CN101280014A - Anti-Cyclin D1 human single-chain antibody - Google Patents
Anti-Cyclin D1 human single-chain antibody Download PDFInfo
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Abstract
The invention relates to an anti-CyclinD1 human single-chain antibody which is composed of IgK leader peptide, anti-CyclinD1 human single-chain antibody, E-tag peptide and KDEL gene coding sequence left on endoplasmic reticulum. The anti-CyclinD1 human single-chain antibody can be specifically combined with CyclinD1 protein after dissoluble expression, purification and being activated. If being recombined in a eucaryon expression vector after the addition of the localization signals in the cell left on the endoplasmic reticulum, the anti-CyclinD1 human single-chain antibody can restrain the proliferation of tumor cells after the transfection of the tumor cells.
Description
Technical field
The present invention relates to a kind of single-chain antibody.A kind of evaluation of anti-Cyclin D 1 human single-chain antibody specifically, born of the same parents' internalization and activity characterization.
Background technology
The appearance of single-chain antibody is the intensification of people's antagonist understanding and the inevitable outcome of scientific technological advance.Since Britain and Japanese scholar have found Diphtheria Antitoxin, and first immune serum has been used for since the passive immunotherapy and serodiagnosis of transmissible disease, antibody technique enjoys people to pay close attention to as a kind of treatment means.
Kohler and Milstein 1975 are at Nature, 1975,256:.495 middle discovery also utilizes hybridoma technology, successfully prepare monoclonal antibody with high specific and avidity, overcome the shortcoming of polyclonal antibody poor specificity, so the appearance of monoclonal antibody gets more and more people's extensive concerning, the various countries scholar puts in the studies on Monoclonal Antibody work one after another, and the effective ways of diagnosis and treatment disease are found in expectation.At present, there has been multiple monoclonal antibody to enter clinical use.But the monoclonal antibody of clinical application at present substantially all is the mouse source, and the progress of human monoclonal antibody is slow.
Though monoclonal antibody technique has obtained very big development, but still exist limitation, wherein the most fatal shortcoming is that monoclonal antibody enters the people as a kind of heterologous protein and knows from experience the initiation immunological rejection, this has not only influenced the performance of monoclonal antibody curative effect, also can disturb its proper distribution in vivo, thereby limit its application in clinical.
After entering the eighties in 20th century, the fast development of Protocols in Molecular Biology has promoted the progress of antibody technique research, and genetic engineering antibody arises at the historic moment, and wherein paid close attention to by people with single-chain antibody.Since Bird in 1988 and Huston, Procatl Acad SciUSA, 1988,5:5875-5879 has successfully developed since first single-chain antibody, succeeded in developing and produced multiple single-chain antibody at present, except that the in-vitro diagnosis that is used for basic medical research and disease, there are some to begin to be used for clinical treatment.As an emerging things, the single-chain antibody technology has become the forward position and the hot issue of antibody Related Research Domain now.It not only can be transformed existing antibody, can also create the non-existent brand-new antibody of nature; Not only can reduce the immunogenicity of antibody, can also strengthen the curative effect of antibody.
Single-chain antibody is the small molecular antibody with the gene engineering method preparation, it is the recombinant antibodies that the variable region of heavy chain (VH) of antibody and variable region of light chain (VL) is formed by connecting by elasticity connection peptides (being generally 12-15 amino acid), its molecular weight only is equivalent to the sixth of original antibody, but single-chain antibody contains whole antigen binding sites, so single-chain antibody has farthest kept the antigen-binding activity of antibody, be minimal segment with parental antibody antigen-binding activity.
After single-chain antibody occurs, people are in the process of research single-chain antibody, find some unrivaled superiority that single-chain antibody has, so people just utilize prior art that single-chain antibody is transformed and modified, with function and the new purposes of development of improving single-chain antibody, the new antibodies that develops based on single-chain antibody that has occurred at present has following several: single-chain antibody polymer, bispecific single-chain antibody, single domain antibody, the single-chain antibody with constant region, intracellular antibody (intracellularantibody or intrabody).Wherein the intracellular antibody technology is in recent years, along with the further investigation of signal conduction in people's antagonist engineering and the cell, derives the antibody technique of important target protein in the brand-new cell capable of blocking.
The intracellular antibody technology can suitably be modified by adding method antagonist molecules such as nucleus signal for locating or ER retention signal, make it directional profile in nucleus, cytoplasm or some organoid, thereby specificity is disturbed or blocking-up is distributed in the activity of some biomacromolecule at this position or processing, secretion process, the a series of bioprocesss that cause cell change, thereby reach the effect of combination and the arbitrary cytoplasmic structure of inactivation.It is an another novel gene treatment approach after technology such as sense-rna, specific ribozyme, the negative sudden change of dominance, suicide gene.Because single-chain antibody has simple in structure, low than the parental antibody immunogenicity, but the affine activity of the antigen that has kept parental antibody preferably, compare with complete antibody, single-chain antibody also has advantages such as the residence time is short in non-target tissue, blood is removed soon, tissue penetration is strong, and the single-chain antibody gene ratio is easier to obtain, and can extract mRNA from hybridoma and carry out RT-PCR or screen from antibody library.Therefore, single-chain antibody becomes the normal antibody mode that adopts of intracellular antibody technology, and reaching its maturity of antibody library technology promoted the development of single-chain antibodies in the born of the same parents, has been penetrated into oncotherapy at present, and a plurality of fields such as nerve degenerative diseases enjoy investigator's favor.
Cell cycle is out of control to be the one of the main reasons that causes the tumour cell malignant proliferation.CyclinD1 albumen is that cell enters first cyclin of cell cycle synthetic, by with CDK4/CDK6 (Cyclin dependent kinase, CDK) being formed with active complex body, promoting the conversion of cell G0 → S phase, is the rate-limiting factor that the G1/S phase changes in the cell cycle progression.The overexpression of CyclinD1 and the generation of kinds of tumors, the development closely related, be confirmed to be a kind of oncogene.Calendar year 2001 Harvard University Yu etc. at Nature, 2001, among the 411:1017 by the CyclinD1 gene knockout mouse model approach that to research and propose anti-CyclinD1 treatment may be treatment mammary cancer high degree of specificity.This result provides experimental basis for CyclinD1 as a target spot of oncotherapy.Because CyclinD1 albumen can not be secreted into the extracellular, therefore, on the design therapeutic strategy, exist difficulty.Although once had investigators in tumour cell, to inject the form that recombinant plasmid that anti-CyclinD1 antibody, antisense oligonucleotide and importing transcribe Antisense Cyclin D1 all can change transformant, suppressed the propagation of tumour cell.But because antisense nucleotide is unstable relatively, and just at the antagonism of Nucleotide, sphere of action is narrower.
Intracellular antibody is being obtained some progress aspect the therapy of tumor research at present.The Curiel study group of Alabama university has carried out aspect the intracellular antibody gene therapy in a large number, systematic research.Found that single-chain antibody can suppress ovarian cancer cell propagation in the anti-erbB-2 born of the same parents, cell death inducing has strong restraining effect to tumour cell clone generation.It is clinical that adenovirus mediated anti-erbB-2 single-chain antibody (AD21) has been used to the I phase at present, Cancer Res.2000, and 6 (8): the result in 3081 shows that it is safe and feasible that AD21 is used for ovarian cancer patients.2000, Proc.Natl.Acad.Sci.USA, 2000, breadboard Rabbitts study group of Britain's molecular biology Medical Res Council is the anti-caspase-3 intracellular antibody that target spot makes up, effectively inducing apoptosis of tumour cell with the molecule relevant with apoptosis among the 97:12266.Domestic less about the research of intracellular antibody therapy of tumor, wherein seminar of Zhen Yong Soviet Union is that target spot has made up the single-chain antibody of expressing in the born of the same parents to shift relevant IV Collagen Type VI enzyme with malignant tumour, studies show that this antibody in intracellular expression, secretion, growth of tumour cell and the external invasive ability of IV Collagen Type VI enzyme had significant inhibitory effect.These results of study more and more demonstrate intracellular antibody as great potential and the application prospect of a class recruit in the Antioncogene treatment.
Cell cycle is out of control to be the one of the main reasons that causes the tumour cell malignant proliferation, and the oncotherapy research that therefore to carry out with crucial regulatory factor of cell cycle be target spot has application promise in clinical practice.CyclinD1 albumen is that cell enters first cyclin of synthetic proliferating cycle, by with CDK4 (Cyclin dependent kinase, CDK) be formed with active complex body CyclinD1/CDK4, promoting the conversion of cell G0/S phase, is the rate-limiting factor that the G1/S phase changes in the cell cycle progression.The overexpression of CyclinD1 gene and the generation of kinds of tumors, development, prognosis are closely related, have been confirmed to be a kind of oncogene.There is the investigator in tumour cell, to inject the recombinant plasmid that Antisense Cyclin D1 is transcribed in anti-Cyclin D1 antibody or importing, aforesaid method has all changed the transformant form, suppress tumor cell proliferation, these results provide experimental basis for Cyclin D1 as a target spot of oncotherapy.But the former is owing to the antibody molecule amount causes immunogenicity stronger greatly, and the latter exists shortcomings such as the interior transformation period weak point of body again.And rising in recent years with the intracellular antibody technology of antibody action effect molecule for addressing the above problem the thinking that provides new.
Summary of the invention
In view of being the problem that exists in the treatment of target spot with Cyclin D1 at present, the purpose of this invention is to provide a kind of efficient, special, stable anti-Cyclin D1 single-chain antibody, and realize that it effectively knocks out the phenotypic function of Cyclin D1 in cell.
To achieve these goals, technical scheme of the present invention is:
A kind of anti-Cyclin D1 human single chain variable fragments antibody, its nucleotide sequence is as described in the Sequence NO.1.
A kind of anti-Cyclin D1 human single chain variable fragments antibody is described, is made up of the leading peptide of IgK, anti-Cyclin D1 human single chain variable fragments antibody, E-tag labelled peptide and endoplasmic reticulum retention peptide KDEL gene coded sequence, and its nucleotide sequence is as described in the Sequence NO.2.
This anti-Cyclin D1 human single chain variable fragments antibody has application in the medicine of antitumor action in preparation.
Described tumour is human breast carcinoma and human cervical carcinoma.
With prokaryotic expression is antigen with the recombinant human cyclinD1 albumen of Ni-NTA Agarose purifying also, from phage antibody library, screen the proteic single-chain antibody of specific anti-human cyclinD1 by " absorption-wash-out-amplification " elutriation process, carry out Analysis and Identification by the ELISA method to the specificity of acquisition antibody with in conjunction with activity, further behind the expression and purification, its activity is carried out vitro characterization, promptly obtain the anti-people cyclinD1 of specific humanized single-chain antibody.Because the endoplasmic reticulum signal for locating can make single-chain antibody correctly be folded to form activated conformation in endoplasmic reticulum, and can make transformation period prolongation in its body, thereby better play a role, therefore, further as template, (PCR) is cloned into ER retention signal by the polymerase chain reaction, the E-tag detection signal, add corresponding restriction enzyme site, promptly obtain single-chain antibody gene in the anti-Cyclin D1 people source born of the same parents, and then with its subclone in carrier for expression of eukaryon pcDNA3.1, thereby obtained the carrier for expression of eukaryon pER-AD κ of single-chain antibody (ER-AD κ) genes in the anti-Cyclin D1 people source born of the same parents of reorganization endoplasmic reticulum retention type.Behind pER-AD κ transfection human breast cancer MCF-7 cell's strain and human hela HeLa cell strain, expression, location situation and external biological activity to single-chain antibody gene in the anti-Cyclin D1 people source born of the same parents of endoplasmic reticulum detention type have been carried out series of studies, the result shows, in the anti-Cyclin D1 people source born of the same parents of endoplasmic reticulum retention type single-chain antibody gene can be in tumour cell effective expression and realize the object location of intracellular antibody.The stably express of the interior single-chain antibody gene of the anti-Cyclin D1 people source born of the same parents of endoplasmic reticulum retention type is cell growth inhibiting and propagation significantly, causes cell-cycle arrest, and obvious cell death inducing.
The present invention has following advantage: what obtained among the present invention is people source anti-Cyclin D1 single-chain antibody, avoided the mouse resource monoclonal antibody to be used for the toxic side effect of human body, in addition can be by introducing nucleus or ER retention signal etc., further recombinate in the carrier for expression of eukaryon, make this single-chain antibody privileged site combination, deactivation Cyclin D1 function in tumour cell, for new way is opened up in oncotherapy.
Description of drawings
Fig. 1 phage antibody and cyclinD1 antigen binding capacity are measured
Fig. 2 soluble single-chain antibody and the proteic activity that combines of cyclin D1
Solubility expression and the purifying of the anti-cyclinD1 single-chain antibody of Fig. 3 AD κ
1.AD κ/HB2151 IPTG induces preceding bacterial sediment; 2.IPTG induce bacterial sediment; 3. go up the sample effluent liquid; 4. ammonium sulphate precipitation supernatant; 5. binding buffer liquid scrub stream fluid; 6. lavation buffer solution scrub stream fluid; 7. the lavation buffer solution washing is flowed out; 8-13. elution buffer wash-out 1,2,3,4,5,6; 14. molecular weight of albumen standard.
The Western blot of the anti-cyclinD1 single-chain antibody of Fig. 4 AD κ analyzes
1.HB2151 empty bacterium; 2. the AD κ of purifying
The inhibition of the anti-cyclinD1 single-chain antibody of Fig. 5 AD κ antagonism cyclin D1 polyclonal antibody
The avidity curve of the anti-cyclinD1 single-chain antibody of Fig. 6 AD κ
Fig. 7 is an ER-AD kappa gene produced in fragments synoptic diagram
Fig. 8 is that recombinant expression vector pER-AD κ makes up schema
Fig. 9 cuts evaluation figure for recombinant expression vector pER-AD κ design of graphics and enzyme
1.1kb?DNA?ladder?marker；2.pcDNA3.1?plasmid；3.pER-ADκ?plasmid；4.pcDNA3.1digested?with?Hind?III+EcoR?I；5.pER-ADκ?digested?with?Hind?III+EcoR?I；6.ER-ADκdigested?with?Hind?III+EcoR?I；7.DL?2000?marker
Figure 10 analyzes the expression of ER-AD κ among MCF-7/pER-AD κ, MCF-7/pcDNA3.1, MCF-7, HeLa/pER-AD κ, HeLa/pcDNA3.1, the HeLa for RT-PCR
Figure 11 is location situation in the born of the same parents of indirect immunofluorescence experiment analysis ER-AD κ, and 1-6 is the transient transfection detected result; 7-12 is the stable transfection detected result
Figure 12 analyzes the expression of ER-AD κ among MCF-7/pER-AD κ, MCF-7/pcDNA3.1, MCF-7, HeLa/pER-AD κ, HeLa/pcDNA3.1, the HeLa for Dot-blot; Left 1.MCF-7; 2.MCF-7/pcDNA3.1; 3.MCF-7/pER-AD κ; Right 1.HeLa; 2.HeLa/pcDNA3.1; 3.HeLa/pER-AD κ
Figure 13 is the growth curve of MCF-7/pER-AD κ, MCF-7/pcDNA3.1, MCF-7, HeLa/pER-AD κ, HeLa/pcDNA3.1, HeLa
Figure 14 is the influence of ER-AD κ to the cell cycle of MCF-7, HeLa cell
Figure 15 is the apoptotic influence of ER-AD κ to MCF-7, HeLa cell
Embodiment
The screening of embodiment 1 specific anti-human cyclin D1 human single chain variable fragments antibody
Cyclin D1 takes place in tumour in order to further investigate, developing mechanism of action, be that target spot is carried out researchs such as tumor treatment, diagnosis better with cyclinD1, the present invention is an antigen with the recombinant human cyclinD1 albumen of Ni-NTA Agarose purifying also with prokaryotic expression, from the phage antibody library of people source, screen the proteic single-chain antibody of specific anti-human cyclinD1 by " absorption-wash-out-amplification " elutriation process, carry out Analysis and Identification to the specificity of acquisition antibody with in conjunction with activity by the ELISA method.Specific as follows:
One, experiment material
Phage single-chain antibody storehouse, people source, E.coli HB2151, XL1-Blue and helper phage VCSM13 are provided by Beijing Navy General Hospital professor Wang Yan, and prokaryotic expression also uses this research department of recombinant human cyclinD1 albumen of Ni-NTA Agarose purifying to prepare; The HRP/Anti-M13 monoclonal antibody is available from Pharmacia company, and anti-V5 monoclonal antibody is an Invitrogen company product; The goat anti-mouse igg of HRP mark is available from Beijing ancient cooking vessel state company; OPD, DAB are available from Sigma company; Other reagent is analytical pure.
Two, experimental technique
1. the screening of phage antibody library
Immobilization antigen immune absorption sieve method is adopted in the screening of antibody library, people CyclinD1 albumen is diluted to 100 μ g/ml with the 0.05mol/L carbonate buffer solution, managed (NUNC company) with the 1ml bag by immunity, with reference to Li Chunying etc. at Chinese skin cypridology magazine, 2005, the antagonist of method described in 19:388-390 storehouse is carried out 4 and is taken turns " absorption-wash-out-amplification " screening.Each is taken turns screening and all measures secondary storehouse titre, and calculates phage input/output than (rate of recovery), as the index of specific phage antibody enrichment.
2. the evaluation of phage antibody
Take turns the bacterium colony culture plate of screening 82 clones of picking at random from the 4th, be seeded among 2 * YT and cultivate, after spending the night with helper virus VCSM13 superingection inducing culture, the collection supernatant is a phage antibody.People cyclinD1 with 10 μ g/ml is antigen coated elisa plate, adds phage antibody supernatant to be measured through 1%BSA sealing back, is two anti-with the HRP/Anti-M13 monoclonal antibody, hatches, washs back OPD substrate and develop the color; The colour developing male clone wrap by elisa plate with purpose antigen (rh-cyclinD1) simultaneously with humanTNF-(rh-TNF) and ovalbumin (OA) again, identify the specificity of its antigen antibody reaction, used two anti-are the HRP/Anti-M13 monoclonal antibody, add the OPD colour developing after, read A
490Value.
3. the expression of solubility ScFv and activity identification
The antigenic phage antibody supernatant of specificity binding purposes is infected the HB2151 bacterial strain of logarithmic phase, hatch coating LB flat board (50 μ g/ml Amp) behind the 30min for 37 ℃.The single bacterium colony of picking next day is transferred with dilution in 1: 100 after the overnight incubation in 2 * YT nutrient solution, cultivates logarithmic phase, and adding IPTG is 1mmol/L to final concentration, and 30 ℃ of inducing culture spend the night, and centrifugal collection supernatant is solubility ScFv.By elisa plate, the sealing back adds expression supernatant to be detected, hatches 1h for 37 ℃ with 2 μ g/ml people cyclinD1 bag, the washing back adds the suitably Anti-V5 monoclonal antibody of dilution, hatches 1 hour for 37 ℃, and the washing back adds the suitably HRP-goat anti-mouse igg of dilution, after adding the OPD colour developing, read A
490Value.
Three, result and analysis
1. the screening of anti-people cyclinD1 phage antibody
Take turns screening through 4, the phage antibody rate of recovery obtains about 100 times of enrichments, prepare phage antibodies from last 1 82 clones that take turns picking, carry out primary dcreening operation with the ELISA method, found that 11 can be cloned with people cyclinD1 antigen bonded, again with TNF-α and ovalbumin in contrast antigen carry out phage E LISA and identify that only 7 clones' phage antibody can combine with people cyclinD1 specificity, nothing to do with antigen does not possess binding characteristic (Fig. 1).
2. the expression of soluble single-chain antibody and combination are active
For the phage antibody that detects acquisition active with combining of people cyclinD1, the phage antibody supernatant of 7 positive colonies is infected the bacterial strain HB2151 that no amber suppresses, IPTG induces the soluble single-chain antibody expression, wrap by elisa plate with people cyclinD1, with solubility expression supernatant, Anti-V5 monoclonal antibody and HRP-goat anti-mouse igg is antibody, carry out ELISA and detect, the result shows to have only 4,5, No. 9 clones to have specific binding activity (Fig. 2).Subsequent experimental result shows that No. 9 clones' light chain is the κ chain, can combine with the CyclinD1 specificity, and have better anti-tumor activity, so we is defined as anti-Cyclin D 1 human single-chain antibody with it, and called after AD κ, and carried out a series of researchs.
Solubility expression, purifying and the evaluation of embodiment 2 anti-Cyclin D1 human single chain variable fragments antibodies
One experiment material
Escherichia coli XL1-Blue, HB2151 are the same, Xylene Brilliant Cyanine G G-250, PMSF, anti-v5 antibody, available from Sigma company, available from ancient cooking vessel state bio-engineering corporation, His Trap HP Kit, PD-10 desalting column are available from Amersham Bioscience available from NeoMARKERS HRP mark goat-anti mouse IGg for anti-cyclin D1 antibody.
Two experimental techniques
The plasmid that will contain anti-CylinD1 single-chain antibody (AD κ) gene transforms expressive host bacterium HB2151.The picking mono-clonal is in 5ml 2YT (100 μ g/ml Amp
+, 10ug/ml ter
+) in 30 ℃ of 180～200rpm incubated overnight, overnight culture with 1: 100 the switching, OD is cultivated in 30 ℃ of continuation
600Be 0.5～1.0 o'clock, adding IPTG induces to final concentration 1mM and spends the night.Centrifugal collection bacterium liquid supernatant, and the low-temperature sludge that albumen is carried out with the ammonium sulfate of 40% (w/v), the centrifugal 20min collecting precipitation of 10000rpm, the 0.01M PBS dissolving of pH7.4, dialysis, desalination.Supernatant behind the desalination carries out western to be analyzed, to determine the accuracy of its product.The residue supernatant is crossed His Trap HP Kit purifying.And collect the effluent liquid of each component.Carrying out SDS-PAGE analyzes.
Three experimental results and analysis
Detect according to ELISA, the IPTG of AD κ/HB2151 that the result is positive induces supernatant to carry out ammonium sulfate precipitation, through the SDS-PAGE analysis revealed, when the amount of the ammonium sulfate that adds is 0.4 with the mass/volume ratio of inducing supernatant, the precipitation foreign protein that can precipitate abundant target protein and exceed helps purifying.Owing to be mixed with ammonium sulfate in the antibody, and there is disadvantageous effect in the existence of ammonium sulfate for purifying antibody and electrophoresis, dialyse more than the 4h, with PBS dissolving back to remove miscellaneous ammonium sulfate in the antibody with the single-chain antibody behind the ammonium sulfate precipitation.Because there is His-Tag in its C-terminal of single-chain antibody of expressing, so can use the Ni-NTA post to carry out purifying, the expression and purification result as shown in Figure 3.The result shows, bacterium after IPTG induces is deposited in tangible protein band about the 33kD place, and do not find the specific proteins band in the precipitation of the bacterium before empty bacterium and IPTG induce, can from the substratum supernatant of ammonium sulfate precipitation, obtain the single-chain antibody of purifying with the Ni-NTA post, with this single-chain antibody called after AD κ.The Western blot that carries out with anti-V5 antibody analyzes, and further proof has obtained the anti-Cyclin D 1 human single-chain antibody AD κ (Fig. 4) of purifying.
The activity characterization of embodiment 3 anti-Cyclin D1 human single chain variable fragments antibodies (AD κ)
One experiment material is the same
Two experimental techniques
1. the competitive ELISA method detects the activity of AD κ
Spent the night by elisa plate and 4 ℃ with the cyclin D1 albumen bag of 1-10 μ g/ml, seal 2h with 0.1%BSA-PBST next day, add 50 μ l AD κ then, add the anti-cyclin D1 of 50 μ l polyclonal antibody (diluting by 1: 200) again as competitive inhibitor with 0.1%BSA-PBST, with not with the anti-cyclin D1 of competitive inhibitor polyclonal antibody as positive control, adding 50 μ l Ami-V5 mouse monoclonal antibodies (diluting by 1: 5000 with 0.1%BSA-PBST) are hatched 2h after hatching 2h, add 50 μ l HRP-goat anti-mouse iggs (diluting by 1: 1000) again with 0.1%BSA-PBST, take OPD as the colour developing of substrate lucifuge, read the value of OD492.Calculate inhibiting rate with following formula:
Inhibiting rate=100% * (A value before suppressing-inhibition back A value)/A value before suppressing
2. the avidity of single-chain antibody AD κ is measured
What single-chain antibody avidity measure to adopt is non-competitiveness enzyme linked immunosorbent assay, and the mensuration of avidity need adopt through the cyclin D1 albumen of accurate quantification and the AD κ albumen behind the purifying, proteic quantitative usefulness be the Bradford method.Use through 2 earlier
nThe cyclin D1 protein 10 0 μ l bag of gradient dilution is spent the night by elisa plate, and inferior daily 0.1%BSA-PBST 200 μ l sealing 2h adds through 2 again
nThe single-chain antibody AD κ protein 10 0 μ l incubated at room 2h of gradient dilution adds the anti-and HRP-goat anti-mouse igg reaction of Anti-V5 mouse monoclonal antibody more respectively, with the colour developing of OPD lucifuge, occurs obvious brown back termination reaction in the hole, reads the value of OD492 then.Then according to formula K=(n-1)/(n[AB
1]-[AB]) calculate the affinity costant of single-chain antibody, n is the extension rate of single-chain antibody, Ab and Ab
1Represent that respectively working as antigen concentration is Ag and Ag
1The time, the antibody concentration of OD50.
Three results and analysis
1. the competitive ELISA method detects the activity of single-chain antibody
Resist being at war with property ELISA reaction with single-chain antibody AD κ behind the purifying and anti-cyclin D1 more, with single-chain antibody AD κ is with reference to antibody, with anti-cyclin D1 polyclonal antibody is competitive antibody, detects the competition combination degree that AD κ antagonism cyclin D1 resists more.When the result is presented at independent adding AD κ antibody, the OD492 value of reaction is 2.407 ± 0.053, after adding competitive antibody, the OD492 value of reaction obviously descends, become 1.627 ± 0.098, according to formula: inhibiting rate=(A value before suppressing-inhibitions back A value)/preceding A value x100% of inhibition can calculate single-chain antibody AD κ competitive inhibition rate and be respectively 25.9%.
The competitive inhibition effect of single-chain antibody AD κ antagonism cyclin D1 polyclonal antibody as shown in Figure 5, the result shows single-chain antibody AD κ can how anti-competition combines with cyclinD1 is proteic with anti-cyclinD1.
2. the avidity of single-chain antibody AD κ is measured
Use noncompetitive ELISA method, the concentration of antibody is X-coordinate during with each antigen concentration, is that ordinate zou is made typical curve as shown in Figure 6 with the OD value of antigen antibody reaction, according to curve and calculation formula
[58]K=(n-1)/(n[AB
1]-[AB]) calculate the affinity costant of single-chain antibody, n is the extension rate of single-chain antibody, Ab and Ab
1Represent that respectively working as antigen concentration is Ag and Ag
1The time, the antibody concentration of OD50.
In the experiment of measuring avidity, each single-chain antibody that need measure has all designed 4 groups of contrasts, when mapping, will generate 4 curves like this, every curve all can draw the numerical value of an avidity according to formula, get the mean value of the avidity of these 4 curves, will draw the average affinity of single-chain antibody.Therefore, be 2.2 * 10 according to the affinity costant of calculating single-chain antibody AD κ
7L/mol, experimental result shows that the single-chain antibody that this experiment obtains has higher avidity, the order of magnitude of avidity has reached 10
7
The clone of single-chain antibody gene and the structure of recombinant eukaryon expression vector in the anti-Cyclin D1 people source born of the same parents of embodiment 4 endoplasmic reticulum retention types
One experiment material
1. plasmid and bacterial strain
(1) pcDNA3.1 (+) plasmid is available from Invitrogen company
(2) intestinal bacteria E.coli DH5 α is available from Beijing ancient cooking vessel state biotech development center
2. the main related reagent of molecular cloning
(1) restriction enzyme Hind III, EcoR I are available from the precious biotechnology in Dalian company limited
(2) T4 dna ligase and corresponding damping fluid thereof are available from the precious biotechnology in Dalian company limited
(3) dNTP is available from Promega company
(4) nucleic acid molecular weight standard (1kb Ladder and DL-2000 DNA Marker) is available from Beijing ancient cooking vessel state biotech development center
(5) plasmid extracts purification kit available from Promega company
(6) DNA reclaims test kit available from Beijing ancient cooking vessel state biotech development center
(7) microbial culture uses Tryptones (Tryptone) and yeast extract (Yeast extract) available from Oxid company
(8) the required agarose of agarose gel electrophoresis, ethidium bromide, Tris etc. are available from Sigma company
(9) other reagent are analytically pure domestic reagent
(10) plastics consumptive materials such as Eppendorf pipe, liquid-transfering gun, rifle point are available from Beijing ancient cooking vessel state biotech development center
3. primer
This experiment of table 2 the primer sequence
Annotate: P1, P2 are for introducing endoplasmic reticulum positioning sequence and detection label institute synthetic primer, and wherein 5 of P1 ' end has been introduced the HindIII restriction enzyme site; 5 of P2 ' end has been introduced the EcoRI restriction enzyme site;
Be translation initiation codon;
Be translation stop codon;
Be 57bp ER type signal peptide gene sequence;
Be 39bp E-tag labelled peptide gene order; Italicized item is and template complementary nucleotide sequence.
P3, the P4 β-actin synthetic primer sequence that increases.P5, P6 amplification ER-AD κ primer sequence, P6 contains and linker complementary sequence, and amplified production is 470bp.Above-mentioned primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Two experimental techniques
(1) the segmental acquisition of ER-AD kappa gene
1. polymerase chain reaction (PCR)
Be built among the carrier for expression of eukaryon pcDNA3.1 for the method by directed cloning will resist people Cyclin D1 single-chain antibody gene, we have designed two primers of P1/P2.Design of primers is according to anti-Cyclin D1 human single chain variable fragments antibody gene order and different ubcellulars district detention type intracellular antibody gene order, and introduces according to PCR design of primers principle and to meet the initiator codon of reading frame and terminator codon and restriction enzyme site accordingly.Wherein P1 introduces Hind III restriction enzyme site, ATG transcription initiation codon, endoplasmic reticulum lead-in signal, and P2 introduces EcoR I restriction enzyme site, ER retention signal, E-tag detection label and terminator codon TAA.Concrete sequence is seen the material part.
With the anti-Cyclin D1 human single chain variable fragments antibody gene order that screens from phage single-chain antibody storehouse, people source is template, with single-chain antibody (ER-AD κ) gene (288 amino acid of encoding) in the anti-people Cyclin of the method amplification endoplasmic reticulum retention type D1 born of the same parents of PCR.
The PCR reaction system:
10×Ex?Taq?buffer 10μl
dNTPs(2.5mM?each) 8μl
P1?and?P2(20μM) 2μl(respectively)
Template(100ng/μl) 0.5μl
ExTaq(5U/μl) 0.5μl
dd?H
2O 77μl
Total 100μl
Schedule of operation: (1) 94 ℃, 4min; (2) 94 ℃, 45sec; (3) 55 ℃, 45sec; (4) 72 ℃, 45sec; (5) 72 ℃, 10min. wherein (2)-(4) carries out 30 circulations.
2.PCR the recovery of amplified production
The PCR product separates, reclaims purification kit recovery and check according to DNA through 1% agarose gel electrophoresis.
(1) after electrophoresis finishes, on the gel imaging instrument, cuts and contain the segmental agarose of purpose with scalpel;
(2) put into load weighted in advance Eppendorf pipe, weigh, smash to pieces, in 1: 3 (weight/mg: ratio adding solution A volume/μ l);
(3) 50 ℃ of metal bath 10min dissolve fully to glue, at room temperature, add solution B, abundant mixing, and solution is transferred to respectively in the centrifugal post and leaves standstill 2min, and the centrifugal 1min of 8500rpm abandons liquid (solution can be centrifugal at twice if once can not add);
(4) abandon liquid, add 500 μ l solution C, 8500rpm is centrifugal, and 1min abandons liquid; Repeat once;
(5) the centrifugal 1min of 12000rpm dries remaining liq, removes residual alcohol;
(6) centrifugal post is placed new centrifuge tube room temperature open and cover 8min, make the ethanol volatilization to the greatest extent;
(7) add the preheating solution D, the centrifugal 10min of 15000rpm, the pipe end, be target DNA.The DNA that takes a morsel carries out 1% agarose gel electrophoresis, and segmental purity and concentration are reclaimed in check.
3. the segmental enzyme of purpose is cut and is reclaimed
The HindIII and the EcoR I double digestion reaction system of the pcr amplified fragment product that reclaims
ddH
2O 12μl
10×Mbuffer 4μl
HindIII 2μl
EcoR?I 2μl
The pcr amplified fragment 20 μ l that reclaim
40μl
Above-mentioned mixed solution is in 37 ℃ of metal bath digestion 1.5-2h, and product carries out 1% agarose gel electrophoresis, reclaims the purpose fragment (method is the same) of about 870bp, and 1% agarose gel electrophoresis is checked its recovering effect, and-20 ℃ of preservations are standby.
(2) acquisition of pcDNA3.1 carrier segments
Plasmid pcDNA3.1 is carried out Hind III and EcoR I double digestion, 37 ℃ of digestion 1.5-2h, 1% sepharose, 98mA electrophoresis 30-40min separates the purpose fragment of about 5.4kb.
It is as follows that the enzyme of vector plasmid pcDNA3.1 is cut system:
dd?H
2O 12μl
10×Mbuffer 4μl
Hind?III 2μl
EcoR?I 2μl
pcDNA3.1 20μl
40μl
Reclaim test kit with DNA and reclaim carrier segments pcDNA3.1, method is the same.1% agarose gel electrophoresis, fragment is reclaimed in check.
(3) being connected of carrier and goal gene: target gene fragment and pcDNA3.1 enzyme are cut big fragment be connected, obtain recombinant plasmid pER-AD κ by the T4 dna ligase.Building process is seen Fig. 1.Linked system is as follows:
dd?H
2O 8.5μl
T
4ligase?buffer 2μl
T
4ligase 1μl
PcDNA3.1 fragment 0.5 μ l
ER-AD κ fragment 8 μ l
20μl
Mixing, 16 ℃ of metal baths connect 12-16h.
(4) preparation of DH5 α competent cell
(1) scrape with the aseptic inoculation ring and get frozenly in the DH5 of-80 ℃ of refrigerators α bacterial classification, streak inoculation is in the LB agar plate that does not contain penbritin (Amp), cultivates about 16h for 37 ℃;
(2) the single colony inoculation of picking is in 5ml liquid LB substratum, and 37 ℃ of 180rpm joltings are cultured to OD
600=0.4;
(3) the microbial culture pipe is taken out, place rapidly more than the ice bath 15min, make culture be cooled to 0 ℃;
(4) under aseptic condition inoculum is transferred to (following operation all needs aseptic) in aseptic, as the to ice precooling 1.5ml Eppendorf pipe, 4 ℃, the centrifugal 10min of 4000rpm abandon supernatant;
(5) with the 0.1mol/L CaCl of 500 μ l ice precooling
2The resuspended bacterial precipitation of solution, ice bath 30min;
(6) 4 ℃, the centrifugal 10min of 4000rpm collect thalline, through the CaCl of 80 μ l ice precooling
2Again the thalline that suspends places 4 ℃ of refrigerator ice baths standby competent cell.
(5) plasmid transforms
(1) every pipe 80 μ l competent cells is joined 20 μ l and connect in the product, mixing gently, ice bath 30min;
(2) pipe is put into 42 ℃ of metal baths, heat-shocked 2min;
(3) transfer in the ice bath cooling 2min fast;
(4) every pipe adds the LB liquid nutrient medium of 37 ℃ of preheatings of 900 μ l, 37 ℃, 150rpm shaking culture 45min;
(5) 4000rpm, 4 ℃ of centrifugal 5min;
(6) abandon 700-800 μ l supernatant,, coat in the LB nutrient agar that contains 100 μ g/ml acillin resistances (Amp), leave standstill 10min with remaining supernatant re-suspended cell; Be inverted for 37 ℃ and cultivate 12～16h, the recon bacterium colony occurs.4 ℃ of preservations of transformed bacteria dish are standby.
(6) evaluation of expression plasmid pER-AD κ
1. a small amount of extraction and purification of expression plasmid pER-AD κ (alkaline lysis extracts plasmid in a small amount)
(1) the single positive colony of picking transformant is inoculated in 5ml and contains 100 μ g/ml Amp
+The LB liquid nutrient medium in, 37 ℃, 180-200rpm shaking culture 12～16h;
(2) bacterium liquid is sub-packed in the 1.5ml Eppendorf pipe, and 15000rpm, 4 ℃ of centrifugal 5min receive bacterial sediment;
(3) add the solution I that 0.1mL ices precooling, blow outstanding precipitation, leave standstill 5min;
(4) add the 0.2mL solution II of now joining, put upside down mixing;
(5) add 0.15mL ice precooling solution III, put upside down mixing gently;
(6) ice bath 15min, 15000rpm4 ℃ of centrifugal 5min;
(7) collect supernatant, add the dehydrated alcohol of 2.5 times of volumes, room temperature leaves standstill 20min;
(8) 4 ℃ of centrifugal 15min of 15000rpm;
(9) abandon supernatant, add 70% washing with alcohol of 2.5 times of volume precoolings, 4 ℃ of centrifugal 5min of 15000rpm;
(10) abandon supernatant, control is done, the rotary evaporation in vacuo drying;
(11) precipitation is dissolved among the 50 μ l TE and (contains 50 μ g/ml RNaseA), 37 ℃ of 30min, and the check of 1% agarose gel electrophoresis ,-20 ℃ of preservations are standby.
2. the enzyme of recombinant plasmid is cut evaluation
The recombinant plasmid that utilizes digestion with restriction enzyme method initial survey to extract.
Recombinant plasmid is carried out Hind III and EcoR I double digestion, 37 ℃ of digestion 1.5-2h, 1% sepharose, 98mA electrophoresis 30-40min observes on the gel imaging analysis instrument.
The Hind III of pER-AD κ plasmid and EcoR I double digestion reaction system:
dd?H
2O 6μl
10×Mbuffer 2μl
Hind?III 1μl
EcoR?I 1μl
pER-ADκ 10μl
20μl
3. the sequencing of recombinant plasmid
Give birth to worker's biotechnology company limited by Shanghai and check order sequence such as Sequence 2.
Three, experimental result and analysis
For single-chain antibody gene in the anti-Cyclin D1 people source born of the same parents that obtain to be positioned endoplasmic reticulum, we are template with the plasmid that is loaded with anti-Cyclin D1 human single chain variable fragments antibody gene order, with the primer that contains ER retention signal, Hind III and EcoR I restriction enzyme site sequence and E-tag labelled peptide sequence, by single-chain antibody gene (ER-AD kappa gene) in the anti-Cyclin D1 people source born of the same parents of method amplification ER type of PCR.The PCR product finds have a tangible DNA band (Fig. 7) to occur at about 870bp place behind agarose gel electrophoresis; Product reclaims rear clone to (Fig. 8) between the restriction enzyme Hind III in the CMV promotor downstream of eukaryon expression plasmid pcDNA3.1 and the EcoR I; Positive recombinant plasmid is through restriction enzyme Hind III and EcoRI double digestion, and agarose gel electrophoresis is found, occurred a tangible DNA band (Fig. 9) respectively at 5400bp and 870bp place.Further by the purpose fragment on T7 and the logical carrier pER-AD κ of the positive and negative two-way survey of BGH universal primer, sequencing result shows that insertion sequence is consistent with experimental design, and reading frame is entirely true; The DNA of ER-AD κ and amino acid sequence analysis result show, have successfully introduced ER retention signal, E-tag detection label etc., ER-AD kappa gene total length 864bp, 288 amino acid of encoding by pcr amplification.This illustrates that we have successfully obtained the carrier for expression of eukaryon pER-AD κ of reorganization intracellular antibody ER-AD kappa gene.
The eukaryotic expression and the positioning analysis of embodiment 5 ER-AD kappa genes
One experiment material
1. molecular biology related reagent and material
2. cell strain
Human breast cancer MCF-7 cell's strain and human hela HeLa cell strain are provided by immunity teaching and research room of Jilin University
3. the required main agents of eukaryotic cell transfection
(1) DMEM, liposome (Lipofectamine 2000) are Invitrogen company product
(2) new-born calf serum is available from Hangzhou folium ilicis chinensis biotechnology company limited
(3) pancreatin is a Sigma company product
(4) EDTA is Beijing Chemical Plant's product
(5) 0.01mol/L PBS (pH7.4): NaCl 8g, KCl 0.2g, Na
2HPO
42H
2O 3.63g, KH
2PO
40.24g distilled water is settled to 1000ml
(6) Tissue Culture Plate is the COSTAR product
Two experimental techniques
1. cell cultures
MCF-7 cell and HeLa cell cultures in the DMEM perfect medium that contains 10% calf serum, 37 ℃ of saturated humidities, 5%CO
2Cultivate in the incubator.The cell attachment growth, every 3-5 days peptic cell, the cultivation of going down to posterity.
2. the transfection preparation of plasmid
(1) inoculum of incubated overnight is transferred in the clean 1.5ml centrifuge tube, and the centrifugal 5min of 15000rpm abandons supernatant, collects bacterial sediment;
(2) add 250 μ l Cell Resuspension Solution, bacterial sediment is hanged;
(3) add 250 μ l Cell Lysis Solution, turn upside down four times;
(4) add 350 μ l Neutralization Solution, turn upside down four times;
(5) the centrifugal 10min of 15000rpm;
(6) sample transfer to is equipped with in the clean collection tube of Spin post, places in the whizzer under room temperature with the centrifugal 1min of 15000rpm;
(7) discard effluent liquid, the Spin post is transferred in the new collection tube, adds 750 μ l Washing Buffer washing pillar, the centrifugal 1min of 15000rpm under the room temperature;
(8) repeat previous step washing column child-operation once with 250 μ l Washing Buffer;
(9) discard effluent liquid, and with the centrifugal void column 1min of 15000rpm to dry base for post matter;
(10) with in posts transfer to the aseptic 1.5ml centrifuge tube, the rnase-free of 100 μ l sterilization deionized water directly is added on the base for post matter, the centrifugal 1min of 15000rpm is with eluted dna;
(11) abandon the recovery post, eluted dna be stored in-20 ℃ standby.
3. liposome-mediated cell transient transfection experiment
3.1 sample preparations
(1) slide glass is cleaned back flowing water flushing with washing powder water logging bubble, and 0.1N HCl soaks 2h, and flushing with clean water is cleaned behind the soaked in absolute ethyl alcohol 12h, 160 ℃ of roasting 4h, place 4 ℃ standby.
(2) slide glass with aseptically process is put in 6 well culture plates, and the MCF-7 cell that changes liquid before the 24h that growth conditions is good is by 5 * 10
5The density in cells/ hole, HeLa cell are by 4 * 10
5The density in cells/ hole is seeded to respectively in 6 orifice plates, 37 ℃ of saturated humidities, 5%CO
2Cultivate in the incubator, cell can be attached on the slide glass naturally.When reaching, the growth of 6 orifice plate inner cells gets final product transfection when 90-95% converges.
3.2 transient transfection experiment
(1) 4 μ g plasmid DNA is not contained antibiotic DMEM with 250 μ l and mix incubated at room 20min gently;
(2) with 10 μ l LipofectAMINE
TM2000 do not contain antibiotic DMEM with 240 μ l mixes incubated at room 5min;
(3) with above-mentioned two kinds of liquid mixing, incubated at room 20min;
(4) cell in 6 orifice plates is not contained antibiotic DMEM nutrient solution with serum-free and wash twice, add said mixture, 37 ℃, 5%CO
2Cultivate;
(5) after transfectional cell is cultivated 6h, change the normal substratum that contains serum into.72h takes out creep plate after transfection, carries out the interior expression of born of the same parents and positioning analysis, the variation of observation of cell form simultaneously of ER-AD κ with the method for immunohistochemical methods.With remaining cell trysinization in the hole, cold PBS washes 2 times, and cell precipitation hangs with Trizol, extracts total RNA, utilizes the RT-PCR method to detect the expression of ER-AD κ.
4.RT-PCR detect
4.1 the extraction of total RNA
MCF-7 cell, the HeLa cell of transfection 72h are digested respectively, after cold PBS washes 2 times, add 0.5ml Trizol extracting solution, room temperature is placed 5min; Add 0.1ml chloroform concuss 15s, room temperature leaves standstill 3min; 4 ℃ of centrifugal 15min of 12000g; The upper strata water changes in the new centrifuge tube, adds the 0.25ml Virahol, mixing, and room temperature is placed 10min; 4 ℃ of centrifugal 10min of 12000g; Precipitation is washed once with 1ml 75% ethanol (preparation of DEPC water); 4 ℃ of centrifugal 5min of 10000g; Air drying adds an amount of DEPC water dissolution (55-60 ℃ of water-bath 10min hydrotropy), be stored in-80 ℃ standby.
4.2 reverse transcription (RT) reaction
With 0.76 μ g mRNA is initiator, according to the explanation of RT-PCR test kit, adds the required various compositions of RT reaction respectively and carries out the RT reaction, and its condition is: 30 ℃ of 10min, 45 ℃ of 30min, 99 ℃ of 5min, 5 ℃ of 5min; 94 ℃ of 2min.
4.3PCR reaction
After the RT reaction finishes, carry out PCR, with the testing goal expression of gene.
(1) from reaction mixture, gets the test tube that 5 μ l put into new PCR, the upstream and downstream primer P3 and the P4 that add 100pmol β-actin successively, 5 μ l PCR damping fluids (10 *), the dNTP of 2.5mmol/L and the archaeal dna polymerase of 5U (Taq DNAPolymerase), final volume are 50 μ l; Place in the PCR instrument and carry out pcr amplification, reaction parameter is 94 ℃ of 30S, 50 ℃ of 30S, 72 ℃ of 1min, 30 circulations; Extend 10min in 72 ℃ at last.Carry out the check of 1% agarose gel electrophoresis after the amplification.
(2) get the test tube that 5 μ l put into new PCR from reaction mixture, add ER-AD κ upstream and downstream primer P5 and the P6 of 100pmol successively, 5 μ l PCR damping fluids (10 *), the dNTP of 2.5mmol/L and the archaeal dna polymerase of 5U, final volume are 50 μ l; Place in the PCR instrument and carry out pcr amplification, reaction parameter is 94 ℃ of 30S, 50 ℃ of 30S, 72 ℃ of 1min, 30 circulations; Extend 10min in 72 ℃ at last.Carry out the check of 1% agarose gel electrophoresis after the amplification.
5. indirect immunofluorescence experiment
(1) cell climbing sheet takes out from 6 well culture plates, and 37 ℃ of D-Hanks wash 5min/ time 2 times;
(2) 4% Paraformaldehyde 96s are 30min fixedly;
(3) dd H
2O washing 3 times, blower fan dries up, and-20 ℃ of preservations are standby;
℃ (4)-20 take out slice, thin piece RT aquation 20min;
(5) PBST washing 10min, PBS washs 3 times * 5min;
(6) 0.01% trysinization 40S, PBS washs 3 times * 5min;
(7) 3%H
2O
2RT blocks 20min, and PBS washs 3 * 5min;
(8) 3%BSA-PBST sealing 20min, PBST washing 3 times, 10min/ time;
(9) add the mouse monoclonal antibody that PBST dilutes the anti-E-tag of (1: 125), 4 ℃ are spent the night;
(10) the PBST washing is 3 times * 10min/ time;
(11) add the FITC-goat anti-mouse IgG that PBST dilutes (1: 100), lucifuge RT 20min;
(12) PBST thorough washing;
(13) glycerine mounting, last machine is observed.
Three experimental results and analysis
ER-AD κ expresses in tumour cell and can product realize that object location is (owing to introduce ER retention signal in order to detect, this albumen should be distributed in the tenuigenin), the transient transfection cell experiment of pER-AD κ has at first been carried out in this research, has carried out the indirect immunofluorescence experiment of RT-PCR and cell climbing sheet then.
1.RT-PCR detect the expression of ER-AD κ
In order to detect the expression of ER-AD κ in tumour cell, we have extracted MCF-7 cell and the HeLa cell total rna of cultivating 72h behind the pER-AD κ transient transfection respectively, carried out the RT-PCR analysis, and in experiment, analyzed as internal reference with β-actin gene.The PCR product is found behind agarose gel electrophoresis, when the corresponding primer with specific amplification β-actin carries out the RT-PCR amplification, all samples all can amplify β-actin cDNA of 350bp, and in every group the band brightness basically identical that amplifies of each sample (Figure 10 A is the MCF-7 groups of cells, Figure 10 C is the HeLa groups of cells), and when the corresponding primer with specific amplification ER-AD κ carries out the RT-PCR amplification, only when being template with the total RNA of pER-AD κ cells transfected, just can amplify the product of about 470bp, (Figure 10 B is the MCF-7 groups of cells all not have the appearance of this band when being template with the RNA of empty carrier pcDNA3.1 cells transfected or ghost, Figure 10 D is the HeLa groups of cells), this result understands that in the horizontal Shanghai Stock Exchange of mRNA ER-AD κ has obtained expression in MCF-7 cell and HeLa cell.
2.ER-AD express and positioning analysis in the born of the same parents of κ
In order to detect the expression of ER-AD κ in transfectional cell, with the cell of cultivating 72h after the transfection is material, after a series of processing such as cell climbing sheet, is one anti-with the mouse monoclonal antibody of anti-E-tag, the FITC-goat anti-mouse IgG is two anti-, carries out the indirect immunofluorescence experiment analysis.Laser confocal microscope is observed and is found, bright yellow-green fluorescence is all sent in the MCF-7 cell of pER-AD κ transfection and the tenuigenin zone of HeLa cell, and a little less than pcDNA3.1 transfection control group do not have fluorescence or fluorescence extremely, this shows that the ER-AD kappa gene obtains effective expression in the tumour cell of transfection, and its expression product is positioned at tenuigenin (Figure 11).
The stably express of embodiment 6 ER-AD kappa genes and extracorporeal anti-tumor biological activity are analyzed
One experiment material
1.RT-PCR required primer P1 P2 P3 P4
2. molecular biology related reagent and material are with embodiment 1 and example 2
3. cell strain is with embodiment 2
4. antibody
(1) anti-E-tag antibody is available from Phamarcia company
(2) FITC mark goat anti-mouse IgG is available from biotech company of China fir Golden Bridge in Beijing
5. the required main agents of cell cultures and stable transfection
Xin Meisu (G418) is a GIBCO company product; DMSO and MTT are Sigma company product; All the other are with embodiment 2
6.Dot-blot required main agents
BSA is available from Beijing ancient cooking vessel state Bioisystech Co., Ltd; DAB and PMSF are available from Sigma company;
7. the preparation of main agents
(1) cell pyrolysis liquid: 150mmol/LNaCl, 10mmol/L Tris-Cl pH 7.4,5mmol/L EDTA, 1%Triton X-100,1mmol/L PMSF
(2) preparation of other related solution is with reference to the molecular cloning experiment guide
Two experimental techniques
1. the foundation of stable transfected cells strain
The extraction and purification of plasmid and the operation of liposome-mediated cell transfecting are the same.72h digests transfectional cell after transfection, according to the cultivation of going down to posterity in 1: 10, adds 600 μ g/ml G 418 simultaneously and carries out resistance screening, changes liquid once in 3-5 days.After about 2 weeks, empty map group cell is all dead, positive colony occurs, progressively reduces G 418 concentration to 200 μ g/ml and keeps screening.The strain of enlarged culturing stable transfected cells, frozen, correlation detection is standby.
2.ER-AD the stably express analysis of κ
2.1RT-PCR detect
2.1.1 the extraction of total RNA
MCF-7 groups of cells, the HeLa groups of cells of the stable transfection that screens are digested respectively, after cold PBS washes 2 times, add 0.5ml Trizol extracting solution, room temperature is placed 5min; Add 0.1ml chloroform concuss 15s, room temperature leaves standstill 3min; 4 ℃ of centrifugal 15min of 12000g; The upper strata water changes in the new centrifuge tube, adds the 0.25ml Virahol, mixing, and room temperature is placed 10min; 4 ℃ of centrifugal 10min of 12000g; Precipitation is washed once with 1ml 75% ethanol (preparation of DEPC water); 4 ℃ of centrifugal 5min of 10000g; Air drying adds an amount of DEPC water dissolution (55-60 ℃ of water-bath 10min hydrotropy), be stored in-80 ℃ standby.
2.1.2 reverse transcription (RT) reaction
With 0.76 μ g mRNA is initiator, according to the explanation of RT-PCR test kit, adds the required various compositions of RT reaction respectively and carries out the RT reaction, and its condition is: 30 ℃ of 10min, 45 ℃ of 30min, 99 ℃ of 5min, 5 ℃ of 5min; 94 ℃ of 2min.
2.1.3PCR reaction
After the RT reaction finishes, carry out PCR, with the testing goal expression of gene.
(1) from reaction mixture, gets the test tube that 5 μ l put into new PCR, the upstream and downstream primer P3 and the P4 that add 100pmol β-actin successively, 5 μ l PCR damping fluids (10 *), the dNTP of 2.5mmol/L and the archaeal dna polymerase of 5U (Taq DNAPolymerase), final volume are 50 μ l; Place in the PCR instrument and carry out pcr amplification, reaction parameter is 94 ℃ of 30S, 50 ℃ of 30S, 72 ℃ of 1min, 30 circulations; Extend 10min in 72 ℃ at last.Carry out the check of 1% agarose gel electrophoresis after the amplification.
(2) get the test tube that 5 μ l put into new PCR from reaction mixture, add ER-AD κ upstream and downstream primer P5 and the P6 of 100pmol successively, 5 μ l PCR damping fluids (10 *), the dNTP of 2.5mmol/L and the archaeal dna polymerase of 5U, final volume are 50 μ l; Place in the PCR instrument and carry out pcr amplification, reaction parameter is 94 ℃ of 30S, 50 ℃ of 30S, 72 ℃ of 1min, 30 circulations; Extend 10min in 72 ℃ at last.Carry out the check of 1% agarose gel electrophoresis after the amplification.
2.2 indirect immunofluorescence experiment
(1) MCF-7 groups of cells, the HeLa groups of cells with the stable transfection that screens prepares cell climbing sheet respectively, it taken out 37 ℃ of D-Hanks washings 2 times, 5min/ time from 6 well culture plates;
(2) 4% Paraformaldehyde 96s are 30min fixedly;
(3) dd H
2O washing 3 times, blower fan dries up, and-20 ℃ of preservations are standby;
℃ (4)-20 take out slice, thin piece RT aquation 20min;
(5) PBST washing 10min, PBS washs 3 times * 5min
(6) 0.01% trysinization 40S, PBS washs 3 times * 5min;
(7) 3%H
2O
2RT blocks 20min, and PBS washs 3 * 5min;
(8) 3%BSA-PBST sealing 20min, PBST washing 3 times, 10min/ time;
(9) add the mouse monoclonal antibody that PBST dilutes the anti-E-tag of (1: 125), 4 ℃ are spent the night;
(10) the PBST washing is 3 times * 10min/ time;
(11) add the FITC-goat anti-mouse IgG that PBST dilutes (1: 100), lucifuge RT 20min;
(12) PBST thorough washing;
(13) glycerine mounting, last machine is observed.
2.3Dot-blot analyze
(1) MCF-7 cell, the HeLa cell of collection stable transfection, cold PBS washes 2 times;
(2) according to 1 * 10
6Cells/100 μ l adds cell pyrolysis liquid (150mmol/L NaCl, 10mmol/L Tris-Cl pH 7.4,5mmol/L EDTA respectively in the cell harvesting pipe, 1%Triton X-100,1mmol/L PMSF), multigelation lysing cell 30min, every the vibration of 10min spiral once;
(3) the centrifugal 10min of 15000rpm, the results supernatant liquor;
(4) utilize the vacuum filtration sample injector that the albumen in the supernatant is transferred on the nitrocellulose filter;
(5) film is placed the slow 15min that shakes of PBST RT, then RT sealing 2h in 1%BSA-PBST;
(6) film places PBST to delay and shakes 3 times * 5min.Then in the anti-E-tag mouse monoclonal antibody of 1%BSA-PBST dilution, the slow 2h that shakes of RT;
(7) film places PBST to delay and shakes 3 times * 5min.The slow 45min that shakes of RT in the goat anti-mouse igg of the HRP mark of 1%BSA-PBST dilution then;
(8) film places PBST to delay and shakes 3 times * 5min;
(9) the DAB colour developing liquid of film being put into new preparation develops the color.
3.ER-AD the external activity of κ
3.1MTT method detects the cells in vitro proliferation activity
(1) inoculating cell: with 0.25% tryptic digestion single-layer culturing cell, be made into the individual cells suspension with the DMEM nutrient solution that contains 10% calf serum, MCF-7 is by 7 * 10
3The cells/ hole; HeLa
By 5 * 10
3The density in cells/ hole is seeded in 96 well culture plates, and 10 parallel holes are established for every kind in 200 μ l/ holes, simultaneously with 200 μ l/ hole nutrient solutions as negative control.
(2) culturing cell: culture plate is moved into CO
2In the incubator, at 37 ℃, 5%CO
2And under the humidity condition, after cultivating 4h, 24h, 48h, 72h, 96h, 120h, take out culture plate respectively, every hole adds 5mg/ml MTT solution 20 μ l, puts back to incubator and continues to cultivate.
(3) after 4h was cultivated in continuation, the careful suction abandoned nutrient solution in the hole.Add DMSO (150 μ l/ hole) dissolving MTT, vibration 10min mixing fully dissolves crystallisate.
(4) colorimetric: select the 570nm wavelength, on enzyme-linked immunosorbent assay instrument, set up each hole absorption value, the record result.As negative control, calculating the average light absorption value of every kind of cell at 570nm wavelength place with nutrient solution hole absorbance value, is transverse axis with time, and the 570nm absorbance value is that the longitudinal axis is drawn cell growth curve.
3.2FACS the analysis of cells cycle
With the cell clone amplification cultivation.Harvested cell is also with cold PBS washing 2 times, use 70% cold ethanol fixedly more than the 2h then, clean ethanol with PBS before detecting, be resuspended among the PBS of PI dye liquor that 200 μ l contain 50 μ g/ml, add RNase A to final concentration 50 μ g/ml, lucifuge is hatched 30min, and the upflowing cytoanalyze detects the changing conditions of cell cycle.
3.3AnnexinV/PI twoly dye the Flow cytometry analysis
(1) cell of collection stable transfection, cold PBS washed cell 2 times collects 5 * 10
5Cell;
(2) draw the 2 * Binding Buffer of 250 μ l and the sterilization deionized water mixing of 250 μ l;
(3) with 1 * Binding Buffer suspension cell of above-mentioned 500 μ l;
(4) add 1 μ l AnnexinV-EGFP mixing, add 5 μ l PI, mixing;
(5) lucifuge reaction 5min;
(6) detect (Ex=488nm with flow cytometer; Em=530nm) apoptosis situation.
Three experimental results and analysis
For the expression that detects ER-AD κ with and the extracorporeal biology activity, with liposome Lipofectame
TM2000 mediation recombinant plasmid pER-AD κ and empty map plasmid pcDNA3.1 transfection human breast cancer cell strain MCF-7 cell and human cervical carcinoma cell strain HeLa cells, through G 418 resistance screenings, obtain the positive cell clone of stable transfection, in order to identify these clones, carry out following experiment after the enlarged culturing.
1.RT-PCR detect the expression of ER-AD κ
In order to detect the stably express of ER-AD κ in tumour cell, we have extracted the total RNA of stable transfected cells respectively, have carried out the RT-PCR analysis, and in experiment with β-actin gene as internal reference.The PCR product is found behind agarose gel electrophoresis, when the corresponding primer with specific amplification β-actin carries out the RT-PCR amplification, all samples all can amplify β-actin cDNA of 350bp, and in every group the band brightness basically identical that amplifies of each sample (Figure 10 E is the MCF-7 groups of cells, Figure 10 G is the HeLa groups of cells), and when the corresponding primer with specific amplification ER-AD κ carries out the RT-PCR amplification, only when being template with the total RNA of pER-AD κ cells transfected, just can amplify the product of about 470bp, (Figure 10 F is the MCF-7 groups of cells all not have the appearance of this band when being template with the RNA of empty carrier cells transfected or ghost, Figure 10 H is the HeLa groups of cells), this result understands that in the horizontal Shanghai Stock Exchange of mRNA the cell clone that we select is the cell strain of stably express ER-AD κ, shows the cell clone MCF-7/ER-AD κ cell strain and the HeLa/ER-AD κ cell strain that tentatively obtain stably express ER-AD κ.
3.2 indirect immunofluorescence experiment analysis
The cell that is positive with RT-PCR result is a material, through a series of processing such as cell climbing sheets, is one anti-with the mouse monoclonal antibody of anti-E-tag, and the FITC-goat anti-mouse IgG is two anti-, carries out the indirect immunofluorescence experiment analysis.Laser confocal microscope is observed and is found, bright yellow-green fluorescence is all sent in the MCF-7 cell of pER-AD κ stable transfection and the tenuigenin zone of HeLa cell, and the tenuigenin zone of the MCF-7 cell of the MCF-7 cell of pcDNA3.1 stable transfection and HeLa cell and empty map and HeLa cell does not have fluorescence (Figure 11), this shows that the ER-AD kappa gene obtains effective expression in the tumour cell of stable transfection, and its expression product is positioned at tenuigenin, has further verified the positive cell strain that we obtained.
3.3Dot-blot analyze
Expression for ER-AD kappa gene in the stable transfected cells that further detects screening, we distinguish the MCF-7 cell of cracking pER-AD κ, pcDNA3.1 stable transfection and the MCF-7 cell and the HeLa cell of HeLa cell and empty map, carry out Dot-blot with the monoclonal antibody of anti-E-tag and detect.The result as shown in figure 12, in MCF-7/pER-AD κ, HeLa/pER-AD κ cell pyrolysis liquid, detect the expression of ER-AD kappa gene, significantly hybridization point promptly on nitrocellulose filter, occurs, and amixia point produces in the cell pyrolysis liquid of empty carrier transfection group and ghost control group.This result has proved the expression of ER-AD kappa gene at protein level, has also further verified the positive cell strain that we obtained simultaneously.
3.4ER-AD κ suppresses growth of tumour cell
We adopt the interior Formazan content of MTT colorimetric method for determining viable cell plastosome to detect tumour cell colony multiplication capacity.The MCF-7, HeLa cell that select logarithmic phase for use are with 5-7 * 10
3The cell concentration in cells/ hole is spread 96 orifice plates, and every kind of cell is divided into 3 groups (pER-AD κ, pcDNA3.1 transfection, ghost groups), point mutually when six of 1-6d are set altogether, the colourimetric number of measuring every day is got the mean number in 10 multiple holes, draws growth curve, carries out the t check between parallel group.The result as shown in figure 13, compare with the ghost group with pcDNA3.1 transfectional cell group, the growing amount prolongation in time of pER-AD κ group cell Formazan significantly reduces, and the growing amount of pER-AD κ group cell Formazan compares with the ghost group with pcDNA3.1 transfection group cell that all there were significant differences (p＜0.01) after the 3rd day.These presentation of results, the expression of ER-AD κ is played significant inhibitory effect to the growth of transfectional cell.
3.5ER-AD κ causes the cell-cycle arrest of tumour cell
In order to detect the ER-ADk expression of gene to the influence of tumour cell cell cycle, we are by dna content in the cells were tested by flow cytometry cell, thus the per-cent of phase when obtaining each cell cycle.At first with the positive cell strain that contains empty carrier pcDNA3.1 of ghost, screening and the positive cell strain of expressing ER-ADk gene amplification cultivation respectively, harvested cell wash, after 70% cold ethanol is fixed, PI dyes, carries out facs analysis through cold PBS.The result expresses the positive cell strain of ER-ADk gene and compares with control group as shown in figure 14, and the cell proportion of G0-G1 phase increases, and S phase cell proportion reduces.In the MCF-7 cell, G0-G1 phase cell is increased to 66.67% by 50.76%, and S phase cell reduces to 11.85% (Figure 14, a left side) by 45.90%; In the HeLa cell, G0-G1 phase cell is increased to 64.56% by 44.33%, and S phase cell reduces to 9.65% (Figure 14, the right side) by 21.06%.The result shows that the expression of ER-AD kappa gene can cause that the tumour cell G1 phase blocks, suppress tumour cell by the G1 phase to S phase transition, thereby suppress tumor cell proliferation.
3.6ER-AD κ can inducing apoptosis of tumour cell
Normocellular phosphatidylserine (PS) is positioned at the cytolemma inboard, goes to the cytolemma outside surface when apoptosis, and this change is considered to specific, and can be used as the mark of apoptotic cell surface modification.Annexin V is a kind of Ca
2+The phospholipids incorporate albumen that relies on has high affinity to PS.So can detect apoptotic cell with combining of fluorescently-labeled Annexin V by the PS that the cell outside exposes.Propidium iodide (PI) is a kind of nucleic acid dye, and it can not see through complete cytolemma.Because the permeability of cell membrane of viable apoptotic cell does not have obvious change, PI can not enter in the cell its nuclear is dyeed; And middle and advanced stage apoptotic cell and non-viable non-apoptotic cell, the permeability of its film takes place obviously to change, and PI can permeate through cell membranes and nucleus is incarnadined.Therefore the two-parameter detection method of Annexin V/PI can be with normal cell (Annexin V
-/ PI
-), viable apoptotic cell (AnnexinV
+/ PI
-), middle and advanced stage apoptotic cell and non-viable non-apoptotic cell (Annexin V
+/ PI
+) distinguish.Sample detects with flow cytometer after treatment, is X-coordinate with Annexin V, and PI is that ordinate zou is made graph discovery, and the natural apoptosis rate of MCF-7 ghost is 6.12%, and the apoptosis rate of expressing the positive cell strain of ER-AD kappa gene is 13.66% (Figure 15, a left side); The natural apoptosis rate of HeLa ghost is 1.83%, and the apoptosis rate of expressing the positive cell strain of ER-AD kappa gene is 6.63% (Figure 15, the right side).The result shows that the expression of ER-AD kappa gene can inducing apoptosis of tumour cell.
Sequence table
＜110〉Jilin University
＜120〉anti-Cyclin D 1 human single-chain antibody
<130>jluligy
<160>2
<170>PatentIn?version?3.2
<210>1
<211>750
<212>DNA
＜213〉people
<400>1
gatattgtga?tgacgcagtc?tccactcccc?ctgcccgtca?cccctggaga?gccggcctcc 60
atctcctgca?ggtctagtca?gagcctcctg?catagtaatg?gatacaacta?tttggattgg 120
tacctgcaga?agccagggca?gtctccacag?ctcctgatct?atttgggttc?taatcgggcc 180
tccggggtcc?ctgacaggtt?cagtggcagt?ggatcaggca?cagattttac?actgaaaatc 240
agcagagtgg?aggctgagga?tgttggggtt?tattactgca?tgcaagctct?acaaactccg 300
tacacttttg?gccaggggac?caaggtggaa?atcaaacgtt?ccggagggtc?gaccataact 360
tcgtagaatg?tatactatac?gaagttatcc?tcgagcggta?cccagctgca?gctgcaggag 420
tctggggctg?aggtgaagaa?gcctggggcc?tcagtgaagg?tctcctgcaa?ggcttctgga 480
tacaccttca?ccggctacta?tatgcactgg?gtgcgacagg?cccctggaca?agggcttgag 540
tggatgggat?ggatcaaccc?taacagtggt?ggcacaaact?atgcacagaa?gtttcagggc 600
agggtcacca?tgaccaggga?cacgtccatc?agcacagcct?acatggagct?gagcaggctg 660
agatctgacg?acacggccgt?gtattactgt?gcgagagatt?ggggcggtat?ggacgtctgg 720
ggccaaggga?ccacggtcac?tgtctcttca 750
<210>2
<211>864
<212>DNA
＜213〉people
<400>2
atgggatgga?gctgtatcat?cctcttcttg?gtatcaacag?ctacagctgt?ccactccgcc 60
gatattgtga?tgacgcagtc?tccactcccc?ctgcccgtca?cccctggaga?gccggcctcc 120
atctcctgca?ggtctagtca?gagcctcctg?catagtaatg?gatacaacta?tttggattgg 180
tacctgcaga?agccagggca?gtctccacag?ctcctgatct?atttgggttc?taatcgggcc 240
tccggggtcc?ctgacaggtt?cagtggcagt?ggatcaggca?cagattttac?actgaaaatc 300
agcagagtgg?aggctgagga?tgttggggtt?tattactgca?tgcaagctct?acaaactccg 360
tacacttttg?gccaggggac?caaggtggaa?atcaaacgtt?ccggagggtc?gaccataact 420
tcgtagaatg?tatactatac?gaagttatcc?tcgagcggta?cccagctgca?gctgcaggag 480
tctggggctg?aggtgaagaa?gcctggggcc?tcagtgaagg?tctcctgcaa?ggcttctgga 540
tacaccttca?ccggctacta?tatgcactgg?gtgcgacagg?cccctggaca?agggcttgag 600
tggatgggat?ggatcaaccc?taacagtggt?ggcacaaact?atgcacagaa?gtttcagggc 660
agggtcacca?tgaccaggga?cacgtccatc?agcacagcct?acatggagct?gagcaggctg 720
agatctgacg?acacggccgt?gtattactgt?gcgagagatt?ggggcggtat?ggacgtctgg 780
ggccaaggga?ccacggtcac?tgtctcttca?ggtgcgccgg?tgccgtatcc?ggatccgctg 840
gaaccgcgta?aggacgagct?gtaa 864
Claims (4)
1. anti-Cyclin D1 human single chain variable fragments antibody is characterized in that: its nucleotide sequence is as described in the SequenceNO.1.
2. according to the anti-Cyclin D1 of claim 1 human single chain variable fragments antibody, it is characterized in that: be made up of the leading peptide of IgK, anti-Cyclin D1 human single chain variable fragments antibody, E-tag labelled peptide and endoplasmic reticulum retention peptide KDEL gene coded sequence, its nucleotide sequence is as described in the Sequence NO.2.
3. anti-Cyclin D1 human single chain variable fragments antibody as claimed in claim 1 or 2 has application in the medicine of antitumor action in preparation.
4. the application of anti-Cyclin D1 human single chain variable fragments antibody as claimed in claim 3, described tumour are human breast carcinoma and human cervical carcinoma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100504173A CN101280014A (en) | 2008-02-29 | 2008-02-29 | Anti-Cyclin D1 human single-chain antibody |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100504173A CN101280014A (en) | 2008-02-29 | 2008-02-29 | Anti-Cyclin D1 human single-chain antibody |
Publications (1)
| Publication Number | Publication Date |
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| CN101280014A true CN101280014A (en) | 2008-10-08 |
Family
ID=40012700
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008100504173A Pending CN101280014A (en) | 2008-02-29 | 2008-02-29 | Anti-Cyclin D1 human single-chain antibody |
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| Country | Link |
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| CN (1) | CN101280014A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103554262A (en) * | 2013-10-25 | 2014-02-05 | 吉林大学 | Anti-cyclooxygenase humanized single-chain antibody |
| CN103848891A (en) * | 2012-11-30 | 2014-06-11 | 北京市结核病胸部肿瘤研究所 | Antigen polypeptide capable of recognizing CCND1 autoantibody |
| CN114395039A (en) * | 2021-12-31 | 2022-04-26 | 河南赛诺特生物技术有限公司 | Monoclonal antibody aiming at human cyclin 1, preparation method thereof, immunoassay reagent and application |
-
2008
- 2008-02-29 CN CNA2008100504173A patent/CN101280014A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103848891A (en) * | 2012-11-30 | 2014-06-11 | 北京市结核病胸部肿瘤研究所 | Antigen polypeptide capable of recognizing CCND1 autoantibody |
| CN103848891B (en) * | 2012-11-30 | 2015-09-09 | 北京市结核病胸部肿瘤研究所 | The antigenic peptide of CCND1 autoantibody identification |
| CN103554262A (en) * | 2013-10-25 | 2014-02-05 | 吉林大学 | Anti-cyclooxygenase humanized single-chain antibody |
| CN114395039A (en) * | 2021-12-31 | 2022-04-26 | 河南赛诺特生物技术有限公司 | Monoclonal antibody aiming at human cyclin 1, preparation method thereof, immunoassay reagent and application |
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