CN102260915A - Method for preparing and constructing significant and innovative library of Ab2beta antibodies against infection with various respiratory tract viruses - Google Patents
Method for preparing and constructing significant and innovative library of Ab2beta antibodies against infection with various respiratory tract viruses Download PDFInfo
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Abstract
The invention relates to a great significant project which has the significant influence in the world. The original immunological test technology aims to solve the problem of human respiratory system acute lethal infectious diseases caused by any virus or variant virus in the world. A large number of effective neutral antibodies can be provided in the shortest time by preparing and constructing library of antibodies for blocking different infection paths of any respiratory tract virus (including unknown virus or variant virus) completely, namely a library of 'Ab2beta' antibodies which consist of specific antibodies capable of blocking virus-target cell binding sites specifically and have special functions and application, so as to prevent and treat any acute viral infectious diseases of the human respiratory systems, and particularly emergent accidents caused by lethal diseases caused by H1N1 influenza, avian influenza, severe acute respiratory syndromes (SARS) biological weapons and the like. More important, the investment for preparing and constructing the antibody library is small (about 2 million RMB), the time is two years at most, and various benefits are difficult to estimate. The 'non-conventional technology' obtained by implementing the project has a great strategic significance in biomedicine.
Description
The present invention relates to prevent and treat the special efficacy " modern weapon " of any acute viral transmissible disease of human respiratory system, promptly all by single-minded blocking virus---the antibody " Ab that form, that have specific function and purposes of respiratory tract target cell binding site
2The β antibody library " preparation and construction process (being particularly related to originality immunological experiment technology of the present invention).
Present respiratory tract mortality virus, the accident that causes as viruses such as " Influenza A H1N1, SARS, bird flus " or its varient has caused serious harm to the people of other countries.Wherein world-renowned " science " magazine is capturing " virus variation " as the first-selected world-famous puzzle of life science.To this antiviral present Research in the present whole world: 1. there is not any specific antiviral drug.2. still there is not any antiviral research project likely.
The present invention compares with the antiviral research in the present whole world, all is diametrically opposite such as the whole sight of things and the theory of knowledge, multidisciplinary intersection guiding theory, invention thinking, research method, technique means etc. in many aspects, and this directly causes different inventive principle.Understand inventive principle for making stakeholder and producer, this paper sets forth with popular language: because human respiratory tract's target cell membrane surface molecular space structure and position are limited, so any approach known or unknown virus invasion human respiratory tract target cell all is limited.Suppose that any virus comprises whole approach total " 1,000 " of variant viral invasion human respiratory tract target cell, so any specific virus must be by several approach invasions of 1-human body cell wherein, outside the virus row.If the science purpose of our project research, be all to arrange corresponding antibody guard at all different structures of respiratory tract target cell membrane surface, so wherein just comprise the corresponding respectively invasion approach that keeps guard over 1,000 different virus of 1,000 different antibody, promptly blocked whole poisoning intrusion approach.Therefore any virus makes a variation howsoever, all can not avoid these different guard antibody and enters human respiratory tract's cell.So how to prepare the novel antibody of guard respiratory tract target cell membrane all different structures of surface (comprising the invasion approach of 1,000 different virus), and, be exactly purpose of the present invention by the antibody library that these antibody constructions become to have specific function and purposes.Say with colloquial language: be exactly the antibody library that makes up a preventing respiratory viruses, the potent antibodies of anti-various viruses is arranged in the storehouse.When tackling any acute viral transmissible disease, only need from antibody library, " angle and get " antibody that combines with this virus specially with Causative virus, just can any acute viral transmissible disease of the most effective control.It is emphasized that, project of the present invention is with the maximum difference of the antibody of all antiviral vaccines generations in the world: antibody of the present invention all is the most effective specificity neutralizing antibody (not having any enhancing antibody or antibodies to "wrong" antigens), is fully to design for the difference invasion approach of blocking different virus.Therefore two great creation points without a peer are arranged: 1. in the virus of Zhi Bei each monoclonal antibody and site all be the space structure in during the most effective, reason is and site be exactly invade human respiratory tract's cell must be through approach, promptly antibody blocking virus---the binding site of target cell.2. make up " antibody library " with specific function and purposes that comprises the whole approach of any poisoning intrusion human respiratory tract target cell of blocking-up.It is very difficult that the research seems, if the forward thinking is only arranged or only come " finding the solution " with traditional research mode, then can not finish this project.Traditional experiment method of the sort of classics, promptly virus or its vaccine difference immune animal and people, screening is only antiviral from antiviral serum then---the experiment method of the neutrality antibody of target cell binding site, be at present general, though feasibility that can this project of side light, but be the most stupid research method certainly, perhaps 100 years also really the acquisition problem " separate ".Imagination of the present invention, once in Chinese doctors'associations net-hot news, to deliver in " life science need be shown great attention to a kind of thoughtcast guilty of heterodoxy " literary composition (partial content in 30,000 words---great preventing respiratory viruses intention), create much of a stir, allow the website staff all be shocked.Domestic and international many specialties and amateur website and university all reprint, tens thousand of doctors and stakeholder have read this paper, without any authority and expert can propose doubtful point both at home and abroad, just think realize this goal too difficult, wish that all I can disclose more innovative contents, but be dragged to now because of privacy problem.Disclose experimental program by application for patent today.
1. theoretical foundation of the present invention
1.1 the indirect theoretical foundation of the present invention:
1.1.1 prepare single-minded blocking virus invasion target cell approach " preferably antibody " theoretical foundation
1.1.1.1 antibody covering theory
Refer to the dependency of antibody fraction of coverage and neutralizing effect, as the neutralization to rabies virus, 200 above IgG molecules of each virion just can occupy its surface tissue (epi-position), make it and can not interact with receptor in target cell.And in another rabies virus and the escape mutants analysis find that each virion is in conjunction with the IgG molecule more than 1000, but still can not be neutralized.
1.1.1.2 the interaction theory of virus " part " and target cell " virus receptor "
Why virus can be adsorbed in cell and begin to infect, its main reasons in structure is the specific site of virus surface structural protein, can corresponding the combination be taken place with specific certain acceptor of topology character and cell surface, this combination has had so as to entering the bridge of cell virus.And in virus infection, why the antiviral specific antibody that body produces can suppress the infection again of virus, article one, the major cause antibody that is exactly can be with specific counter structure, produce specific combination with the site structure of virus surface and acceptor generation keying action, thereby blocking virus and cell receptor combines on topology, therefore stopped the infection of viral pair cell, the mechanism of such nature obviously provides the theoretical basis of searching treatment of viral infections medicine (annotating: comprise pharmaceutical chemicals).
1.1.1.3 antibody and viral interaction theory
HA albumen is one of major antigen of influenza virus, and its head has 5 antigen sites, divides another name A, B, C, D and exit site, and wherein the B site is the receptor in target cell binding site.Particularly find in the HIV-cell-cell interaction in research virus, except that the amino-acid residue that directly contacts between peplos " part " and target cell " virus receptor ", the antibody that other viral positions induce is difficult to define neutrality antibody or non-neutral antibody or enhancing antibody, and reason is: 1. the virus stain that can be neutralized can sport same antibody is had resistance or by this antibody enhanced strain.2. the antibody that produces with a certain strain immune body may be neutralizing antibody to this strain but be to strengthen antibody to other strains.It is 3. clear and definite that only to induce neutralizing antibody and do not induce the peplos district that strengthens antibody be very difficult.Final virus and antibody results of interaction will depend on the ratio that produces neutrality antibody and enhancing antibody, and promptly both are to " only " effect of viral overall function.
1.1.1.4 conclusion: in the antibody and virus key, it is the position (point of application) that need find neutralization virus, and best position is exactly virus---the combining site of target cell, promptly virus " part " binding site, and at virus " part " binding site antibody be exactly best antibody.
1.1.2 make up theoretical foundation with specific function and purposes " antibody library ":
1.1.2, whole approach of 1 virus or its varient invasion human respiratory tract cell
The variation of an amino-acid residue on any known or unknown virus variation such as the influenza virus hemagglutinin albumen just can make virus and institute's bonded sialic acid acceptor type change.The essence of this variation is exactly the new way that increases invasion respiratory tract cell, particularly pointing out this new way all must finish by target cell surface corresponding " virus receptor ", and " virus receptor " is the known or unknown membrane protein molecule in respiratory tract target cell surface, (protein component).Just because various dissimilar " virus receptor " quantity on respiratory tract target cell surface are limited, so the approach of poisoning intrusion respiratory tract also is limited, we can say that therefore any virus all is hidden in these " virus receptors " by the new way that variation produces.This project desired product---make up have specific function and purposes ' antibody library ", its antibody cloning quantity comprise corresponding poisoning intrusion target cell all may or potential invade approach, the big frame of project of the present invention that Here it is.
1.1.2.2 conclusion: make up the antibody library of the whole approach of blocking virus invasion respiratory tract, a kind of preferred approach that solves unknown virus or variant viral world-famous puzzle may be provided.
1.2 direct theoretical foundation of the present invention:
1.2.1 viral part---target cell " acceptor " interaction theory
Experimental results show that, the prerequisite of virus infected cell is to combine with the acceptor of cell surface, this is combined in joining outside structure concerns on reflection and the space conformation of structural protein and the biological chemistry meaning between the acceptor space conformation, has then reflected the phenomenon of viral tissue and cell-specific preferendum on biological meaning.If virus can not enter cell, or be blocked at before the adherent cell acceptor, then virus is minimum to the influence of host cell, and to virus---the research of cell receptor just in time provides a kind of like this possibility.Only for the purpose of the present invention, the so just a kind of similar normal physiological part and the cell biological effect of receptors bind cause virus to enter cell.
1.2.2 antibody idiotype and antiidiotypic antibody analogue antigen theory
Huge Id or the AId antibody of numeral can simulate the whole exotic antigen world, and this is to make up " Ab
2The β antibody library " main theoretical basis.Utilize this theory,, just can prepare all space structures of the easy contact of molecule of simulation target cell membrane surface, comprising the antibody of the whole virus receptors of simulation particularly by inventive application correlation theory and immunological experiment technology.In addition, no matter how ever-changing virus antigen is, the cell surface virus receptor of living is constant, so the present invention simulates the antibody of target cell virus acceptor, just can be with " ten thousand changes of the constant reply virus antigen of antibody ".And the present invention infers that in theory this antibody itself also may more or less be kept in advance can be in conjunction with the required locus of virus variation amino-acid residue.
1.2.3 polymorphism MHC immunity identification is theoretical
The biological function of polymorphism MHC and meaning, determined different strain animals particularly some strain animal have wider and darker immune recognition function, it is theoretical to use polymorphism MHC immunity identification, be for increasing to greatest extent equally to all immunity identification of the easy attaching space structure of molecule of airway epithelial cell film surface, and, make up Ab with this antibody for preparing simulation target cell whole " potential virus acceptor "
2The β antibody library.Polymorphism MHC immunity identification theory is to make up " Ab equally
2The β antibody library " the most important theories basis.
The present invention is from viral part---and derive following important conclusion target cell " acceptor " interaction theory and antibody idiotype and the antiidiotypic antibody analogue antigen theory: the antibody of 1. anti-target cell " virus receptor " is exactly the antibody of simulated virus part; The antibody of antiviral part is simulated the antibody of target cell " virus receptor " exactly.2. viral tissue tropism, i.e. tissue specificity has determined the main target cell of respiratory system---epithelial cell is a research object of the present invention.The present invention adopts originality immunology and cytobiology technology, at first uses the important target cell of Respirovirus---and epithelial cell is a large amount of different animals of immunity respectively, prepare the antibody of the different antigenic determinants of the different protein moleculars in anti-epithelial cell membrane surface.And then utilize these antibody, preparation can simulate the antibody of the different epi-positions of the different protein moleculars of epithelial cell (annotating: wherein comprise the potential virus acceptor), Here it is can any virus of single-minded blocking-up---the antibody of the whole binding sites of target cell.At last form " Ab with specific function and purposes by all this antibody
2β antibody library ".So just can be at any time from the storehouse screening at the specificity neutralizing antibody of any virus (comprising unknown virus) that causes the respiratory system acute infectious disease or its varient.
2. experimental program
2.1 first experimental program:
2.1.1 the preparation of the comprehensive preventing respiratory target cell membrane different epitope antibodies in surface (Ab1 antibody): use intact epithelial cell of human respiratory or corresponding cancer cells, the rabbit and the mouse of respectively immune a large amount of different strains, therefrom extract the AHS of different strain rabbits and mouse, the airway epithelial cell of choosing then absorbs wherein antibody, and with these antibody of novel method wash-out, extraction and purifying, the present invention claims " Ab1 antibody " this antibody.
2.1.2 the B cell of single-minded screening combination " Ab1 antibody " antigen binding site: 1. use routine immunization and learn experimental technique, extract the B cell of a large amount of different people of purifying and different strain rabbits respectively.2. very simple by the present invention, originality immunology New/Experimental Techniques, " the Ab1 antibody " that the people that manipulative above-mentioned test obtains, the B cell of rabbit and 2.1.1 test obtain, solve B cell and " Ab1 antibody " interaction relationship problem between the two, promptly disposing fully from these B are fine can not any irrelevant B cell of bonded with " Ab1 antibody " antigen-binding site itself, and purpose is to keep other remaining B cell related to the present invention.The B cell related to the present invention that 3. will keep mixes with " Ab1 antibody " that the 2.1.1 test obtains, again by flow cytometry (FCM), filters out the B cell of single-minded combination " Ab1 antibody " antigen-binding site, " Ab promptly required for the present invention
2β antibody " the generation cell.Specify be " Ab1 antibody " itself be antibody be again antigen, the antigen-binding site that the key of noting this test is " Ab1 antibody " as unique antigen in conjunction with the BCR--B cell, in exempt from service scientific principle opinion and actual the combination, can not forget this point, otherwise can not understand this test---the single-minded B cell that filters out combination " Ab1 antibody " antigen-binding site how.
2.1.3 make up elementary " Ab with specific function and purposes
2The β antibody library ": can carry out vitro culture with Ab1 antibody antigen combining site bonded B cell, in specific different time, by 2 approach: 1., prepare phage " Ab by phage antibody library technique with the B cell of vitro culture
2β antibody ".2. obtain secretion property " Ab by vitro culture B cell technology
2β antibody ".Obtain whole " Ab of the different epi-positions of the different protein moleculars in anthropomorphic dummy's airway epithelial cell film surface (annotating: wherein comprise the potential virus acceptor) as far as possible
2β antibody ", and make up elementary " Ab with this
2β antibody library "
2.1.4 verify elementary " Ab
2The β antibody library " function and effect: utilize specific virus or attenuated live vaccine etc. from elementary " Ab
2" middle screening antibody extracts and the single-minded antibody that resists specific Respirovirus-target cell binding site of purifying the β antibody library, promptly simulates the Ab of target cell " virus receptor "
2β antibody is checked elementary " Ab with this
2The β antibody library " function and effect.
2.1.5 virus neutralization tests: utilize some known viruse or attenuated live vaccine etc. from " Ab
2The β antibody library " middle screening specific antibody, and carry out virus neutralization experiment, conclusive evidence " Ab
2β antibody " function and effect, preparation makes up the " Ab with specific function and purposes at the specificity neutralizing antibody (specific immunoglobulin) of some known specific virus
2β antibody library ".
2.1.6 accompanying drawing is the technological line figure of this experimental program.
2.2 second experimental program:
2.2.1 the preparation of the comprehensive preventing respiratory target cell membrane different epitope antibodies in surface (Ab1 antibody): use intact epithelial cell of human respiratory or corresponding cancer cells, the rabbit and the mouse of respectively immune a large amount of different strains, therefrom extract the AHS of different strain rabbits and mouse, the airway epithelial cell of choosing then absorbs wherein antibody, and with these antibody of novel method wash-out, extraction and purifying, the present invention claims " Ab1 antibody " this antibody.
2.2.2 single-minded screening is in conjunction with the phage " Ab of " Ab1 antibody " antigen binding site
2β antibody ", make up elementary phage " Ab
2The β antibody library ": utilize the ready-made commodity in market---phage antibody library, and by the phage antibody library triage techniques, single-minded screening is in conjunction with the phage " Ab of " Ab1 antibody " antigen binding site
2β antibody "; this process of the test needs two steps: 1. adopt originality triage techniques of the present invention; disposing can not any irrelevant phage antibody of bonded with " Ab1 antibody " antigen-binding site itself, and purpose is to keep other phage antibody related to the present invention.2. adopt classical screening method, utilize " Ab1 antibody " antigen-binding site own as unique antigen, from the phage antibody related to the present invention that keeps, the single-minded phage " Ab that filters out in conjunction with this position
2β antibody ", and make up elementary phage " Ab with this
2β antibody library ".
2.2.3 verify elementary phage " Ab
2The β antibody library " function and effect: utilize specific virus or attenuated live vaccine etc. from elementary phage " Ab
2" middle screening antibody extracts and the single-minded specific phage antibody that resists specific Respirovirus-target cell binding site of purifying the β antibody library, promptly simulates the Ab of target cell " virus receptor "
2β antibody is checked elementary phage " Ab with this
2The β antibody library " function and effect.
2.2.4 virus neutralization tests: utilize some known viruse or attenuated live vaccine etc. from elementary phage " Ab
2The β antibody library " middle screening specific phage antibody, and carry out virus neutralization tests, conclusive evidence phage " Ab
2β antibody " function and effect.Preparation makes up the phage " Ab with specific function and purposes at the specificity neutralizing antibody (specific immunoglobulin) of some known specific virus
2β antibody library ".
2.3 remarks:
2.3.1 the present invention only adopts first experimental program, the confirmatory test that this experimental program detects anti-different virus is successful more, and " Ab is described
2The β antibody library " structure perfect more, the objective " Ab that says
2The β antibody library " structure do not have failure, have only constantly perfect.
2.3.2 because second experimental program success ratio is relatively low, the present invention does not adopt, and does not therefore mark the relevant art route map yet.For no other reason than that predicted a kind of like this experimental technique, be protection the present invention, the spy sets forth out in the present invention.
3. 2 years and budgetary resources are tested in expection
3.1 1 year, one of Primary Construction had the " Ab in the people source and the rabbit source of specific function and purposes
2The β antibody library ", the extraction from antibody library that certain effect can be arranged is at the specific antibody of certain specific virus.Funds in need 80-100 ten thousand Renminbi.
3.2 1 year, further improve above-mentioned " Ab
2β antibody library ".Utilize some known Respirovirus or attenuated live vaccine etc. from antibody library, to screen specific antibody, and carry out the virus neutralization and test, the function and the effect of conclusive evidence antibody, and preparation at the specificity neutralizing antibody (specific immunoglobulin) of specific virus with standby, make up " specificity Ab simultaneously at some known breath road virus
2β antibody library ".Funds in need 90-110 ten thousand Renminbi.
4. the important trial test of proof project feasibility of the present invention
Having under immunology normal experiment condition and the skilled scientific research and testing personnel situation, only need the trial test of 150,000 Renminbi, in one-month period, just can detect and verify whether the present invention has the problem of feasibility technically.As in one-month period, the anti-people CD4 of the animal source molecule monoclonal antibody of buying with different biotech firms is as antigen-like material, immunological testing technology by the present invention's original creation, screen single-minded BCR in conjunction with the antigen-binding site of CD4 monoclonal antibody own---the B cell, and obtain the anti-antibody-Ab of anti-CD4 molecule with this
2β antibody.This process of the test is exactly the important trial test of proof project feasibility of the present invention.What is interesting is this trial test of selecting especially, itself be worth just relevant with the super antibody of the single-minded blocking-up HIV (human immunodeficiency virus) infection approach of preparation.If trial test successfully except that explanation project of the present invention is feasible, also has other its own strategic significance on biomedicine.A present invention's statement only trial test itself just has important use and meaning.
5. expected result---the important use and the meaning of project of the present invention
Well-known SARS period, the antiviral serum that the famous medical officer in Beijing utilizes Guangdong to infect SARS virus recovery from illness patient is saved oneself and is obtained newborn story, project desired product-specific immunoglobulin of the present invention, than on state strong hundred times of antiviral serum, thousand times.With people's infective virus or inoculated the antiviral serum that extracts behind the vaccine and compared, overcome the four major problem that antiviral serum exists: 1. safety issue, may there be virus in the antiviral serum, may increase the chance of receptor's infective virus thus, and owing to exist numerous materials also may bring anaphylaxis in the allogeneic serum to the receptor.2. validity problem, antiviral antibody in the antiviral serum is impure, and there is a reason of different immunne response general layouts owing to Different Individual, the antibody that virus or vaccine produce in host has the branch of " good " antibody and " bad " antibody, and real antivirus action depends on " only " effect of " good " antibody and the effect of " bad " antibody.3. antigen " original sin " problem, this problem is very important, after promptly infecting a certain specific virus or having inoculated the vaccine of a certain specific virus, once more contact infection after " varient " (virus variation) of this specific virus, some individuality does not produce immunne response to variant viral probably, if this immune delay phenomenon really is not so good as not vaccination originally, so vaccine inoculation has principle and condition.4. " antibody library " screening and a large amount of preparation specific antiviral antibodies from having specific function and purposes, embodied principle many, fast, good and that economize, can effective and rapidly particularly prevent and treat the accident that any Respirovirus causes, this be that any virus vaccines and antiviral serum can't be accomplished, also be any antiviral substance comprise pharmaceutical chemicals can not accomplish.
5.1 project desired product of the present invention-" Ab
2The β antibody library " can provide in a large number potent antibodies, any acute viral transmissible disease of control human respiratory system in the shortest time.The particularly accident that causes as mortality diseases such as H1N1 influenza, bird flu, SARS or biological weapon.
5.2 use from " Ab
2The β antibody library " middle screening specificity antiviral antibody, the preparation antibody chip.
5.3 inventive technique of the present invention is also relevant with the super antibody of preparation double blocking HIV (human immunodeficiency virus) infection especially, as the main target molecule of preparation simulation HIV---the Ab of CD4 and SIGN " virus receptor "
2β antibody, even using anticancer field, to prepare the single-minded anticancer cell of high accuracy CTL that kills and wounds cancer cells (annotate: screen the unprecedented a large amount of different clones' that extract from polymorphism MHC colony the CTL cells) relevant.
5.4 the preparation antibody vaccine is like being the recombinant vaccine of carrier with the strain of the bovine vaccine the Temple of Heaven.
5.5 " Ab
2The β antibody library " can prevent and treat the attack of biological weapon or gene target weapon to specific ethnic group.
5.6 the immunological testing technology of the present invention original creation can promote virus with a definite target in view---research such as both interaction relationships and the action rule and the space structure of " part and acceptor " between target cell.Also can promote the RESEARCH ON CELL-BIOLOGY of normal physiological part and receptors bind in addition.
5.7 particularly point out at last: the immunological testing technology of the present invention original creation, life science for high resolution between any material fine difference an important method is provided.
Owing to the secret reason of the present invention and input funds are few, so do not apply for local and national research funding.The inventor is not for money, only for doing some cause.The all trades and professions knowledgeable people that welcome wants to do vigorous cause cooperates (as needs I can provide strength R﹠D institution cooperate) with me with producer of any businessman, let us is created great achievement throughout the world, makes a stir all over the world!
Claims (10)
1. one kind prepares and makes up the great novelty Ab that resists various respiratory virus infections
2The method of β antibody library is characterized in that first experimental program: comprise that the preparation of the comprehensive preventing respiratory target cell membrane different epitope antibodies in surface (Ab1 antibody), single-minded screening are in conjunction with the B cell of " Ab1 antibody " antigen binding site, the elementary " Ab that structure has specific function and purposes
2The β antibody library ", the checking elementary " Ab
2The β antibody library " function and effect, five steps of virus neutralization tests: promptly use intact epithelial cell of human respiratory or corresponding cancer cells on the one hand; the rabbit and the mouse of a large amount of different strains of immunity respectively; therefrom extract the AHS of different strain rabbits and mouse; the airway epithelial cell of choosing then absorbs wherein antibody; and with novel method wash-out, extraction and these antibody of purifying; the present invention claims " Ab1 antibody " this antibody. use routine immunization experimental technique on the other hand, extract the B cell of a large amount of different people of purifying and different strain rabbits respectively.Very simple by the present invention then, originality immunology New/Experimental Techniques, the people that manipulative above-mentioned two tests obtain, B cell and " the Ab1 antibody " of rabbit, solve B cell and Ab1 antibody interaction relationship problem between the two, promptly disposing fully from these B are fine can not any irrelevant B cell of bonded with " Ab1 antibody " antigen-binding site itself, and purpose is to keep other remaining B cell related to the present invention.The B cell related to the present invention that then will keep mixes with " Ab1 antibody ", again by flow cytometry (FCM), filters out the B cell of single-minded combination " Ab1 antibody " antigen-binding site, " Ab promptly required for the present invention
2β antibody " the generation cell.Specify be " Ab1 antibody " itself be antibody be again antigen, the antigen-binding site that the key of noting this test is " Ab1 antibody " as unique antigen in conjunction with the BCR--B cell, in exempt from service scientific principle opinion and practical application, can not forget this point, otherwise can not understand this test---the single-minded B cell that filters out in conjunction with Ab1 antibody antigen combining site how.On above-mentioned experimental basis, can carry out vitro culture, in specific different time, by 2 approach: 1., prepare phage antibody by phage antibody library technique with the B cell of vitro culture with " Ab1 antibody " antigen-binding site bonded B cell.2. obtain secretion property antibody by vitro culture B cell technology.Obtain whole " Ab of the different epi-positions of the different protein moleculars in anthropomorphic dummy's airway epithelial cell film surface (annotating: wherein comprise the potential virus acceptor) as far as possible
2β antibody ", make up elementary " Ab with this
2β antibody library ".Utilize specific virus or attenuated live vaccine etc. from elementary " Ab then
2The β antibody library " middle screening antibody, extract and the single-minded antibody that resists specific Respirovirus-target cell binding site of purifying, promptly simulate the Ab of target cell " virus receptor "
2β antibody is checked elementary " Ab with this
2The β antibody library " function and effect.Utilize some known viruse at last and from elementary " Ab
2The β antibody library " the middle corresponding specific antibody that screens; carry out virus neutralization tests; prove conclusively the function and the effect of antibody with this, preparation makes up the " Ab with specific function and purposes at the specificity neutralizing antibody (specific immunoglobulin) of some known specific virus
2The β antibody library ",
2. one kind prepares and makes up the great novelty Ab that resists various respiratory virus infections
2The method of β antibody library, it is characterized in that second experimental program: comprise the phage antibody of the preparation of the comprehensive preventing respiratory target cell membrane different epitope antibodies in surface (Ab1 antibody), single-minded screening combination " Ab1 antibody " antigen binding site, make up elementary phage " Ab
2β antibody library ", verify elementary phage " Ab
2The β antibody library " function and effect, four steps of virus neutralization tests: promptly use intact epithelial cell of human respiratory or corresponding cancer cells on the one hand; the rabbit and the mouse of a large amount of different strains of immunity respectively; therefrom extract the AHS of different strain rabbits and mouse; the airway epithelial cell of choosing then absorbs wherein antibody; and with novel method wash-out, extraction and these antibody of purifying, the present invention claims " Ab1 antibody " this antibody.Utilize the ready-made commodity in market---phage antibody library on the other hand, and by the phage antibody library triage techniques, single-minded screening is in conjunction with the phage antibody of above-mentioned " Ab1 antibody " antigen binding site, this test needs two processes: 1. adopt originality triage techniques of the present invention, disposing can not any irrelevant phage antibody of bonded with " Ab1 antibody " antigen-binding site itself, and purpose is to keep other phage antibody related to the present invention.2. adopt classical screening method, utilize " Ab1 antibody " antigen-binding site own as unique antigen, from the phage antibody related to the present invention that keeps, the single-minded phage antibody that filters out in conjunction with this position, and obtain whole phage " Ab of the different epi-positions of the different protein moleculars in anthropomorphic dummy's airway epithelial cell film surface (annotating: wherein comprise the potential virus acceptor) as far as possible
2β antibody ", make up elementary phage " Ab with this
2β antibody library ".Utilize specific virus or attenuated live vaccine etc. from elementary phage " Ab then
2The β antibody library " middle screening antibody, extract and the single-minded specific antibody that resists specific Respirovirus-target cell binding site of purifying, promptly simulate the phage Ab of target cell " virus receptor "
2β antibody is checked elementary phage " Ab with this
2The β antibody library " function and effect.Utilize some known viruse at last and from phage " Ab
2The β antibody library " the middle corresponding specific phage antibody that screens, carry out virus neutralization tests, conclusive evidence phage " Ab
2β antibody " function and effect.Preparation makes up the phage " Ab with specific function and purposes at the specificity neutralizing antibody (specific immunoglobulin) of some known specific virus
2β antibody library ".
3. the great novelty Ab of a kind of preparation according to claim 1 and the anti-various respiratory virus infections of structure
2The method of β antibody library is characterized in that the rabbit and the mouse of a large amount of different strains of the described respectively immunity of first experimental program, with immunity respectively a large amount of other not the animal of system of the same race replace.
4. the great novelty Ab of a kind of preparation according to claim 1 and the anti-various respiratory virus infections of structure
2The method of β antibody library, it is characterized in that described routine immunization of first experimental program experimental technique, extract the B cell of a large amount of different people of purifying and different strain rabbits respectively, learn experimental technique with routine immunization, extract respectively purifying a large amount of other not of the same race be that the B cell of animal replaces.
5. the great novelty Ab of a kind of preparation according to claim 1 and the anti-various respiratory virus infections of structure
2The method of β antibody library is characterized in that intact epithelial cell of the described human respiratory of first experimental program or corresponding cancer cells, with alveolar cell or corresponding cancer cells, endotheliocyte, red corpuscle etc. replace.
6. the great novelty Ab of a kind of preparation according to claim 1 and the anti-various respiratory virus infections of structure
2The method of β antibody library is characterized in that first experimental program is described 1. with the B cell of vitro culture, prepares phage antibody by phage antibody library technique., prepare rrna antibody by rrna antibody library technology and replace the B cell of vitro culture with 1..
7. the great novelty Ab of a kind of preparation according to claim 2 and the anti-various respiratory virus infections of structure
2The method of β antibody library is characterized in that the rabbit and the mouse of a large amount of different strains of the described immunity respectively of second experimental program, with immunity respectively a large amount of other not the animal of system of the same race replace.
8. the great novelty Ab of a kind of preparation according to claim 2 and the anti-various respiratory virus infections of structure
2The method of β antibody library is characterized in that the phage " Ab of second described single-minded screening of experimental program in conjunction with " Ab1 antibody " antigen binding site
2β antibody ", make up elementary phage " Ab
2β antibody library ".With the rrna " Ab of single-minded screening in conjunction with " Ab1 antibody " antigen binding site
2β antibody ", and make up elementary rrna " Ab with this
2The β antibody library " replace.
9. the great novelty Ab of a kind of preparation according to claim 2 and the anti-various respiratory virus infections of structure
2The method of β antibody library is characterized in that the phage " Ab of second described single-minded screening of experimental program in conjunction with " Ab1 antibody " antigen binding site
2β antibody ", make up elementary phage " Ab
2β antibody library ".With single-minded screening phage random polypeptide fragment, and make up elementary " phage random peptide library " with this and replaced in conjunction with " Ab1 antibody " antigen binding site.
10. the great novelty Ab of a kind of preparation according to claim 2 and the anti-various respiratory virus infections of structure
2The method of β antibody library is characterized in that the phage " Ab of second described single-minded screening of experimental program in conjunction with " Ab1 antibody " antigen binding site
2β antibody ", make up elementary phage " Ab
2β antibody library ".With single-minded screening all cpds, and make up elementary " compound library " with this and replaced in conjunction with " Ab1 antibody " antigen binding site.
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| CN2011101039664A CN102260915A (en) | 2011-04-26 | 2011-04-26 | Method for preparing and constructing significant and innovative library of Ab2beta antibodies against infection with various respiratory tract viruses |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618939A (en) * | 2012-03-13 | 2012-08-01 | 王胜军 | Method for preparing and constructing Ab2 beta antibody library capable of resisting all kinds of known and unknown respiratory virus infection |
| CN102628191A (en) * | 2012-03-29 | 2012-08-08 | 王胜军 | Method for preparing and constructing Ab2belta antibody library capable of resisting infection of various known and unknown respiratory viruses |
| CN112321685A (en) * | 2018-11-05 | 2021-02-05 | 共鳞实业(深圳)有限公司 | Agent for preventing or treating CyHV-2 infection of fish and application thereof |
-
2011
- 2011-04-26 CN CN2011101039664A patent/CN102260915A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102618939A (en) * | 2012-03-13 | 2012-08-01 | 王胜军 | Method for preparing and constructing Ab2 beta antibody library capable of resisting all kinds of known and unknown respiratory virus infection |
| CN102628191A (en) * | 2012-03-29 | 2012-08-08 | 王胜军 | Method for preparing and constructing Ab2belta antibody library capable of resisting infection of various known and unknown respiratory viruses |
| CN112321685A (en) * | 2018-11-05 | 2021-02-05 | 共鳞实业(深圳)有限公司 | Agent for preventing or treating CyHV-2 infection of fish and application thereof |
| CN112321685B (en) * | 2018-11-05 | 2022-02-18 | 深圳技术大学 | Agent for preventing or treating CyHV-2 infection of fish and application thereof |
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