CN1970573A - Neutrality monoclonal antibody 3E1 for resisting ricin, its preparation method and uses - Google Patents
Neutrality monoclonal antibody 3E1 for resisting ricin, its preparation method and uses Download PDFInfo
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Abstract
The invention discloses a neutral monoclonal antibody 3E1 of anti-castor toxin and making method and application in the castor toxin detecting agent, vaccine and or immune toxic drug, which is characterized by the following: cloning antibody, heavy-chain variable area gene; obtaining correct coded antibody variable area of mouse; expressing multi-molecular gene engineering antibody; crosslinking multiple biological activity molecule based on the peptide or protein coded by gene.
Description
Technical field
The present invention relates to a kind of antiricin neutralizing monoclonal antibody 3E1, also relate to this MONOCLONAL ANTIBODIES SPECIFIC FOR method and the application in preparing Ricin vaccine and Ricin detection, diagnosis, medicine.
Background technology
Ricin (Ricin) is a kind of ribosome inactivating protein, it almost can with all eukaryotic cell combinations, be cytotoxicity.A part Ricin enters in the cell, and is dead with regard to being enough to make synthetic the stopping of whole cell protein.Its toxicity is 6000 times of prussiate.The Ricin aerosol enters in the body, can cause serious alveolar inflammation and pulmonary edema, because of blocking anoxic, lung causes death (1.GriffithsGD at last, Lindsay CD, Allenby AC, et al.Protection against inhalation toxicity ofricin and abrin by immunisation.Hum Exp Toxicol 1995; 14:155-64.2.BrownRFR.White DE.Ultrastructure of rat lung following inhalation of ricin aerosol.IntJ Exp Pathol 1997; 78:267-76.).Intravenously administrable per kilogram of body weight 3-5 microgram can be fatal.Up to now, also be not applicable to both at home and abroad the special-purpose specifics people, that poison at Ricin.Ricin raw materials for production castor seeds wide material sources, it is easy to extract the preparation method, is easy to be utilized by the terrorist, therefore studies its antagonist and has important practical significance.
Antibody has been used for the treatment of poisonous snake, malicious scorpion is bitten, and has good curative effect always since 19 end of the centurys reports can be used for treating disease.Studies show that in a large number: treat sudden Ricin with the neutralizing antibody of Ricin and poison respond well.Reports such as Furukawa-Stofferdeng: injection contains the culture supernatant of Ricin neutralizing antibody and injects 0.5g Ricin (lethal dose) simultaneously to mouse peritoneal; can protect and be tried the mouse attack (T.Furukawa-Stoffer that fights toxins; D.C.W.Mah; J.Cherwonogrodzkyet al.A novel biological-based assay for the screening of neutralizing antibodies toricin.Hybridoma; 1999,18 (6): 505-511).The success or failure of Antybody therapy depend on Ricin absorbed what and enter intravital path (B.M.J.Foxwell, S.I.Detre, T.A.Donovan.et al.The useof anti-ricin antibotied to protect mice intoxicated with ricin.Toxicology, 1985,34:79-8834.).Be 4: 1 o'clock at the monoclonal antibody of Ricin living chain and the molecular ratio of toxin, antibody external can in and Ricin to the cytotoxicity of EL-4 lymphoma cell.N.F,USP MANNITOL has increases the function of macromolecular substance by hemato encephalic barrier, and the toxic action of Ricin increases twice than intravenous injection after the use N.F,USP MANNITOL, at the neurotoxic of Ricin, has the protection effect same with intravenous injection behind the intracranial injection antibody.
At present, both at home and abroad at the report of Ricin monoclonal antibody, be mainly the monoclonal antibody that detects usefulness and have active two classes of neutralization.Report with the active monoclonal antibody of neutralization is mainly antiricin A chain, B chain and A, three kinds of form (1.P.V.Lemley of B chain, P.Amanatides, D.C.Wright.Identification and characterization of a monoclonal antibody thatneutralizes ricin toxicity in vitro and in vivo.Hybridoma, 1994,13 (5): 417-421.2.Maddaloni M, Cooke C, Wilkinson R, Stout AV, Eng L, Pincus SH.Immunological characteristics associated with the protective efficacy of antibodiesto ricin.J Immunol.2004,15; 172 (10): 6221-8.).At the early-stage (1. Song Yun raises to research fields such as the detection of Ricin biological warfare agent and medical protections in China, Liu Juan, Ying Tianyi, Deng. the sandwich immunoassay PCR method detects Ricin. Journal of Immunology, 2002,18 (3): 229-231.2. Hao Lan group, Guo Shengqing, Guo Zhenquan, Deng. antiricin MONOCLONAL ANTIBODIES SPECIFIC FOR and application. cell and molecular immunology magazine, 2003,19 (5): 517-518), still do not have supporting diagnostic reagent and effective immunotherapy and preventive means at the attack of terrorism of Ricin, therefore press for detection and protective articles that foundation and development have independent intellectual property right based on antibody.At the monoclonal antibody of Ricin semi-lactosi binding site, still there is not similarly report at present.
Summary of the invention
For detection or the treatment means that provides a kind of Ricin to poison, the invention discloses a kind of monoclonal antibody 3E1 of antiricin.
Light, the heavy chain protein matter molecule of antiricin neutralizing monoclonal antibody 3E1 disclosed by the invention, shown in sequence in the sequence table 3, sequence 4, its encoding gene is respectively shown in sequence in the sequence table 1, sequence 2 respectively for its aminoacid sequence.Light, the heavy chain protein matter molecule amino acid sequences of this monoclonal antibody is respectively shown in sequence in the sequence table 5, sequence 6.Complementary determining region CDR1, the CDR2 of the light chain protein matter molecule variable region of this monoclonal antibody, the aminoacid sequence of CDR3 are respectively shown in sequence in the sequence table 7, sequence 8, sequence 9.Complementary determining region CDR1, the CDR2 of the heavy chain protein matter molecule variable region of this monoclonal antibody, the aminoacid sequence of CDR3 are respectively shown in sequence in the sequence table 10, sequence 11, sequence 12.
The invention also discloses the preparation method of said monoclonal antibody 3E1, main contents are as follows:
1. the structure of antiricin semi-lactosi binding site monoclonal antibody hybridoma cell strain
At first Ricin is carried out attenuation treatment with formaldehyde, immune then Balb/c mouse, ordinary method is carried out cytogamy.With indirect elisa method screening, positive cell clone is subclone repeatedly again, and detecting up to all Hybridoma Cell Culture supernatants is 100% positive.Hybridoma 3E1 has been carried out chromosome karyotype analysis, and hybridoma 3E1 karyomit(e) average number is 106, experimental results show that with the two-phase agar diffusion the secreted immunoglobulin (Ig) hypotype of 3E1 hybridoma is IgGl.
2. the screening and the evaluation of the strain of antiricin semi-lactosi binding site monoclonal antibody hybridoma cell
Use mtt assay, with regard to Ricin murine myeloma cell SP2/0 has been carried out cellulotoxic experiment, the result calculates the mld IC50=0.31ng/ml of Ricin to the Sp2/0 cell.According to IC50, filter out neutrality antibody 3E1, Western-blotting has done evaluation to specificity.The result shows that 3E1 is the Ricin neutrality antibody, can be in experiment in vitro, in and Ricin to the toxic action of SP2/0 cell, but the linear epi-position of the B chain of neutralizing monoclonal antibody 3E1 specific recognition Ricin.
3. monoclonal antibody 3E1 is to the provide protection of BALB/C mice
Affinity column purifying Balb/c mouse hybridoma cell ascites, ultraviolet spectrophotometer is measured, and calculates protein content.Give injected in mice various dose Ricin, calculate mld LD50.Give injected in mice various dose Ricin, after the different time sections, injection neutralizing monoclonal antibody 3E1 record growing state.The result shows that Ricin is 10.4 μ g/kg to the LD50 of mouse, and Ricin neutralizing monoclonal antibody 3E1 still has provide protection to mouse at the Ricin of 10 times of deadly meterings of injected in mice after 20 minutes.
4. light, the angling of heavy chain gene of hybridoma 3E1 got
Extract neutralizing monoclonal antibody 3E1 cell RNA,, angle the weight chain gene of getting antibody with two pairs of Auele Specific Primers through RT-PCR.Conventional method connects into carrier, and the transformed competence colibacillus bacterium is cultivated the single bacterium colony of back picking, extracts and carries out the dna sequencing analysis after plasmid PCR identifies.
By this part experiment, made up and contained that Ricin neutralizing antibody 3E1 is light, the carrier of heavy chain gene, through sequential analysis, comparison, encoding sequence is that mouse immuning ball protein is light, heavy chain gene (sequence 1 and sequence 2 in the sequence table).
5. light, heavy chain variable region gene sequence of monoclonal antibody 3E1 and aminoacid sequence determines
With
Www.expasy.orgOnline software will be encoded, and Ricin neutralizing monoclonal antibody 3E1 is light, the weight chain variable region nucleotide sequence is translated as its amino acid sequence coded, and monoclonal antibody 3E1 is light, heavy chain amino acid sequence is shown in sequence in the sequence table 3 and sequence 4.According to Kabat database (ElvinA.Kabat. " Sequences of Proteins of Immunological Interest " .1991) determine the light chain variable region sequence shown in sequence in the sequence table 5 and weight chain variabl area sequence shown in sequence in the sequence table 6.Determine that according to the Kabat database complementary determining region CDR1, CDR2 in the light chain variable region sequence and its aminoacid sequence of CDR3 are respectively shown in sequence in the sequence table 7, sequence 8 and sequence 9.Complementary determining region CDR1, CDR2 in the weight chain variabl area sequence and its aminoacid sequence of CDR3 are respectively shown in sequence in the sequence table 10, sequence 11 and sequence 12.
The primer that the present invention uses an Analysis of Nested Design has cloned from the Ricin semi-lactosi binding site neutralizing monoclonal antibody 3E1 hybridoma of cultivating successfully that antibody is light, heavy chain variable region gene.The mouse antibodies variable region that gained light chain and heavy chain variable region gene codified are correct.Monoclonal antibody of the present invention is light based on above-mentioned Ricin semi-lactosi binding site of being cloned into and neutralizing monoclonal antibody 3E1, heavy chain variable region gene, can make up and express multiple small molecules genetic engineering antibody, as single-chain antibody, single domain antibody, chimeric antibody, Fab antibody, antibody fusion protein etc.; Based on said gene encoded polypeptide or protein, can crosslinkedly multiple bioactive molecules, prepare vaccine and be used for diagnosis or the medicine that Ricin detection, Ricin and immunotoxin are poisoned, have boundless application prospect.
Description of drawings
Fig. 1 is an immunity Ricin SDS-PAGE collection of illustrative plates as a result, and wherein swimming lane 1 is the non-reduced attitude Ricin of purifying; Swimming lane 2 is the non-reduced attitude Ricin through the formaldehyde detoxification treatment; Swimming lane 3 is gone back the ortho states Ricin for purifying; Swimming lane 4 is for going back the ortho states Ricin through the formaldehyde detoxification treatment; Swimming lane 5 is protein Marker.
Fig. 2 is hybridoma chromosome karyotype analysis figure as a result.
Fig. 3 is the cytotoxicity figure of Ricin to the Sp2/0 cell, and wherein X-coordinate is that the concentration ordinate zou of Ricin is the kill rate of Ricin Sp2/0 cell, calculates the IC50=0.31ng/ml of Ricin to the Sp2/0 cell according to this result.
Fig. 4 is the cytotoxicity figure of monoclonal antibody 3E1 opposing Ricin to the Sp2/0 cell, wherein X-coordinate is the kind of monoclonal antibody, ordinate zou is for after adding monoclonal antibody 3E1, and Ricin is represented with the survival rate of cell the cytotoxicity of Sp2/0 cell.
Fig. 5 is that the binding characteristic of neutralizing monoclonal antibody 3E1 is identified figure, and wherein swimming lane 1 is the SDS-PAGE result of Ricin, and top is the B chain, and following is the A chain; Swimming lane 2 is protein Marker, and swimming lane 3 is the Western-blot result of neutralizing monoclonal antibody 3E1.The result shows that neutralizing monoclonal antibody 3E1 can discern Ricin B chain (semi-lactosi binding site) linear epitope.
Fig. 6 is the tomographic results figure of neutralizing monoclonal antibody 3E1 through Protein A purifying, and wherein peak 1 is last sample peak, and peak 2 is the antibody purified peak.
Fig. 7 be the SDS-PAGE of monoclonal antibody 3E1 behind Protein A purifying as a result collection of illustrative plates wherein swimming lane 1,2,3 be neutralizing monoclonal antibody 3E1 through Protein A purifying, swimming lane 4 is protein Marker, A is a heavy chain, B is a light chain.
Fig. 8 is that Ricin neutralizing monoclonal antibody 3E1 hybridoma RNA extracts collection of illustrative plates as a result.
Fig. 9 is the PCR figure as a result of Ricin neutralizing monoclonal antibody 3E1 hybridoma weight chain gene, and wherein swimming lane 1 is a light chain gene; Swimming lane 2 is DL2000; Swimming lane 3 is a heavy chain gene.The gene size is approximately 660bp.
Embodiment
Can more easily understand content of the present invention by consulting following embodiment, these embodiment are just for further specifying, and do not mean that qualification scope of the present invention.
The structure of embodiment one antiricin monoclonal antibody hybridoma cell strain
One, material:
Fu Shi Freund's complete adjuvant and Freund, TMB:Sigma company product
20% foetal calf serum: Beijing Heng Shengma of unit biotechnology research institute product
Serum-free RPMI 1640:Gibco company product
SP2/0 cell: ATCC introduces, and preserve in this laboratory.
The Balb/c mouse: Military Medical Science Institute's Experimental Animal Center provides
All the other reagent are commercial.
Two, methods and results:
1. Ricin (the Nicolson of the attenuation treatment of Ricin behind purifying, G.L., Blaustein, J., 1972.The interaction of Ricinus com-munis agglutininwith normal and tumor cell surfaces.Biochim.Biophys.Acta 266,543-547.) adding final concentration among the 2mg/ml is 1% formaldehyde, handles 72h for 37 ℃, the PH7.20.01M PBS 72h that dialyses, SDS-PAGE detects (Fig. 1).
2.Balb/c mouse immune is selected 6 of the female Balb/c mouse in age in 4-6 week for use, with the subcutaneous immunity of 100 μ gricin inguinal regions, first pin adds the Fu Shi Freund's complete adjuvant, and the 2nd pin adds freund 's incomplete adjuvant, per 3 week immunity 1 time, immune 3 times altogether.The 3rd immunity back tail vein blood, indirect ELISA detects the antibody production, merges preceding 3 days, with 100 μ g ricin abdominal cavity booster immunizations once, merges in the 3rd day.
3. cytogamy is taken off neck execution after mice immunized is plucked eyeball, and the aseptic mouse boosting cell of winning carries out cytogamy according to a conventional method.Concrete grammar: take off neck execution after 1. mice immunized being plucked the eyeball bloodletting, 75% alcohol-pickled 3min, aseptic taking-up spleen grinds the individual cells suspension with 200 order steel meshes, and serum-free RPMI 1640 washes twice and numeration; 2. collect the SP2/0 cell of logarithmic phase, wash twice and numeration with serum-free RPMI 1640; 3. press the SP2/0 cell: two kinds of cells of mixed of splenocyte=1: 5, wash 1 time with RPMI 1640, abandon most supernatant, gently cell is broken up; 4. slowly add 1ml 50%PEG (Mw 1500) solution in the time at 1min, put 37 ℃ of water-bath 1min; 5. add serum-free RPMI1640 1ml, 5ml, 10ml, 10ml in the time at 1min, 2min, 2min, 5min; 6. the centrifugal 7min of 800r/min abandons supernatant, as far as possible gently cell is hanged; 7. add HAT (the Sigma)-RPMI RPMI-1640 that contains 20%FCS, adjusting cell concn is 2 * 10
6/ ml behind the mixing, drips and is being covered with nurse cell (1 * 10
4Cells/well) in 96 well culture plates (Gibco), 100 μ l/ holes, 37 ℃ of 5%CO
2Cultivate in the incubator.
4. indirect ELISA screening antiricin monoclonal antibody hybridoma cell (1), is spent the night and seals in 4 ℃ by elisa plate with 10 μ g/mlricin bag.Add cell culture supernatant to be measured (37 ℃ of 1h, PBST wash plate 4 times) successively, and the 50 μ l HRP-GAM (37 ℃ of 45min, PBST wash plate 4 times) of dilution in 1: 500.After the tmb substrate colour developing, measure the A value in the 450nm wavelength.
5. the positive cell clone of hybridoma cell clone indirect elisa method screening subclone repeatedly again, detecting up to all Hybridoma Cell Culture supernatants is 100% positive.The hybridoma cloning limiting dilution assay: (1) prepared nurse cell on the same day or preceding 1 day of cloning: take off neck and handle kunming mice, 75% alcohol-pickled sterilization skin, the aseptic skin of abdomen of peeling off, syringe extracts the 5ml RPMI-1640 and injects mouse peritoneal, wash sucking-off abdominal cavity, back washing lotion repeatedly, with splashing into 96 orifice plates, the about 0.1ml in every hole after the dilution of 20% foetal calf serum RPMI-1640.(2) get a little and wait that the hybridoma of doing cloning moves in another sterile test tube, and accurate counting.(3) limiting dilution assay carries out subclone.(4) culture plate is placed 5%CO2, cultivate in 37 ℃ of incubators, can be observed cell clone at microscopically about 5 days.Change liquid in good time, detect, get the positive monoclonal cell strain and carry out enlarged culturing, freeze-stored cell strain in time.
6. the chromosome karyotype analysis of hybridoma adds colchicine in the hybridoma of logarithmic phase, final concentration is 0.02 μ g/ml, in incubator, continue to cultivate collecting cell after 4 hours, (add pre-temperature through hypotonic cracking to 37 ℃ 0.075M KCL8ml, beat even gently, 37 ℃ 30-40 minute), pre-fix (methyl alcohol: Glacial acetic acid=fresh preparation in 3: 1, beat even gently, the centrifugal supernatant of abandoning) and 3 times fixing (the 1st time fixing: add the 6ml stationary liquid, beat even gently, room temperature 30 minutes, the centrifugal supernatant of abandoning; The 2nd time is fixing: add the 6ml stationary liquid, beat gently even, room temperature 15 minutes, the centrifugal supernatant of abandoning; The 3rd time is fixing: add the 6ml stationary liquid, beat gently even, room temperature 15 minutes, the centrifugal supernatant of abandoning) handle the back and prepare cell suspension with a small amount of stationary liquid, drip on the slide that frozen water soaks, microscopy (Fig. 2) is washed in slow fire oven dry back 10%Giemsa dyeing 15-20 minute.The result shows: through the statistical average value of 30 division phase cells, the hybridoma chromosome number changes between 85-116 (n=30), and average number is 106, and kinetochore, 1-2 middle part karyomit(e) is arranged.Known Balb/c mouse boosting cell chromosome number is 40, and Sp2/0 cell chromosome number is the 60-70 bar, and the hybridoma chromosome number after the fusion should be the 90-120 bar, and pointing out the antibody secreting cell that is obtained thus is hybridoma.
7. hybridoma immunoglobulin (Ig) hypotype is definite with sheep anti-mouse igg 1, IgG2a, IgG2b and IgG3, culture supernatant after concentrating with regard to hybridoma is done the experiment of two-phase agar diffusion, proves that the secreted immunoglobulin (Ig) hypotype of 3E1 hybridoma is IgG1.
By this part work, screen Ricin semi-lactosi binding site monoclonal antibody 3E1.
Embodiment two has Ricin semi-lactosi binding site and neutralizing monoclonal antibody
The screening of hybridoma cell strain and evaluation
One, material:
The same.
Two, methods and results:
1.ricin make its concentration be respectively 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml, 0.3125ng/ml, 0.156ng/ml, 0.078ng/ml with RPMI-1640 dilution ricin to the definite of murine myeloma cell SP2/0 mld (IC50), add respectively in the 96 porocyte culture plates, each concentration 3 multiple hole, 100 μ l/ holes, the SP2/0 cell concn of adjusting logarithmic phase is adjusted into 5 * 105/ml, 100 μ l/ holes.Cultivate 24h in 37 ℃ of 5%CO2 incubators, every hole adds 10 μ l MTT, cultivates 6h in 37 ℃ of 5%CO2 incubators, and the centrifugal 10min of 2000r/min abandons supernatant, and it is dissolving crystallized to add 100 μ l DMSO, measures the A value in the 570nm wavelength.By formula: cell mortality %=[1-(experimental group A value one blank group A value)/(control group A value one blank group A value)] * 100% estimate cytotoxicity, and calculate IC50 (Fig. 3).Use mtt assay, with regard to Ricin murine myeloma cell SP2/0 has been carried out cellulotoxic experiment, the result calculates IC50=0.31ng/ml.
2. the screening of monoclonal antibody 3E1 and neutralization are active makes its concentration be respectively 40ng/ml, 20ng/ml, 10ng/ml, 5ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml, 0.3125ng/ml, 0.156ng/ml, 0.078ng/ml with RPMI-1640 dilution ricin, add respectively in the 96 porocyte culture plates, each concentration 3 multiple hole, 100 μ l/ holes.Add 4 kinds of cell culture supernatant to be measured 50 μ l/ holes successively respectively.The SP2/0 cell concn of adjusting logarithmic phase is adjusted into 1 * 10
6/ ml, 50 μ l/ holes.37 ℃ of 5%CO
2Cultivate 24h in the incubator, every hole adds 10 μ l MTT, 37 ℃ of 5%CO
2Cultivate 6h in the incubator, the centrifugal 10min of 2000r/min abandons supernatant, and it is dissolving crystallized to add 100 μ l DMSO, measures the A value in the 570nm wavelength.By formula: cell survival rate %=[(experimental group A value-blank group A value)/(control group A value-blank group A value)] * 100% estimate cytotoxicity (Fig. 4).
3. monoclonal antibody 3E1 specificity is identified Ricin and to be dripped the PBS that contains 5% milk powder and to spend the night in 4 ℃ of sealings through 120g/L reduction SDS-PAGE and be transferred on the nitrocellulose filter, washes film 3 times with the PBS that contains 0.5ml/LTween-20.Film is cut into same widths, drips Ricin neutralizing monoclonal antibody 3E1 successively and hand over oncocyte culture supernatant (37 ℃ in conjunction with 1h, PBST washes film 3 times, again with the TBS washing that contains 0.5ml/L Tween-20 3 times).And HRP-GAMIgG (37 ℃ in conjunction with 1h, wash film 3 times) with the DAB colour developing after, observations (Fig. 5).
4. induce preceding 10 days of Monoclonal Antibody Ricin monoclonal antibody 3E1 inoculation hybridoma in the animal body, give Balb/c mouse (♂, 8～12 weeks) abdominal injection 0.5ml liquid petrolatum earlier.Collect the hybridoma of logarithmic phase, the centrifugal 7min of 1500r/min abandons supernatant, and physiological saline is washed 2 times, at last cell concn is adjusted into 4 * 10
6/ ml, every mouse peritoneal injection 0.5ml.After 7～10 days, visible mouse web portion obviously expands, and the tincture of iodine and cotton ball soaked in alcohol sterilization skin of abdomen extract ascites.With the centrifugal 10min of ascites 3000r/min that extracts, collect supernatant, add equivalent glycerine ,-20 ℃ are frozen standby.
Work by this part, the Ricin semi-lactosi binding site neutralizing monoclonal antibody that has obtained called after 3E1 is handed over oncocyte, in experiment in vitro, 3E1 can in and Ricin to the toxic action of SP2/0 cell, but neutralizing monoclonal antibody 3E1 specific recognition Ricin B chain (semi-lactosi binding site) linear epitope.
The Protein A purifying of embodiment three monoclonal antibody 3E1 reaches
Protectiveness experiment to BALB/C mice
One, material:
Protein A Sepharose CL 4B post albumen post: this yuan Zhenyang, Beijing Bioisystech Co., Ltd product; Surplus the same.
Two, methods and results:
1. add 1 milliliter of PH8.0 in 2 milliliters of mouse ascites, the 0.1moL/L phosphoric acid buffer is also used PH9.0, and it is 9 that 1moL/L TRIS-HCL adjusts PH.The mouse ascites adding has been used in the good Protein A Sepharose CL 4B post albumen post of 0.1moL/L phosphoric acid buffer PH 8.0 balances, washed pillar, in effluent liquid, do not detected till the foreign protein with above-mentioned damping fluid.With the citrate buffer solution wash-out of PH 3.0, collect effluent liquid, and, use PH7.2, the 0.01M BS 72h that dialyses immediately with the neutralization of 1moL/L TRIS-HCL PH 8.5 damping fluids.0D260 is surveyed in sampling on ultraviolet spectrophotometer, 0D280 calculates protein content, and freeze-drying-20 ℃ preservation (Fig. 6, Fig. 7).
2. selecting age in 4-6 week, body weight for use is 38 of Balb/c mouse about 20g, be divided into 4 groups, male and female half and half, survey abdominal injection 0 μ g, 10 μ g, 20 μ g, 40 μ g Ricins by per kilogram of body weight respectively at a left side, the normal raising, every 1h observes once, the record growing state, observed for 2 weeks, calculate mld LD50, see Table 1.
3. selecting age in 4-6 week, body weight for use is the Balb/c mouse about 20g, and male and female half and half are divided into 3 groups of antibody group, PBS group and normal controls, amount to 5 batches.Survey abdominal injection 20 μ g, 40 μ g, 60 μ g, 100 μ g, Ricin by per kilogram of body weight respectively at a left side, respectively at behind 0min, 10min, 20min, the 30min in the neutralizing monoclonal antibody 3E1 of right side abdominal injection purifying 100 μ g, the normal raising, every 1h observes once, the record growing state, observed for 2 weeks, the results are shown in (table 2, table 3, table 4).
The result shows that Ricin is 10.4 μ g/kg to the LD50 of mouse, and Ricin neutralizing monoclonal antibody 3E1 still has provide protection to mouse at the Ricin of 10 times of deadly meterings of injected in mice after 20 minutes.
Table 1. Ricin is determined the medium lethal dose of Balb/c mouse
| Dosage of ricin(μg/kg) | number of surviving mice | number of |
| 0 10 20 40 | 6/6 9/14 1/11 1/7 | 0/6 5/14 10/11 6/7 |
Can calculate LD50 from the result is 10.4 μ g/kg
Table 2. Ricin semi-lactosi binding site monoclonal antibody is to the provide protection (6 LD50) of mouse
| Antibody | Number of dead mice |
| No protection(control) 60μg/kg ricin Intraperitoneal injection Ricin B chain McAb(3E1)10 mins later | 3/3(X＝18h) 0/3 |
Table 3. Ricin semi-lactosi binding site monoclonal antibody is to the provide protection (10LD50 is after 20 minutes) of mouse
| Antibody | Number of dead mice |
| No protection(control) 100μg/kg ricin Intraperitoneal injection Ricin B chain McAb(3E1)20 mins later | 3/3(X＝16h) 0/3 |
Table 4. Ricin semi-lactosi binding site monoclonal antibody is to the provide protection (10LD50 is after 30 minutes) of mouse
| Antibody | Number of dead mice |
| No protection(control) 100μtg/kg ricin Intraperitoneal injection Ricin B chain McAb(3E1)30 mins later | 3/3(X＝14.3h) 3/3(X＝95.4h) |
Embodiment four Ricin semi-lactosi binding site neutralizing monoclonal antibodies
Hybridoma 3E1 is light, angling of heavy chain gene got
One, material:
Primer: PHs1: see sequence 13 in the sequence table; PHs2: see sequence 14 in the sequence table; PLs1: see sequence 15 in the sequence table; PLa1: see sequence 16 in the sequence table; PHa1: see sequence 17 in the sequence table.Dna fragmentation purification kit: OMEGA biotechnology company product; T4 dna ligase: NewEngland Biolabs product; Carrier PGEM Teasy:Promega company product; Competence bacterium JM109: available from Promega company.Surplus the same.
Two, methods and results
1. the neutralizing monoclonal antibody 3E1 cell 5 * 10 of taking the logarithm vegetative period
6-10
7Individual, centrifugal removal supernatant is evenly upspring cell.Adding 1ml TRIzol (Invitrogen) blows and beats repeatedly and makes the abundant cracking of cell, vibrate after 5 minutes, add the 0.2ml chloroform, vibrated 15 seconds, room temperature was placed 2-3 minute, 2-8 ℃ of 12000r/min, centrifugal 15 minutes, get supernatant in another new pipe, add behind the 500 μ l Virahol mixings room temperature and placed 10 minutes, centrifugal 10 minutes of 2-8 ℃ of 12000r/min.75% washing with alcohol precipitation after the drying, does not have the deionized water dissolving precipitation (Fig. 8) of RNA enzyme with 20 μ l.
2. get the solution that contains the total RNA of 1 μ g, add AMV5 * damping fluid 4 μ l successively, Oligo (dT) (500ng/ μ l) 0.5 μ l, 2.5mmol/L dNTP2 μ l, Rnasin (50U/ μ l) 0.5 μ l, deionized water mend to 20 μ l, ThermoScript II 2-5U, and 42 ℃ were extended 1 hour.95 ℃ of sex change 5 minutes are put in the ice bath, and product is cDNA first chain.With 2 couples of Auele Specific Primer PHs1, PHal and PLs1, PLa1, in 20 μ l PCR reaction systems, add reverse transcription product 2 μ l respectively, Taq enzyme 10 * buffer2 μ l, each 1 μ l of upstream and downstream primer, 2.5mmo1/L dNTP 1 μ l, add Taq enzyme 1-2U, deionized water is mended to 20 μ l.95 ℃ of sex change 2 minutes, loop parameter is: 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, totally 30 circulations are extended 10 minutes (Fig. 9) after 72 ℃.
3. with isolating the dna fragmentation that desire reclaims, downcut the blob of viscose that contains target DNA fragment under long wave ultraviolet light, put into centrifuge tube, add the long-pending change glue of three times of colloids, blob of viscose is dissolved in 55 ℃ of water-baths fully.With the dna fragmentation purification kit reclaim dna fragmentation and with the dna fragmentation of purifying in the aqueous solution, with the PCR product that reclaims in T4 dna ligase damping fluid by after 2: 1 the ratio (mol ratio) and carrier PGEMTeasy mixing, the T4 dna ligase that adds 0.5U spends the night in 16 ℃ of connections, and the cumulative volume of ligation is 10 μ L.
4. get and connect liquid 10 μ l, add among the 200 μ l competence bacterium JM109 and soft mixing ice bath 30 minutes, 42 ℃ of water-bath heat-shockeds 90 seconds, changed ice bath rapidly over to 2 minutes, add 800 μ l LB substratum, change 37 ℃ of constant temperature shaking tables over to, shook 45 minutes with 150 rev/mins speed, centrifugal 1 minute of 4000r/min, discard 800 μ l supernatants, get precipitation and coat the solid LB flat board that contains Amp (final concentration is 100 μ g/ml), flat board was inverted in 37 ℃ of incubators 12～18 hours.
5. the single clone of picking in above-mentioned flat board is inoculated in the LB substratum that contains acillin (100 μ g/ml).37 ℃ of constant temperature shaking table 170rpm, the concussion overnight incubation.Get 3ml bacterium liquid and add in the 1.5mlEppendorf pipe, the centrifugal 1min of 10000rpm abandons supernatant.To precipitate thalline and be resuspended in the 100 μ L solution I, and add freshly prepared solution II 200 μ L, light and slow ground is put upside down up and down for several times, to liquid become limpid till.Subsequently, add 150 μ l solution III again, gently turning upside down makes liquid mixing, this moment a large amount of white flockss occur for several times.4 ℃, the centrifugal 5min of 12000rpm gets supernatant and adds in another Eppendorf pipe, adds the saturated phenol of isopyknic Tris-HCl, and behind the concuss, the centrifugal 5min of 12000rpm moves to upper water in the one new pipe mutually.Add 500 μ L chloroforms again, extracting once again., carefully draw upper strata water, move in the new pipe, add the dehydrated alcohol mixing of 2 times of volumes, place 3h in-20 ℃ thereafter.4 ℃, the centrifugal 10min of 12000rpm abandons supernatant, washes precipitation 2 times with 70% ethanol, and drying at room temperature 20min with the dissolving of 40 μ L aseptic double-distilled waters, carries out PCR and identifies and the dna sequencing analysis.
Having made up and contained that Ricin neutralizing antibody 3E1 is light, the carrier of heavy chain gene, is that mouse immuning ball protein is light, heavy chain gene (sequence 1, sequence 2 in the sequence table) through sequencing analysis, sequence alignment.
Sequence table
＜110〉Institute of Basic Medical Sciences, Academy of Military Medical Sciences, PLA
＜120〉a kind of neutralizing monoclonal antibody 3E1 of antiricin, Preparation Method And The Use
<130>
<160>17
<170>PatentIn version 3.3
<210>1
<211>638
<212>DNA
<213>
<400>1
ccagatgacc cagtctccat cctcactgtc tgcatctctg ggaggcaaag tcaccatcac 60
ttgcaaggca agccaagaca ttaaaaagta tatagcttgg taccaacaca agcctggaaa 120
aggtcctagg ctgctcatac attacacatc tacattacag ccaggcatcc catcaaggtt 180
cagtggaagt gggtctggga gagattattc cttcagcatc agcaacctgg agcctgaaga 240
tattgcaact tattattgtc tacagtatga taatctgtac acgttcggag gggggaccaa 300
gctggaaata aaacgggctg atgctgcacc aactgtatcc atcttcccac catccagtga 360
gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg aacaacttct accccaaaga 420
catcaatgtc aagtggaaga ttgatggcag tgaacgacaa aatggcgtcc tgaacagttg 480
gactgatcag gacagcaaag acagcaccta cagcatgagc agcaccctca cgttgaccaa 540
ggacgagtat gaacgacata acagctatac ctgtgaggcc actcacaaga catcaacttc 600
acccattgtc aagagcttca acaggaatga gtgttaat 638
<210>2
<211>654
<212>DNA
<213>
<400>2
tctggagctg agctgatgaa gcctggggcc tcagtgaaaa tttcctgcaa ggctactggc 60
tacacattca gtagctactg gatagagtgg ataaagcaga ggcctggaca tggccttgag 120
tggattggag acattttacc tggaagtggt agtactaact acaatgagaa gttcaagggc 180
aaggccacat tcactgcaga tacatcctcc aacacagcct acatgcaact cagcagcctg 240
acatctgagg actctgccgt ctattactgt tcaagatctt tctactataa ttacgacggg 300
gcctactttg cttactgggg ccaagggact ctggtcactg tctctgcagc caaaacgaca 360
cccccatctg tctatccact ggcccctgga tctgctgccc aaactaactc catggtgacc 420
ctgggatgcc tggtcaaggg ctatttccct gagccagtga cagtgacctg gaactctgga 480
tccctgtcca gcggtgtgca caccttccca gctgtcctgc agtctgacct ctacactctg 540
agcagctcag tgactgtccc ctccagcacc tggcccagcg agaccgtcac ctgcaacgtt 600
gcccacccgg ccagcagcac caaggtggac aagaaaattg tgcccaggga ttgt 654
<210>3
<211>211
<212>PRT
<213>
<400>3
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Gly Lys
1 5 10 15
Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Lys Tyr Ile Ala
20 25 30
Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile His Tyr
35 40 45
Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly
50 55 60
Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp
65 70 75 80
Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr Phe Gly
85 90 95
Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val
100 105 110
Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser
115 120 125
Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys
130 135 140
Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp
145 150 155 160
Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu
165 170 175
Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu
180 185 190
Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg
195 200 205
Asn Glu Cys
210
<210>4
<211>218
<212>PRT
<213>
<400>4
Ser Gly Ala Glu Leu Met Lys Pro Gly Ala Ser Val Lys Ile Ser Cys
1 5 10 15
Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr Trp Ile Glu Trp Ile Lys
20 25 30
Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Asp Ile Leu Pro Gly
35 40 45
Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Phe
50 55 60
Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Met Gln Leu Ser Ser Leu
65 70 75 80
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ser Arg Ser Phe Tyr Tyr
85 90 95
Asn Tyr Asp Gly Ala Tyr Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala
115 120 125
Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu
130 135 140
Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly
145 150 155 160
Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp
165 170 175
Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro
180 185 190
Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys
195 200 205
Val Asp Lys Lys Ile Val Pro Arg Asp Cys
210 215
<210>5
<211>108
<212>PRT
<213>
<400>5
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Lys Lys Tyr
20 25 30
Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile
35 40 45
His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Tyr Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala
100 105
<210>6
<211>122
<212>PRT
<213>
<400>6
Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile
35 40 45
Gly Asp Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Ser Phe Tyr Tyr Asn Tyr Asp Gly Ala Tyr Phe Ala Tyr Trp
100 105 110
Gly Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210>7
<211>12
<212>PRT
<213>
<400>7
Lys Ala Ser Gln Asp Ile Lys Lys Tyr Ile Ala Trp
1 5 10
<210>8
<211>7
<212>PRT
<213>
<400>8
Tyr Thr Ser Thr Leu Gln Pro
1 5
<210>9
<211>8
<212>PRT
<213>
<400>9
Leu Gln Tyr Asp Asn Leu Tyr Thr
1 5
<210>10
<211>10
<212>PRT
<213>
<400>10
Gly Tyr Thr Phe Ser Ser Tyr Trp Ile Glu
1 5 10
<210>11
<211>17
<212>PRT
<213>
<400>11
Asp Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe Lys
1 5 10 15
Gly
<210>12
<211>13
<212>PRT
<213>
<400>12
Ser Phe Tyr Tyr Asn Tyr Asp Gly Ala Tyr Phe Ala Tyr
1 5 10
<210>13
<211>22
<212>DNA
<213>
<400>13
aggtccagct tctcgagtca gg 22
<210>14
<211>22
<212>DNA
<213>
<400>14
aggtccaact gctcgagtct gg 22
<210>15
<211>32
<212>DNA
<213>
<400>15
ccagttccga gctccagatg acccagtctc ca 32
<210>16
<211>34
<212>DNA
<213>
<400>16
gcgccgtcta gaattaacac tcattcctgt tgaa 34
<210>17
<211>30
<212>DNA
<213>
<400>17
aggcttacta gtacaatccc tgggcacaat 30
Claims (7)
1. an antiricin neutralizing monoclonal antibody 3E1 is characterized in that light, heavy chain protein matter molecule has the aminoacid sequence shown in sequence in the sequence table 3, sequence 4 respectively.
2. light, the heavy chain protein matter molecule encoding gene of the described monoclonal antibody 3E1 of claim 1 is characterized in that having respectively the nucleotide sequence shown in sequence in the sequence table 1, sequence 2.
3. the variable region in light, the heavy chain protein matter molecule of the described monoclonal antibody 3E1 of claim 1 is characterized in that having respectively the aminoacid sequence shown in sequence in the sequence table 5, sequence 6.
4. complementary determining region CDR1, CDR2, the CDR3 of the light chain protein matter molecule variable region of the described monoclonal antibody 3E1 of claim 3 is characterized in that having respectively the aminoacid sequence shown in sequence in the sequence table 7, sequence 8, sequence 9.
5. complementary determining region CDR1, CDR2, the CDR3 of the heavy chain protein matter molecule variable region of the described monoclonal antibody 3E1 of claim 3 is characterized in that having respectively the aminoacid sequence shown in sequence in the sequence table 10, sequence 11, sequence 12.
6. the preparation method of the described antiricin neutralizing monoclonal antibody of claim 1 3E1 comprises the steps:
(1) structure of antiricin monoclonal antibody hybridoma cell strain;
(2) screening of antiricin monoclonal antibody hybridoma cell strain and evaluation;
(3) monoclonal antibody 3E1 is to the provide protection of mouse;
(4) light, the angling of heavy chain gene of hybridoma 3E1 got;
(5) light, heavy chain variable region gene sequence of monoclonal antibody 3E1 and aminoacid sequence determines.
7. the described antiricin neutralizing monoclonal antibody of claim 1 3E1, diagnosis of poisoning at the detection of preparation Ricin, vaccine and Ricin and immunotoxin or the application in the medicine.
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| CN2005101237941A CN1970573B (en) | 2005-11-24 | 2005-11-24 | Neutrality monoclonal antibody 3E1 for resisting ricin, its preparation method and uses |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463087B (en) * | 2009-01-13 | 2011-05-18 | 中国人民解放军海军总医院 | Anti-ricin antibody |
| CN103214554A (en) * | 2012-01-20 | 2013-07-24 | 中国人民解放军海军总医院 | Mucosal immunoadjuvant inducing Th1 immune response, and application thereof |
| CN103589687A (en) * | 2012-12-03 | 2014-02-19 | 中国人民解放军海军总医院 | Tetrodo toxin neutralizing monoclonal antibody 5E7 hybridomas, genes of light-chain and heavy-chain variable regions thereof and applications |
| CN108484760A (en) * | 2018-05-04 | 2018-09-04 | 中国人民解放军第三0二医院 | A kind of antiricin immunoglobulin F（ab’）2And preparation method thereof |
| CN109852673A (en) * | 2019-01-17 | 2019-06-07 | 北京市疾病预防控制中心 | A kind of gold/quantum dot nano probe and its application for detecting active ricin (WA) in complex matrices |
| CN114560933A (en) * | 2022-01-25 | 2022-05-31 | 北京弘进久安生物科技有限公司 | Monoclonal antibody specifically binding to ricin and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5626844A (en) * | 1991-11-04 | 1997-05-06 | The United States Of America As Represented By The Secretary Of The Army | Monoclonal antibody against ricin A chain |
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2005
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| CN101463087B (en) * | 2009-01-13 | 2011-05-18 | 中国人民解放军海军总医院 | Anti-ricin antibody |
| CN103214554A (en) * | 2012-01-20 | 2013-07-24 | 中国人民解放军海军总医院 | Mucosal immunoadjuvant inducing Th1 immune response, and application thereof |
| CN103214554B (en) * | 2012-01-20 | 2014-10-15 | 中国人民解放军海军总医院 | Mucosal immunoadjuvant inducing Th1 immune response, and application thereof |
| CN103589687A (en) * | 2012-12-03 | 2014-02-19 | 中国人民解放军海军总医院 | Tetrodo toxin neutralizing monoclonal antibody 5E7 hybridomas, genes of light-chain and heavy-chain variable regions thereof and applications |
| CN103589687B (en) * | 2012-12-03 | 2016-01-06 | 中国人民解放军海军总医院 | Tetraodotoxin neutralizing monoclonal antibodies hybridoma 5E7 and light, heavy chain variable region gene thereof and application |
| CN108484760A (en) * | 2018-05-04 | 2018-09-04 | 中国人民解放军第三0二医院 | A kind of antiricin immunoglobulin F（ab’）2And preparation method thereof |
| CN109852673A (en) * | 2019-01-17 | 2019-06-07 | 北京市疾病预防控制中心 | A kind of gold/quantum dot nano probe and its application for detecting active ricin (WA) in complex matrices |
| WO2020147735A1 (en) * | 2019-01-17 | 2020-07-23 | 北京市疾病预防控制中心 | Gold/quantum dot nanoprobe for detecting active ricin in complex matrix and use thereof |
| US11391711B2 (en) | 2019-01-17 | 2022-07-19 | Beijing Center For Disease Prevention And Control | Gold/quantum dot nanoprobe for detecting active ricin in complex matrix and application thereof |
| CN114560933A (en) * | 2022-01-25 | 2022-05-31 | 北京弘进久安生物科技有限公司 | Monoclonal antibody specifically binding to ricin and application thereof |
| CN114560933B (en) * | 2022-01-25 | 2023-01-31 | 北京弘进久安生物科技有限公司 | Monoclonal antibody specifically binding to ricin and application thereof |
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| Publication number | Publication date |
|---|---|
| CN1970573B (en) | 2011-02-02 |
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