET-21a (+) with the SalI restriction enzyme site by BamHI, the plasmid of structure is pET-hPPGLBD.Same amplification PCR segment is connected with pCGMT, and the plasmid of structure is pCGMT-LBD.
The expression of PPAR γ LBD in intestinal bacteria and anti--LBD and anti-pD Polyclonal Antibody Preparation
Behind the pET-hPPGLBD plasmid transformation escherichia coli BL21 (DE3), IPTG induces PPAR γ LBD to express, bacterium liquid supernatant (S) after ultrasonication is handled and precipitation (P) are carried out SDS-PAGE and are analyzed, the result as shown in Figure 4, at the about 31kDa of molecular weight place significant protein band (the 4th road) is arranged in the cellular lysate liquid precipitate, and the corresponding position soluble recombining protein content in the supernatant lysate fewer (the 3rd road), illustrate PPAR γ LBD in intestinal bacteria mainly with insoluble inclusion body formal representation.PPAR γ LBD has reached more than 95% in whole inclusion body protein, and the centrifugation inclusion body protein is used for immune mouse.
After the p171Bio3 vector plasmid transformed BB4, IPTG induced the pD protein gene expression on the plasmid, and after the SDS-PAGE electrophoretic separation, rubber tapping back multigelation reclaims pD albumen, extracting to albumen be used for immune mouse.
Utilize PPAR γ LBD that purifying obtains and pD albumen repeatedly behind the immune mouse, after anti--LBD that obtains respectively and anti-pD polyclonal antibody serum utilize indirect elisa method to measure to tire, standby.
The lambda particles phage display systems is more suitable for the proteic presenting and expressing in PPAR γ LBD than filobactivirus gp3 system
PPAR γ LBD respectively amalgamation and expression phagemid pCGMT coding the gp3 protein N terminal and the pD PROTEIN C end of plasmid p171Bio3 coding, IPTG induces back expressed fusion protein LBD-gp3 (55kDa) and pD-LBD (46kDa) respectively.Thalline utilizes ultrasonic disruption after the IPTG inducible protein is expressed.The ultrasonic treatment thing carries out the SDS-PAGE electrophoresis, utilizes anti-LBD polyclonal antibody to carry out Western blot and detects (Fig. 5).T, S, P represent the albumen in the cleer and peaceful cellular lysate liquid precipitate on fusion rotein bacterial protein, the cellular lysate liquid respectively among the figure.As seen the gp3-LBD fusion rotein of solubility mainly is distributed in the precipitation, exists with the form of insoluble inclusion body.And inductive pD-LBD fusion rotein is present in the last cleer and peaceful precipitation of ultrasonic treatment thing simultaneously under the same terms, and the pD-LBD albumen of some exists with soluble form.Because helping its assembling, the foreign protein of solubility is shown in the phage particle.
Therefore from then on as can be seen, the lambda particles phage display systems is more suitable for the proteic presenting and expressing in PPAR γ LBD than filobactivirus gp3 system.
Part is expressed in host's mycetocyte matter with soluble form with the PPAR γ LBD that pD albumen merges
Further to the pD-LBD fusion rotein in the host bacterium, induce with non-inductive condition under expression carry out Western blot and analyze.
The results are shown in Figure 6, show as the carrier p17 1Bio3 transformed bacteria of blank induce with non-inductive condition under all do not have the proteic expression of pD-LBD.And the bacterial clone that transforms recombinant plasmid p171-LBD has new raw albumen pD-LBD solubility expression (the 6th road) at the 46kDa place under non-inductive condition, and the corresponding position does not have band appearance (the 7th road) in the cracking precipitation.After IPTG induces, pD-LBD Expression of Fusion Protein amount significantly increases (the 8th road) in the lysate supernatant, simultaneously because the solvability of pD albumen raising foreign protein is limited, along with IPTG induces a large amount of pD-LBD Expression of Fusion Protein in back, fusion rotein mainly is present in (the 9th road) in host's mycetocyte matter with insoluble inclusion body form.Wherein the pD-LBD of solubility accounts for 30% of its total expression amount.This result further illustrates pD albumen and can promote to a certain extent to keep dissolved state with the albumen of its fusion, though most of fusion rotein of expressing is still to have (the 9th road) with the inclusion body form, this small portion keeps the fusion rotein of dissolved state enough to be used for the displaying of phage.S, P represent the albumen in the cleer and peaceful cellular lysate liquid precipitate on the cellular lysate liquid respectively among the figure.
PPAR γ can be assembled in the lambda particles phage capsid protein
Carrier p171Bio3 and p171-LBD transform lysogen BB4 (λ D1180) respectively, thermal induction lambda particles phage cracking assembling and release, transfer on the pvdf membrane behind the λ p171Bio3 of PEG precipitation gained and the λ p171-LBD SDS-PAGE electrophoresis, utilize anti-LBD polyclonal antibody (Fig. 7 A) and anti-pD polyclonal antibody (Fig. 7 B) to detect.Among Fig. 7 A, an obvious band appears in λ p171-LBD at the 46kDa place, and in contrast the no corresponding band in λ p171Bio3 place, the albumen of this band should be the pD-LBD that is assembled in the lambda particles phage.Further use the antibody test of anti-lambda particles phage coat protein pD, λ p171-LBD is except that the pD-LBD at 46kDa place protein band, also occur a pD albumen (Fig. 7 B) in addition at the 11kDa place, it derives from the pD albumen of the gpD genes encoding in the prophage λ D1180 genome of molten source in host bacterium BB4.
The ratio of pD albumen and pD-LBD fusion rotein has reacted the ability that foreign protein PPAR γ LBD is assembled into lambda particles phage.Usually each wild-type lambda particles phage contains the pD albumen of 405 copies, by scanning quantitative analysis to two bands among Fig. 7 B, the ratio that can draw pD-LBD and pD is about 0.4, therefore the quantity that merges the pD that foreign protein is arranged accounts for 28% of pD quantity altogether, is assembled with 115 PPAR γ LBD molecules thereby can calculate general each lambda particles phage.
Amalgamation and expression and the catching of PPAR γ LBD on λ p171-LBD phage by anti-LBD antibody recognition
Phage by being assembled with PPAR γ LBD and the interaction between its antibody determine that foreign protein is assembled into the position in the lambda particles phage.Utilize anti-PPAR γ LBD coated elisa plate hole, combine with λ p171Bio3 and λ p171-LBD phage respectively, and simultaneously with the enzyme plate hole of the mice serum bag quilt of no antibody (Fig. 8) in contrast.The λ p171-LBD quantity of catching in the enzyme plate hole of anti-LBD bag quilt is four times of the λ p171Bio3 quantity of catching, and the statistical analysis explanation has significant difference (P＜0.01) between the two.Both difference derives from has assembled PPAR γ LBD albumen among the λ p171-LBD, illustrate that anti-LBD serum exists intensive to combine with PPAR γ LBD albumen.The amount that λ p171-LBD is caught in the enzyme plate hole of not having antibody mice serum bag quilt simultaneously significantly descends (P＜0.01), and strong the combination mainly between anti-LBD antibody in the antibody serum and the PPAR γ LBD fusion rotein that anti-LBD serum and PPAR γ LBD albumen exist is described.
λ p171-LBD can be illustrated that PPAR γ LBD is expressed in the lambda particles phage surface by antibody capture.Simultaneously illustrated that also the PPAR γ LBD that is assembled in the lambda particles phage is the outside surface of presenting and expressing at lambda particles phage, and kept can be by antibody recognition and bonded constitutional features.
Carry out application with the PPAR γ LBD that is assembled in the lambda particles phage surface based on presenting and expressing recombinant protein qualitative and quantitative detection
First phage: the capsid surface-assembled has the lambda particles phage of PPAR γ LBD.
Second phage: the capsid surface is not assembled with the lambda particles phage of PPAR γ LBD.
Candidate substances is contacted with second phage with above-mentioned first phage respectively, observe the situation that combines of candidate substances and first phage and second phage.
Analyze the result that combines of candidate substances and first and second phages respectively, debond is in second phage if a kind of candidate substances can be incorporated into first phage, and it is exactly the ligand binding domain bonded material with nuclear receptor or nuclear receptor.
Discuss
(1) proteic solubleness makes moderate progress behind the foreign protein of poorly soluble and the pD amalgamation and expression
Intestinal bacteria are easy to operate and be easy to advantage such as cultivation because of it, make it the protein expression system that remains main, but eukaryotic protein often forms insoluble protein inclusion body when expression in escherichia coli, and it is albumen non-correct folding and protein intermediate product of forming in the expression process.Protein function research can not directly utilize inclusion body to carry out, and common way is earlier the inclusion body protein that is separated to be carried out renaturation, can follow but protein renaturation there is no certain rules, so protein renaturation also has very big difficulty under current technical qualification.Another method of walking around the protein renaturation problem is that by reducing inclusion body generation in the protein expression process, the raising albumen solubility is realized.Usual method is by optimizing the protein expression condition or improving proteic solubleness with the protein fusion expression that can improve solubleness, as glutathione S-transferase (Glutathione S-Transferase, GST), maltose binding protein (Maltose Binding Protein, MBP), thioredoxin (Thioredoxin, Trx) etc.
The ligand binding domain of nuclear receptor and nuclear receptor (as PPAR γ LBD) is owing to contain and hydrophobic small molecules part bonded interface, the content of the hydrophobic amino acid that it is whole is higher, therefore be easier to form inclusion body in the expression process, the inventor's test-results has also confirmed this point, PPAR γ LBD is when expression in escherichia coli, and most of expressed proteins all is to be present in the bacterium with insoluble form.Even after merging GST albumen, the solvability of PPAR γ LBD does not improve a lot.And after lambda particles phage capsid protein pD and the PPAR γ LBD fusion, can significantly improve the latter's solvability, illustrate that utilizing pD albumen is a kind of better selection.
(2) albumen maintenance solvability is the prerequisite that is assembled in the phage
Though display technique of bacteriophage need not proteic purifying, when expressing, foreign protein keeps certain dissolubility in the host bacterium, and this also is that foreign protein is able to the prerequisite of presenting and expressing to phage surface.Though do not need foreign protein to keep solvable state all, need part energy solubility expression at least.The existing solubility that improves foreign protein that studies show that comprises and short molten protein fusion expression or coexpression chaperone molecule, can improve the efficient that foreign protein is assembled into phage surface.From this aspect, select pD albumen as carrier proteins, be suitable for those form the insolubility inclusion body protein easily in the expression process presenting and expressing.
(3) to be limited in the lambda particles phage display systems be not key constraints to the albumen size
The inventor's the bigger foreign protein of result's demonstration can be expressed at phage display.Other report also shows, the albumen of macromolecule can be assembled in the lambda particles phage as beta-galactosidase enzymes, β-Nei Xiananmei, proteotoxin high copy, and these presentation of results lambda particles phages limit the broad that compares for its albumen of showing size.Therefore utilizing the lambda particles phage display systems is relatively to be fit to show large molecular weight protein.
(4) the lambda particles phage display systems is the multivalence display systems
Lambda particles phage is a multivalence display systems, relatively is suitable for screening the less aglucon of avidity.Screening for the high-affinity aglucon, can select the monovalent filobactivirus gp3 system of widespread use, also can regulate and control to merge the proteic expression of pD that foreign protein is arranged by regulating promotor, the ratio of the molecular amounts of the pD by changing wild-type pD albumen and fusion, thus reach the valence mumber that regulation and control are assembled into foreign protein in the lambda particles phage.
(5) other nuclear receptor or nucleus receptor ligand binding domain equally can presenting and expressing at phage surface
Other nuclear receptor is structurally very similar, the ligand binding domain size all about 250 amino acid, is more or less the same, and owing to be to combine with fat-soluble part all, therefore have strong-hydrophobicity, when expression in escherichia coli, all be easy to form insoluble inclusion body.For the presenting and expressing of foreign protein at phage surface, main restriction derives from the solubility limits and the molecular size restriction of foreign protein, according to embodiments of the invention, PPAR γ LBD can presenting and expressing at phage surface, therefore can infer, for other similar nuclear receptor or nuclear receptor land equally also can presenting and expressing at phage surface.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. phage, constitute by capsid and the bacteriophage nucleic acid that is positioned at capsid, it is characterized in that, described phage is a lytic phage, merge on the capsid surface of described phage the nuclear receptor of external source or the ligand binding domain of nuclear receptor, described lytic phage is selected from: lambda particles phage, T7 phage.
2. phage as claimed in claim 1 is characterized in that, the capsid protein pD of described phage goes up to merge the nuclear receptor of external source or the ligand binding domain of nuclear receptor.
3. phage as claimed in claim 1 is characterized in that, described nuclear receptor is selected from down group:
1. steroid hormone receptor;
2. pth receptor;
3. orphan nuclear receptor.
4. phage as claimed in claim 1 is characterized in that described phage is a lambda particles phage.
5. phage as claimed in claim 1 is characterized in that, described bacteriophage nucleic acid coding phage capsid protein and the nuclear receptor of external source or the ligand binding domain of nuclear receptor.
6. the purposes of phage as claimed in claim 1 is characterized in that, is used to screen the ligand binding domain bonded material with nuclear receptor or nuclear receptor.
7. the method for the ligand binding domain bonded material of a screening and nuclear receptor or nuclear receptor is characterized in that, comprises step:
(a) candidate substances is contacted with second phage with first phage respectively, wherein said first phage is the described phage of claim 1, and described second phage is ligand binding domain and the phage same kind of the described phage of claim 1 that does not have the external source nuclear receptor or the nuclear receptor of fusion on the capsid surface;
(b) observe the situation that combines of candidate substances and first phage and second phage,
Wherein, be incorporated into first phage but debond is exactly a ligand binding domain bonded material with nuclear receptor or nuclear receptor in the candidate substances of second phage.
8. method as claimed in claim 7 is characterized in that, described ligand binding domain bonded material with nuclear receptor or nuclear receptor is selected from part, antibody, polypeptide or micromolecular compound.
9. method as claimed in claim 7, it is characterized in that, observe combining of candidate substances and first phage and second phage in the step (b) by the following method: phage and candidate substances in conjunction with after, reclaim phage or measure the bonded phage titre by the competition wash-out by directly infecting the host bacterium.
10. a nucleic acid molecule is characterized in that, it contains following element: promotor, operon, replication site, selection markers site, bacteriophage coat protein coding region, phage foreign protein coding region; And, the nuclear receptor of described phage foreign protein coding region encoding exogenous or the ligand binding domain of nuclear receptor; Described phage is a lytic phage.
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| 许正平等.噬菌体展示系统的研究进展.生命的化学16 3.1996,16(3),29-34. |
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