CN107982526A - A kind of screening technique of more transmembrane region protein antibodies and its application - Google Patents
A kind of screening technique of more transmembrane region protein antibodies and its application Download PDFInfo
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- CN107982526A CN107982526A CN201711181648.3A CN201711181648A CN107982526A CN 107982526 A CN107982526 A CN 107982526A CN 201711181648 A CN201711181648 A CN 201711181648A CN 107982526 A CN107982526 A CN 107982526A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39516—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum from serum, plasma
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
Abstract
The present invention provides a kind of screening technique of more transmembrane region memebrane protein antibody, the described method includes the antibody that intact proteins are directed to by the use of more transmembrane region albumen as antigen selection, then using the baculoviral containing more transmembrane region albumen as antigen, antibody of the identification for more transmembrane region protein extracellulars.The invention further relates to the antibody and its application obtained with the method.
Description
Technical field
Screening technique and its application the present invention relates to a kind of more transmembrane region protein antibodies, more particularly to for more cross-films
The screening technique of area protein extracellular domain conformation antibody and application.
Background technology
More transmembrane region integrated membrane protein, including g protein coupled receptor are referred to as across the albumen of biomembrane more than twice, from
Subchannel, transmembrane transporter etc..Integrated membrane protein accounts for the 30% of total protein of cell, is the target spot of more than 2/3rds medicines.
Such as people's copper ion transport protein (hCtr1), belong to more transmembrane region integrated membrane protein.It is the main cross-film of needed by human body copper ion
One of transport protein.In addition Cisplatin is also to carry out transdermal delivery by hCtr1 albumen.Cisplatin is a kind of
For treating the chemotherapeutics of various cancers, limitation of the cell for Cisplatin transmembrane transports limits the medicine validity
One of approach.The transfer efficiency for improving hCtr1 albumen is to release the medicine caused by the limitation of Cisplatin transmembrane transports
One of effective means of reduction of validity.
Antibody drug created the sales volume in 65,000,000,000 dollars of the whole world in 2012, accounted for the 51.8% of bio-pharmaceuticals, by 2015,
Antibody drug sales volume is up to 98,000,000,000 dollars.But the antibody drug for more transmembrane region albumen is not developed so far.This be by
Still lack effective means in the research and development of more transmembrane region protein antibodies, including the antibody for lacking suitable more transmembrane region albumen
Screening technique.
More transmembrane region albumen are divided into extracellular region, intracellular region and film inner region.Wherein film inner region is mainly by hydrophobic amino acid
Composition, is the chief component of more transmembrane region albumen, in more transmembrane region albumen of purifying, film inner region is mainly covered by detergent
Lid.Each more transmembrane region albumen all possesses detergent the selectivity and dependence of height.Therefore in the screening process of antibody,
The presence of detergent is depended on always.What the preliminary screening of antibody was usually used in addition is enzyme-linked immunoassay（ELISA）：First
By antigen coat on 96 hole microwell plates, detection antibody is added, and the antibody that envelope antigen combines can be contained antiantibody
The colour developing enzyme carried on knot merga pass antiantibody develops the color substrate, so as to be screened out.More transmembrane region albumen due to
Mainly it is made of hydrophobic amino acid, and by detergent dynamic encompassing, is not easy to combine with microwell plate, therefore its antibody produced is not
It can be detected with ELISA.And any region of the albumen can be directed to using more transmembrane region albumen as antibody prepared by antigen.Due to
Antibody cannot pass through cell membrane, and the antibody drug of more transmembrane region albumen can only be combined by the extracellular region with albumen and play work
With.
In cell membrane surface, the extracellular region of only more transmembrane region albumen can be identified by its specific antibody, but directly
It is obvious to connect the shortcomings that coated cell carries out ELISA, that is, 1）The cell concentration accommodated in each micropore is very limited
, the amount of purpose antigen is also few, it is impossible to is effectively detected；2）Cell attachment is not very close, and strong in antibody test is washed
Easily come off under the conditions of washing, further have impact on the detection of target antibody.
The content of the invention
The purpose of the present invention is to solve：1）Antigen is unable to direct coated；2）Selection for the detergent of board-washing；3）
Just for the selection of extracellular domain antibodies., can be effective the present invention provides the screening technique of the alphab-integrin antibodies of transmembrane region more than one
The antibody in more transmembrane region protein extracellulars domain can be identified by filtering out.This method is made of two steps：A) indirect ELISA sieves
Select the antibody of specific recognition target protein；B) the purposeful albumen of expression and the bar containing N or C-terminal FLAG-tag are isolated
Shape virus, antigen selection target antibody is used as using virus instead of purifying protein.
It is an object of the present invention to provide a kind of fat Emission in Cubic, and it includes more transmembrane region albumen.
Preferably, more transmembrane region albumen are the memebrane proteins for traversing cross-cell membrane more than 2 times.
Specifically, more transmembrane region albumen can be g protein coupled receptor, ion channel, transmembrane transporter etc..
It is further preferred that more transmembrane region albumen are copper ion transport protein.
It is highly preferred that more transmembrane region albumen are CTR1.
Most preferably, more transmembrane region albumen are to contain such as SEQ ID NO：The albumen of amino acid sequence shown in 1.It is excellent
Selection of land, the baculoviral are recombinant baculovirus.
Specifically, the fat Emission in Cubic includes glyceride and phosphatide.
More specifically, the fat Emission in Cubic includes olein and phosphatide A.
It is a further object to provide a kind of preparation method of the fat Emission in Cubic, include the following steps：
Step（1）, synthetic DNA sequence is designed according to the amino acid sequence of more transmembrane region albumen and is cloned into expression system；
Step（2）, the expression system is cultivated and is inoculated with baculoviral, continue culture to cell survival rate decline
During to 70% ~ 80%, cell is harvested, the cell is washed, is crushed, centrifugal treating, collects film component；
Step（3）, the film component is diluted, centrifuges, be incubated, purifies and obtains protein solution；
Step（4）, the raw material of the protein solution and fat Emission in Cubic is mixed to form fat Emission in Cubic.
Preferably, step（1）In, merge anti-FLAG antibody in the C-terminal of the amino acid sequence of more transmembrane region albumen
The epitope of identification, then redesigns the synthesis DNA sequence dna.
Preferably, step（2）The baculoviral of middle inoculation is the third generation baculoviral that infection multiplicity is 0.5.
Preferably, step（4）Embodiment be：Glyceride is melted, by step（3）Obtained protein solution and
Phosphatide forms mixed solution, is then 1 ~ 2.5 by volume by the glyceride after melting and the mixed solution:1 is mixed
Close and form the fat Emission in Cubic, wherein, the mass ratio of albumen described in the mixed solution and the phosphatide for 15 ~
25:1.It is further preferred that the glyceride melted is added in syringe, it is molten that the mixing is added in another syringe
Liquid, after two syringes are connected with special joint, the content in above-mentioned two syringe is mixed, until forming transparent people
Work biofilm system.
It is further preferred that the volume ratio of the glyceride and the mixed solution is 1.5:1.
It is further preferred that the mass ratio of the albumen and the phosphatide described in the mixed solution is 20:1.
Third object of the present invention is to provide a kind of fat Emission in Cubic through broken manufactured minitype particle.
Fourth object of the present invention is to provide a kind of immune composition, it includes the fat Emission in Cubic or described micro-
Type particle.
The 5th purpose of the present invention is to provide the use of the fat Emission in Cubic or the minitype particle as immunogene
On the way.
The 6th purpose of the present invention is to provide a kind of using the fat Emission in Cubic, the minitype particle or described
Immune composition is through the immune antibody obtained.
Preferably, the antibody has special binding ability with more transmembrane region albumen and its extracellular region, and can promote
Into the divalent cation transporter function of more transmembrane region albumen.
The 7th purpose of the present invention is to provide a kind of preparation method of the antibody, using the fat Emission in Cubic,
Animal is immunized in the minitype particle or the immune composition, obtains the antibody.
The 8th purpose of the present invention is to provide a kind of screening technique of more transmembrane region protein antibodies, it includes step as follows
Suddenly：
Step（1）, by the use of more transmembrane region albumen as antigen and the antibody be combined antibody of the screening for intact proteins,
Then
Step（2）, be combined as antigen and the antibody using the baculoviral containing more transmembrane region albumen, identify pin
To the antibody of more transmembrane region protein extracellulars.
Preferably, the baculoviral containing more transmembrane region albumen is containing the rod-shaped of more transmembrane region protein gene
Virus, more transmembrane region protein gene coding copper ion transport proteins.
Preferably, in step（1）Described in more transmembrane region albumen and/or step（2）In baculoviral with indirect form
It is coated with carrier.
It is further preferred that the carrier is elisa plate.
It is further preferred that described indirect form coating can be to pass through the energy connected on more transmembrane region albumen
Carried out by the part of specific recognition.
It is highly preferred that the part can be specific amino acid sequence, such as FLAG-tag or His-tag, correspondingly
More transmembrane region albumen or baculoviral containing more transmembrane region albumen can be by identifying the sequence antibody etc. indirectly
On coating to carrier, such as elisa plate.
It is highly preferred that the part can also be compound.The compound can be added by way of modifying in vitro
Into more transmembrane region albumen, most common modification is by more transmembrane region protein biotinylations.Correspondingly, more transmembrane region albumen
Or the baculoviral containing more transmembrane region albumen can be coated with to carrier, such as enzyme indirectly by identifying the material of the compound
On yoke plate.
In scheme is further carried out, when using the baculoviral as antigen, the bar described in screening
Shape virus first and antibody binding, is then screened.
The 9th purpose of the present invention is to provide is directed to more transmembrane region albumen born of the same parents using what the screening technique screened
The antibody of outskirt.
The tenth purpose of the present invention is to provide use of the antibody in the ancillary drug for the treatment of of cancer is used to prepare
On the way.
The 11st purpose of the present invention is to provide a kind of pharmaceutical composition, and it includes the antibody.
As described above, integrated membrane protein is the target spot of more than 2/3rds medicines.So antibody screening using the present invention
More transmembrane region protein antibodies that method obtains for example can be used for preparing medicine, such as be used to prepare the adjuvant for the treatment of of cancer
Thing.
Due to the implementation of above technical scheme, the present invention has the following advantages that compared with prior art：
The concentration of memebrane protein is high in the fat Emission in Cubic of the present invention.Antibody produced by the present invention can effectively identify albumen and albumen
Extracellular space, and the transport of protein mediated ion can be significantly improved.The screening technique of the present invention can be filtered out and can had
The antibody of the extracellular space of effect identification albumen.
Brief description of the drawings
Fig. 1 is the topological diagram of hCtr1 albumen.
Fig. 2 is the SDS-PAGEhCtr figures of the hCtr1 albumen after purifying, TEV digestions and the Dionex liquid of purifying protein
Analysis of hplc figure.
Fig. 3 shows the result of ELISA antibody.
Fig. 4 shows the result of cell surface fluorescence staining analysis antibody.
Fig. 5 shows influence of the fluorescence analysis antibody for the transdermal delivery of copper ion protein mediated hCtr1.
Embodiment
Term " more transmembrane region integrated membrane protein " used herein and " more transmembrane region albumen " are used interchangeably, it is intended that horizontal
Across the albumen of biomembrane more than twice, the example includes g protein coupled receptor, ion channel, transmembrane transporter etc., such as copper
Ion transporter etc..
More transmembrane region albumen are divided into extracellular region, intracellular region and film inner region.Wherein film inner region is mainly by hydrophobic amino acid group
Into, be more transmembrane region albumen chief component.The extracellular region of more transmembrane region albumen is located at outside cell membrane, for example, connects more
The part of transmembrane region protein transmembrane sequence and the albumen are located at N-terminal or C-terminal part outside cell membrane.For people's copper ion
For transport protein hCTR1, extracellular region is SEQ ID NO：1 to 30th, the amino of the 50 to 60th and the 80 to 90th in 1
Acid sequence.
It is to allow those skilled in the art to more fully understand, implement the present invention, be not intended to limit to provide following embodiments
The scope of the present invention processed.
Embodiment 1 is used for the expression strategy of the copper ion transport protein CTR1 of antibody screening
People's copper ion transport protein three times, belongs to membrane-spanning proteins across cell membrane, is made of 270 amino acid, the protein
N-terminal contain divalent cation binding site, the purifying available for metal chelate affinity chromatography.In addition we merge in its C-terminal
One section（DYKDDDDKG）The epitope of anti-FLAG antibody identification.Whole DNA is synthesized by invitrogen companies of U.S. full genome,
And U.S. invitrogenBac-to-Bac is cloned into expression system.The preparation of recombinant baculovirus in strict accordance with
The operation sequence of invitrogen settings carries out.SEQ ID NO in sequence table：1 is hCTR1 amino acid sequences, SEQ ID NO：2
For the DNA sequence dna of hctr1, SEQ ID NO：3 and SEQ ID NO：4 be the amino in the addition of people's copper ion transport protein C-terminal
Sour and corresponding DNA sequence dna.
The expression and purifying of 2 hCTR1 albumen of embodiment
The expression and purifying of hCTR1 albumen are divided into following steps.
1st, the expression of hCTR1 albumen and the separation of cell membrane component
The expression system Sf9 that embodiment 1 is obtained is cultivated in the shaking flask containing 900 II culture mediums (Invitrogen) of SF
In, cell density is 2.0 × 106Cells/ml culture mediums, the third generation baculoviral that inoculation plural number is 0.5.After
The continuous rotating speed with 100 rpm continues to cultivate cell at 27 DEG C.When the survival rate of cell drops between 70%~80%（It is logical
It is often 70~72 hours of infection）Harvest cell.Cells rinsed with PBS is once suspended in 50 mM HEPES afterwards（pH 7.5）'s
In buffer solution, with EmulsiFlex-C5 under the pressure of 10000 psi smudge cells.Cell lysate is first in low temperature bar
Under part（4 ℃ ）1000g centrifuges 10 min and removes uncracked cell and nucleus.Supernatant fraction by 4 DEG C, 100,
000g centrifuges 1 h and collects film component, and film component is suspended in the buffer solution of 50 mM HEPES, pH 7.5, and -80 DEG C freeze.
2nd, the structure of the purifying of hCTR1 albumen and LCP
Film component is diluted to (every 30 ml buffer solutions in the 50 mM HEPES buffer solutions containing 200 mM NaCl and 1% DM
For the film component from 1 liter of culture), sample is centrifuging 45 min in 4 DEG C, 100,000g after 1 h.Supernatant adds
Enter respective amount TalonTM25 DEG C continue be incubated 1 it is small when, be fitted into chromatographic column.With equivalent to TalonTM50 times of volume contains
The 50mM HEPES buffer solutions that 200mM sodium chloride, the pH of 0.15% DM, 15mM imidazoles are 7.4 clean chromatographic column.Finally with containing
The above-mentioned buffer solution for having 300mM imidazoles elutes destination protein.The buffer solution of obtained albumen is converted into containing with PD10 desalting columns
There is 200mM sodium chloride, in the HEPES buffer solution of 20 mM that the pH of 0.15% DM is 7.4.Phase is added in above-mentioned protein solution
The nitrogen end of equivalent contains the TEV enzymes of 6 histidines, after 4 DEG C of processing overnight, adds TalonTMRemove TEV enzymes.Will after digestion
Protein solution is concentrated to 1 milliliter, and with 200 10/300 gel columns of Supedex into purifying, buffer solution is to contain 0.15% DM's
50mM phosphate buffers（PBS）.Major protein peak is collected, 5 mg/ml are concentrated to the inspissator of 100K.Fig. 2 left figures are shown
Use sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）Result to the analysis of protein isolated and purified.Band 1
It is TalonTMThe albumen of purifying, band 2 are the protein products after TEV is handled and eliminates TEV enzymes.Fig. 2 right figures be through
The Dionex high pressure liquid phase molecule sieve chromatography chromatograms that TEV is handled and eliminated the hCtr1 after TEV enzymes are crossed, RI is reflection system
Number, UV is Ultroviolet absorptivity, and RALS is scattering coefficient, and protein expression is single symmetry absorbing proteins peak, is shown pure
It is homogeneous to change the physical property of hCtr1, by calculating protein presence in the form of tripolymer in detergent DM.
The preparation of 3 antigen of embodiment and animal immune
Glycerin mono-fatty acid ester is melted at 40 DEG C, and is added in a Haminton syringe, is noted in an other Haminton
The obtained hCtr1 protein solutions after TEV is handled and eliminates TEV enzymes of embodiment 2 are added in emitter（5 mg/ml）With
And the phosphatide A of 1mg/ml forms mixed solution（The volume ratio of protein solution and phosphatide A are 4:1；Glycerin mono-fatty acid ester is molten with mixing
The volume ratio of liquid is 3:2）, two syringes connected with special joint after by two syringes content mix, until
Form transparent fat Emission in Cubic（LCP）.The fat Emission in Cubic of preparation is suspended in PBS so that in every milliliter of phosphate buffer
Containing the albumen that concentration is 2 milligrams every milliliter, then fat Emission in Cubic is divided twice with 20Kpsi using a small amount of high pressure broken cell devices
Dissipate into minitype particle.
With fat Emission in Cubic obtained above and isometric Freund's complete adjuvant mixing and emulsifying, immunizing rabbit, first immunisation
When according to every 600 μ g protein of rabbit dosage.Back part is subcutaneous and groin multi-point injection：Every 200 μ g albumen
Matter.Booster immunization amount of antigen is the half of initial immunity.Carry out within 2 weeks after previous be immunized.14 after last time booster immunization
Its bloodletting, takes serum.Immune serum obtains anti-hCTR1 polyclonal serums with Protein G purified.
4 enzyme linked immunological of embodiment（ELISA）Detection
Indirect ELISA using purifying protein as antigen
The mouse 96 hole elisa plates of anti-FLAG monoclonal antibodies coating for amounting to 2 μ g with 50 μ l are stayed overnight.50 μ l are added per hole within second day to be total to
5 μ g are counted through TalonTMThe hCTR1 albumen of purifying, 37 DEG C be incubated 1 it is small when after with the PBS containing 0.15% DM wash 3 times, Ran Houjia
Enter confining liquid（PBS containing 5% BSA and 0.15% DM）A hour is handled, after removing confining liquid, 50 μ l is added per hole and are used
Anti- hCTR1 polyclonal serums made from the embodiment 3 of confining liquid doubling dilution, each 2 multiple holes of sample design, 37 DEG C are incubated 1
Hour, washed 5 times with the PBS containing 0.15% DM, 50 μ l sealing liquids 1 are added per hole:5000 diluted horseradish peroxidases
Enzyme (HRP) mark secondary antibody, 37 DEG C be incubated 1 it is small when, equally wash 5 times then add OPD colour developing, with sulfuric acid terminate react, 490
Nm readings.ELISA experiments show that the antibody obtained with fat cube phase method can identify the hCtr1 albumen isolated and purified and be somebody's turn to do
The extracellular space of albumen（Legend shown in protein in Fig. 3）.
Baculoviral is the indirect ELISA of antigen
When having infected the survival rate of cell of the recombinant baculovirus containing hCTR1 and having dropped between 70%~80%（Typically
Infect 70~72 hours）Harvest cell, cell harvest after remaining supernatant with 50000g continue centrifugation 1 it is small when.Precipitation（It is i.e. sick
Poison）It is suspended in after washed once with PBS in the PBS buffer containing 5% BSA, is prepared into every milliliter of 5 milligrams of total proteins, takes
100ul, adds the anti-hCTR1 polyclonal serums made from the embodiment 3 of confining liquid doubling dilution, be incubated 1 it is small when after, with containing
The PBS of 1% BSA is washed twice, and is suspended to 100 microlitres, and adds 1% DM, is incubated after ten minutes, precipitation is removed in centrifugation.Supernatant
Be added to and be coated with the elisa plate of FLAG antibody, be incubated 1 it is small when, remaining steps and using purifying protein as the indirect of antigen
ELISA is consistent, filters out the antibody for the extracellular space that can identify albumen.ELISA test datas are referring to the viral institute in Fig. 3
Diagram example.
5 cell fluorescence of embodiment is immunized
In order to determine that can antibody selected by embodiment 4 correct hCTr1 albumen of the recognition expression in cell membrane surface（That is outside
Point）, sf9 cells are cultivated in 24 orifice plates of circular coverslip are covered with first, with the recombinant baculovirus containing hCTr1 with 0.2
When a MOI units infection 60 is small（For in cells show and cell inner expression destination protein）.Metainfective cell is used first
PBS containing 5% serum is closed one hour, is then used as first antibody using the antibody prepared by embodiment 4（1:500）And cell
Be incubated 1 it is small when, then how anti-developed the color with the FITC sheep anti mouses marked.With confocal fluorescence microscope staining conditions.
The results show（Fig. 4）：With by LCP（Fat Emission in Cubic）HCtr1 after reconstruct is that the antibody that antigen is immunized can be identified effectively
The extracellular space of albumen.
Influence of the antibody for hCtr1 protein functions selected by 6 embodiment 4 of embodiment
The film reconstruct and the addition of fluorescence indicator of hCtr1 albumen
The hCtr1 albumen of purifying with by artificial synthesized lipid (POPC:POPE:POPG;Weight ratio is 3:3:1) lipid of composition
Body is reconstructed：The albumen of purifying（0.25 mg/ml）With the liposome of detergent stabilization removal（10 mg/ml）Using volume ratio as
1:After 4 ratio mixing, hCtr1 liposomes are obtained after removing detergent with bio-beads SM2 (Bio-Rad).HCtr1 fat
Plastid is then suspended in containing 200 μM of indicator Phen Green SK（GSK）25mM HEPES buffer solution in
（PH7.4 is in (20 mM HEPES, 100mM NaCl, pH 7.5)）, after the cyclic process by 3 frozen section-thawings,
The indicator for not entering into hCtr1 liposomes is removed using 10 DG of Econo-Pac.What is obtained embedded in the hCtr1 of indicator
140,000 g centrifuge 30 min washings 3 times to liposome again（Use HEPES buffer solution）.
The measure of the transmembrane transport of copper ion protein mediated hCtr1
The measurement of Phen Green SK fluorescence is carried out using MGW-microplate reader.150 μ l contain GSK's
HCtr1 proteoliposomes and nonprotein liposome are added separately in 96 orifice plates.Film transhipment measure 25 °C into
OK.The copper ion of the 0.2 mM Fresh of 10 μ l, the effect of antibody are automatically added to during the startup of film transhipment by instrument
And it is injected into the 1 of 10 μ l by instrument:100 antibody measurement.By detecting 380 nm of excitation wavelength, diverging light wave
The change of the fluorescence of long 460 nm determines whether copper ion enters liposome interior by hCtr1 albumen（Copper ion and GSK
The fluorescence of the latter specific can be quenched after contact）.The results show：The protein mediated copper of hCtr1 can be significantly improved after adding antibody
The transport of ion.
Claims (17)
- A kind of 1. fat Emission in Cubic, it is characterised in that：It includes more transmembrane region albumen.
- 2. fat Emission in Cubic according to claim 1, it is characterised in that：More transmembrane region albumen transport egg for copper ion In vain.
- 3. fat Emission in Cubic according to claim 2, it is characterised in that：More transmembrane region albumen are CTR1.
- A kind of 4. preparation method of fat Emission in Cubic as claimed any one in claims 1 to 3, it is characterised in that：Including as follows Step：Step（1）, synthetic DNA sequence is designed according to the amino acid sequence of more transmembrane region albumen and is cloned into expression system；Step（2）, the expression system is cultivated and is inoculated with baculoviral, continue culture to cell survival rate decline During to 70% ~ 80%, cell is harvested, the cell is washed, is crushed, centrifugal treating, collects film component；Step（3）, the film component is diluted, centrifuges, be incubated, purifies and obtains protein solution；Step（4）, the raw material of the protein solution and fat Emission in Cubic is mixed to form fat Emission in Cubic.
- 5. the preparation method of fat Emission in Cubic according to claim 4, it is characterised in that：Step（1）In, in the multispan The C-terminal of the amino acid sequence of film area albumen merges the epitope of anti-FLAG antibody identification, then the redesign synthesis DNA sequences Row；Step（2）The baculoviral of middle inoculation is the third generation baculoviral that infection multiplicity is 0.5.
- A kind of 6. minitype particle made of fat Emission in Cubic as claimed any one in claims 1 to 3 is through crushing.
- A kind of 7. immune composition, it is characterised in that：It include fat Emission in Cubic as claimed any one in claims 1 to 3 or Minitype particle as claimed in claim 6.
- 8. fat Emission in Cubic as claimed any one in claims 1 to 3 or minitype particle as claimed in claim 6 are as immune Former purposes.
- 9. one kind uses fat Emission in Cubic as claimed any one in claims 1 to 3, minitype particle as claimed in claim 6 Or immune composition as claimed in claim 7 is through the immune antibody obtained, the antibody and more transmembrane region albumen and its Extracellular region has special binding ability, and can promote the divalent cation transporter function of more transmembrane region albumen.
- A kind of 10. preparation method of antibody as claimed in claim 9, it is characterised in that：Appoint using in such as claims 1 to 3 Fat Emission in Cubic, minitype particle as claimed in claim 6 or immune composition as claimed in claim 7 described in one are immunized Animal, obtains the antibody.
- A kind of 11. screening technique of more transmembrane region protein antibodies, it is characterised in that：It includes the following steps：Step（1）, by the use of more transmembrane region albumen as antigen and antibody as claimed in claim 9 be combined screening for complete The antibody of albumen, thenStep（2）, carry out by antigen and antibody as claimed in claim 9 of the baculoviral containing more transmembrane region albumen With reference to antibody of the identification for more transmembrane region protein extracellulars.
- 12. screening technique according to claim 11, it is characterised in that：It is described to contain the rod-shaped of more transmembrane region albumen Virus is the baculoviral containing more transmembrane region protein gene, more transmembrane region protein gene coding copper ion transport proteins.
- 13. screening technique according to claim 11, it is characterised in that：In step（1）Described in more transmembrane region albumen and/ Or step（2）In baculoviral be coated with indirect form on carrier.
- 14. screening technique according to claim 11, it is characterised in that：When using the baculoviral as antigen When, the baculoviral described in screening first and antibody binding, is then screened.
- What 15. a kind of screening technique using any one of claim 11 to 14 screened is directed to more transmembrane region albumen The antibody of extracellular region.
- 16. purposes of the antibody as described in claim 9 or 15 in the ancillary drug for the treatment of of cancer is used to prepare.
- A kind of 17. pharmaceutical composition, it is characterised in that：It includes the antibody as described in claim 9 or 15.
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