CN102309755A - Design scheme for compound type dual duplex locating type intracellular vaccine - Google Patents
Design scheme for compound type dual duplex locating type intracellular vaccine Download PDFInfo
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Abstract
The invention relates to a routine technology applying gene protein engineering and antibody protein engineering, in particular to a design scheme for an anti-cancer and anti-acquired immure deficiency syndrome (AIDS) biological missile of a compound type dual duplex locating type intracellular vaccine. The design scheme is characterized in that: compared with the common biological missile, the biological missile mainly adopts a dual imported biological missile technology for jointly using imported protein transactivator of transcription (TAT) and a nuclear localization sequence (NLS), and can directly feed an anti-cancer and anti-AIDS medicinal warhead into cytoplasm and nucleuses, a general formula of the molecular structure of the biological missile is given, and a specific molecular structural formula of the anti-cancer and anti-AIDS biological missile is established on the basis of the general formula.
Description
The present invention relates to one type about applying gene protein engineering technology, make the technical method invention of the design of " compound type duplication double localization type intracellular vaccine " biological missile.It is the anticancer disease that proposed in 2002 of the inventor and the method patent of invention " duplication double localization type intracellular vaccine " of AIDS resisting; And in 2007 in order further to improve and expansion for above-mentioned patent; And on the basis of the method patent of invention " compound type duplication double localization type intracellular vaccine " that proposes, a kind of art designs scheme of being invented with practical implementation property.The inventor proposed " duplication double localization type intracellular vaccine " patent application early than 2002 to China national Patent Office; Application number is 02100099.9; Be distributed on the 19th volume the 32nd interim state Gazette of Patent for Invention in 2003; Proposed " compound type duplication double localization type intracellular vaccine " patent application in 2007 to China national Patent Office again, application number is 200710086430.X, be published in volume 10 days the 24th JIUYUE in 2008 (on) in No. 37 Chinese invention patent communique.
Through computer search, also find to have the technical method invention with the similar biological missile design of patent of the present invention both at home and abroad, have initiatively, therefore, this method patent of invention ought to receive the protection of Patent Law.
At first, need to prove that the basic mechanism or the basic skills of compound type vaccine and the effect of traditional vaccine treatment compound type are different.
Traditional vaccine is the specific antigen that utilizes artificial culture, and the startup human immune system produces immune antibody and kills virus; Or diseased cells; The compound type vaccine then is the biochemical drug of the fusion rotein of one type of targeting type, and it can directionally be sent biochemical drug in the cell physiological metabolism transferring or regulate in the diseased cells and react and treat diseased cells, for example; The compound type vaccine can be with the biochemical drug that can start cancerous cell gene apoptosis program; Be sent in the cancerous cell, start the self-destruction program of cancerous cell, cancerous cell is killed; Or the biochemical drug that can restrain reverse transcriptase is sent in the human immunocyte T4 cell, checks hereditary material---the reverse transcriptase of the AIDS in the T.
This shows that composite type vaccin is not traditional vaccine.So; Why be vaccine again? This is because when invention dual duplex vaccine in 2002; Not having can be with reference to title, and simultaneously, the effect and the traditional vaccine of biological missile that acts on extracellular fusion rotein is very alike; Therefore be called vaccine, should be called intellectual drug in fact or biological missile is more suitable.
Guided missile is the modernized war weapon, has powerful directed lethality.So, what is " biological missile "? " biological missile " also is made up of carrier and bullet as guided missile, is a kind of modern weapon that is used to treat tumor.Utilize " biological missile " treatment tumor to be referred to as " targeting therapy " or " biological missile " therapy.Along with the quadroma Progress in technique, can prepare the antineoplastic monoclonal antibody specific at present.People with anticancer material, as " bullet ", are prepared into " biological missile " like chemotherapeutics, isotope or Lysin (Lysin) being carrier as " guidance system " with the bonded monoclonal antibody of tumor cell.Bullet (chemotherapeutics, isotope or Lysin) is transported to the tumor locus of body through carrier (monoclonal antibody).Its killing tumor cell, and do not kill and wound the normal tissue cell of body, have very big superiority.Animal experiment study proves that " biological missile " has the good anticancer effect.
Biological missile " fusion rotein ", wherein " directed albumen " is called as " guidance device ", and with " antibody protein " or restricted enzyme that " directed albumen " merges mutually, can be described as " killing and wounding device " or " bullet ".This orientation class fusion rotein can be made up of the protein or the polypeptide short-movie of two sections or three sections, wherein the A section be by can with a certain type persona certa somatic cell; For example hepatocellular surface receptor, or human cell " wide spectrum receptor ", for example hemagglutinin (hemgg Iutinin); Highly single-minded complementary antibody or matter white matter are used as " guidance device ", are responsible for killing and wounding the device or the protein drug of bullet; Or the chemicals of other kinds is for example, will have to check intracytoplasmic viral genetic, such as; Can check the reverse transcriptase medicine of HIV; Also have the various repressor proteins that check the viral DNA that is incorporated into the human cell DNA that have, and the repressor protein that can check the oncogene in the cell chromosome, be sent in the human body cell.Here it is " compound type duplication double localization type intracellular vaccine " biological missile.
But common biological missile is general only to be made up of antibody with " guidance device " function and the medicine bullet with killing ability, and their shortcoming is can't be with getting in the cell; Therefore; Medical function is little, and can constitute a threat to intravital other healthy cells of machine, and toxicity is bigger.And " compound type duplication double localization type intracellular vaccine " biological missile owing to used a kind of dual " gatherer " than common biological missile more; Can the medicine bullet directly be sent in Cytoplasm and the nucleus, therefore overcome the weak point of common biological missile, can accurately act on the target spot of interior viral genetic of diseased cells matter or cell nuclear dna; Therefore; Medical function is big, and can not constitute a threat to intravital other healthy cells of machine, and toxicity is little.This is " dual lead-in " biological missile.
Meanwhile; " compound type duplication double localization type intracellular vaccine " biological missile can also produce respectively a weight " gatherer " only is installed, and only can get into intracytoplasmic biological missile; Perhaps produce dual " gatherer " is installed, can get into endonuclear biological missile.They belong to " twin " biological missile.
First characteristic of Here it is " compound type duplication double localization type intracellular vaccine " biological missile also is its main characteristic or essential characteristic.
This just this patent be called the basic reason of " compound type duplication double localization type intracellular vaccine " biological missile, be two big characteristics (dual and duplex) of the creationary invention of this patent.
Why " compound type duplication double localization type intracellular vaccine " biological missile has the function of dual " gatherer ", is closely-related for the proteic progress of TAT with in recent years.
Human immunodeficiency virus type 1 (HIV1) gene a kind of trans activating transcription factor (transactivator of transcription, albumen TAT) of being called as of can encoding.It is through (transactivating response, TAR) the RNA sequence combines to transcribe with initial gene with trans-activating response.TAT albumen and REV albumen as the regulatory factor of HIV1, play a part very important in the gene expression of HIV together.Discover that TAT albumen can pass cell membrane [1,2], arrive the extracellular, (it is worn the film sequence and is made up of 11 aminoacid for cellmembrane penetrating peptide, CPP) activity to have the cell membrane penetrating peptide.This characteristic has very important using value, because it provides other albumen of transhipment or medicine to pass cell membrane, gets into the approach of cell.Wear film sequence and other proteic fusion genes through the design coding, express the fusion rotein that the back obtains, under the leading of TAT, can get into cell.
Vive etc. [3] find that having the sequence of wearing the film function in the TAT albumen is to be positioned at 11 aminoacid (YGRKKRRQRRR) of 47～57, is rich in basic amino acid (8), be called as protein transduction domain (protein transduction domain, PTD).TAT albumen has the film of wearing function, its PTD can with other macromolecular substances, wear film together like albumen etc. and get into cell.Utilize this character to make it to bring into play biological action with not getting into cell through this approach transduction by normal synthetic protease in the body.Delom etc. [14] find that first the TAT albumen foreign protein of can transduceing gets into the enterocyte of Caenorhabditis elegans (Caenorhabditis elegans).The film function of striding of this explanation PTD does not have species specificity.In recent years, the proteic PTD of TAT received attention more and more widely, and many scientists are engaged in correlational study utilizing it to wear the film function.[it is auspicious that " HIV TAT albumen progress " " Jiangsu University's journal (medicine) " 2008 the 18th volumes the 03rd phase ancestor raises brave Shao Qi].
Existing in addition research confirms; TAT is except to intracellular transport albumen; Also portability nucleotide, liposome even metal particle get into nearly all tissue and cell, and it is fast to have speed, does not rely on temperature, energy, cell membrane antibody; And pair cell characteristics such as do not damage, and TAT is considered to a kind of up-and-coming vehicle.[" TAT EGFP Expression of Fusion Protein and active " in the transduction of PC12 cell "
The Four army medical university's journals "2007 the 28th volume the 10th phase author: Wang Haizhen are permitted to give Ming Lee and are increased Fu Yang Jing Zhangyunhan.】
More than, be exactly can be with albumen or polypeptide drugs, or first weight " gatherer " the TAT albumen or the PTD polypeptide of the biological missile of other chemicals transfered cell matter.
Can't get into nucleus because molecular weight is higher than the macromole of 15kd, this just needs second weight " gatherer "-" appraising and deciding the position order " (NLS).
This be because, eukaryotic nuclear is cingens by nuclear envelope, nuclear envelope is then by two membranes---interior nuclear membrane with outer nuclear membrane---forms, during separated by examining all spaces.Outer nuclear membrane and rough endoplasmic reticulum are successive, and examining all spaces is successive with the cavity of ER then.All proteins in the nucleus all is synthetic by free ribosome in Cell sap.How does other protein in archaeal dna polymerase class, RNA polymerase class, histone class and the nuclear cross nuclear envelope? On this obstacle selectively opening is arranged, be called nucleopore, the about 70A of its diameter.Little protein (～15kd) can get into rapidly like histone, big protein (＞～90kd) then be rejected as an outsider, only if they have special signal.Sort signal also can quicken small protein matter and get into.The transhipment of originally this larger protein is gone into nucleus and is decided by a kind of existence of appraising and deciding the position order, and this contains five successive positively charged residues in proper order, and------bad---bad---essence---relies---figured silk fabrics to dried meat.This polypeptide sequence is attached on a kind of protein that is positioned usually in the Cell sap, can make it get into nucleus? Once made overtesting with pyruvate kinase, this is positioned in the cytoplasm by the tetramer that the subunit of 58kd is formed usually.Through recombinant DNA technology, with coding put in place the order cDNA with the coding kinase whose cDNA be connected together.The plasmid that will contain this chimeric cDNA with microinjection then injects mammiferous cell.Containing the pyruvate kinase that this sv40 appraises and decides position order almost only is present in the nucleus.In contrast, having the reformed pyruvate kinase of appraising and deciding position order (threonine has replaced lysine on position 128) then rests in the Cell sap.This has just proved exist (" biochemistry " [U.S.A] LVBER STRYER BJ University Press P871 page or leaf in 1992) of appraising and deciding the position order.
It may be noted that for some specific cell to also have some specific " gatherers "; For example, HIV relies on gp41 albumen (transmembrane protein) exactly; Cooperate gp120 albumen, import to viral hereditary material in the Cytoplasm of human immunity T cell, therefore; Relevant AIDS resisting biological missile also can use gp41 albumen rather than TAT albumen is used as a weight " gatherer ", and still, this belongs to particular case.
It more than is explanation to first characteristic of " compound type duplication double localization type intracellular vaccine " biological missile.
Second characteristic of " compound type duplication double localization type intracellular vaccine " biological missile used " HAART " exactly and used biological missile, and is duplex and usefulness.
Here, special needs to be pointed out is: also the reverse transcriptase with AIDS is the same owing to telomerase, also is a kind of reverse transcriptase; Therefore, can use RTI fully, be used as cancer therapy drug; For example, and application telomerase inhibitor azidothymidine AZT (Azidothymidine, AZT).Simultaneously, telomerase inhibitor and cancer standard care medicine carboplatin (carboplatin) and cisplatin (cisplatin) also have extra anti-proliferative effect when being used in combination.Like this, just can accomplish at one stroke what should be carried through by stages, AIDS resisting and cancer therapy drug are integrated, thereby significantly reduce the fund and the time of developing this two Chinese medicine.
Therefore; Can unite simultaneously to use and used telomerase inhibitor and come the co-therapy cancer with the biological missile of having used cancer standard care medicine carboplatin (carboplatin) and cisplatin (cisplatin) as the biological missile of bullet; Wherein, used telomerase inhibitor and only used a weight " gatherer " TAT or PTD as the biological missile of bullet; And used the biological missile of cancer standard care medicine carboplatin (carboplatin) and cisplatin (cisplatin), then use TAT or NLS to be used as double " gatherer " simultaneously.
Here it is a kind of twin biological missile; Wherein, Having used telomerase inhibitor only is responsible for bullet is sent in the Cytoplasm as the biological missile (weight " gatherer ") of bullet; Act on the hereditary material of intracytoplasmic HIV, used telomerase inhibitor and then be responsible for bullet is sent in the nucleus, act on the target spot of DNA as the biological missile (double " gatherer ") of bullet.
The 3rd characteristic of " compound type duplication double localization type intracellular vaccine " biological missile; Using " bonding albumen " exactly cooperatively interacts with " biological cross-linking agent "; Be used as " combined unit " of biological missile, so that stick together the bullet of biological missile and guidance device.In the ordinary course of things; Be to use " biological cross-linking agent " this chemical method to stick together the bullet of biological missile and guidance device, still, because " compound type duplication double localization type intracellular vaccine " biological missile has one heavy or double " guidance device "; Simple biology " cross-linking agent " this chemical method of using; The bullet of biological missile is bound up on " guidance device " of biological missile by error, therefore, is necessary to use " bonding albumen " to be used as " combined unit ".
" bonding albumen " is the notion that this patent people is proposed in " compound type duplication double localization type intracellular vaccine ".So-called " bonding albumen ", use a kind of being called as the exactly protein or the polypeptide (being generally antibody protein) of " bonding albumen ", one to one at first with a certain " guidance device " albumen; Be combined into " bonding type fusion rotein " jointly; Then, make this " bonding type fusion rotein " again, come and nucleotide or other chemical synthetic drug; Equally also be to be bonded together naturally one to one, thereby obtain a kind of novel " bonding type " biological missile.Here, at first need filter out earlier a kind of have can bind a certain nucleotide or the antibody protein of other chemical synthetic drug.
But; This " bonding type " biological missile has dual mode: a kind of is " pure bonding type " biological missile; Its feature be earlier can with the adherent antibody of biological missile and " guidance device " albumen, for example certain antibody and TAT unite and process " antibody-TAT-anitibody type " fusion rotein.Then, again with this " antibody-TAT-anitibody type " fusion rotein directly and biological missile stick together; Another kind then is " bonding type indirectly " biological missile.It is that first application " cross-linking agent " is bound up the bullet of biological missile and TAT.Meanwhile; Again with certain " guidance device " albumen; Certain hormone----LHRH for example; Be luteinizing hormone releasing hormone and certain also can with adherent antibody combined being made into of TAT " hormone---anitibody type " fusion rotein, then, again with this " hormone---anitibody type " fusion rotein directly and the TAT in the biological missile bullet stick together and get final product.Certainly; If the biological missile bullet is certain protein medicaments; P53 albumen for example; So, just do not need to use " bonding albumen ", to process " antibody---TAT---P53 protein type " fusion rotein just passable and directly " guidance device " antibody of biological missile is united with TAT and P53 albumen.
Biological cross-linking agent is the small molecule chemical compound; Molecular weight is generally between 200-600; Have 2 or more reactive terminal to specific groups (amino, sulfydryl etc.), thus can with 2 or more molecules respectively coupling these molecules are combined.In life science, use cross-linking agent that a lot of work are made a breakthrough dexterously.
Biological cross-linking agent is widely used in: membrane structure research, protein structure research, protein-protein interaction research; Biological missile research; Carrier protein is connected with haptenic, the immobilization of protein or other molecules, the labelling of antibody; Labelling shifts, and protein is connected with nucleic acid.
Bind proteic making, use be antibody library technology.
The dominant ideas of antibody library technology are that all antibody variable genes with certain animal are cloned in plasmid or the phage and express, and utilize different antigens to filter out and carry the clone of specific antibody gene, thereby obtain corresponding specific antibody.
The multiple important antibody of screening from antibody library is like the antibody of memebrane protein antigen, autoantigen, virus antigen.These demonstrate antibody library The Application of Technology potentiality.
The antibody library technology not only can the simulated animal immuning system generating antibody process, also have the advantage of many uniquenesses, make hybridoma technology be difficult to compare.The antibody library technology need not immunity, theoretically, and 10
6～10
8Storage capacity just possibly contain all antibody.Utilize antigen directly from non-immune animal antibody library, to filter out specific antibody, and can screen antibody to this species autoantigen.From people's antibody library, can be the McAb in people source fully, have been overcome the obstacle that is difficult to obtain people source McAb with hybridoma technology.In addition,, cultivate with low costly, be beneficial to a large amount of preparation high-purity antibody, carry out albumin crystal structural research and application because bacterial cell propagation is fast.Therefore, the antibody library technology will play important impetus to the development of biology and medical science.
The antibody library technology comprises following main process: clone a complete set of variable region gene of antibody, connect with relevant carrier, import the recipient bacterium system; Utilize conditions such as recipient bacterium albumen synthesis secretion; These gene expressions on surfaces such as antibacterial, phagies, are screened and increased, set up antibody library.
The four characteristics of " compound type duplication double localization type intracellular vaccine " biological missile is exactly to use " proto-oncogene antibody " (proto-oncogene lg), is used as " killing and wounding device " or " bullet " of biological missile.
Scfv or Fab that applications exploiting phage antibody library technique in oncotherapy is produced are little because of molecular weight, and tissue penetration property is good, gather in that tumor locus is dense, advantage, can filter out and can organize the antibody of holding back proto-oncogene, be used as the bullet of biological missile.And the scfv in total man source or Fab have avoided human body to produce the anti-Mus immunoreation of people especially fully.But also there are some problems in phage antibody library technique at present: the screening effeciency like phage antibody library is lower, and the phage of some high specifics is lost in screening process easily; Phage antibody particularly scfv only contains more than ten aminoacid, make some radioactive substances (as ' 9mTc) labelling difficulty, labeling effciency is low; Because the phage antibody molecular weight is little, decomposes in vivo and too fast it is restricted in the application aspect the RIT of tumor; The endocytosis of some phage antibodies possibly suppress the tumor killing cell effect of tumour medicine.
The antibody library triage techniques mainly comprises phage antibody library and ribosomal display technology.Phage antibody library technique: from immunity or not by separation antibody variable region gene the B cell of immunity; The a complete set of genetic fragment (like VH, VL) of pcr amplification antibody is cloned into respective carrier at random with VH, the VL genetic fragment of amplification in vitro, forms combinatorial library; Assortment of genes library is inserted the phage encoded memebrane protein gene III (g3) or gene VIII (g8) guide's series near downstream, the polypeptide that makes exogenous gene expression with the presented of fusion rotein coat protein gp III or-the N end of gp VIII.Direct, convenient, simple and direct with immobilization antigen through " affine combination-eluting-amplification " several circulations, filter out the antibody phage storehouse that expression specificity is good, affinity is strong efficiently.
The ribosomal display technology: genotype and phenotype are linked together, and the DNA of encoding proteins transcribes and translates external, because DNA has been carried out special processing and modification; As, remove 3 ' terminal termination codon, when ribosome is translated the mRNA end; Because lack termination codon, the 3 ' end that rests on mRNA does not break away from, thereby form protein-ribose-2mRNA trimer; With the specific aglucon immobilization of target protein; As: be fixed on ELISA micropore or magnetic bead surfaces, the ribosome trimer that contains target protein just can be filtered out in the elisa plate hole or on the magnetic bead, and the complex that screening and separating is obtained decomposes; The mRNA that discharges carries out reverse transcriptase chain polymerization reaction (RT-PCR); The PCR product gets into the next round circulation, through repeatedly circulation, finally can make the gene order of target protein and its coding obtain enrichment and separate.
Utilize the antibody library triage techniques, can filter out and to organize the antibody of holding back proto-oncogene.The proto-oncogene antibody that is screened can comprise common proto-oncogene family antibody, for example, src family antibody, ras family antibody, myc family antibody, or the like.
The 5th characteristic of " compound type duplication double localization type intracellular vaccine " biological missile; Used " polyethyleneglycol modified pharmaceutical grade protein matter and polypeptide drugs " packing technique exactly; Exist problem when the medication to solve " compound type duplication double localization type intracellular vaccine " biological missile; For example, because the molecular weight of protein and polypeptide drugs is not high, easily by glomerular filtration; Exist a lot of protease in the blood, pharmaceutical grade protein is decomposed and lose drug effect.These problems can cause the half-life of medicine short.In addition,, can produce antibody in the human body, make it lose drug effect with protein bound, and cause side effect for the exogenous protein medicine.Because the half-life of protein and polypeptide drugs is short, when medicine had just got in the body, concentration once can be too high, produces stronger side effect.But drug level can drop to active drug again immediately with below the concentration subsequently, and does not have therapeutical effect, therefore; Necessary frequent drug administration, for example rhG-CSF needs to inject every day 1 time, for this reason; Need pack it, this thing happens to avoid, for example; Can utilize Polyethylene Glycol that protein and polypeptide drugs are modified, the intravenous formulations of preparation long-acting protein and polypeptide drugs.Has lot of advantages with polyethyleneglycol modified pharmaceutical grade protein: (a) increase the molecular weight of pharmaceutical grade protein, reduce, improve the medicine half-life in vivo by the frequency of glomerular filtration; (b) protected protein matter is avoided the degraded of protease; (c) immunogenicity of shielding exogenous protein reduces side effect; (d) still can keep certain activity after protein is modified.
Polyethylene Glycol is that ethane via epoxyethane is polymerized, and is made up of multiple oxyethylene group.Not only has good water-solubility; Also can be dissolved in organic solvents such as dichloromethane, N`N`-dimethyl formamide, benzene, acetonitrile and ethanol; Chain structure with linearity (relative molecular weight 5000～30000) or branching (average molecular measures one's own ability 40000～60000), linear PEG molecular formula are H-(O-CH2-CH2) n-OH.Respectively there is a hydroxyl at common Polyethylene Glycol two ends; If an end then obtains methoxy poly (ethylene glycol) (mPEG) with the methyl sealing; The molecular formula of linear mPEG is CH3-(O-CH2-CH2) n-OH, and what application was maximum in polypeptide and proteinic Pegylation modification research is the derivant of mPEG.
The polyethyleneglycol modified of medicine is PEGization, is activated polyglycol is coupled on albumen, polypeptide, micromolecule organic drug and the liposome through chemical method.What wherein study at most is that proteinic PEG modifies, and starting from is the seventies in 20th century.Medicine is after PEG modifies; Tend to have the following advantages: 1, less 4, less enzyme degradation 5 of longer half-life 2, lower maximum plasma concentration 3, blood concentration fluctuation, less immunogenicity and antigenicity 6, less toxicity 7, better dissolubility 8, medicine frequency reduce 9, improve patient's compliance; Improve the quality of living, reduction medical expense 9, liposome have stronger passive target effect to tumor.The modification approach mainly contains amido modified (comprising the alkylation modification that the amino acidylate of acidylate modification, lysine side-chain is modified, the N end is amino that the N end is amino), carboxyl modified, sulfydryl modification to albumen and polypeptide; Also have other mPEG-NH2 to be transferred on the proteinic glutamine side chain like the imidazole group of the histidine side chain in the control pH realization SC-mPEG selective modification protein with glutamine transaminage; Realization is to the selective modification of glutamine; Wherein mainly be that N-terminal or lysine side-chain amino are carried out the acidylate modification; Because perhaps the more there are a plurality of amino in albumen in the peptide structure; So control and definite decorating site and degree of modification are the difficult points in albumen and polypeptide polyethyleneglycol modified always, can realize pointed decoration through adopting the proper protection strategy in peptides synthetic to amino, and the PEG of organic molecule medicine modification approach mainly with on PEG and these small-molecule drugs-OH;-NH2; The coupling of-COOH phase does not possess these functional groups like small-molecule drug to be finished, can introduce through chemical method.[" the polyethyleneglycol modified and research and the progress of application of medicine ".State person health.Beijng?Guoren?Yechology?Co.，LTD】。If in peptides synthetic, can control and definite decorating site and degree of modification through adopting the proper protection strategy, realize pointed decoration to amino, so, just can adopt " albumen adhesive " again.
The 6th characteristic of " compound type duplication double localization type intracellular vaccine " biological missile; Be exactly in the method for designing of " compound type duplication double localization type intracellular vaccine " biological missile; Or in the method for preparing, aspect engineering, adopt ripe and complete GFP engineering and antibody technique fully; Aspect anticancer disease and AIDS drug; Adopt existingly fully, be widely used in the clinical anticancer disease and the medicine of AIDS resisting, make the bullet of " compound type duplication double localization type intracellular vaccine " biological missile.This also is to make two basic principles that " compound type duplication double localization type intracellular vaccine " biological missile is followed.This two basic principles have been arranged, just guaranteed that " compound type duplication double localization type intracellular vaccine " biological missile has the feasibility of operation and the safety of use.
At first; So-called genetic engineering (genetic engineering) is the complex technology of on molecular level, gene being operated; Be with exogenous gene through importing in the recipient cell after the vitro recombination, make that this gene can duplicate, transcribes, the operation of accurate translation in recipient cell.It is with artificial method with needed a certain donor biological hereditary material---the DNA macromole extracts, under isolated condition with suitable
ToolenzymeAfter cutting, it and as
CarrierDna molecular couple together, import in a certain recipient cell that more is prone to growth, breeding with carrier then, to let allogenic material " settle down " therein, duplicate normally and express, thereby obtain new proteinic a kind of brand-new technology.Protein merges that a kind of proteinic portion gene of will encode is transplanted on the another kind of protein gene or with the fragment combination of different proteins gene together, through gene clone and expression, and the fused protein that generation makes new advances.This method can concentrate on the characteristic of different proteins on a kind of protein, changes proteinic characteristic significantly.The characteristics of fusion rotein are to express different proteic functional activities territory; The synergism of each component makes the fusion rotein performance optimised; Even producing new BA. fusion protein technology moves towards clinical practice from basic research, and is progressively ripe, improve and innovation to some extent. and the artificial protein of novel Multidomain has potential using value at many clinical fields. and this paper is to the main contents of fusion protein technology, development and in the application effect aspect the treatment of dna vaccination, multifunctional enzyme, targeted drug, thrombolytics, antibacterial peptide, ischemia reperfusion injury.. fusion protein technology also comprises many now what is called " chimeric antibody " and " popularity antibody " technology of research.
Antibody technique comprises humanized construction method of murine antibody and full humanization antibody technique.
The humanization modified main method of murine antibody comprises that chimeric antibody, surface reinvent antibody, reshaped antibody.
Chimeric antibody: utilize the DNA recombinant technique that light, the heavy chain variable region gene insertion of Mus monoclonal antibody are contained in the expression vector of people's antibody constant region; Transformed mammalian cell gives expression to human mouse chimeric antibody; Its humanization degree reaches about 70%; The variable region that has intactly kept the allos monoclonal antibody has kept its affine activity to greatest extent, has reduced immunogenicity.But, can bring out the anti-mice reaction of people HAMA because human mouse chimeric antibody has still kept 30% Mus source property.
Antibody is reinvented on the surface: carry out humanization modified to murine antibody surface amino groups acid residue.The principle of this method is only to replace and the tangible zone of people's antibody SAR difference, is keeping antibody activity and is taking into account to reduce and select the aminoacid replacement similar with people's antibody surface residue on the heterologous basis for use; In addition, the section of being replaced should be not too much, for influencing side chain size, electric charge, hydrophobicity, do not replace thereby maybe possibly form the residue that hydrogen bond has influence on complementary antibody determining area (CDR) conformation as far as possible.
Reshaped antibody:, comprise the transplanting of CDR district, portion C DR transplanting and specific determining area (SDR) transfer by combining relevant residue and people's antibody to splice structure again with antigen in the heterologous antibody.
Full humanization antibody technique.The formation of fully human antibodies starts from nineteen nineties, and obtaining full humanization antibody method at present has antibody library triage techniques, genetic engineering mice to prepare human antibody, transfection chromosome cattle.It comprises the antibody library triage techniques, phage antibody library technique, and ribosomal display technology.
Secondly; The bullet of " compound type duplication double localization type intracellular vaccine " biological missile all uses conventional clinical medicine, to guarantee safety; Or be proved to be effective cancer therapy drug; For example the carboplatin, the cisplatin that are proposed of this paper page 5 also has the P53 albumen of the 16th page of made human cancer cell apoptosis that is proposed in the back, and; Bullet with " compound type duplication double localization type intracellular vaccine " biological missile of AIDS resisting function, use be exactly the medicine AZT and the IDV of " HAART ".
Be the explanation of biological missile being used the relevant parameter of conventional biochemical product below.
The parameter of AZT:
[Chinese] zidovudine
[molecular formula] C
10H
13N
5O
4=267.242
[structural formula]
[another name] azidothymidine AZT; AZT; Azidothymidine; Zidovudine; ZDV; Retrovir
[pharmacological action] these article are nucleoside family congener, are the medicines of drugs approved by FDA treatment AIDS.After medicine absorbs; In the cell that is infected by HIV; But these article phosphorylation becomes triphosphoric acid azidothymidine AZT (AZTTP) and the viral reverse transcriptase of normal thymidylic acid competitive inhibition, and the deoxythymidine triphosphoric acid replaces the DNA of virus, makes the DNA chain growth stop; Can not continue to duplicate, thereby hinder synthesizing and viral breeding of core protein.Because AZTTP is stronger 100 times than cell dna polymerase to the affinity of viral reverse transcriptase, so its antivirus action is selective.
The parameter of IDV (indinavir sulfate)
CAS number: 157810-81-6
Indinavir sulfateEnglish name Indinavir sulfate
-2-hydroxyl-1H-indenes-1-
Base)-5-[2-[[(1, the 1-diformazan
The base ethyl) amino] carbonyl
]-4-(3-picolyl)-1-
Piperazinyl]-2-(benzyl
)-D-erythro form-pentanamide sulfur
Hydrochlorate; Indinavir sulfate; Sulfur
That dimension of acid indenes ground; That dimension of indenes ground
CBNum CB9123498
ber：
Molecular formula: C36H49N5O8S
Molecular weight: 711.87
MOL
157810-81-6.mol
File：
Parameter with " compound type duplication double localization type intracellular vaccine " biological missile " guidance device " gp120 of AIDS resisting function:
About 120 nanometers of HIV's diameter, roughly spherical in shape.Outer virionic membrane is a phospholipid bilayer, from host cell, and is embedded with viral albumen gp120 and gp41; Gp41 is a transmembrane protein, and gp120 is positioned at the surface, and combines through noncovalent interaction with gp41.Inwardly be the sphere matrix (Matrix) that forms by albumen p17, and the half-cone capsid of albumen p24 formation.Gp120 and gp41 are that the structural protein Env by HIV forms.
At first Env is the albumen of 160kD, and through glycosylation, the N that on agedoite, is coupled with 25 to 30 complicacies connects sugar chain, becomes gp160 in Golgi body; This glycosylation process is necessary to infectivity.Afterwards, host cell a protease is cut to gp41 and gp120 with gp160.
For another example: bullet carboplatin (carboplatin) and the parameter of cisplatin (cisplatin) with " compound type duplication double localization type intracellular vaccine " biological missile of anticancer disease function:
The parameter of Cisplatin
Common name: cisplatin for inj
Former name:
Trade name:
English name: Cisplatin for Injection
The Chinese phonetic alphabet: Zhusheyong shunbo
These article chemical name is: (Z)-two hydrazine dichloride platinum.
Its structural formula is
Molecular formula: PtCl
2(NH
3)
2
Molecular weight: 300.05
[character]
These article are glassy yellow or orange-yellow crystalline powder
[pharmacological toxicology]
These article are the metal complex of platinum, and effect is like alkylating agent, and main action target spot is DNA, acts on interlinkage in DNA interchain and the chain, forms DDP～DNA complex, disturbs dna replication dna, or combines with nucleoprotein and plasmosin.Non-specific medicine of genus cycle.
The parameter of carboplatin
Common name: carboplatin
Trade name: English name: CarboplatinforInjection
Chinese phonetic alphabet ZhusheyongKabo
These article chemical name is: cis-1,1-CBDCA CBDCA diamino platinum.
Structural formula is: molecular formula: C6H12N2O4Pt molecular weight: 371.2
[character]
These article are loose block of white or off-white color lyophilizing or powder
Also have, as the parameter of the P51 of protein medicaments:
The P53 gene is positioned at human No. 17
ChromosomeContain 11 exons, the wild type P53 albumen of its transcription and translation coding is by 393
Amino acid residueForm, comprise a plurality of functional domains.(activtiondomain, AD) AD1, AD2 are positioned at amino acid/11-50 to the terminal transcriptional activation domain of N-, combine with general transcription factor TF11D and bring into play transcriptional activation function.TF11D is the complex by TBP (TATAbinding protain) and TAF (TBPassociatedfactor) be combined into, and P53 combines with TAF among the TF11D, acts on the TATA box in the downstream gene promoter, reaches transcriptional activation function.P53 gene growth inhibited domain is positioned at aminoacid 65-90 position, and proline rich contains 5 multiple pxxp sequences, can with the protein interaction that contains the SH3 domain, P53 and means of information transmission are coupled together.
The P53 gene also has: sequence-specific DNA binding structural domain is positioned at amino acid/11 00-300 interdigit; Nuclear localization signal NLS is positioned at amino acid residue 316-325; Tetramer oligomerization domain is positioned amino acid residue 334-356; The terminal non-single-minded DNA adjustment structure of C-territory, when running into DNA damage, P53 possibly replenish other protein to damage location, and the DNA damage signal is provided simultaneously.P53 is not to combine with DNA specifically with the binding ability of DNA, participates in the bonded other structure of core space and DNA and regulates, and when running into DNA damage, P53 possibly replenish other protein to damage location, and the DNA damage signal is provided simultaneously.
Being noted that P53 albumen is self-contained nuclear localization signal NLS arranged of value like this, when making anticancer disease biological missile, just do not needed second weight " gatherer " NLS.The experiment proof: aspect cancer research, the accent that has utilized PTD to carry the protein induced cancerous cell of P53 is died.Simultaneously; The health cell is also possessed little P 53 albumen, therefore, as long as accomplish mode with precise guidance; P53 albumen is induced one in the cancerous cell, exactly [" trans-activation is transcribed the progress of fusion rotein " " Yunnan medicine " the 28th volume the 6th phase Yuan Shunhui in 2007 work] of safety.
The proteic parameter of protein medicaments P105.
The Rb gene is the tumor suppressor gene of finding the earliest, is found in child's retinoblastoma the earliest, therefore is called the Rb gene.In case when loss of function of Rb gene or congenital deficiency, abnormality proliferation then appears in retinoblastoma cell, form retinoblastoma.The Rb gene inactivation also is shown in kinds of tumors, has certain popularity.The Rb genetic comparison is big, and coded protein is P105, and being positioned has two kinds of forms of phosphorylation and non-phosphorylating in the nuclear, and the non-phosphorylating form is claimed active form, can promote cell differentiation, suppresses cell proliferation.The inhibitory action of Rb gene pairs tumor is relevant with transcription factor (E-2F).E-2F is the activated protein of a Class Activation Transcription, and in G0, G1 phase, the Rb albumen and the E-2F of low phosphorylation type are combined into complex, make E-2F be in the disactivation state; In the S phase, Rb albumen is dissociated with E-2F by phosphorylation, and the E-2F of bonding state becomes free state, and cell gets into the multiplicative stage immediately.When disappearance or sudden change take place the Rb gene, lose the ability that combines, suppresses E-2F, so cell proliferation is active, cause tumor to take place.
Common oncogene family:
(1) src family
They all contain similar gene code structure, and product has the protein kinase activity that makes tyrosine phosphorylation, are positioned the after birth inner face or stride film to distribute.
(2) ras family
Family member's gene order difference is big, but coded protein is P21, is positioned at the cytoplasma membrane inner face, and P21 can combine with GTP, and the GTP enzymatic activity is arranged, and participates in the adjusting of cAMP level.
(3) myc family
The coding nuclear dna is conjugated protein, and the effect of direct other genetic transcriptions of adjusting is arranged.
(4) sis family
Sis family has only member of sis gene, and coding P28 with human blood platelets source somatomedin similar, stimulates mesenchymal tissue cell division propagation.
(5) myb family
Coding nucleoprotein can combine with DNA, is a kind of transcription factor in examining.
[" oncogene, antioncogene and somatomedin " " Chinese education is online " www.eol.cn]
As for " guidance device " aspect, the biochemical product of many routines is arranged also.
(1) antibody class
1。Monoclonal antibody (McAb) is an antibody with the most use in the current targeted therapy because of its specificity is high.Can be divided into Mus source property McAb and humanized McAb.
2。The antibody that genetic engineering antibody is processed the effective fragment in the anti-tumour antibody with engineered method can divide three kinds: people-mouse chimeric antibody, reshaping antibody and total manization antibody.
1。The recombiant protein of interleukin-2 (IL-2) and diphtheria toxin, diphtherotoxin (common name Denileukin diftitox, trade name Ontak): recombiant protein contains 387 aminoacid of diphtheria toxin, diphtherotoxin and 133 aminoacid of interleukin-2 (IL-2).Recombiant protein is at expression in escherichia coli, and its molecular weight is 58kD.Interleukin-2 (IL-2) can be incorporated into the interleukin-2 receptor, make diphtheria toxin, diphtherotoxin part among the Ontak special kill the cell of expressing the interleukin-2 receptor.
2, anti-CD 20 antibodies: CD20 be people's B cell differentiation antigen (B-lymphocyte-restricteddifferentiationantigen, Bp35).CD20 is expressed in immaturity and sophisticated bone-marrow-derived lymphocyte (pre-B andmature B lymphocytes) surface, but is not expressed on hematopoietic stem cell, normal plasma cell, pro-B cell and other normal structures.B cell non-Hodgkin's more than 90% (Non-Hodgkin ' s Lymphoma, NHL) express CD20, so closely related with non_hodgkin lymphoma and other B cell differentiation property disease (B-cellLymphoproliferative diseases)
3。Anti-CD 33 antibody: CD33 antigen is the adhesion protein that a kind of sialic acid relies on, and (acute myeloid leukemia AML) expresses on patient's the leukemic blasts acute leukemia 80%.CD33 also is expressed in bone marrow colony forming cell (myeloid colony-forming cells) normal and that cancerate, but is not expressed on dried hematopoietic stem cell of multipotency and the non-hematopoietic stem cell.(Gemtuzumab Ozogamicin, trade (brand) name Mylotarg) by the development of Wyeth-Ayerst company, are gone on the market by the FDA approval in May, 2000 with the bonded anti-CD 33 antibody of immunotoxin calicheamicin.
4, be the monoclonal antibody of target molecule with EGF-R ELISA (EGFR).
(2) hormones
Mainly be luteinising hormone-releasing hormo (LHRH).The luteinising hormone-releasing hormo receptor can be used as the target of some malignant cell. and the binding site of luteinising hormone-releasing hormo (LHRH) all has higher statement in human breast carcinoma, ovary and endometrial tumors and prostate gland cancer cell; And therefore rare expression in people's normal structure becomes the nude mice tumor growth that a kind of effective malignant tumor specific receptor .LHRH recombinant toxin can effectively suppress to inoculate LHRH receptor positive tumor. [" the Chinese biological goods are learned magazine＞2007 year 10 phases " " luteinising hormone-releasing hormo and targeting toxins thereof " author Zheng Yan Zheng Yan of army rosy clouds Zhang Guoli]
(3) epidermal growth factor subclass
With EGF-R ELISA (EGFR) is the monoclonal antibody of target molecule.
EGF-R ELISA (Epidermal Growth Factor Receptor EGFR) is the expression product of proto-oncogene c-erb-B1.It crosses expression and mutant all has substantial connection with tumor development, becomes the novel targets of present oncotherapy based on this advantage.
EGFR is a kind of transmembrane glycoprotein, and molecular weight is 170KD.Be distributed widely on each cells of tissues film of human body
[1,2]Its in most of tumors (like bladder cancer, nonsmall-cell lung cancer, ovarian cancer; Incidence scale cancer, breast carcinoma, glioma, cancer of pancreas, gastric cancer, carcinoma of prostate, the esophageal carcinoma etc.) cross to express with (or) sudden change, make the out of control and malignization of cell growth by means of signal transduction
[3]Expression rate is different in various tumor tissues, and with differentiation degree, the grade malignancy of tumor and to invade the profit degree closely related.
Abgenix company uses the XenoMouse technology to produce humanization IgG2 monoclonal antibody ABX-EGF.With EGFR higher affinity (5 ★ 10-11M) is arranged, and long half time.Can stop the combining of human tumor cell line of epidermal growth factor EGF, transforming growth factor TGF α and multiple expression EGFR, suppress the activation that EGF relies on tumor cell.ABX-EGF stops the generation [5] of tumor in nude mice and human body, and the negative tumor of EGFR is not had influence.[" EGF-R ELISA EGFR monoclonal antibody and oncotherapy progress " " Chinese tumor biotherapy journal 2003, the 10th volume the 3rd phase P221-222].
More than be exactly the explanation to the six big characteristics of " compound type duplication double localization type intracellular vaccine " biological missile, they also set up the basis of " compound type duplication double localization type intracellular vaccine " biological missile design just.
Set up the design of " compound type duplication double localization type intracellular vaccine " biological missile, set up the design of the molecular structure of this biological missile exactly.
At first, need set up the general formula of the molecular structure of " compound type duplication double localization type intracellular vaccine " biological missile, with pattern as design.
If: with the first capitalization of the Chinese phonetic alphabet of the Chinese character title of every kind of molecule; Represent this molecule, for example, " guidance device (ZH_ZH) "; " bind albumen (N_D) "; " kill and wound device or bullet (SH_ZH), and represent the use cross-linking agent, connect with the fusion rotein mode with " " expression with " * ".
(1) AIDS resisting " compound type duplex duplication double localization type intracellular vaccine " biological missile general formula of molecular structure:
《ZH_ZH+TAT+N_D》+SH_ZH
General formula unit: molecule.
General formula representes earlier by guidance device and TAT, and can with kill and wound the adherent protein antibodies of device (bullet) and constitute fusion rotein, then, bind mutually with bullet automatically again.
(2) anticancer disease " compound type duplex duplication double localization type intracellular vaccine " biological missile general formula of molecular structure:
(1) the anticancer disease of protide " compound type duplex duplication double localization type intracellular vaccine " biological missile general molecular formula:
(a)《ZH_ZH+TAT+SH_ZH》
(b)《ZH_ZH+TAT+N_D》+SH_ZH×NLS
(2) biochemical type of anticancer disease " compound type duplex duplication double localization type intracellular vaccine " biological missile general molecular formula:
《ZH_ZH+TAT+N_D》+SH_ZH
General formula unit: molecule.
The molecular structural formula of (a) formula of (1) in (one) formula; The anticancer disease biological missile of expression protide can be directly by guidance device and TAT; And as the protein medicaments of bullet, for example, P53 albumen constitutes fusion rotein. the molecular structural formula of (b) formula of (1) formula; Expression b type biological missile is represented earlier by guidance device and TAT; And can with kill and wound the adherent protein antibodies of device (bullet) and constitute fusion rotein, then, the combination molecule with NLS that uses biological cross-linking agent to be bound up and protein medicaments bullet binds mutually again.This is because the employed protide bullet of b type biological missile does not comprise appraises and decides the position order.
The molecular structural formula of (2) formula, biochemical type of biological missile of expression represent earlier by guidance device and TAT, and can with kill and wound the adherent protein antibodies of device (bullet) and constitute fusion rotein, then, more automatically and the bullet as biochemical drug bind mutually.
Below, again according to above general formula of molecular structure, provide several concrete designs, or concrete molecular structural formula.Need to prove that especially concrete molecular structural formula and specific molecular structural formula are two different concept: the specific molecular structure formula is to be made up of specific molecule fully, its a kind of prescription patent medicine; And concrete molecular structural formula had both comprised specific molecule, comprised a certain type of drug molecule, the for example structural formula of antibody class molecule again.
(1) device of AIDS resisting biological missile guidance
(1) guidance device of AIDS resisting biological missile: gp120 albumen.
(2) the AIDS resisting biological missile kill and wound device: AIDS resisting " cocktail " medicine of therapy, AZT, IDV or the like.
3) importing of AIDS resisting biological missile dress: import albumen TAT.
(4) special conglutinating antibody albumen (Bonding Ig) N_D of AIDS resisting biological missile
(2) design of AIDS resisting biological missile
| ← fusion rotein → | molecule/unit
(3) guidance device of anticancer biological missile
(1) anticancer biological missile guidance device
(a) protein antibodies class: can with embryonal antigen---the antibody (AFP-Ig) that the receptor of alpha-fetoprotein (AFP) combines, anti-CD 20 antibodies, anti-CD 33 antibody, or the like.
(b) hormones: luteinising hormone-releasing hormo (LHRH)
(c) somatomedin class: humanization IgG2 monoclonal antibody ABX-EGF
(2) anticancer biological missile kills and wounds the several types of device
(a) albumen and polypeptide class: P53 albumen, P105 albumen, proto-oncogene antibody (proto-oncogene lg), or the like.
(b) anti-reverse transcription enzyme medicine class, purpose are that anti-telomerase reverses: AIDS resisting " cocktail " medicine of therapy, AZT, IDV and anticancer disease drug: carboplatin carboplatin and cisplatin cisplatin, or the like.
(3) gatherer of anticancer biological missile
(a) Cytoplasm conduction device: import albumen TAT.
(b) nucleus conduction device: appraise and decide position order NLS.
(4) the pharmaceutical carrier device of anticancer biological missile
Nano-carrier, tree-shaped polymer () T-PEG-PAMAM). or the like.
(4) design of the fusion rotein of anticancer biological missile
(1) design of the fusion rotein of protide or the anticancer biological missile of polypeptide class
| ← fusion rotein district → | molecule/unit
Since P53 albumen self-contained appraise and decide a position order NLS, therefore,, just no longer added NLS here.
| ← fusion rotein district → | molecule/unit
The situation that has occurred " the double antibody connection " of two kinds of heterogeneity antibody here.
(2) design of the fusion rotein of the anticancer biological missile of biochemical molecular class
| ← fusion rotein district → | molecule/unit
More than the medicine of anticancer biological missile, comprised two types of design of the fusion rotein of the anticancer biological missile of biochemical molecular class, a kind of its bullet is with to appraise and decide the position order crosslinked, another kind of with appraise and decide the position and exchange couplet in proper order.They can be copied ' HAART ' to mix and use.
For the device that kills and wounds of the fusion rotein of the anticancer biological missile of biochemical molecular class,, just need not add and not appraise and decide the position order if they can be advanced in the nucleus certainly.Meanwhile, for the stability of the fusion rotein of protecting anticancer biological missile, all should be attached on the pharmaceutical carrier device.For example, can use " polyethyleneglycol modified pharmaceutical grade protein matter and polypeptide drugs " packing technique.
In sum; Because the design of " compound type duplex duplication double localization type intracellular vaccine " biological missile; Abideed by in two illustrated big basic principles of the 6th characteristic, therefore, the medicine of the biological missile of making according to this design has just had the assurance of feasibility and safety.Further in the reality of clinical practice, it is just passable to test for these needs.Because there are several hundred million cancer patients and several ten million AIDS VICTIMSs in the whole world, therefore, the biological missile of being developed according to this patent design must have wide application market meanwhile!
Claims (10)
1. the routine techniques that should plant with gene protein engineering and antibody protein engineering; According to the basic skills by " compound type dual duplex locating type intracellular vaccine " of the independent development of patent inventor institute, the design of the state-of-the art anticancer disease AIDS resisting biological missile of being invented.It is characterized by: it has adopted a kind of dual " gatherer " with respect to common biological missile more; Be a kind of the associating to use a technology that imports albumen TAT and appraise and decide " dual lead-in " biological missile of position order NLS, can anticancer disease and AIDS drug bullet directly be sent in Cytoplasm and the nucleus, therefore overcome the weak point of common biological missile; Can accurately act on the target spot of interior viral genetic of diseased cells matter or cell nuclear dna; Therefore, medical function is big, and toxicity is little; Can not constitute a threat to intravital other healthy cells of machine; For this reason, used a kind of the associating therein and used biological cross-linking agent and bind the albumen new technique, and used " polyethyleneglycol modified pharmaceutical grade protein matter and polypeptide drugs " packing technique simultaneously.Meanwhile, this patent or in the method for preparing, aspect engineering, adopts ripe and complete GFP engineering and antibody technique fully in the method for designing of " compound type duplication double localization type intracellular vaccine " biological missile; Aspect anticancer disease and anticancer disease and AIDS drug, adopt existingly fully, be widely used in the clinical anticancer disease and the medicine of AIDS resisting, make " compound type duplication double localization type intracellular vaccine " biological missile, these two basic principles.This two basic principles have been arranged, just guaranteed that " compound type duplication double localization type intracellular vaccine " biological missile has the feasibility of operation and the safety of use.On the basis of the above, this patent has proposed the general general formula of molecular structure of " compound type duplication double localization type intracellular vaccine " biological missile, and the concrete molecular structural formula of anticancer disease and AIDS resisting biological missile.
2. according to right 1; This patent has creatively designed a kind of dual " gatherer ", can the medicine bullet directly be sent in Cytoplasm and the nucleus, has therefore overcome the weak point of common biological missile; Can accurately act on the target spot of interior viral genetic of diseased cells matter or cell nuclear dna; Therefore, medical function is big, and can not constitute a threat to intravital other healthy cells of machine; Toxicity is little, it is characterized by: the TAT albumen of first weight " gatherer " or PTD polypeptide.Can't get into nucleus because molecular weight is higher than the macromole of 15kd, this just needs second weight " gatherer " " to appraise and decide the position order " (NLS).Here it is " dual lead-in " biological missile.Meanwhile; " compound type duplication double localization type intracellular vaccine " biological missile; Can also produce respectively a weight " gatherer " only is installed, only can get into intracytoplasmic biological missile, perhaps produce dual " gatherer " is installed; Can get into endonuclear biological missile, be a kind of " twin " biological missile.
3. according to right 1; This patent has creatively been used " HAART " and has been used biological missile; And be duplex and usefulness; It is characterized by: use telomerase inhibitor and come the co-therapy cancer with the biological missile of having used cancer standard care medicine carboplatin (carboplatin) and cisplatin (cisplatin), wherein, used telomerase inhibitor and only used a weight " gatherer " TAT or PTD as the biological missile of bullet as the biological missile of bullet; The biological missile of having used cancer standard care medicine carboplatin (carboplatin) and cisplatin (cisplatin) then; Use TAT or NLS to be used as double " gatherer " simultaneously; Meanwhile, it still is a kind of twin biological missile, wherein; Having used telomerase inhibitor only is responsible for bullet is sent in the Cytoplasm as the biological missile (weight " gatherer ") of bullet; Act on the hereditary material of intracytoplasmic HIV, used telomerase inhibitor and then be responsible for bullet is sent in the nucleus, act on the target spot of middle A among the DN as the biological missile (double " gatherer ") of bullet.
4. according to right 1; This patent is creatively used " bonding albumen " and is cooperatively interacted with " biological cross-linking agent "; Be used as " combined unit " of biological missile, so that stick together the bullet of biological missile and guidance device, it is characterized by: in the ordinary course of things; Be to use " biological cross-linking agent " this chemical method to stick together the bullet of biological missile and guidance device; But because " compound type duplication double localization type intracellular vaccine " biological missile has one heavy or double " guidance device ", simple biology " cross-linking agent " this chemical method of using may make the bullet of biological missile be bound up on by error on " guidance device " of biological missile; Therefore, be necessary to use " bonding albumen " to be used as " combined unit ".Biological cross-linking agent is the small molecule chemical compound, and molecular weight generally between 200-600, has 2 or more reactive terminal to specific groups (amino, sulfydryl etc.); Thereby can with 2 or more molecules respectively coupling these molecules are combined; Biological cross-linking agent is widely used in: membrane structure research, protein structure research, protein-protein interaction research; Biological missile research, carrier protein is connected with haptenic.
5. according to right 1; This patent is creatively used " proto-oncogene antibody " (proto-oncogene lg); Be used as biological missile " killing and wounding device " or " bullet "; It is characterized by: utilize antibody library technology antibody library triage techniques, filter out and to organize the antibody of holding back proto-oncogene, be used as the bullet of biological missile.For example, can filter out common proto-oncogene antibody: src family antibody, ras family antibody, myc family antibody, or the like.
6. according to right 1; This patent creatively used biological missile protective package technology; It is characterized by: adopted " polyethyleneglycol modified pharmaceutical grade protein matter and polypeptide drugs " packing technique, existed problem to solve " compound type duplication double localization type intracellular vaccine " biological missile when the medication, for example; Because the molecular weight of protein and polypeptide drugs is not high, easily by glomerular filtration; Exist a lot of protease in the blood, pharmaceutical grade protein is decomposed and lose drug effect.These problems can cause the half-life of medicine short.In addition,, can produce antibody in the human body, make it lose drug effect with protein bound, and cause side effect for the exogenous protein medicine.The polyethyleneglycol modified of medicine is PEGization, is activated polyglycol is coupled on albumen, polypeptide, micromolecule organic drug and the liposome through chemical method.The modification approach mainly contains amido modified (comprising the alkylation modification that the amino acidylate of acidylate modification, lysine side-chain is modified, the N end is amino that the N end is amino), carboxyl modified, sulfydryl modification to albumen and polypeptide; Also have other mPEG-NH2 to be transferred on the proteinic glutamine side chain like the imidazole group of the histidine side chain in the control pH realization SC-mPEG selective modification protein with glutamine transaminage; Realization is to the selective modification of glutamine; Wherein mainly be that N-terminal or lysine side-chain amino are carried out the acidylate modification; Because perhaps the more there are a plurality of amino in albumen in the peptide structure; So control and definite decorating site and degree of modification are the difficult points in albumen and polypeptide polyethyleneglycol modified always, can realize pointed decoration through adopting the proper protection strategy in peptides synthetic to amino, and the PEG of organic molecule medicine modification approach mainly with on PEG and these small-molecule drugs-OH;-NH2; The coupling of-COOH phase does not possess these functional groups like small-molecule drug to be finished, can introduce through chemical method.
7. according to right 1; This patent has creatively proposed to make the method for designing of " compound type duplication double localization type intracellular vaccine " biological missile have feasibility; The safety of medicine that should follow and reliable these two basic principles of technology; It is characterized by: in method for preparing, aspect engineering, adopt ripe and complete GFP engineering and antibody technique fully; Aspect anticancer disease and AIDS drug, adopt existingly fully, be widely used in the clinical anticancer disease and the medicine of AIDS resisting, make the bullet of " compound type duplication double localization type intracellular vaccine " biological missile.This also is to make two basic principles that " compound type duplication double localization type intracellular vaccine " biological missile is followed.This two basic principles have been arranged, just guaranteed that " compound type duplication double localization type intracellular vaccine " biological missile has the feasibility of operation and the safety of use.
8. according to right 1; This patent has creatively designed the general formula of the molecular structure of " compound type duplication double localization type intracellular vaccine " biological missile, and the pattern with as design is characterized by: establish: with the first capitalization of the Chinese phonetic alphabet of the Chinese character title of every kind of molecule; Represent this molecule; For example: " guidance device (ZH_ZH) ", " bind albumen (N_D) ", " kill and wound device or bullet (SH_ZH); and represent the use cross-linking agent with " * ", represent to connect with " " with the fusion rotein mode.
(1) AIDS resisting " compound type duplex duplication double localization type intracellular vaccine " biological missile general formula of molecular structure:
《ZH_ZH+TAT+N_D》+SH_ZH
General formula unit: molecule.
General formula representes earlier by guidance device and TAT, and can with kill and wound the adherent protein antibodies of device (bullet) and constitute fusion rotein, then, bind mutually with bullet automatically again.
(2) anticancer disease " compound type duplex duplication double localization type intracellular vaccine " biological missile general formula of molecular structure:
(1) the anticancer disease of protide " compound type duplex duplication double localization type intracellular vaccine " biological missile general molecular formula:
(a)《ZH_ZH+TAT+SH_ZH》
(b)《ZH_ZH+TAT+N_D》+SH_ZH×NLS
(2) biochemical type of anticancer disease " compound type duplex duplication double localization type intracellular vaccine " biological missile general molecular formula:
《ZH_ZH+TAT+N_D》+SH_ZH
General formula unit: molecule.
The molecular structural formula of (a) of (1) formula; The anticancer disease biological missile of expression protide can be directly by guidance device and TAT; And as the protein medicaments of bullet, for example, P53 albumen constitutes fusion rotein. the molecular structural formula of (b) of (1) formula; Expression b type biological missile is represented earlier by guidance device and TAT; And can with kill and wound the adherent protein antibodies of device (bullet) and constitute fusion rotein, then, the combination molecule with NLS that uses biological cross-linking agent to be bound up and protein medicaments bullet binds mutually again.This is because the employed protide bullet of b type biological missile does not comprise appraises and decides the position order.
Biochemical type of biological missile of (2) formula represent earlier by guidance device and TAT, and can with kill and wound the adherent protein antibodies of device (bullet) and constitute fusion rotein, then, more automatically and the bullet as biochemical drug bind mutually.
9. according to right 1, this patent is the general formula of the molecular structure of design consideration " compound type duplication double localization type intracellular vaccine " biological missile creatively, has provided the concrete design of AIDS resisting biological missile, it is characterized by:
(1) device of AIDS resisting biological missile
(1) guidance device of AIDS resisting biological missile: gp120 albumen.
(2) the AIDS resisting biological missile kill and wound device: AIDS resisting " cocktail " medicine of therapy, AZT, IDV or the like.
(3) gatherer of AIDS resisting biological missile: import albumen TAT.
(4) special conglutinating antibody albumen (Bonding Ig) N_D of AIDS resisting biological missile
(2) design of AIDS resisting biological missile
| ← fusion rotein district → | molecule/unit
10. according to right 1, this patent is the general formula of the molecular structure of design consideration " compound type duplication double localization type intracellular vaccine " biological missile creatively, has provided the concrete design of anticancer biological missile, it is characterized by:
(1) device of anticancer biological missile
(1) anticancer biological missile guidance device
(a) protein antibodies class: can with embryonal antigen---the antibody (AFP-Ig) that the receptor of alpha-fetoprotein (AFP) combines, anti-CD 20 antibodies, anti-CD 33 antibody, or the like.
(b) hormones: luteinising hormone-releasing hormo (LHRH)
(c) somatomedin class: humanization IgG2 monoclonal antibody ABX-EGF
(2) anticancer biological missile kills and wounds the several types of device
(a) albumen and polypeptide class: P53 albumen, 105 albumen, proto-oncogene antibody (proto-oncogene lg), or the like.
(b) anti-reverse transcription enzyme medicine class, purpose are that anti-telomerase reverses: AIDS resisting " cocktail " medicine of therapy, AZT, IDV and anticancer disease drug: carboplatin carboplatin and cisplatin cisplatin, or the like.
(3) gatherer of anticancer biological missile
(a) Cytoplasm conduction device: import albumen TAT.
(b) nucleus conduction device: appraise and decide position order NLS.
(4) the pharmaceutical carrier device of anticancer biological missile
Nano-carrier, tree-shaped polymer OT-PEG-PAMAM). or the like.
(2) design of the fusion rotein of anticancer biological missile
(1) design of the fusion rotein of protide or the anticancer biological missile of polypeptide class
| ← fusion rotein district → | molecule/unit
| ← fusion rotein district → | molecule/unit
The phenomenon that has occurred " the double antibody connection " of two kinds of heterogeneity antibody here.
(2) design of the fusion rotein of the anticancer biological missile of biochemical molecular class
| ← fusion rotein district → | molecule/unit
More than the medicine of anticancer biological missile, comprised two types of design of the fusion rotein of the anticancer biological missile of biochemical molecular class, a kind of its bullet is with to appraise and decide the position order crosslinked, and is another kind of with to appraise and decide the position order crosslinked.They can be copied ' HAART ' to mix and use.
For the device that kills and wounds of the fusion rotein of the anticancer biological missile of biochemical molecular class, if they can be advanced in the nucleus certainly, for example, P53 albumen just need not add and not appraise and decide the position order.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110075319A (en) * | 2019-04-29 | 2019-08-02 | 白涛 | A kind of anti-hereditary disease biological missile |
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| CN1896230A (en) * | 2006-06-22 | 2007-01-17 | 复旦大学 | Fi-C31 integrase modified TAT and NLS polypeptide and its use |
| CN101259267A (en) * | 2007-03-09 | 2008-09-10 | 白涛 | Compound type dual duplex locating type intracellular vaccine |
| KR100902634B1 (en) * | 2007-05-30 | 2009-06-15 | 한양대학교 산학협력단 | Nucleic acid delivery complex comprising recombinant hmgb-1 peptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104689339A (en) * | 2015-03-17 | 2015-06-10 | 白涛 | Direct-transfer type multipurpose biological missile and manufacturing method thereof |
| CN110075319A (en) * | 2019-04-29 | 2019-08-02 | 白涛 | A kind of anti-hereditary disease biological missile |
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