CN108611368A - A kind of preparation method of PLS3-ABD2 recombinant proteins - Google Patents
A kind of preparation method of PLS3-ABD2 recombinant proteins Download PDFInfo
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Abstract
The invention belongs to biotechnologies, specifically disclose a kind of preparation method of PLS3 ABD2 recombinant proteins.The present invention is by the cloned dna molecule containing sequence shown in SEQ ID NO.4 to carrier for expression of eukaryon, and it will be cultivated in the eukaryotic expression vector transfection to eukaryotic host cell system, so that the PLS3 ABD2 recombinant proteins being prepared using this method, compared with prior art for (prokaryotic expression), the exogenous proteins that reprocessing modification generates after being translated by eukaryocyte, prokaryotic expression system is outclassed in terms of activity, closer to native protein, can be to be subsequently used for laying the foundation with the protein screening of PLS3 protein-interactings and the polypeptide combined with PLS3 protein-specifics screening.
Description
Technical field
The invention belongs to biotechnologies, specifically, being related to the preparation of PLS3-ABD2 recombinant proteins.
Background technology
Actin fasciclin PLS3 is a member in Plastin protein families, and be otherwise known as T-plastin, gene
It is positioned at Xq23, the protein coding regions mRNA (Coding sequence, CDS) includes 1893 nucleotide, and albumen is by 630
Amino acid forms.It can be combined with tri- kinds of actins of α, β, γ, and be expressed in a variety of solid tissue cells, in peripheral blood
In do not express.PLS3 plays key player during tumour occurrence and development.Studies have shown that PLS3 can regulate and control regulating cell
Movement, the survival and migration of tumour cell, and energy modulate tumor cell are to the sensibility of the chemotherapeutics such as cis-platinum or UV.Except this it
Outside, PLS3 may be a kind of potential tumor markers：Its unconventionality expression can be as the mark of skin T cell lymphoma
Object；The expression of PLS3 is suffered from colorectal cancer in colorectal cancer circulating tumor cell (Circulating tumor cells, CTCs)
The far-end transfer of person is related to prognosis.In conclusion the occurrence and development of PLS3 and tumour are closely related, and clear PLS3 is in tumour
Function in occurrence and development all has very important meaning to the diagnosis, treatment and prognosis of tumour.
Although PLS3 has very important effect, the preparation method of PLS3 albumen to still have problem.PLS3 albumen by
EF-2hands and 4 CH (Calponin-homology) structural domain composition, wherein CH3 and CH4 are main functional domain,
The two is known as ABD2 (Actin-binding domain 2).The PLS3 albumen of commercialization is the recombinant protein of ABD2 mostly, should
Structural domain is that PLS3 antibody is predominantly targeting structural domain there are multiple conserved positions.The PLS3 albumen of commercialization is mostly protokaryon table
Expressed by system, lack the natural modifications of eukaryotic system protein synthesis.Recombination egg expressed by prokaryotic expression system
The PLS3 albumen that Bai Keneng is expressed in space conformation etc. with eukaryotic system has differences, to be subsequently used for and PLS3 albumen phases
The protein screening of interaction and the polypeptide screening zone combined with PLS3 protein-specifics are come difficult.
Invention content
In order to solve the problems in the existing technology, the object of the present invention is to provide a kind of PLS3-ABD2 recombinant proteins
Preparation method so that the PLS3-ABD2 recombinant proteins being prepared using this method, compared with prior art (prokaryotic expression) and
Speech, can be formed stable disulfide bond by eukaryocyte, correctly be modified after protein translation, generated foreign protein
Matter is with more natural activity rather than is degraded or is formed inclusion body, and prokaryotic expression system is outclassed in terms of activity, is more connect
Be bordering on native protein, can be subsequently used for the protein screening of PLS3 protein-interactings and with PLS3 protein-specific knots
The polypeptide screening of conjunction lays the foundation.
The PLS3 albumen being commercialized at present is mostly to lack eukaryotic system protein synthesis expressed by prokaryotic expression system
Disulfide bond and glycosylation modified, there are larger differences with natural PLS3 albumen on space structure.And it is carrying out and PLS3 albumen
In the protein screening of interaction and the polypeptide screening process combined with PLS3 protein-specifics, some conserved positions are due to day
The presence of the space structure of right PLS3 albumen and be folded in active site of protein, so it is difficult to screening the egg combined with these sites
White or polypeptide.But by the obtained albumen of prokaryotic expression system, due to lacking complicated glycosylation modified, part binding site
By being exposed for mistake, the protein screening of Thermodynamic parameters and the polypeptide screening of specific binding cause puzzlement.And this hair
Bright provided method can correctly fold the albumen of expression other than high efficient expression PLS3-ABD2 albumen, and
Carry out complicated glycosylation modified, protein active is close to native protein, therefore for the egg with PLS3 protein-interactings
The white polypeptide screening screened and combined with PLS3 protein-specifics has some superiority.
In order to realize the object of the invention, technical scheme is as follows：
The present invention provides a kind of preparation method of PLS3-ABD2 recombinant proteins, the method includes：SEQ ID will be contained
The cloned dna molecule of sequence shown in NO.4 is and thin to eucaryon host by the eukaryotic expression vector transfection to carrier for expression of eukaryon
It is cultivated in born of the same parents system.
The present invention is by using carrier for expression of eukaryon and eukaryotic host cell system so that generated exogenous proteins have more
There is natural activity rather than be degraded or formed inclusion body, prokaryotic expression system is outclassed in terms of activity, closer to day
Right protein, can be subsequently used for being combined with the protein screening of PLS3 protein-interactings and with PLS3 protein-specifics it is more
Peptide screening lays the foundation.
Further, the carrier for expression of eukaryon include but not limited to pIRES2-ZsGreen1, pcDNA3.1, pEGFP or
PCMV7.1 etc..
The process of the cloned dna molecule to carrier for expression of eukaryon can be used to cloning process commonly used in the art, for example,
Utilize restriction enzyme site digestion connection etc..
Further, the preferably described carrier for expression of eukaryon of the present invention is pIRES2-ZsGreen1 plasmids.It is described
PIRES2-ZsGreen1 plasmids contain IRES elements, and multiple cloning sites are separated, and can build while express the weight of two genes
Group plasmid.When using the plasmid transfection eukaryocyte, target gene is transcribed into a mRNA altogether with reporter gene ZsGreen1,
But translate respectively, can monitor whether to transfect successfully and reporter gene protein influence target gene albumen structure and work(
Energy；ZsGreen1 is the highest green fluorescent protein of brightness, is suitble to flow cytometer and other fluorescent screenings；pIRES2-
ZsGreen1 plasmids are high copy number plasmid, and containing screening-gene Neo genes, the stabilization that can be used in screening expression PLS3 albumen is thin
Born of the same parents system.Therefore the structure of PLS3-ABD2 segments is carried out present invention preferably employs pIRES2-ZsGreen1 carriers, compares other
Carrier for expression of eukaryon has more advantage.
Property illustrates as an example, and the present invention is directed to expresses PLS3-ABD2 using pIRES2-ZsGreen1 plasmid constructions
Carrier for expression of eukaryon scheme, a kind of specific construction method is provided：
(1) with PLS3 recombinant protein eukaryotic expressions constructed in the Chinese patent application of Publication No. CN107446949A
Plasmid is template, is primer using sequence shown in SEQ ID NO.1 and SEQ ID NO.2, introduces restriction enzyme site and 6 × His
Segment, the cDNA segments (SEQ ID NO.3) that structure restriction enzyme site is EcoRI-His-PLS3-ABD2-BamHI；
(2) the bis- enzymes of EcoRI and BamHI are carried out to pIRES2-ZsGreen1 plasmids and step (1) the cDNA segments respectively
It cuts, the cDNA segments after digestion is then cloned into pIRES2-ZsGreen1 plasmids, structure obtains to express PLS3-ABD2's
Carrier for expression of eukaryon is named as pIRES2-ZsGreen1-His-PLS3-ABD2 eukaryon expression plasmids.
Further, by by the carrier for expression of eukaryon transformed competence colibacillus Escherichia coli of above-mentioned expression PLS3-ABD2, utilizing
Escherichia coli self mechanism carries out the amplification of plasmid, can prepare a large amount of eukaryon expression plasmids.
Further, the eukaryotic host cell system includes but not limited to CHO-K1,293T, MCF-7, T-24 etc..
Transfection side commonly used in the art is can be used into the process of the eukaryotic expression vector transfection to eukaryotic host cell system
Method.For example, transfecting 6 by the eukaryotic expression vector transfection to eukaryotic host cell system for expressing PLS3-ABD2 using Lipo2000
Liquid is changed after hour, transfection collects cell after 48 hours.
Further, the preferably described eukaryotic host cell system of the present invention is CHO-K1 cell lines.The CHO-K1 cells
System has destination protein expression high relative to other eukaryotic host cell systems, and foreign protein is less, more conducively obtain purity compared with
The advantages of high destination protein.
Further, described after it will be cultivated in the eukaryotic expression vector transfection to eukaryotic host cell system
The preparation method of PLS3-ABD2 recombinant proteins further includes the purifying to PLS3-ABD2 recombinant proteins.
For the ease of being purified to PLS3-ABD2 recombinant proteins, when building aforementioned carrier for expression of eukaryon, preferably in weight
His-tag (6 × His segments) is added before the encoding gene of histone, that is, by the DNA molecular of sequence shown in SEQ ID NO.3 or
Cloned dna molecule containing sequence shown in SEQ ID NO.3 is to carrier for expression of eukaryon.
After eukaryotic expression vector transfection eukaryotic host cell system, the recombinant protein purification specifically comprises the following steps：
(1) it collects the eukaryotic host cell after culture and is cracked：
Collect the eukaryotic host cell after culture, lysate be added, after multigelation, centrifuging and taking supernatant, after filtering plus
Enter lysate dilution, obtains cell pyrolysis liquid.
Preferably, when the eukaryotic host cell system is CHO-K1 cell lines, the CHO-K1 cells after transfection are existed
After being cultivated 48 hours in 37 DEG C of constant temperature cell incubators, outwells culture medium and the remaining culture medium of PBS cleanings is added, cracking is added
Liquid (300mM NaCl, 20mM imidazoles, 20mM PB, pH7.0) then uses scraper to collect in cell to centrifuge tube；In liquid nitrogen and
Multigelation is three times in 37 DEG C of water-baths；By the liquid after multigelation in 4 DEG C, 2000rpm, 5min is centrifuged；It is upper after centrifugation
The dilution of 10 μ L lysates is added in clear liquid after 0.22 μm of membrane filtration, that is, preliminary cell pyrolysis liquid is made.
(2) preliminary purification：
The PLS3-ABD2 with His-tag contained in above-mentioned cell pyrolysis liquid is recombinated into egg using nickel-agarose Gel column
It is isolated and purified in vain.
Preferably, using nickel-agarose Gel column (Ni-NTA Resin) to band His- contained in cell pyrolysis liquid
The PLS3-ABD2 recombinant proteins of tag are isolated and purified, and concrete operations are as follows：
Fill column：Appropriate medium is added chromatographic column, stood by re-suspending medium according to protein content to be purified；
Balance：With 5-10 times of column volume equilibration buffer chromatographic column；
Loading：Sample buffer should be as consistent with equilibration buffer as possible, and to avoid blocking chromatographic column, sample should be through centrifugation
Or it is filtered using 0.45 μm of filter；
Washing：After loading, with 5-10 times of column volume equilibration buffer solution chromatographic column, efflux is collected；Balance is slow
Fliud flushing formula：300mM NaCl, 20mM imidazoles, 20mM PB, 10mM Tris base, pH7.0.The efflux being collected into carries out
SDS-PAGE electrophoresis is verified, and determines which destination protein have higher purity under the conditions of, is laid the foundation to be subsequently secondarily purified.
Elution：The imidazoles of various concentration is prepared with equilibration buffer, carries out gradient elution.
(3) secondarily purified：
The purified product of step (2) is carried out to secondarily purified, to be purified PLS3-ABD2 recombinations using liquid chromatograph
Albumen.
Property explanation as an example：It is secondarily purified using Waters e2695 liquid chromatographs (Alliance) progress,
Purification step is carried out according to instrument specification.The efflux for collecting 15min-18min, verifies through liquid phase, obtains simple spike, passes through
Western blot and mass spectrum are verified as PLS3-ABD2 recombinant proteins.
The present invention relates to raw material or reagent be ordinary commercial products, the operation being related to is unless otherwise specified
This field routine operation.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can be combined with each other, obtain specific embodiment party
Formula.
The beneficial effects of the present invention are：
For the 385-629 amino acids of actin fasciclin PLS3 albumen, a kind of relative ease, eukaryon are provided
The method of expression system prepares PLS3-ABD2 recombinant proteins, and then purifying obtains its single albumen, real by Western Blot
It is verified, the PLS3-ABD2 recombinant proteins purified.
The present invention provides a kind of PLS3-ABD2 recombinant proteins, be for PLS3 protein 385-629 amino acids into
The protein that row is prepared and purified, preparation process are mainly to build the carrier for expression of eukaryon of PLS3-ABD2 recombinant proteins, are transferred to
Eukaryotic host cell is detached using nickel-agarose Gel column and liquid chromatograph and purifies PLS3-ABD2 recombinant proteins, and led to
Wesern Blot are crossed to be verified.PLS3-ABD2 recombinant proteins after purification can be used for sieving with PLS3 protein interactive proteins
Choosing and the screening of the polypeptide combined with PLS3 protein-specifics.
Description of the drawings
Fig. 1 is the plasmid map of eukaryotic expression vector pIRES 2-ZsGreen1-His-PLS3-ABD2.
Fig. 2 is the electrophoresis result of the PLS3-ABD2cDNA segments expanded in embodiment 1；Wherein, Ladder:2000bp
DNA Marker。
Fig. 3 is the pIRES2-ZsGreen1-His- built with EcoR I and BamH I double digestions in the embodiment of the present invention 1
The electrophoresis result of PLS3-ABD2 carrier for expression of eukaryon；Wherein, M:5000bp DNA Marker；1:The sample of plasmid double digestion.
Fig. 4 is that the single bacterium colony chosen in the embodiment of the present invention 1 carries out bacterium colony PCR, obtained electrophoresis result；Wherein,
Ladder:2,000bp DNA Marker。
Fig. 5 is the plasmid transfection CHO-K1 cells with fluorescent protein report gene filtered out in the embodiment of the present invention 2
It is to excite green fluorescence under fluorescence after 48h.
Fig. 6 is the result of Western blot verifications after Ni-NTA Resin preliminary purifications.
Fig. 7 is the result of the first pure sample of Alliance liquid chromatographs analysis.
Fig. 8 is the gross protein Western blot testing result figures for transfecting PLS3-ABD2 plasmids.
Specific implementation mode
With reference to embodiment the present invention will be further explained explanation.It will be appreciated that following embodiment provides
Merely to playing the purpose of explanation, it is not used to limit the scope of the present invention.Those skilled in the art is not
In the case of spirit of the invention and spirit, the present invention can be carry out various modifications and be replaced.
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J ＆ Russell DW,
Molecular Cloning:A Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
The main agents that are used in following embodiment, instrument：
Main agents：CHO-K1 cell lines；DME/F-12(Hyclone)；FBS(Gibco)；pIRES2-ZsGreen1
(Yrbio)；Bacillus coli DH 5 alpha (Tiangeng biochemistry Co., Ltd)；DL5000 (Tiangeng biochemistry Co., Ltd)；Small amount plasmid is extracted
Kit (Tiangeng biochemistry Co., Ltd)；Ago-Gel DNA QIAquick Gel Extraction Kits (Tiangeng biochemistry Co., Ltd)；DL2000
(Takara)；Restriction enzyme EcoR I (Takara)；BamH I(Takara)；T4 ligases (Takara)；Archaeal dna polymerase
(Takara)；QIAquick PCR Purification kit(MN)；Imidazoles (Aladdin)；Agarose (Biowest)；Pancreas egg
White peptone (Oxide)；Yeast extract (Oxide)；NaCl (Aladdin)；Kanamycins (Coolaber)；Gelred
(BIOTIUM)；Reverse Transcriptase kit (Roche)；Ni-NTA RESIN(TRAN)；NaH2PO4·12H2O(Amresco)；Tris
(Amresco)。
Key instrument：37 DEG C of constant-temperature shaking incubators (Taicang Science ＆ Teaching Instrument factory)；Thermal cycler (Thermo
Fisher)；Mini desktop vortex oscillator (VORTEX)；37 DEG C of constant incubators (Taicang Science ＆ Teaching Instrument factory)；Horizontal strip electrophoresis
Instrument (Bio-Rad)；Gel imager (Shanghai Qin Xiang scientific instrument Co., Ltd)；Ultraviolet specrophotometer (MALCOM, e-
spect)；Waters e2695 liquid chromatographs (Alliance)；Biological super-clean bench (Beijing Dong Lianhaer instruments)；Cell culture
Case (Thermo Fisher).
The preparation method of 1 His-PLS3-ABD2 fusion proteins of embodiment
1. experimental method
(1) structure of PLS3 eukaryon expression plasmids
According to PLS3 recombinant protein eukaryotic expressions constructed in the Chinese patent application of Publication No. CN107446949A
Plasmid is template, according to PLS3 gene orders (https://www.ncbi.nlm.nih.gov/gene/5358), with SEQ ID
Sequence shown in NO.1 and SEQ ID NO.2 is primer, and restriction enzyme site and 6 × His segments is added, and structure restriction enzyme site is EcoR
The cDNA segments of I-His-PLS3-ABD2-BamH I；Then by digestion integrated enzyme reaction, the cDNA segments of acquisition are cloned into
PIRES2-ZsGreen1 plasmids are built into pIRES2-ZsGreen1-His-PLS3-ABD2 eukaryon expression plasmids；
A) PCR amplification system is as follows：
B) PCR programs are as follows
98 DEG C of pre-degeneration 4min；98 DEG C of denaturation 10s, 58 DEG C of annealing 10s, 72 DEG C of extension 2min, 35 recycle；72 DEG C of extensions
7min；4 DEG C of preservations.
C) agarose gel electrophoresis
1 × TAE electrophoretic buffers that 0.5g agaroses are dissolved in 50mL are weighed, the Ago-Gel of 1% (g/mL) is configured to
The GelRed (BIOTIUM companies of the U.S.) of 5 μ L is added in micro-wave oven, is uniformly mixed for solution after heating 3min.It is subsequently poured into and puts
20min or more is solidified in the gel slot of good comb, and then gel slot is put into the electrophoresis tank of the electrophoretic buffer containing 1 × TAE, according to
Secondary addition DNA ladder and sample.Electrophoretic voltage is adjusted to 80V, 30min.It is taken pictures using gel imager.And ultraviolet
Electrophoretic band is observed under lamp, judges amplified production whether to be wanted segment according to clip size.
D) the purifying of PCR product
It is purified from PCR single, double chain DNAs obtained by the reaction using kit QIAquick PCR Purification kit
Segment.By PCR samples and PB buffer 5：PB buffer, and mixing is added in 1 ratio.QIAquick adsorption columns are put into
In 2mL collecting pipes.Sample is added in adsorption column, 12,000g centrifugation 1min.The liquid of filtration is discarded, 0.75mL PE are added
In buffer to column, 1min is centrifuged.Filtered solution is discarded, then centrifuges 1min, uncaps and is placed at room temperature for 10min to adsorbed film exsiccation.It will
QIAquick columns are put into new 1.5mL centrifuge tubes, are added on 30 μ L water to QIAquick films, and 1min, 12,000g centrifugations are placed
1min.It can be in -20 DEG C of preservations.
E) enzyme connection reaction, reaction system are as follows
The molal quantity of DNA fragmentation should control within the scope of 3-10 times of carrier DNA molal quantity.5-6h is connected under room temperature.
F it) converts
Enzyme-linked product is added in 50 μ L DH5 α bacterium solutions, mixing is gently rotated.DNA volumes used do not exceed competence
/ 10th of cell suspension volume.30min is placed in ice bath.In 42 DEG C of heat shock 45s, it is quickly transferred in ice bath.Ice bath
2min is stood, which not shake centrifuge tube.The 500 μ L of LB culture mediums without kanamycins are added, in 37 DEG C of constant temperature incubations
1h is shaken in 150rpm/min, it is therefore an objective to so that thalline is recovered, related resistance marker gene on plasmid is made to express in case.Take 100-
200 μ L culture solutions are added on the LB agar plates containing kanamycins (100 μ g/mL), lightly will with sterile triangle spreading rod
Cell is uniformly spreadable.Tablet is placed in room temperature, until liquid is absorbed, dries tablet.Culture 10-16h, observation are inverted at 37 DEG C
Whether resistant clones are grown；Time cannot be long, otherwise will appear satellite colony.
G) bacterium colony PCR
Bacterium colony is chosen using toothpick method.Go out 16 small lattice of pros in the LB agar plate reverse side marker strokes of the benzyl containing ammonia,
As spare.PCR reaction system total volumes are that 5.5 μ L aqua sterilisas are added in advance in 10 μ L, PCR tubules, use autoclave sterilization
Toothpick gently choose the monoclonal colonies on tablet, row dry oblique line on the solid medium of the small lattice of pros, then small in PCR
Gently turn toothpick tip in pipe, that is, that completes bacterium colony selects work.0.5 μ L primer and 5 μ L are added in PCR tubules
primer STAR Hs.(PCR procedure references step B) by PCR product into row agarose gel electrophoresis, observation is electric in the UV lamp
Swimming band, judges whether the bacterium colony is purpose bacterium colony according to clip size.With toothpick on the small lattice surface of the corresponding bacterium colonies of positive PCR
Bacterium is gently chosen, is added in suitable fluid nutrient medium containing kanamycins and does plasmid extraction preparation.Then it is tried according to plasmid extraction
Agent box specification extracts plasmid
I) digestion verification
It is as follows to prepare double digestion system：
Carry out agarose electrophoresis, according to the band of DNA ladder indicate, judge digestion products band whether with mesh
Band coincide.
(2) eukaryotic expression of PLS3 albumen
The CHO-K1 cells after antibiotic-free culture for 24 hours, cell confluency degree is taken to carry 70% or so, transfected.Take two
The sterile centrifugation tube of 1.5mL takes in one pipe 3 μ g plasmids in 200 μ L without in the DME/F-12 culture mediums of serum and antibiotic,
Mixing；Another centrifuge tube is added in the DME/F-12 culture mediums of 7.5 μ g plasmids and 200 μ L without serum and antibiotic, mixing；
By the liquid blending in two centrifuge tubes after standing 5min, 20min is stood.Then it takes out from cell incubator and is ready in advance
Cell, discard original culture medium, after being cleaned using PBS, the serum free medium of 5mL be added, then will before mixing it is quiet
The liquid set is added.After standing 6h in 37 DEG C of cell incubators, cell changes liquid, and culture mediums of the 10mL containing serum is added.Then exist
After cultivating 48h in cell incubator, fluorescence microscope cell transfecting efficiency is used.Such as Fig. 5 it is observed that PLS3-ABD2
The Successful transfection of plasmid and expression.
(3) purifying of PLS3 albumen
A cell pyrolysis liquid) is prepared
CHO-K1 cells after transfection discard culture medium, and 1mL lysates (300mM NaCl, 20mM imidazoles, 20mM is added
PB, pH7.0), then scraper is used to collect in cell to 1.5mL centrifuge tubes；The multigelation three in liquid nitrogen and 37 DEG C of water-baths
It is secondary；By the liquid after multigelation in 4 DEG C, 2000rpm, 5min is centrifuged；Supernatant after centrifugation is through 0.22 μm of membrane filtration
After be added 10 μ L lysates dilution, that is, preliminary cell pyrolysis liquid is made.
B) the purifying of PLS3-ABD2 recombinant proteins
Using Ni-NTA Resin to step A) PLS3-ABD2 with His labels contained in gained cell pyrolysis liquid weighs
Histone is isolated and purified.Fill column：Appropriate medium is added chromatographic column, stood by re-suspending medium according to protein content to be purified；
Balance：With 5-10 times of column volume equilibration buffer chromatographic column；Loading：Sample buffer should as far as possible with equilibration buffer one
It causes, to avoid blocking chromatographic column, sample should be through centrifuging or using 0.45 μm of filter filtering；Washing：After loading, 5-10 is used
Times column volume equilibration buffer solution chromatographic column collects efflux；Elution：The imidazoles of various concentration is prepared with equilibration buffer,
Carry out gradient elution.
Equalizing and buffering formula of liquid：300mM NaCl, 20mM imidazoles, 20mM PB, 10mM Tris base, pH7.0.
C it) after pure at the beginning of the Ni-NTA Resin, is purified using Alliance liquid chromatographs, purification step is according to instrument
Device specification carries out.The efflux for collecting 15min-18min, verifies through liquid phase, obtains simple spike, through Western blot and matter
Spectrum is verified as PLS3-ABD2 recombinant proteins.
2. experimental result and analysis
The plasmid map of quasi- structure is as shown in Figure 1, be named as pIRES2-ZsGreen1-PLS3-ABD2.What amplification obtained
PLS3-ABD2cDNA purpose bands about in 750bp or so, take forward position most bright wisp band to be cut (Fig. 2).Glue after cutting carries out
Glue recycles.CDNA segments after glue recycling carry out EcoR I and BamH I double digestions with pIRES2-ZsGreen1 plasmids.Double digestion
Product is respectively into row agarose gel electrophoresis (Fig. 3), and gel extraction, then the two is with certain proportion progress enzyme connection.Connection production
Object converts DH5 α competence, and bacterium solution is uniformly smeared to the tablet containing kanamycins, and next day picking monoclonal carries out bacterium colony PCR
Experiment.By PCR product into row agarose gel electrophoresis, electrophoretic band is observed in the UV lamp, which is about 200bp,
It coincide (Fig. 4) with purpose band.Next, the method using double digestion is verified.It is indicated according to the band of DNA ladder,
Detection has two master tapes, respectively in 5000bp and 800bp or so, band and pIRES2-ZsGreen1 with PLS3-ABD2cDNA
The segment of empty plasmid band is coincide.After digestion verification passes through, generation bidirectional sequencing is carried out, testing result shows and sequence SEQ
ID NO.3 are consistent.Thus the present invention successfully builds the eukaryon expression plasmid of pIRES2-ZsGreen1-PLS3-ABD2.
Contain IRES elements in pIRES2-ZsGreen1 carriers, multiple cloning sites are separated, can build while express two
The recombinant plasmid of a gene.When using the plasmid transfection eukaryocyte, target gene is recorded with reporter gene ZsGreen1 corotation
It for a mRNA, but translates respectively, can monitor whether to transfect successfully and reporter gene protein influences target gene albumen
Structure and function；ZsGreen1 is the highest green fluorescent protein of brightness, is suitble to flow cytometer and other fluorescent screenings；
PIRES2-ZsGreen1 plasmids are high copy number plasmid, containing screening-gene Neo genes, can be used in screening expression PLS3 albumen
Stable cell lines.Therefore the structure of PLS3-ABD2 segments, phase are carried out in the present invention using pIRES2-ZsGreen1 carriers
Advantage is had more than other eukaryotic vectors.It respectively can by comparing fluorescence intensity to 2 μ g of CHO-K1 cell line transfections, 4 μ g plasmids
To find that 4 μ g plasmid-transfected cells are more efficient (Fig. 5).
To transfecting the CHO-K1 cell lines after the plasmid, mild cell cracking is carried out with Cell Lysis buffer, it will
Supernatant after cracking crosses the nickel column equipped with Ni-NTA, by changing the concentration of imidazoles in eluent, detaches and purifies PLS3-
ABD2 albumen.As shown in fig. 6, the PLS3-ABD2 protein content biggers of the CHO-K1 cell lines expression after transfection purpose plasmid；And it passes through
After Ni-NTA Resin preliminary purifications, when 200mM elutions, most of foreign protein has removed, only remaining purpose band and
The foreign protein of 70KD or so.Next 200mM eluents are purified through Alliance liquid chromatographs, purification step foundation
Instrument specification carries out.The efflux for collecting 15min-18min, verifies through liquid phase, obtains simple spike (Fig. 7).Through Western
Blot and mass spectrum are verified as PLS3-ABD2 recombinant proteins.
2 pIRES2-ZsGreen1-PLS3-ABD2 of embodiment is overexpressed in CHO-K1 cell lines
Whether can to detect the eukaryotic expression vector pIRES 2-ZsGreen1-PLS3-ABD2 built in embodiment 1
Successful expression His labels and PLS3 albumen, the present invention use the expression of the method detection fusion albumen of Western blot.
1. experiment material
Main agents：
Protein pre-dyed Marker：Fermentas companies of the U.S.；Antibody information is as follows：anti-PLS3(Santa Cruz,
CA,USA)；Cell pyrolysis liquid and His-Tag (Cell Signaling Technology)；β-Actin (Bo Aoyijie companies)；
PVDF and ECL luminescent solutions are purchased from Millipore；BCA determination of protein concentration kit (Beyotime)；Protease
Inhibitor Cocktail(Calbiochem)。
Key instrument：
Gel imager and vertical electrophoresis apparatus are purchased from Bio-rad.
2. experimental method
(1) CHO-K1 cells are transfected
In transfection method such as embodiment 1 shown in method, respectively with unloaded and structure PLS3-ABD2 plasmid transfections CHO-K1
Cell line.After transfectional cell culture 48h, total protein is extracted.
(2) measurement of protein content
A it) takes 1.2mL protein standards to prepare liquid to be added in a pipe standards albumen (30mg, BSA), fully be prepared after dissolving
At the protein standard solution of 25mg/mL.- 20 DEG C of long-term preservations can be sub-packed in after preparation immediately.
B appropriate 25mg/mL protein standards) are taken, final concentration of 0.5mg/mL is diluted to.According to sample size, by 50 volumes
BCA reagent As add 1 volume BCA reagents B (50:1) appropriate BCA working solutions, are prepared, are mixed well.BCA working solutions room temperature 24 hours
Interior stabilization.
C) standard items are added to by 0,1,2,4,8,12,16,20 μ L in the standard sample wells of 96 orifice plates, add standard dilutions
It supplies to 20 μ L.Add in proper volume sample to the sample well of 96 orifice plates, adds standard dilutions to 20 μ L.
D) each hole is added 200 μ L BCA working solutions, 37 DEG C place 20-30 minutes after measure.When measurement, using microplate reader,
Each hole OD values are measured at 570nm, draw standard curve, calculate protein content, are needed to dispense protein sample according to experiment, and protect
In the presence of spare in -80 DEG C of refrigerators.
(3)Western blot
A cell sample) is rinsed with 1 × PBS of precooling, suitable cell pyrolysis liquid is added, 1 is pressed in lysate:100 are added
Protease inhibitors.Scraping cell with cell scraper immediately makes it be sufficiently mixed with lysate, ice bath 30min.After cracking, in 4
DEG C, 12,000rpm centrifugation 15min collect supernatant in new EP pipes.With BCA kit measurement protein contents.By sample and 5
× sample-loading buffer presses 1:4 mixing, boil 10min, centrifuging and taking supernatant.The applied sample amount of total protein is 15 μ g.It is drawn with liquid-transfering gun
The sample handled well, is added slowly in well.Start electrophoresis, waits for that sample completes concentration step, 120V is adjusted to by 80V.
B) after electrophoresis, transferring film.Clip corresponds to the pvdf membrane of size, impregnates 1min in methyl alcohol, then be placed in distilled water
In.Gel will be put just as possible, and bubble removing is removed with glass bar (careful operation pays attention to not breaking gel into pieces).According to albumen marker
Judge the sequence of loading, and pvdf membrane is completely covered by gel, cuts off one jiao as label.Leaching is stacked on transfer film successively
The three layers of filter paper moistened and one layer of sponge close transferring film folder.Whole process should avoid introducing bubble.It is set according to destination protein property
Determine transferring film electric current and transferring film time, start transferring film, the general transferring film time is the transferring film 2h at 200mA.
C the closing of 5% skimmed milk power) is prepared, shaking table is incubated 0.5h or more or is incubated overnight.According to albumen marker instructions
Molecular weight cuts pvdf membrane.Pvdf membrane is put into hybridization bag, and the primary antibody solution diluted with 1%BSA, 4 DEG C of incubator overnights are added
Or 2h under room temperature.Secondary daily PBST is rinsed 3 times, each 10min.Two corresponding anti-solution (the dilution diluted with 1%BSA is added later
Ratio is 1:3000), shaking table is incubated 1h.PBST is rinsed 3 times, each 10min.ECL luminescent solutions are added dropwise on film, illustrate according to reagent
It is operated, passes through chemiluminescence imaging System Gel imager capture images.
3. experimental result and analysis
After transfecting CHO-K1 cells 48h, albumen is extracted, detects protein content.Sample applied sample amount is adjusted according to albumen concentration,
Carry out Western blot experiments, use respectively anti-His (purchased from Cell Signaling Techonology, 12698) and
Anti-PLS3 antibody (being purchased from Santa Cruz Biotechnology, sc-166208) is detected.The results are shown in Figure 8,
Under internal reference β-Actin expression quantity unanimous circumstances, compared with unloaded group, the high expression His labels of PLS3 groups success and PLS3 eggs
In vain, show plasmid construction and transfect success.The His label proteins band of expression is in 28KD or so, with PLS3-ABD2 protein bands
It is in the same size, show that PLS3-ABD2 recombinant proteins being capable of successful expression.
The screening of 3 PLS3-ABD2 albumen high-affinity peptide fragments of embodiment
1. experiment material
Main agents：N-methylmorpholine, N,N-dimethylformamide (DMF), dichloromethane (DCM), hexahydropyridine (Beijing
Chemical plant), Fmoc protected amino acids (Shanghai gill biochemistry Co., Ltd), TentaGel resins (Rapp Polymere), magnetism
Microballoon (thinks happy chromatographic technique development centre in Tianjin) again, streptomysin (Gbicol)；
Key instrument：
High performance liquid chromatograph (Waters e2695), ground substance assistant laser dissociation-time of flight mass spectrometry (MALDI-
TOF-MS, BIFLEX III, Bruker Daltonics Inc. Germany).
2. experimental method
A) peptide library synthesizes
The synthesis of peptide library is carried out using Tentagel resins as solid phase carrier.The resin of 150mg is taken, it is molten using DMF
Then swollen resin 30min cleans three times resins with DMF；Deprotection, is added 20% hexahydropyridine, on room temperature shaking table 10min into
Row deprotection；Deprotection is examined, is alternately cleaned using DMF and DCM, is cleaned 6 times altogether, last takes few all over centainly being cleaned with DMF
The PE that resin is measured in 1.5ml is managed, and each drop of vitamin C, stupid aldehyde, ninhydrin is added, 30s, observation tree are then boiled in boiling water
Whether fat changes colour, if discoloration, illustrates that resin is deprotected successfully；After the completion of deprotection, it is sequentially added into Fmoc protection ammonia successively
Base is sour, 2-3h on room temperature shaking table；It is cleaned 3 times using DMF, carries out deprotection inspection again, if resin is non-discolouring, illustrate amino acid
It has connected；The connection for carrying out deprotection and next amino acid after amino acid is connected, above step is repeated, to the last one
A amino acid；After the deprotection of the last one amino acid, amino acid is started to shrink at, is first cleaned 3 times with DMF, then is cleaned 3 times with DCM,
Methanol cleans 3 times, can take out resin after draining, be stored in -20 DEG C.
B) screening of PLS3 specific polypeptides and Mass Spectrometric Identification
Egg is recombinated using ChromaLink Biotin Labeling Kit biotin labeling reagent boxes label PLS-ABD2
In vain；Peptide library 2h is swollen using PBS and clean 3 times simultaneously；With 5% skim milk blocking peptide pearl non-specific sites, 37 DEG C, mix
Outstanding instrument rotation is cleaned 3 times after being incubated 2h with PBS；The albumen of 2ml marked by streptavidin is added, 1h is incubated on 37 DEG C of suspension instrument；
Picking has the peptide pearl of combination under the microscope, then carries out second order ms sequencing, finally obtains polypeptide with Mascot software spectrum unscramblings
Sequence.
3. experimental result is expected
The polypeptide specifically bound with PLS3-ABD2 is screened, is verified from cellular level and animal level.
Comparative example 1
The CHO-K1 cells in eukaryotic host cell 293T alternative embodiments 1 commonly used in the art are respectively adopted in this comparative example
The pIRES2-ZsGreen1-His-PLS3-ABD2 eukaryon expression plasmids that the structure of embodiment 1 obtains are transfected into above-mentioned by system respectively
In different eukaryotic host cell systems, and PLS3-ABD2 recombination eggs are isolated and purified using purification process same as Example 1
In vain.
By comparing the Western Blot after destination protein initial purification as a result, as shown in fig. 6, can by experimental result
Know, will can express the eukaryotic expression vector transfections of PLS3-ABD2 recombinant proteins to CHO-K1 cell lines, relative to transfection to other
Eukaryotic host cell system has destination protein expression quantity higher, the relatively small number of advantage of foreign protein.
It should be understood that after the dosage of above-described embodiment agents useful for same or raw material is carried out equal proportion expansion or is reduced
Technical solution, it is substantially identical with above-described embodiment.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>State Nanometer Science Center
<120>A kind of preparation method of PLS3-ABD2 recombinant proteins
<141> 2018-05-03
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> DNA
<213>Artificial primer (Artificial Sequence)
<400> 1
cggaattcat gcatcatcac catcatcata accaggatat 40
<210> 2
<211> 30
<212> DNA
<213>Artificial primer (Artificial Sequence)
<400> 2
cgcggatcct tatctcttca ttcccctgcc 30
<210> 3
<211> 771
<212> DNA
<213>Artificial primer (Artificial Sequence)
<400> 3
gaattcatgc atcatcacca tcatcataac caggatattg actggactct attagaagga 60
gaaactcgtg aagaaagaac cttccgtaac tggatgaact ctcttggtgt caatcctcac 120
gtaaaccatc tctatgctga cctgcaagat gccctggtaa tcttacagtt atatgaacga 180
attaaagttc ctgttgactg gagtaaggtt aataaacctc catacccgaa actgggagcc 240
aacatgaaaa agctagaaaa ctgcaactat gctgttgaat tagggaagca tcctgctaaa 300
ttctccctgg ttggcattgg agggcaagac ctgaatgatg ggaaccaaac cctgacttta 360
gctttagtct ggcagctgat gagaagatat accctcaatg tcctggaaga tcttggagat 420
ggtcagaaag ccaatgacga catcattgtg aactgggtga acagaacgtt gagtgaagct 480
ggaaaatcaa cttccattca gagttttaag gacaagacga tcagctccag tttggcagtt 540
gtggatttaa ttgatgccat ccagccaggc tgtataaact atgaccttgt gaagagtggc 600
aatctaacag aagatgacaa gcacaataat gccaagtatg cagtgtcaat ggctagaaga 660
atcggagcca gagtgtatgc tctccctgaa gaccttgtgg aagtaaagcc caagatggtc 720
atgactgtgt ttgcatgttt gatgggcagg ggaatgaaga gataacctag g 771
<210> 4
<211> 735
<212> DNA
<213>Artificial primer (Artificial Sequence)
<400> 4
aaccaggata ttgactggac tctattagaa ggagaaactc gtgaagaaag aaccttccgt 60
aactggatga actctcttgg tgtcaatccc cacgtaaacc atctctatgc tgacctgcaa 120
gatgccctgg taatcttaca gctatatgaa cgaattaaag ttcctgttga ctggagtaag 180
gttaataaac ctccataccc gaaactggga gccaacatga aaaagctaga aaactgcaac 240
tatgctgttg aattagggaa gcatcctgct aaattctccc tggttggcat tggagggcaa 300
gacctgaatg atgggaacca aaccctgact ttagctttag tctggcagct gatgagaaga 360
tataccctca atgtcctgga agatcttgga gatggtcaga aagccaatga cgacatcatt 420
gtgaactggg tgaacagaac gttgagtgaa gctggaaaat caacttccat tcagagtttt 480
aaggacaaga cgatcagctc cagtttggca gttgtggatt taattgatgc catccagcca 540
ggctgtataa actatgacct tgtgaagagt ggcaatctaa cagaagatga caagcacaat 600
aatgccaagt atgcagtgtc aatggctaga agaatcggag ccagagtgta tgctctccct 660
gaagaccttg tggaagtaaa gcccaagatg gtcatgactg tgtttgcatg tttgatgggc 720
aggggaatga agaga 735
Claims (10)
1. a kind of preparation method of PLS3-ABD2 recombinant proteins, which is characterized in that the method includes：SEQ ID will be contained
The cloned dna molecule of sequence shown in NO.4 is and thin to eucaryon host by the eukaryotic expression vector transfection to carrier for expression of eukaryon
It is cultivated in born of the same parents system.
2. preparation method according to claim 1, which is characterized in that the carrier for expression of eukaryon is pIRES2-
ZsGreen1 plasmids.
3. preparation method according to claim 1 or 2, which is characterized in that the eukaryotic host cell system is that CHO-K1 is thin
Born of the same parents system.
4. according to claims 1 to 3 any one of them preparation method, which is characterized in that the sequence shown in SEQ ID NO.4
His-tag is added before DNA molecular.
5. preparation method according to claim 4, which is characterized in that by the eukaryotic expression vector transfection to eucaryon host
After being cultivated in cell line, collects eukaryotic host cell and isolate and purify PLS3-ABD2 recombinant proteins；
The isolation and purification method of the PLS3-ABD2 recombinant proteins includes the following steps：
(1) it collects the eukaryotic host cell after culture and is cracked：
The eukaryotic host cell after culture is collected, lysate is added, after multigelation, centrifuging and taking supernatant is added after filtering and splits
Liquid dilution is solved, cell pyrolysis liquid is obtained；
(2) preliminary purification：
Using nickel-agarose Gel column by the PLS3-ABD2 recombinant proteins with His-tag contained in above-mentioned cell pyrolysis liquid into
Row isolates and purifies；
(3) secondarily purified：
The purified product of step (2) is carried out to secondarily purified, to be purified PLS3-ABD2 recombination eggs using liquid chromatograph
In vain.
6. preparation method according to claim 5, which is characterized in that in the step (1), the eukaryotic host cell system
For CHO-K1 cell lines, after the CHO-K1 cell culture 48 hours after transfection, culture medium is outwelled, PBS cleaning cells are added and add
Enter lysate, lysate is added, collects in cell to centrifuge tube；After multigelation, by gained liquid centrifuging and taking supernatant, warp
Lysate dilution is added after 0.22 μm of membrane filtration, obtains the cell pyrolysis liquid.
7. preparation method according to claim 6, which is characterized in that the pH of the lysate is 7.0, contains 300mM
NaCl, 20mM imidazoles, 20mM PB.
8. preparation method according to claim 5, which is characterized in that in the step (2), using balance as following formula
Buffer solution washs nickel-agarose Gel column after loading：
Equalizing and buffering formula of liquid：300mM NaCl, 20mM imidazoles, 20mM PB, 10mM Tris base, pH7.0.
9. the recombinant protein that claim 1~8 any one of them preparation method is prepared.
10. the recombinant protein described in claim 9 is in PLS3 interactions between protein protein screening or PLS3 protein-specific combination polypeptides
Application in terms of screening.
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| CN107446949A (en) * | 2017-07-25 | 2017-12-08 | 国家纳米科学中心 | PLS3 recombinant proteins eukaryon expression plasmid and its construction method and application |
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| CN101765774A (en) * | 2007-07-19 | 2010-06-30 | 生物梅里埃公司 | The I-fimbrin assay method of in-vitro diagnosis colorectal cancer |
| CN107446949A (en) * | 2017-07-25 | 2017-12-08 | 国家纳米科学中心 | PLS3 recombinant proteins eukaryon expression plasmid and its construction method and application |
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| CN110025766A (en) * | 2019-04-22 | 2019-07-19 | 南京医科大学 | The medical usage of fimbrin T |
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