CN1911963A - SARS neutralization antibody and application - Google Patents
SARS neutralization antibody and application Download PDFInfo
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- CN1911963A CN1911963A CN 200510028637 CN200510028637A CN1911963A CN 1911963 A CN1911963 A CN 1911963A CN 200510028637 CN200510028637 CN 200510028637 CN 200510028637 A CN200510028637 A CN 200510028637A CN 1911963 A CN1911963 A CN 1911963A
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Abstract
The present invention discloses one kind of high affinity SARS neutralizing monoclonal antibody and its preparation process. The present invention also provides mouse hybridoma cell lines N-176-15 CCTCC-C200507 and S-9-11CCTCC-C200515 producing the said SARS neutrality antibody. The SARS neutrality neutralizing antibody of the present invention has excellent SARS virus neutralizing effect.
Description
Technical field
The present invention relates to a kind of atypical pneumonia virus (SARS-CoV) neutrality antibody, especially SARS neutralizing monoclonal antibody and preparation method thereof.
Background technology
Severe acute respiratory syndrome (SARS) epidemic situation that autumn in 2002 was economized outburst in Chinese Guangdong is once serious public health crisis.In a few short months in the time, the SARS rapid spread is to surpassing 25 countries and regions, brought for people's healthy and orthobiosis and seriously influences, and caused massive losses for Chinese economic construction and development.Spring in 2004,, cause people's very big concern because laboratory scientific research personnel's misoperation causes the atypical pneumonia Anhui in Beijing once more two places personnel that cause to infect.Up to the present, SARS causes altogether about 8000 people in the whole world and infects, dead people more than 800, and mortality ratio reaches about 10%.
Known at present, atypical pneumonia is caused by a kind of newfound coronavirus (SARS-CoV).Coronavirus is to gain the name because of its shape such as crown under electron microscope.This unique outward appearance is that the bar-shaped membranin by its film surface is formed.SARS virus promptly is the novel coronavirus of a class, mainly by forming with the lower section: lipid bilayer and on bar-shaped membranin S, envelope protein E, membrane glycoprotein M and the inner capsid protein N of film and by the geneome RNA of N parcel.Wherein the proteic main effect of S is to combine with the host cell membrane surface receptor, thereby the mediation virus particle is invaded cell, has identified that the functional receptor that has obtained on the SARS virus host cell is an angiotensin conversion enzyme 2 (ACE2) at present.
From the infection immunity angle, utilize the neutrality antibody of virus is applied the method that treatment is the most effective at present known treatment SARS to the patient of acute HIV infection.Its mechanism is to utilize the intrusion of neutrality antibody blocking virus to normal host cell, or blocks the biological function of its key protein, thereby reaches the purpose that treatment is infected.When SARS is popular, the methods of treatment that contains the convalescence patients serum of a large amount of neutrality antibodies by injection was once arranged, make patient's state of an illness alleviate and cure the huge effect that has proved that the anti-SARS of neutrality antibody infects rapidly.But the patients serum originates limited, and has problems such as security.So the neutrality antibody medicine at SARS just becomes the focus that countries in the world are paid close attention to.
At present known directly and the section of host cell receptor contact on S Protein S 1 subprovince between about 318-510 amino acid, be named as receptor binding domain (Receptor Binding Domain) and contain important viral neutralizing epitope.Preparation is at the monoclonal antibody of this section, and possible blocking virus particle and cells contacting reach anti-infectious purpose.
Though can directly adopt the immunity of RBD peptide section, but the antibody that produces may be because the difference that albumen total length and partial peptide section structurally exist makes antibody produce defective discerning on the complete natural SARS virus S albumen, though perhaps may obtain some can identification receptor land RBD but do not have the monoclonal antibody of keying action with SARS S albumen total length intact proteins, therefore the efficient that not only obtains potent antibodies is low, and is difficult to obtain active very high neutrality antibody.
In sum, this area still obtains active very high, specificity and is incorporated into the proteic antibody of SARS virus S.Therefore this area presses for SARS neutrality antibody, the especially monoclonal antibody that exploitation has the high-affinity of the viral effect of good neutralization.
Summary of the invention
The neutrality antibody that the purpose of this invention is to provide a kind of high-affinity at atypical pneumonia (SARS), and preparation method thereof.
In a first aspect of the present invention, a kind of immunoglobulin (Ig) is provided, it has following characteristic:
(a) be incorporated into the receptor binding domain of SARS virus specifically, described receptor binding domain is the proteic 318-510 of SARS virus S position;
(b) specifically in and SARS virus;
(c) to tire be 1 for described antibody and receptor binding domain bonded nude mice ascites: 50000-1: 1000000 (preferably be 1: 100000-1: 800000, more preferably be 1: 200000-1: 600000).
(d) to tire be 1 for described antibody and SARS virus neutral nude mice ascites: 4000-1: 10000 (preferably 1: 5000-1: 8000).
In another preference, described immunoglobulin (Ig) at receptor binding domain have the aminoacid sequence shown in the SEQ ID NO:1.
In another preference, described immunoglobulin (Ig) is a monoclonal antibody.
In another preference, described immunoglobulin (Ig) is produced by the cell that is selected from down group: mouse hybridoma cell is N-176-15, CCTCC No.:C200507; Or mouse hybridoma cell is S-9-11 CCTCC No.:C200515.
In a second aspect of the present invention, a kind of immune conjugate is provided, and this immune conjugate contains the above-mentioned immunoglobulin (Ig) of the present invention and is incorporated into the coupling part of organizing under being selected from of described immunoglobulin (Ig) specifically: medicine, toxin, cytokine, radionuclide, enzyme.
In a third aspect of the present invention, a kind of hybridoma that produces monoclonal antibody is provided, it is selected from down group: mouse hybridoma cell is N-176-15, CCTCC No.:C200507; Or mouse hybridoma cell is S-9-11 CCTCC No.:C200515.
In a fourth aspect of the present invention, a kind of composition is provided, it contains above-mentioned immunoglobulin (Ig) of the present invention or above-mentioned immune conjugate and acceptable carrier.Usually, the content of immunoglobulin (Ig) or above-mentioned immune conjugate is the 0.001-99.99wt% of composition total weight, preferably is 0.01-90wt%, more preferably is 0.1-80wt%.
In another preference, described composition is a pharmaceutical composition.
In a fifth aspect of the present invention, provide the purposes of immunoglobulin (Ig) of the present invention, during they are used to prepare and the composition of SARS virus.
In a fifth aspect of the present invention, provide preparation monoclonal antibody method of the present invention, it comprises step: (1) uses the reorganization pseudovirus vaccine immune mouse of atypical pneumonia virus (SARS-CoV); (2) behind the splenocyte and rat bone marrow tumour cell fusion with immune mouse, (3) hybridoma of atypical pneumonia (SARS) the virus receptor land bonded monoclonal antibody of screening secretion and S protein 31 8-510 amino acid section, build strain and obtain monoclonal antibody (4) utilization cytogamy and viral neutralized system, screening obtains having the monoclonal antibody of the virus neutralization of high-affinity from these monoclonal antibodies.
In another preference, described preparation monoclonal antibody method comprises step:
(a) the reorganization pseudovirus vaccine immune mouse of usefulness atypical pneumonia virus;
(b) behind the splenocyte and rat bone marrow tumour cell fusion with immune mouse, obtain hybridoma:
(c) from the hybridoma of step (b), select the hybridoma of the atypical pneumonia virus receptor binding domain bonded monoclonal antibody of secretion and S protein 31 8-510 amino acid section, obtain to combine hybridoma with receptor binding domain;
(d) from the hybridoma of step (c), selecting that the monoclonal antibody of generation and SARS virus bonded nude mice ascites tires is 1: 50000-1: 1000000 hybridoma;
(e) from the hybridoma of step (d), make monoclonal antibody.
In another preference, described hybridoma cell line is that mouse hybridoma cell is N-176-15, and its hypotype is IgG1, k type.
In another preference, described hybridoma cell line is that mouse hybridoma cell is S-9-11, and its hypotype is IgG2a, k type.
Description of drawings
Fig. 1 is the synoptic diagram of cytogamy blocking experiment.
Fig. 2 is the synoptic diagram of reporter gene system.
Fig. 3 has shown that antibody that the S-9-11 cell strain produces can block the fusion of SARS virus and cell.
Fig. 4 has shown that antibody that the N-176-15 cell strain produces can block the fusion of SARS virus and cell.
Embodiment
The inventor is through extensive and deep research, the proteic reorganization pseudovirus of discovery SARS virus S vaccine can be induced the neutrality antibody that obtains high titre, therefore be particularly suitable as the original preparation of immunity SARS neutralizing monoclonal antibody, find that simultaneously the proteic receptor binding domain of S (RBD) section has important neutrality site.On the above-mentioned research basis, the inventor utilizes hybridoma technology to prepare the monoclonal antibody of the high anti-people SARS of avidity, thereby has finished the present invention.
As used herein, term " SARS receptor binding domain (RBD) " or " RBD " are used interchangeably, and all refer to contain SARS virus S albumen 318-510 amino acids polypeptide of sequence.A kind of preferred SARS receptor binding domain (RBD) is made of SARS virus S albumen 318-510 amino acids sequence.More preferably, described RBD has the amino acid shown in SEQ ID NO:1 or the gi:30173397.
SARS neutrality monoclonal antibody of the present invention or its fragment can be used for the immunodetection SARS virus, in also can be used for and SARS virus or be used for the treatment of SARS and infect, thereby for example make it can block the fusion of SARS virus and cell by directly or indirectly SARS neutrality monoclonal antibody or its fragment being linked to each other with chemotherapeutic or radiotherapeutic agents.
The present invention includes: have the corresponding aminoacid sequence of SARS neutrality monoclonal antibody monoclonal antibody, have the monoclonal antibody of SARS neutrality monoclonal antibody variable region chain, and other protein or protein conjugate and fusion expressed product with these chains.Particularly, the present invention includes and have the hypervariable region of containing (complementary determining region, any protein of light chain CDR) and heavy chain or protein conjugate and fusion expressed product (being immune conjugate and fusion expressed product), as long as identical or at least 90% homology of hypervariable region of this hypervariable region and light chain of the present invention and heavy chain, preferably at least 95% homology.
As is known to the person skilled in the art, immune conjugate and fusion expressed product comprise: medicine, toxin, cytokine (cytokine), radionuclide, enzyme and other diagnosis or treatment molecule and SARS neutrality monoclonal antibody or its fragment bonded and the conjugate that forms.The present invention also comprises and SARS neutrality monoclonal antibody or its fragment bonded cell surface marker thing or antigen.
As used herein, " immunotoxin " refers to target cell is had the material of specificity avidity and lethality, and for example immune conjugate and fusion expressed product comprise: medicine, toxin, cytokine, radionuclide or other treatment molecule and SARS neutrality monoclonal antibody or its fragment bonded and the conjugate that forms.Concrete example for example has
131I-SARS neutrality antibody conjugate etc.
For SARS neutrality monoclonal antibody heavy chain of the present invention and sequence of light chain, can measure with ordinary method.The hypervariable region of SARS neutrality monoclonal antibody V chain or complementary determining region (complementarity determining region, CDR) interesting especially, because relate to conjugated antigen to small part in them.Therefore, the present invention includes those the light chain immunoglobulins and the molecule of weight chain variable chain, as long as its CDR and SARS neutrality monoclonal antibody CDR have the homology of (preferably more than 95%) more than 90% with band CDR.
The present invention not only comprises complete monoclonal antibody, also comprises having immunocompetent antibody fragment, as Fab or (Fab ')
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody, as have the murine antibody binding specificity but keep antibody from people's antibody moiety.
The present invention also provides the DNA of coding RBD.The antigenic Nucleotide full length sequence of SARS neutrality monoclonal antibody of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be according to the nucleotide sequence of RBD, especially open reading frame sequence designs primer, prepares template by ordinary method well known by persons skilled in the art, obtains relevant sequence by amplification.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
The present invention also provides above-mentioned immunoglobulin (Ig) or its segmental dna molecular.The sequence of these dna moleculars can as above be used routine techniques, and utilizing mouse hybridoma cell is that SARS neutrality (CCTCC No.:C200507 or 200515) obtains.In addition, also the encoding sequence of light chain and heavy chain can be merged, form single-chain antibody.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The invention still further relates to the carrier that comprises above-mentioned suitable dna sequence dna and suitable promotor or control sequence.These carriers can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS7,293 cells or Bowes melanoma cells etc.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, handle the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, supersound process, super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods with protein precipitant.
In addition, the present invention also provides a kind of test kit that detects SARS virus, and it contains above-mentioned immunoglobulin (Ig) or immune conjugate, or its active fragments.
The present invention also provides a kind of pharmaceutical composition, and it contains above-mentioned immunoglobulin (Ig) or immune conjugate, and pharmaceutically acceptable carrier.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intraperitoneal, intravenously or topical.
During pharmaceutical composition of the present invention can be directly used in and SARS virus (in particular for the fusion of blocking-up SARS virus and cell), also can be used for the treatment of SARS virus.In addition, also can use the other treatment agent simultaneously, as antibody and Interferon, rabbit, interleukin-etc.
Pharmaceutical composition of the present invention contains above-mentioned immunoglobulin (Ig) of the present invention of safe and effective amount or above-mentioned immune conjugate and pharmaceutically acceptable carrier or vehicle.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as injection, solution should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that SARS neutrality immune conjugate with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
In an example of the present invention, atypical pneumonia (SARS) S albumen reorganization pseudovirus vaccine immune mouse with artificial preparation, behind the splenocyte and rat bone marrow tumour cell fusion with immune mouse, screening has obtained atypical pneumonia (SARS) the virus receptor land bonded hybridoma with S protein 31 8-510 amino acid section then.From described and the some strain of hybridoma of RBD bonded, identify the monoclonal antibody that obtains having neutralizing effect.
The preferred hybridoma cell line of two strains is difference called after: S-9-11 and N-176-15; Wherein S-9-11 is that splenocyte and SP2/0 myeloma cell's fusion form, and N-176-15 is that splenocyte and the fusion of NS-1 myeloma cell strain form.Identify that through hypotype S-9-11 is IgG2a, k type, N-176-15 is IgG1, k type.Cytogamy and virus neutralization detect proof, and above-mentioned two strain mouse hybridoma cell strains have good virus neutralization.
Major advantage of the present invention is:
(a) reorganization pseudovirus vaccine can be induced the neutrality antibody at RBD of high titre, is suitable as very much the immunogen of preparation SARS neutrality antibody.
(b) neutrality antibody of the present invention has extremely excellent neutralization activity to SARS virus, and it is 1 that nude mice ascites is tired: 50000-1: 1000000 or higher.
(c) compare with the SARS virus receptor binding domain peptide section RBD immunity of adopting and with the method for its screening monoclonal antibody, the efficient height that method of the present invention has, the monoclonal antibody that promptly screens as long as and RBD in conjunction with the SARS S albumen that just must discern under the complete native state, and in the vigor height.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: animal immune
Antigen is SARS pseudovirus vaccine, for Ankara virus of attenuation obtains in its genome by reorganization SARS S full-length proteins gene.This viromembrane surface expression SARS virus S albumen, concentration: 2 * 10
9Pfu/ml, Balb/c mouse immune dosage: 5 * 10
7Every of pfu is each.The muscle multi-point injection.Dilute with PBS or physiological saline during use.Immune programme for children: time immunity 0,3,6 Wednesdays.Merge first three sky and get 5 * 10
7Pfu vaccinogen liquid PBS is diluted to the 0.5ml abdominal injection, and doing to recall stimulates.
The brief introduction of SARS reorganization pseudovirus vaccine: vector virus is improved attenuation alive Ankara vaccine virus (MVA), the preparation method is as follows: use ordinary method, SARS virus S albumen full-length gene is inserted the genomic III number deletion district (Deletion III Region) of this virus and becomes improved SARS vaccine, and improved SARS reorganization pseudovirus vaccine is named as ADS-MVA.This vaccine antigen can be induced the neutrality antibody of high titre in animal bodies such as mouse, rabbit, monkey.And show after deliberation neutralizing antibody at epi-position have and be positioned at greatly the RBD zone.
Embodiment 2: the structure of hybridoma cell strain and MONOCLONAL ANTIBODIES SPECIFIC FOR
Recall to stimulate and did fusion in back three days.Get the spleen cell of immunity back mouse and the fusion method of rat bone marrow tumour cell NS-1 and SP2/0 employing polyoxyethylene glycol (PEG) routine and do fusion.Add the HAT selective medium after the fusion, non-hybridoma is all killed.The concrete steps aftermentioned.
Then, carry out the screening operation of hybridoma.ELISA method (concrete steps aftermentioned) is adopted in screening, and used screening antigen is the SARS virus receptor binding domain that is positioned at S protein 31 8-510 amino acid section (Receptor Binding Domain) (connecing human IgG constant region fc section as the albumen label) of eukaryotic expression.Cell conditioned medium in the hybridoma sample well that grows is taken out, do the ELISA experiment, see whether contain and RBD bonded antibody as sample.If have, then further do hybridoma cloning.
The cloning process adopts limiting dilution assay, concrete experimental procedure aftermentioned.
After three successive ELISA detections and cloning, obtain some strains and RBD antigen bonded monoclonal antibody.Further do analytical test.Investigate from avidity and functional experiment aspect, screening at last obtain two strain antibodies be found to be avidity high, in and experiment effect very good, called after: S-9-11 and N-176-15 respectively.Wherein S-9-11 is that splenocyte and SP2/0 myeloma cell's fusion form, and N-176-15 is that splenocyte and the fusion of NS-1 myeloma cell strain form.Identify that through hypotype S-9-11 is IgG2a, k type, N-176-15 is IgG1, k type.Two strain antibodies have prepared ascites respectively according to a conventional method.Ascites is tired after testing, and about 1: 256000-1: 512000.
Experimental technique
(a) cytogamy:
1. the preceding preparation of experiment is carried out in aseptic technique;
2. eyeball of mouse is got blood.Bubble is gone in the alcohol cylinder.Be fixed in and dissect on the plate.Cut off the mouse crust with cover scissors tweezers and upwards tear and expose chest, abdominal cavity, cut off peritonaeum with another set of scissors tweezers, and the taking-up spleen.Place in 6 orifice plates that fill training liquid in advance;
3. with the nylon wire on the RDF training liquid band nylon wire beaker, shred, push with the tweezers scissors spleen is placed on it, cell filtration is gone in the beaker under the nylon wire;
4. splenocyte in the beaker is placed in the 50ml centrifuge tube centrifugal 1500rpm, 3min;
5. wash with serum-free medium, counting divides 5 * 10 again
7Some parts of cell, standby;
6. collect NS-1 and SP2/0 cell, centrifugal 1500rpm, 3min;
7.RDF nutrient solution is washed, counting is got the myeloma cell, every kind 10
7
8. a kind of myeloma cell splenocyte is a and that get mixes, and is centrifugal, 2000rpm, and 5min, loss of gloss training liquid blots residual liquid in the centrifuge tube with aseptic filter paper, bullet pine cell lump;
9. add PEG0.8-1ml, need to add within the 1min, the limit edged rocks;
10. static 1.5min;
11. add the ready RDF training of 10ml liquid, add in the 2.5min, the limit edged rocks;
12. static 5min;
13. centrifugal, 1000rpm, 3min;
14. with dropper with the supernatant liquor sucking-off;
15. add 25ml, the nutrient solution of 12%BS is inhaled gently and is beaten mixing;
16. carry out in 96 orifice plates of feeder layer the 50ul/ hole with volley of rifle fire adding.
(b)ELISA：
1. antigen coated: get antigen and be mixed with the coating buffer of 0.05mol/L concentration with carbonate buffer solution (pH9.6), mixing adds plastics 96 hole check-out consoles, and the 50ul/ hole places in 4 ℃ of refrigerators and spends the night;
2.Block: wash plate, add Block liquid (diluent is the 10% new-born calf serum+0.1%Tween20 or 3% gelatin of PBS preparation), 100ul/ hole, 37 ℃ of incubator incubations 2 hours;
3. add one anti-: wash plate, anti-(being the hybridoma supernatant) solution of handling well is added in the hole, 50ul/ hole, 37 ℃ of incubator incubations 2 hours;
4. add two anti-: wash plate, (antibody of goat anti-mouse igg (H+L) HRP enzyme labelling Calbiochem-Novabiochem) adds in the hand-hole good two anti-of dilution, 50ul/ hole, 37 ℃ of incubations 1 hour;
5. colour developing: wash plate, prepare tmb substrate, add in the hand-hole, the 50ul/ hole, colour developing 5-10min adds 2M sulfuric acid, 50ul/ hole, termination reaction;
6. detect: the OD (450nm) that detects hole solution with microplate reader (Thermo) is worth, and obtains data.
(c) limiting dilution assay experimental procedure:
1. carefully draw piping and druming positive hole to cell with suction pipe and sneak into nutrient solution fully;
2. add 1 to a glass centrifuge tube, add 9 HT nutrient solutions, mixing, all the other are added in the front-seat several holes of 96 orifice plates (carrying out feeder layer);
3. dilution for the first time: with the nutrient solution sucking-off of mixing, add to second and be with in the graduated 10ml glass centrifuge tube, add 9 training liquid, mixing;
4. dilution for the second time: all the other add front-seat a few hole, and sucking-off training liquid adds 2 again in this centrifuge tube, add HT training liquid to 10ml, add behind the mixing in 96 orifice plates, and the 50ul/ hole, 96 orifice plates place in the incubator.
Embodiment 3: the cytogamy blocking experiment
Present embodiment is used for the film fusion process of simulated virus and cell with the cytogamy blocking experiment, thereby detects the neutrality effect of antibody.Transfection and the membranin Spike and the corresponding host cell membrane receptor ACE2 thereof that express SARS virus realize receptors ligand cell fusion mediated process respectively by pair cell in this system, and represent its syncretizing effect by the reporter gene systematic quantification that cotransfection just take place to be expressed after cytogamy, i.e. what of the cell that merges take place in expression.Add antibody or polypeptide in this system, if its pair cell merges blocking effect is arranged, then the big I of this blocking effect is represented (as Fig. 1) by the decline degree of reporter gene expression amount.PSpike, pACE2 are respectively the expression plasmid that contains S albumen and ACE2 protein gene total length, and p13-luc and p15-1 are for forming two kinds of plasmids of reporter gene system, and rellina is a kind of confidential reference items plasmid of expressing the sea urchin luciferase.
The synoptic diagram of cytogamy blocking experiment as shown in Figure 1.Transfected cell is a HEK293T clone.Be divided into two groups of equivalent, one group of cotransfection S albumen (Spike), p13-luc and rellina confidential reference items plasmid; Another group cotransfection ACE2 and p15-1 plasmid (plasmid increases by Shanghai biochemical cell research teacher Zhu Xueliang of institute favour).Wherein Spike and ACE2 mediated cell film merge, and p15-1 and p13-luc are the reporter gene system, express reporter gene luciferase luciferase.The Rellina plasmid is confidential reference items.
What the reporter gene system adopted is the tsiklomitsin adjusting reporter gene system (Tet-Off system) of BD Bioscience company.Expression mechanism is: p15-1 is for regulating plasmid, and background is expressed the albumen tTA of a kind of regulating effect under the effect of pCMV promotor, and this albumen is formed by two kinds of protein protomer TetR and VP16 amalgamation and expression.It is attached to induces the downstream promotor to start to express luciferase albumen (as Fig. 2) on tsiklomitsin (Tet) operon of target plasmid p13-luc.Detect the two channels Luciferase detection system that adopts Promega company.
Experiment flow is:
(1) by two groups of 293T cells of experimental design transfection, 37 ℃ of cultivations;
Receive cell after (2) 24 hours, counting is regulated density respectively.The Spike group adds ascites 10ul, 20ul, 40ul/0.5ml system, hatches on ice 30 minutes.Two groups of cell balanced mix, 37 ℃ of cultivations;
After (3) 24 hours, press to handle shown in the explanation of the two channels Luciferase of Promega company detection system and detect Luciferase.
2, experimental result
S-9-11 cell strain ascites cells merges blocking experiment:
Contrast ascites: the ascites of anti-mouse T4 antigen cell strain, its concentration is identical with S-9-11 cell strain ascites concentration after testing.
+ group: no antibody blocking-up group
-group: do not change ACE2 acceptor group
Serum: antigen immune mouse polyclonal antiserum
The result of S-9-11 cell strain ascites cells fusion blocking experiment as shown in Figure 3.Ascites pair cell to be detected as can be seen merges tangible blocking effect, and it act as dose dependent, can judge that S-9-11 cell strain ascites has barrier effect to this film fusion process.
N-176-15 cell strain ascites cells merges blocking experiment:
Contrast ascites: the ascites of anti-mouse T4 antigen cell strain, its concentration is identical with N-176-15 cell strain ascites concentration after testing.
+ group: no antibody blocking-up group
-group: do not change ACE2 acceptor group
Serum: antigen immune mouse polyclonal antiserum.
N-176-15 cell strain ascites cells merges the blocking experiment result as shown in Figure 4.N-176-15 cell strain ascites cell membrane fusion process also has blocking effect preferably as can be seen.And the antibody of S-9-11 and N-176-15 two cell strains has synergy.
Embodiment 4: virus neutralization experiment
Virus neutralization experiment:
1, SARS virus strain GZ50 obtains by separating in the infected patient's sample in the SARS epidemic situation in 2003 of Guangzhou.Sequence number on the GenBank is AY304495.Antibody ascites is done the twice dilution (1: 80 to 1: 10,240) of series.Respectively with 100TCID
50The SARS virus (GZ50) of (viral 50 3nfective dose) was mixed, 37 ℃ of incubations 1 hour.Then it is added to respectively in the FRhK-4 cell training liquid.Training liquid is done serial dilution and and viral the mixing as positive control.Each extent of dilution is done 10 multiple holes.The poroid attitude of observation sample every day, cytopathic effect (CPE) node reads after adding viral 3 days.The TCID of each sample well
50Calculate by lining De-Ming Qi (Reed-Muench) method.The titre of neutrality antibody is by suppressing the greatest dilution decision (having 5 holes at least in 10 multiple holes) that virus causes the odd contradictive hydroperitoneum of CPE.
The result is as follows:
| Extent of dilution | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| 80 | - | - | - | - | - | - | - | - | - | - | ||
| 160 | - | - | - | - | - | - | - | - | - | - | ||
| 320 | - | - | - | - | - | - | - | - | - | - | ||
| 640 | - | - | - | - | - | - | - | - | - | - | ||
| 1280 | - | - | - | - | - | - | - | - | - | - | ||
| 2560 | - | - | - | - | - | - | - | - | - | - | ||
| 5120 | - | - | - | - | - | - | - | - | - | - | ||
| 10240 | + | + | - | + | + | + | - | - | + | + |
By in the table as seen, antibody ascites sample 1: 5120 when dilution each sample well do not have the CPE effect, and by 7 holes the CPE effect appears when diluting at 1: 10240, the antiviral greatest dilution of interpret sample ascites is about 1: 5120.
After testing, S-9-11 clone's ascites has tangible viral neutralization.Its dilution is tired and can be reached 1: 5000.In addition, tire can be greater than 1: 5000 for N-176-15 dilution.The invasion of blocking virus pair cell very effectively of these antibody is described, plays a protective role.
Embodiment 5: separation and purification antibody
The method of purifies and separates antibody: the saturated ammonium sulphate method can obtain antibody purified (IgG) in conjunction with Protein G affinity chromatography from ascites or cell conditioned medium, method is as follows:
Concrete steps:
1. saturated ammonium sulphate: 1 volume ascites or cell conditioned medium sample add 1 volume PBS or normal saline buffer solution, dropwise add the saturated ammonium sulphate solution of 2 volumes under the situation of vibration, centrifugal must the precipitation, and ion is removed in dialysis;
2.Protein G affinity chromatography:
PBS with 5 times of column volumes washes post;
(2) add the sample of having dialysed;
(3) wash post with 5 times of column volume PBS;
(4) with 1-3 times of column volume glycine wash-out antibody;
(5) with Tris renaturation antibody-solutions;
(6) 20% ethanol regeneration Protein G post;
(7) detect antibody concentration, preserve.
Protein G chromatography column: Hi-Trap Protein G Column (Pharmacia Biotech#17-0404-01)
Required reagent is:
PBS damping fluid-1.084g NaH
2PO
4, 3.273g Na
2HPO
4.7H
2O, ddH
2O is settled to 1 liter of 0.1M glycine, pH 2.8-3.75g Glycine 1.4ml HCl, ddH
2O is settled to 1 liter of 1M Tris-141.1g Tris base ddH
2O.Be settled to 1 liter
20% ethanol
The result:
Obtained purity greater than 95% monoclonal antibody S-9-11 and N-176-15.
Embodiment 6: the composition that contains the SARS neutralizing antibody: SARS treats injection
Preparation before the experiment:
1. the processing of empty ampoule: ampoule should be earlier through experiments such as visual inspection, degree of cleaning experiment, resistance toheat experiment, neutral experiment, alkali-resistants before saw kerf.Eligible can cut, round mouth, and washing then is in 100 above dry for standby;
2. the processing of experiment apparatus: before the allotment utensil uses, scrub with soap, washing powder etc. earlier, glass wares can be handled with washing lotion, washes with tap water then, uses distilled water flushing again, drains, and faces to swing with preceding fresh water for injection and washes.New rubber, soft rubber ball boiled 30 minutes with 0.5-1% sodium hydroxide earlier, and then boiled 30 minutes with 1% hydrochloric acid, washing, and adding distil water boiled 30 minutes, with the distillation washing, can use with the water for injection flushing at last again.
Prescription: SARS neutralizing antibody immunoglobulin (Ig) (normal saline solution) 50g of axenic purification, add stroke-physiological saline solution and be settled to 100ml, regulating the pH value with HCl is 7.Be filtered to clear and brightly with No. 3 remelting glass funnels, be poured into the 2ml peace and cut open, sealing by fusing, promptly.
Finished product is done clarity, thermal source, content of drug, is contained inspections such as bacterium, and products obtained therefrom should meet that " Chinese pharmacopoeia and the countries concerned's standard are to the requirement of injection medicine.
Culture presevation
Mouse hybridoma cell of the present invention is that N-176-15 and mouse hybridoma cell are S-9-11, be preserved in Chinese typical culture collection center (CCTCC on June 29th, 2005, China, the Wuhan City), preserving number is Chinese typical culture collection center (CCTCC, China, the Wuhan City) CCTCC No.:C200507 and C200515.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉SARS neutrality antibody and application
<130>055503
<160>1
<170>PatentIn version 3.1
<210>1
<211>193
<212>PRT
＜213〉sars coronavirus (SARS coronavirus)
<400>1
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys
1 5 10 15
Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys Val
20 25 30
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys
35 40 45
Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser Asn
50 55 60
Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln Ile
65 70 75 80
Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
85 90 95
Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile Asp
100 105 110
Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His
115 120 125
Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe Ser
130 135 140
Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp Pro
145 150 155 160
Leu Asn Asp Tyr Gly Phe Tyr Thr Thr Thr Gly Ile Gly Tyr Gln Pro
165 170 175
Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr
180 185 190
Val
Claims (10)
1. an immunoglobulin (Ig) is characterized in that, it has following characteristic:
(a) be incorporated into the receptor binding domain of SARS virus specifically, described receptor binding domain is the proteic 318-510 of SARS virus S position;
(b) specifically in and SARS virus;
(c) to tire be 1 for described antibody and receptor binding domain bonded nude mice ascites: 50000-1: 1000000;
(d) to tire be 1 for described antibody and SARS virus neutral nude mice ascites: 4000-1: 10000.
2. immunoglobulin (Ig) as claimed in claim 1 is characterized in that described receptor binding domain has the aminoacid sequence shown in the SEQID NO:1.
3. immunoglobulin (Ig) as claimed in claim 1 is characterized in that it is a monoclonal antibody.
4. immunoglobulin (Ig) as claimed in claim 1 is characterized in that, it is produced by the cell that is selected from down group: mouse hybridoma cell is N-176-15, CCTCC No.:C200507; Or mouse hybridoma cell is S-9-11 CCTCC No.:C200515.
5. an immune conjugate is characterized in that, this immune conjugate contains the described immunoglobulin (Ig) of claim 1 and is incorporated into the coupling part of organizing under being selected from of described immunoglobulin (Ig) specifically: medicine, toxin, cytokine, radionuclide, enzyme.
6. a hybridoma that produces monoclonal antibody is characterized in that, it is selected from down group: mouse hybridoma cell is N-176-15, CCTCC No.:C200507; Or mouse hybridoma cell is S-9-11 CCTCC No.:C200515.
7. a composition is characterized in that, it contains the described immunoglobulin (Ig) of claim 1 or described immune conjugate of claim 5 and acceptable carrier.
8. composition as claimed in claim 7 is characterized in that described composition is a pharmaceutical composition.
9. the purposes of immunoglobulin (Ig) as claimed in claim 1 is characterized in that, is used for preparing the composition with SARS virus.
10. one kind prepares monoclonal antibody method, it is characterized in that it comprises step:
(a) the reorganization pseudovirus vaccine immune mouse of usefulness atypical pneumonia virus;
(b) behind the splenocyte and rat bone marrow tumour cell fusion with immune mouse, obtain hybridoma:
(c) from the hybridoma of step (b), select the hybridoma of the atypical pneumonia virus receptor binding domain bonded monoclonal antibody of secretion and S protein 31 8-510 amino acid section, obtain to combine hybridoma with receptor binding domain;
(d) from the hybridoma of step (c), selecting that the monoclonal antibody of generation and SARS virus bonded nude mice ascites tires is 1: 50000-1: 1000000 hybridoma;
(e) from the hybridoma of step (d), make monoclonal antibody.
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| CNB2005100286372A CN100519583C (en) | 2005-08-10 | 2005-08-10 | SARS neutralization antibody and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104628848A (en) * | 2013-11-14 | 2015-05-20 | 清华大学 | Monoclonal antibody MERS-27, as well as coding gene and application thereof |
| CN112535730A (en) * | 2020-11-27 | 2021-03-23 | 常州文松生物技术有限公司 | Novel coronavirus polypeptide vaccine and application thereof |
| WO2021169255A1 (en) * | 2020-02-24 | 2021-09-02 | 成都威斯克生物医药有限公司 | Anti-sars-cov-2 infection protein and vaccine |
| CN113945714A (en) * | 2020-07-16 | 2022-01-18 | 南京蓬勃生物科技有限公司 | Method for detecting neutralizing capacity of novel coronavirus neutralizing antibody drugs |
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| CN112210004B (en) * | 2020-07-20 | 2021-06-29 | 江苏集萃医学免疫技术研究所有限公司 | Coronavirus COVID-19 detection antibody and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1319596C (en) * | 2004-05-20 | 2007-06-06 | 南京大学 | Prepn process of S190 polypeptide vaccine and its application in resisting SARS virus |
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2005
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104628848A (en) * | 2013-11-14 | 2015-05-20 | 清华大学 | Monoclonal antibody MERS-27, as well as coding gene and application thereof |
| CN104628848B (en) * | 2013-11-14 | 2018-01-23 | 清华大学 | Monoclonal antibody MERS 27 and its encoding gene and application |
| WO2021169255A1 (en) * | 2020-02-24 | 2021-09-02 | 成都威斯克生物医药有限公司 | Anti-sars-cov-2 infection protein and vaccine |
| CN113945714A (en) * | 2020-07-16 | 2022-01-18 | 南京蓬勃生物科技有限公司 | Method for detecting neutralizing capacity of novel coronavirus neutralizing antibody drugs |
| CN113945714B (en) * | 2020-07-16 | 2023-01-31 | 南京蓬勃生物科技有限公司 | Method for detecting neutralizing capacity of novel coronavirus neutralizing antibody drugs |
| CN112535730A (en) * | 2020-11-27 | 2021-03-23 | 常州文松生物技术有限公司 | Novel coronavirus polypeptide vaccine and application thereof |
| CN112535730B (en) * | 2020-11-27 | 2021-09-17 | 常州文松生物技术有限公司 | Novel coronavirus polypeptide vaccine and application thereof |
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| CN100519583C (en) | 2009-07-29 |
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