CN102719443B - Carboxyl-terminal specific anti-human amyloid protein monoclonal antibody gene and its encoded polypeptide and application - Google Patents
Carboxyl-terminal specific anti-human amyloid protein monoclonal antibody gene and its encoded polypeptide and application Download PDFInfo
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Abstract
The invention discloses a carboxyl-terminal specific anti-human amyloid protein monoclonal antibody gene and its encoded polypeptide and an application, the gene comprises a heavy chain variable region gene and a light chain variable region gene, wherein a sequence of the heavy chain variable region is showed as SEQ ID NO:1, and a sequence of the light chain variable region is showed as SEQ ID NO:3. According to the gene provided by the invention, because a Abeta 42 oligomer can be specifically recognized, and Abeta 40 and Abeta 42 oligomers which have the difference of two amino acid can be distinguished, in an immunotherapy experiment, the combination of monoclonal antibody gene and Abeta 40 can be avoided, thereby the side reaction caused by the non specific recognition can be reduced; the polypeptide obtained according to the gene coding is amyloid protein carboxyl-terminal polypeptide, the immunization effect is good, the mice immunization serum has high valence, and its antigen recognition capability has conformational characteristics, the invention can be used for AD early stage diagnosis.
Description
Technical field
The present invention relates to gene engineering technology field, more specifically, the present invention relates to the special anti-human amyloid monoclonal antibody gene of a kind of C-terminal and its coded polypeptide and application.
Background technology
In alzheimer's disease (Alzheimer ' s disease, AD) patient's brain, producing beta-amyloyd plaque deposition is this sick typical cytopathic.A β 40 and A β 42 are main components of beta-amyloyd patch in AD patient's brain, and the difference of the two is that A β 42 C-terminals are than more than 40 two of A β, and wherein A β 42 is more easily gathered into the polymkeric substance of multiple ranks, is the main virulence factor of AD.Therefore, more meaningful than A β 40 for diagnosis and the treatment research of A β 42.
At present existing people carries out the development of A β 42 specific antibodies, but does not also obtain A β 42 monoclonal antibodies of conformational epitope.Can not specific recognition A β 2 oligomer, therefore the target spot effect of differential diagnosis and treatment is undesirable, need to have C-terminal specific recognition and the monoclonal antibody in conjunction with A β 2 oligomer.
Summary of the invention
The present invention is intended at least one of solve the problems of the technologies described above.
For this reason, one object of the present invention be to propose a kind of have specific recognition and in conjunction with the antibody activity fragment of A β 42 oligomer and with the gene of A β 40 no cross reactions.
The anti-human amyloid monoclonal antibody gene special according to the C-terminal of the embodiment of the present invention, comprise heavy chain variable region gene and chain variable region gene, wherein, the sequence of described heavy chain variable region gene is as shown in SEQ ID NO:1, and the sequence of described chain variable region gene is as shown in SEQ ID NO:3.
The anti-human amyloid monoclonal antibody gene special according to the C-terminal of the embodiment of the present invention, owing to can specific recognition A β 42 oligomer distinguishing, only differ 2 amino acid whose A β 40 and A β 42 oligomer, in immunotherapy experiment, can avoid 40 combinations with A β, thereby reduce the side reaction that non-specific identification causes; According to the polypeptide of this genes encoding gained, be amyloid C-terminal polypeptide, good immune effect, mouse immune serum titer is high, and its antigen recognition ability has conformational characteristic, can be for AD early diagnosis.
In addition, the anti-human amyloid monoclonal antibody gene that C-terminal according to the above embodiment of the present invention is special, can also have following additional technical characterictic:
The long 405bp of described heavy chain variable region gene, the long 393bp of described chain variable region gene, can be used for expression specificity identification and the antibody activity fragment in conjunction with A beta oligomers after two gene recombination.
Described heavy chain variable region gene and described chain variable region gene are hybridoma cell strain (the called after BJNA of the mouse A β 42 oligomer specific monoclonal antibodies from secreting greater activity, by Beijing Jiaotong University's life science and bio-engineering research institute, set up and preserve, its immunogen is A β 35-42 polypeptide, and the antibody molecule hypotype of its secretion is IgG
2a) middle clone, described hybridoma cell strain has carried out preservation, and its preservation information is as follows:
Culture title: hybridoma cell strain BJNA
Deposit number: CCTCC C201155
Depositary institution: Chinese Typical Representative culture collection center (CCTCC)
The preservation time: on July 1st, 2011.
Its concrete operation step can be: first, adopt Trizol method to extract the total RNA of hybridoma, total RNA synthesizes cDNA by reverse transcription, then take cDNA as masterplate, according to antibody (IgG) conserved sequence design primer, by the method amplification antibody weight chain variable region gene of PCR, just can obtain described heavy chain variable region gene and described chain variable region gene.
By sequential analysis, show, described heavy chain variable region gene and described chain variable region gene sequence reach 90% and 92% with the mouse IgG variable region sequences homology of submitting to, can determine that obtained sequence is light chain of antibody and weight chain variabl area sequence, but not the sequence of other gene.The mouse antibodies variable region that gained VH and VL gene codified are correct.
Another object of the present invention is to propose a kind of according to the polypeptide of the special anti-human amyloid monoclonal antibody gene coding of C-terminal described in above-described embodiment, and the aminoacid sequence of the polypeptide of encoding according to described SEQ ID NO:1 is as shown in SEQ ID NO:2; The aminoacid sequence of the polypeptide of encoding according to described SEQ ID NO:3 is as shown in SEQ ID NO:4.
The clone of the polypeptide that the invention allows for the clone that contains the special anti-human amyloid monoclonal antibody gene of described C-terminal and contain the anti-human amyloid monoclonal antibody gene coding special by described C-terminal.
The cultural method of described clone can be: the chain variable region gene of anti-human amyloid monoclonal antibody gene special described C-terminal and the nucleotide sequence of heavy chain variable region gene are cloned into corresponding expression vector, transfection/transform corresponding recipient cell (including, but not limited to bacterium, mammalian cell and yeast), can obtain the clone of antibody gene described in high expression level.
The protein product crystal that the special anti-human amyloid monoclonal antibody gene of described C-terminal obtains after expressing can also be for the preparation of the medicine of alzheimer's disease.
Its concrete operations can be: the chain variable region gene of anti-human amyloid monoclonal antibody gene special C-terminal and heavy chain variable region gene are expressed at different expression systems, after the albumen obtaining or polypeptide product purifying, can be made into crystal.This crystal, when preparing medicine, can be used as drug target, for the structure design of new drug.
Based on above-mentioned described heavy chain variable region gene and the chain variable region gene being cloned into, can adopt the method for gene recombination, build and express the genetic engineering antibodies such as multiple micromolecular chimeric antibody, single-chain antibody and Fab, for basic and clinic studies and the application of diagnosis, prevention and the treatment of alzheimer's disease, for example can apply preparing in the special anti-human amyloid monoclonal antibody of C-terminal, apply preparing in diagnosis of Alzheimer disease reagent, also can apply building in gene engineering monoclonal antibody expression system.
The invention allows for the application of polypeptide epitope A β 37-42 in the preparation anti-human amyloid protein oligomer monoclonal antibody special with screening C-terminal.
Additional aspect of the present invention and advantage in the following description part provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage accompanying drawing below combination is understood becoming the description of embodiment obviously and easily, wherein:
Fig. 1 is heavy chain variable region gene, the chain variable region gene schematic diagram after C-terminal specificity anti-amyloid monoclonal antibody purifying;
Fig. 2 is the sensitivity schematic diagram that C-terminal specificity anti-amyloid monoclonal antibody detects;
Fig. 3 is monoclonal antibody BJNA and the A8 different ability schematic diagram to A β 40 and A β 42 oligomer identifications respectively;
Fig. 4 is spot marking result schematic diagram;
Fig. 5 is Morris determined with Morris water result schematic diagram;
Fig. 6 is that the C-terminal specificity anti-amyloid monoclonal antibody of RT-PCR amplification is light, the agarose gel electrophoresis figure of heavy chain variable region gene.
Embodiment
By specific embodiment, further describe the present invention below, the following example is used for illustration purpose, but not for limiting the scope of the invention.The test method of unreceipted actual conditions in the following example, can operate according to the condition described in common molecular cloning handbook.Test materials used in the following example, if no special instructions, is from routine biochemistry reagent company and buys gained.
The preparation of the special anti-human amyloid monoclonal antibody of embodiment 1 C-terminal
One, test materials
1, (MVGGVVIA) (Shanghai Sheng Gong biotechnology company limited is synthetic) of A β (35-42)
2, A β 42 peptides (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) and A β 40 peptides (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) (Shanghai Sheng Gong biotechnology company limited is synthetic)
3, hexafluoroisopropanol (1,1,1,3,3,3-hexafluoro-2-propannol, HFIP) (purchased from Sigma company)
4, anhydrous dimethyl sulphoxide (DMSO) (purchased from Sigma company)
5, F12 substratum (purchased from Sigma company)
6, Freund's complete adjuvant (purchased from Sigma company)
7, freund 's incomplete adjuvant (purchased from Sigma company)
8, RPMI1640 nutrient solution (purchased from Sigma company)
9, PEG4000 (polyethylene glycol) (purchased from Sigma company)
10, foetal calf serum (Hangzhou Sijiqing Biological Engineering Material Co., Ltd. provides)
11, HAT (purchased from Sigma company)
12, mycillin (purchased from Sigma company)
13, HT (purchased from Sigma company)
14, mouse IgG isotype identification kit (purchased from Sigma company)
15, pristane (Pristane) (purchased from Sigma company)
16, BCA test kit (production of U.S. PIERCE company)
Two, testing sequence
1. the preparation of immunogenic design, synthetic and detectable antigens
Immunogenic design is with synthetic: get A β (35-42), at aminoterminal, add 1 Cys, then, with the directed coupling of KLH, obtain KLH-A β (35-42), set it as immunogen.
The preparation of detectable antigens A β oligomerization mixture: first A β 42 peptide 1mg are dissolved in the HFIP of ice precooling, make A β 42 peptide monomers, room temperature, makes HFIP volatilize totally after 1h.Then with DMSO20 μ l, dissolve A β 42 monomers, finally be placed on F12 substratum or phosphate buffer (the corresponding 1ml of supplying of volume), under 4 ℃ of temperature condition, cultivate 24h, make its natural polymerization, obtain antigen A β oligomerization mixture, and with Western blot, detect the preparation situation of A β 42 oligomerization mixtures.
2. the immunization of mouse
Get 5 of female BALB/c mouse in age in 6-8 week.
With A β oligomerization mixture, carry out altogether 4 immunity respectively: after by a 50 μ g/ dosage, A β oligomerization mixture and equal-volume Freund's complete adjuvant being mixed first, abdominal injection.Interval duplicate injection after 2 weeks, is used freund 's incomplete adjuvant, and coupling polypeptide dosage is 30 μ g/.Repeated once after 2 weeks at interval; Two weeks, interval again, carries out booster immunization in spleen with pure antigen 50 μ g, gets spleen and merge after 3-4d.
3. the preparation of feeder cell
Get 6-8 BALB/c mouse in age in week, the execution of craning one, under aseptic condition, RPMI1640 nutrient solution rinses abdominal cavity for several times, by washing fluid 2000rpm, centrifugal 10min.Abandon supernatant, with the RPMI1640 nutrient solution containing 20% foetal calf serum, suspend and precipitate, cell counting, adjusting cell concn is 10
5individual/ml, adds in 96 orifice plates, and 100 μ l/ holes, are placed in 37 ℃, 5%CO
2in incubator, hatch.
4. cytogamy
Get the mouse of reinforced immunological 3-4d, after eyeball bloodletting, collect serum, draw neck to put to death, aseptic taking-up spleen, make after single cell suspension, in the ratios of 10: 1 by splenocyte with in the Sp2/0 of logarithmic phase cell (through 8-anaguanine, be 8-Azaguanine, be called for short 8-AG screening) mix, the centrifugal 10min of 1000rpm, abandon supernatant, flicking pipe makes sedimentation cell in the pasty state in the end, in 37 ℃ of water-baths, add 0.7ml50%PEG4000, limit edged rotating centrifugal pipe, make cell keep mixing state, 1min adds, standing 90s in 37 ℃ of water-baths, slowly add immediately 20ml RPMI1640 liquid, the centrifugal 8min of 800rpm, abandon supernatant, add containing 20% foetal calf serum, 2%HAT, the RPMI-1640 of 1% mycillin, mix gently, adjusting cell concn is 2 × 10
6individual/ml, adds in 96 orifice plates that have feeder cell, and 100 μ l/ holes, are placed in 37 ℃, 5%CO
2in incubator, hatch, observation of cell growing state day by day, adds the RPMI1640 nutrient solution containing 20% foetal calf serum, 1%HT, 1% mycillin after 1 week, by following formula, calculate clonal growth rate.
Clonal growth rate (%)=(cell clone growth hole/inoculation hole) × 100%
5. the screening of positive colony and subclone
Until cell clone, grow to (the microscope magnification 40 ×) in the visual field at 1/3 o'clock, the careful cell conditioned medium liquid of drawing, 10 μ g/mlA β oligomerization mixtures are envelope antigen, ELISA method detects the antibody (concrete grammar and judging criterion are with reference to " embodiment 2 indirect ELISA experiments ") in supernatant, by following formula, calculates clone's positive rate.
Clone's positive rate (%)=(the cell clone growth hole of antibody positive hole/detection) × 100%
Adopt limiting dilution assay antagonist to secrete positive hole and carry out cloning repeatedly, extremely all mono-clonals hole supernatant antibody positive rate is 100%.
6. the foundation of hybridoma cell strain
The positive colony of cloning is moved into 24 orifice plates, 6 orifice plates and T25 Tissue Culture Flask, and enlarged culturing 60-90d also builds strain.
7. the subgroup identification of the special anti-human amyloid monoclonal antibody of C-terminal
IgG subclass according to the special anti-human amyloid monoclonal antibody of the mouse IgG isotype identification kit operation hybridoma cell strain secretion C-terminal of Sigma company is IgG
2a.
8. the epitope analysis of the special anti-human amyloid monoclonal antibody of C-terminal
Use Inhibition ELISA to measure the antigen recognition epi-position of monoclonal antibody: by each peptide section (A β 35-42 of monoclonal antibody and concentration gradient, A β 36-42, A β 37-42) hatch to join after 1h and on the enzyme plate that is coated with A beta oligomers, carry out indirect ELISA detection.Result shows that its epi-position is C end polypeptide A β 37-42.
9. a large amount of preparations and the purifying of the special anti-human amyloid monoclonal antibody of C-terminal
Inoculation hybridoma is to the pretreated BABL/c mouse peritoneal of pristane, 1 × 10
7individual hybridoma/only, after 7-10 days, extract ascites, use AKTA explore100 protein chromatography system by Protein A affinity column monoclonal antibody purification, shown in Fig. 1, wherein A represents heavy chain variable region gene, B represents chain variable region gene, and M is molecular weight protein marker (Marker), and L is heavy chain variable region gene and the chain variable region gene of the special anti-human amyloid monoclonal antibody of C-terminal.
By BCA kit measurement antibody concentration, be 3mg/ml.
One, test materials
1, enzyme plate (production of SUNRISE company)
2, the sheep anti-mouse igg of HRP mark (purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge)
Two, test method
1, coated: get 10 μ g/ml A β 40 and A β 42 oligomerization mixtures respectively as envelope antigen, be dissolved in coated damping fluid (pH9.6,0.05mol/L carbonate buffer solution), add in enzyme plate, every hole 100 μ l spend the night under 4 ℃ of temperature environments.
2, PBS-T (takes NaCl8g, KCl0.2g, Na
2hPO
41.44g, KH
2pO
40.44g, Tween-200.05ml, mends ddH
2o to 1L, regulates pH to 7.2～7.4) wash plate: 3 times, each 5min.
3, sealing: add the PBS-T containing 0.2%BSA, every hole 100 μ l.At 37 ℃, process 2h.
4, by anti-human amyloid the monoclonal antibody special C-terminal with PBS-T doubling dilution, add in 96 orifice plates every hole 100 μ l.At 37 ℃, process 2h.Establish negative control and positive control (using respectively SP2/0 cells and supernatant or antibody diluent, monoclonal antibody A8) simultaneously.
5, PBS-T washes plate: 3 times, and each 5min.
6, add the sheep anti-mouse igg of the HRP mark of dilution in 1: 10000, every hole 100 μ l.At 37 ℃, process 1h.
7, PBS-T washes plate: 3 times, and each 5min.
8, add substrate TMB, every hole 100 μ l, after 20min, 450nm wavelength detects A value.
Three, judgement mark
Positive judging criterion is as follows:
(sample well A value-blank A value)/(negative control hole A value-blank A value) >=2
What the greatest dilution detecting was this antibody tires.
Four, test-results
The special anti-human amyloid monoclonal antibody of application C-terminal can the coated A β 42 oligomerization mixtures of specific recognition, and nonrecognition A β 40 the results are shown in Table 1.Lowest detection is limited to: 30ng/ml, test-results is shown in Fig. 2.
The specificity (indirect ELISA) of table 12D7 identification A β 42
The above results explanation: the A β 42 oligomerization mixtures that the special anti-human amyloid monoclonal antibody of C-terminal can specific recognition assembled in vitro, its reaction sensitivity better and with A β 42 no cross reactions.
One, test method
The preparation of detectable antigens A β oligomerization mixture: as described in Example 1.
Get 5～10 μ g samples, 5 × sample buffer, mixes rear loading, first with 100V voltage, makes albumen pass through concentrated glue.When sample enters separation gel, regulating voltage makes it constant in 120V.When tetrabromophenol sulfonphthalein swimming is to gel when bottom, finish electrophoresis, take off gel, conventional with the dyeing of coomassie brilliant blue R_250 staining; Gel and nitrocellulose filter are put into respectively to balance 10min in the container that trace damping fluid is housed, putting into filter paper, gel, NC film, filter paper successively, become " sandwich " shape; pour transferring film damping fluid into; glue faces negative pole, and NC face is towards positive pole, the bubble of carefully avoiding and rush.Switch on power, make the continuous transferase 12 h of constant current 80mA, cut off the electricity supply.
After transferring film finishes, with Ponceau S staining fluid (10 × Ponceau S stock solution compound method is: take Ponceau S 2g, trichoroacetic acid(TCA) 30g, semi-annular jade pendant base Whitfield's ointment 30g, adds water to 100ml; Ratio according to 1: 10 during use is diluted with deionized water) determine protein band position, do corresponding mark.With confining liquid, sealing nitrocellulose filter (takes skim-milk 5g, is dissolved in 0.1mol/L PBST (NaCl8g, KCl0.2g, Na
2hPO
41.44g, KH
2pO
40.44g, Tween-200.05ml, mends deionized water to 1L, pH7.2～7.4) 100ml), 4 ℃ of sealings are spent the night.With confining liquid liquid dilution monoclonal antibody BJNA, concentration is generally 0.2～1 μ g/ml, hatches 12～14h in 4 ℃, or hatches 2h in 20～37 ℃.With 0.1mol/L PBST washing nitrocellulose filter 4 times, each 5～10min.With PBS, dilute two of HRP mark and resist, extent of dilution is 1: 1000, incubated at room 1h.With 0.1mol/L PBST washing nitrocellulose filter 4 times, each 5min.According to the explanation of PIERCE chemical luminescence reagent kit, A liquid and B liquid equal-volume are mixed, be added on nitrocellulose filter, after 2～5min, use X-ray exposure imaging, observations.
Two, test-results
Western Blot result, the special anti-human amyloid monoclonal antibody of C-terminal of purifying is mainly identified A β 2 oligomerizations, there is no cross reaction with A β 40, result is (WB after Native-PAGE) as shown in Figure 3, wherein C represents the anti-human amyloid monoclonal antibody that C-terminal is special, D represents monoclonal antibody A8, and 1 and 2 represent the special anti-human amyloid monoclonal antibody nonrecognition A β 40 (1) of C-terminal, only identify A β 42 (2); 3 and 4 represent that monoclonal antibody A8 can identify A β 40 (3) and A β 42 oligomerizations (4) simultaneously; 2 represent that detecting antibody is A8; 3 represent that detecting antibody is 6E10.
The above results explanation, the special anti-human amyloid monoclonal antibody of C-terminal has resolving power to only differing 2 amino acid whose A β 42 oligomerization mixtures and A β 40.Sample after the special anti-human amyloid monoclonal antibody of C-terminal can separate Native-PAGE, the A β 42 that utilizes Western Blot to carry out sample detects and differentiates.The identification that shows the special anti-human amyloid monoclonal antibody of C-terminal has conformation selection feature.
One, test method
1, the NC film of cutting suitable size soaks 5min in deionized water.
2, the NC film soaking is dried to 30min at room temperature naturally.
3, A β 42 (0.5mg/mL) and A β 40 (0.5mg/mL) are put respectively to 2 μ L to NC film, two repetitions of each sample.
4, by after slightly dry the NC film after point sample, be immersed in 5% skimmed milk confining liquid, 4 ℃ of sealings are spent the night.
5, the first antibody (the special anti-human amyloid monoclonal antibody of C-terminal after purifying) diluting by NC film and with 5% skimmed milk, incubated at room 2h, then TBST damping fluid is washed film 3 times, each 3min.
6, sheep anti-mouse igg (1: 5000) the incubated at room 1h with the HRP mark with 5% skimmed milk dilution by film, TBST damping fluid is washed film 3 times, each 5min.
7, in darkroom, prepare chemical luminous substrate (by 1: 1 mixed substrates liquid).Luminous substrate is added on NC film equably, after NC film is wrapped with preservative film, puts into magazine and make egative film exposure, the egative film after exposure is put into after developing solution develops and put into stop bath photographic fixing again.
Two, test-results
In Nucleolar skeleton experiment, the special anti-human amyloid monoclonal antibody of C-terminal of purifying is mainly identified A β 2 oligomerizations, there is no cross reaction with A β 40, see Fig. 4, show A β 42 on the special anti-human amyloid monoclonal antibody specificity identification NC film of C-terminal, and nonrecognition A β 40.
The above results explanation, the special anti-human amyloid monoclonal antibody of C-terminal to be adsorbed on NC film only differ 2 amino acid whose A β 42 oligomerization mixtures and A β 40 has resolving power.Point out the identification of the special anti-human amyloid monoclonal antibody of C-terminal to there is conformation selection feature.
The test of embodiment 5Morris determined with Morris water
One, test objective
This test objective is in order to detect the therapeutic action of the special anti-human amyloid monoclonal antibody of C-terminal to rapid ageing Model of Dementia mouse.
Two, experimental animal
Double transgenic mouse APPswe/PS1 Δ E9, purchased from Institute of Experimental Animals, Chinese Academy of Medical Sciences, entrusts Department Of Medicine, Peking University's medical experiment animal center to raise.Grouping situation is as follows:
According to injectable drug difference, be divided into the special anti-human amyloid mab treatment group of C-terminal (being called for short BJNA treatment group), mouse non-specific IgG injection group and APPswe/PS1 Δ E9 blank group, 12 every group.
Three, test method
With special anti-human 6 monthly age of the amyloid monoclonal antibody abdominal injection male APPswe/PS1 Δ E9 mouse (400 μ g/ only) of C-terminal, 1 time weekly, after treating 4 weeks, carry out Morris determined with Morris water (described test completes in Military Medical Science Institute's prevention and control of diseases institute's toxicological evaluation research centre).
Its concrete testing sequence is:
1, designing special water maze is mainly comprised of the platform of a cylinder shape pond and a removable position.The high 70cm in pond, diameter 80cm, platform diameter 8cm, overhead, pond is connected with computer by a digital camera.
2, in pond, inject in advance clear water, depth of water 15cm, is black at the bottom of pool wall and pond, makes Chi Shui become opaque black, and platform surface is black, and mouse can not be seen, the water surface exceeds platform surface 0.5cm.
3, water temperature is controlled at 22 ± 0.5 ℃, the place of entry in pond subscript phasing same point as each experiment mice.On sidewall corresponding to each quadrant, paste difform sign.Platform is placed in from place of entry in the middle of the quadrant away from, and experimentation keeps position of platform constant.
4, each experiment is carried out in the room of sound insulation, pond, and light source, the position of the each objects in laboratory such as mouse cage remains unchanged.
5, treatment starts training on the 3rd day after finishing, if animal is found platform in 60s, is allowed to condition on platform and stops 20s, if animal is not found platform, places it on platform and makes it stop 20s.
6, each experiment is limited with 60s, notes down each each treated animal and can arrive the mouse number of elements of platform.If do not find platform in setting-up time, computer stops following the tracks of, and be 60s writing time.
7,4,5,6,7,8 days continue studyings and test after treatment finishes, note is done the 0th, 1,2,3 and 4 day respectively, records every group of mouse and find the latent period of platform.
Four, test-results
BJNA treatment group learning and memory improves situation and is obviously better than mouse non-specific IgG injection group and APPswe/PS1 Δ E9 blank group, there is notable difference latent period, test-results is shown in that (X-coordinate is latent period to Fig. 5, unit is second (s), and ordinate zou is the time of test, and unit is day d), wherein, E represents BJNA treatment group, and F represents the non-specific IgG injection of mouse group, and G represents APPswe/PS1 Δ E9 blank group.
The above results shows, the special anti-human amyloid monoclonal antibody of C-terminal is improved the effect of APPswe/PS1 Δ E9 model mice ability of learning and memory.
The clone of the special anti-human amyloid variable region of mab gene of embodiment 6 C-terminals
Get in the BJNA of logarithmic phase hybridoma (5 × 10
6), adopt Trizol reagent method to extract total RNA of hybridoma cell strain, take a morsel and carry out ultraviolet spectrophotometer quantitatively and 1% denaturing formaldehyde agarose gel electrophoresis.Utilize the method for RT-PCR increase light, the heavy chain variable region gene of the special anti-human amyloid monoclonal antibody gene of anti-A beta oligomers C-terminal.Follow respectively according to mouse IgG light chain and heavy chain conserved sequence design primer.According to test kit specification sheets, take total RNA as template, by random primer, use synthetic cDNA the first chain of SuperScrip II ThermoScript II.
Reverse transcription reaction scheme is: total RNA5 μ g, and random primer 100n mol/L, adds the L without Rnase water to 18 μ, puts immediately on ice bath after hatching 10min for 70 ℃; Continuation adds 10 × buffer2.5 μ L in reaction solution, 25m mol/L MgCl
22.5 μ L, 10m mol/L dNTP1 μ L, 42 ℃ add 1 μ L200U/ μ L SuperScrip II ThermoScript II after hatching 1min, and 42 ℃ of reaction 50min, hatch 10min for 70 ℃.After reverse transcription, add RNase hydrolysis RNA, make only to contain in product cDNA the first chain.
Respectively in order to HF atgggactccaggcttcaatttagttttcctt and HR cagtggatagaccgatggggg and LF atgaagattgcctgttaggctgttggtgctg and LR actggatggtgggaagatgg two to primer take cDNA the first chain as template amplification light chain of antibody with heavy chain (VL, VH) variable region gene.Amplified production VH (about 406bp) and VL (about 393bp) identify through sepharose (1.5%) electrophoresis, electrophoretogram as described in Figure 6, in figure, M is DL-2000DNA molecular weight Marker, 1 is the special anti-human amyloid monoclonal antibody heavy chain variable region gene of C-terminal, and 2 is the special anti-human amyloid monoclonal antibody chain variable region gene of C-terminal.
With after PCR product purification test kit (production of Omega company) recovery purifying, object fragment is cloned into T carrier, sequencing analysis, analytical results is as shown in sequence table SEQ ID NO:1～SEQ ID NO:4.
Wherein, pcr amplification reaction carries out according to ordinary method: take above-mentioned product as template, use respectively that heavy chain and light chain primer amplification antibody are light, heavy chain variable region gene.Reaction system is: cDNA5 μ L, the each 1 μ L of upstream and downstream primer (10mmol/L), 10m mol/L dNTP2 μ L, 10 × buffer5 μ L, add distilled water to 50 μ L, Ex Taq archaeal dna polymerase 1.25 μ l, add water to 50 μ l, mix, instantaneous centrifugal after, put reaction in PCR instrument.PCR reaction conditions is: 95 denaturation 5min, and loop parameter is 94 sex change 40s, 55 annealing 40s, 72 extend 1min, totally 30 circulations, last 72 extend 15min.
Object fragment T carrier cloning order-checking scheme is as follows: after PCR product is reclaimed with test kit (production of Omega company) purifying, be connected into pMD18-T carrier.Ligation system is: pMD18-T carrier 1 μ l, PCR product purification heavy chain (or light chain) 4 μ l, connect damping fluid 5 μ l, mix rearmounted 4 ℃ and spend the night, and transform bacillus coli DH 5 alpha, screening recombinant clone order-checking.
Specifically describe the application of the special anti-human amyloid monoclonal antibody gene of C-terminal according to the present invention below.
Particularly, light, the heavy chain variable region gene of the special anti-human amyloid monoclonal antibody of described C-terminal can be applied in the reagent (box) for the preparation of diagnosis, prevention and treatment AD and medicine.
With the special protein drug that anti-human amyloid monoclonal antibody is light, heavy chain variable region gene reconstitutes certain forms of C-terminal of the present invention, can be directly used in diagnosis and the immunotherapy of AD.The anti-human amyloid monoclonal antibody special with C-terminal involved in the present invention is light, the polypeptide of heavy chain variable region gene coding, and the novel antibody that can reassemble into mainly contains following several form:
(1) chimeric antibody: be to be formed by connecting as people-mouse chimeric antibody with the V district of mouse MAb and the C district of human IgG.Due to its specificity and avidity of intactly having preserved mouse MAb, the untoward reactions such as HAMA have been reduced, therefore demonstrate good effect in immunotherapy simultaneously;
(2) humanized antibody: humanization modified for variable region gene structure, comprise that CDR transplants, surface amino groups acid residue is modified, the exchange of skeleton district, location retain and epi-position guided selection etc., thereby the mouse that has not only reduced variable region has kept again specificity and the avidity of mouse MAb simultaneously;
(3) small molecular antibody: mainly contain the Fab antibody that formed with VL-CH1 by VH-CH1, with a polypeptide (Gly4Ser) 3 joints be connected single-chain antibody that VH gene and VL gene form, by VH and VL with non covalent bond be combined into Fv fragment antibody, the single domain antibody being formed by VH or functional domain of VL, the atom being formed by single CDR etc.;
(4) multivalence miniantibody: mainly contain double-chain antibody (ScFv)
2, Flex miniantibody, LD miniantibody, F (ab ')
2, F (ab ')
3, (ScFv)
4deng.Owing to there being polyvalent antigen binding site, avidity is high, and molecular size is moderate, and the clearance rate in kidney has higher clinical value compared with slow feature;
(5) bi-specific antibody: be the antibody that a class has dual specificity and dual-use function, claim again bifunctional antibody;
(6) recombinant antibody fusion proteins: gene fragments such as Fab or Fv, there is the specific biological activity recombinant protein of target with other biologically functional molecules such as the nucleic acid of non-antibody or enzyme are connected to form;
(7) phage antibody: the V district gene of Ig is connected after transfecting host bacterium with the upper gene III of filobactivirus DNA or gene VIII, make its fusion protein product at film surface outer casing protein expression Fab or ScFv, by this product being taken turns more to the affine absorption of related antigen, therefrom filter out required specific antibody.
The anti-human amyloid monoclonal antibody special with C-terminal involved in the present invention is light, the polypeptide of heavy chain variable region gene coding, and reconstitute the antibody of certain forms, carry out the marks such as various enzymes, vitamin H, fluorescence (Cy3, Cy5 etc.).
On the basis of above-mentioned test, can utilize the synthetic or A β of prokaryotic expression and the A β of oligomerization as standard antigen sample, set up the suitableeest coated antibody and the concentration of sandwich ELISA method analysis, the suitableeest biotinylated detection antibody and concentration, simultaneously take the avidin of horseradish peroxidase-labeled and TMB as detection system, obtain solubility A β 42 oligomer sandwich ELISA analytical procedures, set up the laboratory standard of AD method of early diagnosis.
With above-mentioned polypeptide restructuring or derivative antibody molecule light by the special anti-human amyloid monoclonal antibody of C-terminal, heavy chain variable region gene coding, can be assembled into AD and AD relative disease diagnostic kit.
Although illustrated and described embodiments of the invention, those having ordinary skill in the art will appreciate that: in the situation that not departing from principle of the present invention and aim, can carry out multiple variation, modification, replacement and modification to these embodiment, scope of the present invention is limited by claim and equivalent thereof.
Claims (7)
1. a hybridoma cell strain BJNA, is characterized in that, the deposit number of described hybridoma cell strain BJNA is CCTCC C201155, and depositary institution is Chinese Typical Representative culture collection center (CCTCC), and the preservation time is on July 1st, 2011.
2. the special anti-human amyloid monoclonal antibody heavy chain variable region gene of C-terminal, is characterized in that, the sequence of described heavy chain variable region gene is as shown in SEQ ID NO:1.
3. the variable region of heavy chain polypeptide that the special anti-human amyloid monoclonal antibody gene of C-terminal is encoded according to claim 2, is characterized in that, the aminoacid sequence of the polypeptide of encoding according to described SEQ ID NO:1 is as shown in SEQ ID NO:2.
4. gene according to claim 2 or polypeptide claimed in claim 3 are in the application of preparing in the special anti-human amyloid monoclonal antibody of C-terminal.
5. gene according to claim 2 or polypeptide claimed in claim 3 are in the application of preparing in diagnosis of Alzheimer disease reagent.
6. gene according to claim 2 or polypeptide claimed in claim 3 are in the application of preparing in therapeutic agent for alzheimer's disease.
7. gene according to claim 2 or polypeptide claimed in claim 3 are in the application building in gene engineering monoclonal antibody expression system.
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| CN115925923B (en) * | 2022-09-26 | 2023-09-15 | 吉林大学 | Single-chain antibody catalyzing degradation of Abeta 42 oligomer, single-chain antibody gene and application thereof |
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| Title |
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| 包福祥等.老年性痴呆抗体药物研究进展.《中国生物工程杂志》.2008,第28卷(第12期),107-111. |
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