CN101607984A - A kind of multi-copy polypeptide and application thereof - Google Patents
A kind of multi-copy polypeptide and application thereof Download PDFInfo
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Abstract
The invention relates to a kind of multi-copy polypeptide and application thereof.This peptide molecule structure is Xn-Linker-Y, wherein X and Y represent biological function different but the polypeptide or the oligopeptides fragment of tool dependency respectively, by the Linker that forms by Methionin multiple copied X is connected with single copy Y, by the biological activity of the raising of the biological synergetic effect between two kinds of fragments product, Zhi Bei polypeptide is applicable to biomedicine field in this way.
Description
Technical field
The present invention relates to by artificial design with two kinds of polypeptide with different biological function or the oligopeptides fragment connects and using artificial synthetic mode is prepared the polypeptide product with high biological activity effect.Can be used as polypeptide drugs, peptide para-immunity toughener or polypeptide vaccine with the polypeptide product of the manner preparation is applied to treatment of diseases and prevention.The invention belongs to biomedicine field.
Technical background
The gene of organism are stored on the polynucleotide chain, and gene are being encoded and carried out the protein of biological function.Biological intravital protein is varied, and they are exercising the activity that earns a bare living of various biological functions.Though proteinic kind is unequal to more lift, the natural amino acid that they are all existed by 20 natures is basically formed.Because amino acid whose composition and the difference that puts in order have caused proteinic varying.In general, contain that amino acid whose molecule is called as protein more than 50, as surpass 10 amino acid whose peptide chains and be called as polypeptide, be less than 10 amino acid whose peptide chains and be referred to as oligopeptides.Find that at present the minimum little peptide of function has only 2 amino acid, the common little peptide of the function more than 4 comparatively commonly.
Because finishing and carrying out that the human protein batch total is drawn of the Human Genome Project will have increasing protein function fragment and be found and be used as medicinal application to biomedicine field.The polypeptide drugs of now having developed and being applied clinically have " pitocin ", " Thymosin alpha 1 ", " thymopeptide-5 " etc., the polypeptide drugs " Sostatin " that have basic enterprising pedestrian's wage reform body plan at natural peptide chain to become fully to be used for the treatment of digestive tract hemorrhage and acromegaly and " HIRULOG " of blood coagulation resisting function etc.Function fragment in the protein usually can screen the polypeptide fragment that contains tens or be so small to have only two amino acid compositions, and they drop into to use for the artificial synthetic polypeptide function fragment and with their and lay a good foundation.
Ectogenic pathogen comprises that toxin that various bacteriums, virus, mycoplasma, chlamydozoan, various animal or plant produce, plant pollen etc. enter the people and know from experience and cause body and produce immunological rejection.The tumour cell that morphs in the human body carries tomour specific albumen or the unusual tumor correlated albumen that produces that normal cell does not possess equally.These foreign peoples's protein moleculars are excitating organism immune system recognition and the molecular basis that repels external source pathogen and endogenous varient.
Along with development of biology, the differential protein molecule of many pathogens and tumor cell surface is identified that characteristics such as the avtive spot in these molecules, functional domain, antigenicity fragment are progressively found.Can utilize the method for polypeptide synthetic they synthetic preparing according to these characteristics and prior art.The polypeptide or the oligopeptides that will possess special biological according to artificial design in advance link together, the technology new or highly active functional polypeptide product that produces through artificial process and remould is referred to as " polypeptide engineering ", and it is after " genetically engineered " and the true tumor technical field of being expanded.Along with finishing and the deep development of " polypeptide engineering " of the Human Genome Project, will have increasing polypeptide drugs and enter people's lives.
Summary of the invention
Main purpose of the present invention is to provide a kind of structure design mode of new polypeptide drugs, technical problem to be solved is to make two kinds to have the different biological function, but segmental one of them form with multiple copied of the polypeptide of tool dependency or oligopeptides is connected with each other, thereby the synergy of utilizing the generation of two kinds of different biological function fragments can be brought into play the effect of function fragment better, make the synthetic polypeptide product improve corresponding biological action, be suitable for practicality more, and have the utility value on the industry.
The object of the invention to solve the technical problems realizes by the following technical solutions.Different but the polypeptide function fragment of tool dependency is designed to the frame mode of Xn-Linker-Y with two kinds according to theory of the present invention, wherein, X is " first function fragment ", is polypeptide or the oligopeptides fragment with biological action or function; N represents the copy number of X; Y is " second function fragment ", is biologically active polypeptides or oligopeptides fragment different with above-mentioned " first function fragment " but the tool dependency; But Linker serves as reasons and has two amino Methionin (Lys of the amino acid whose activation of condensation, or K) forms and represent with＞K-, the N-terminal amino of the Linker chain that the carboxyl of X fragment by its carboxyl terminal of above-mentioned tool n copy and Methionin are constituted is done condensation and is connected, and the carboxyl on the carboxyl terminal of Linker and the aminoterminal that singly copies Y condensation mutually are connected to form the structure of Xn-Linker-T.The objective of the invention is for solving but the polypeptide fragment of tool dependency is prepared the design of highly active polypeptide product by the mode of connection of multiple copied two kinds of difference in functionalitys, it may be used in the biomedicine field, and its technical problem can be applied to the following technical measures to achieve further.
Invent described Xn-Linker-Y polypeptide structure, the wherein said Linker structure that is made of Methionin (Lys or K) can be:＞K-,＞K
2＞K-or＞K
4＞K
2＞K-,＞K-representative has two can be for the amino Methionin of the amino acid whose activation of condensation.Can utilize " second function fragment " middle N end inherent Methionin of Y representative to open the connection of beginning to do multiple copied.
Invent the polypeptide product of described Xn-Linker-Y structure, the copy number n of wherein said " first function fragment " X can be 2,4 or 8.On the amino acid side chain group in the polypeptide product of Xn-Linker-Y structure, the aminoterminal of product or carboxyl terminal carry out hydroxylation, carboxylated, carbonylation, methylate, acetylize, phosphorylation, esterification, glycosylation, interpolation amino acid or other any chemical elements obtain product.In multi-copy polypeptide, in the Linker that forms by Methionin, insert the product that arbitrary amino acid or other any chemical elements obtain.Polypeptide product according to design preparation of the present invention can be polypeptide drugs, immunostimulant or polypeptide vaccine.
In the human immune system, be applied as object lesson with peptide molecule, the design and the application of polypeptide product involved in the present invention is illustrated.The immunity system of human body mainly contains the humoral immunization two parts that cellular immunization that the T lymphocyte bears and bone-marrow-derived lymphocyte bear and forms.They are resisted the infringement of external pathogen and play important effect safeguarding the health of body.
The fabricius bursa is the peculiar immune organs of birds, is the place that bone-marrow-derived lymphocyte takes place the earliest, and B cells whose development, maturation and transport are played an important role.People were purified into a kind of three peptide materials of being made up of 3 amino acid, Methionin-Histidine-G-NH2 (Lys-His-Gly-Amide, or KHG-NH first from the chicken bursa extract in 1986
2), be named as capsule element (Bursin).The capsule element mainly is synthetic and excretory by fabricius bursa follicular epithelial cell.The capsule element does not exist only in the fabricius bursa of birds, in the thymus gland Hassall corpusculum, in the cloacal bursa, but also extensively be present in the marrow and liver and gall epithelial cell of mammal (comprising the people), it has important promoter action to birds and mammiferous humoral immunization and cellular immunization, differentiation with the B lymph precursor that promotes to comprise people and poultry, propagation, improve the phagocytic activity of scavenger cell, significantly improve bone-marrow-derived lymphocyte and reply and improve the effect of immunologic function (Jiang Qian etc. " progress of capsule element ", China's animal and veterinary digest 2007,36 pages of the 3rd phases).
The 4 peptide biological activity molecules " stimulin " that the human spleen produces are by Thr-Lys-Pro-Arg (Thr-Lys-Pro-Arg (Tuftsin), or write a Chinese character in simplified form TKPR) form, experimental results show that it has the lymphocytic differentiation of the T of promotion, propagation and kill capability have the effect (Nishioka K et al " Tuftsin:an immunomodulating peptide hormone and itsclinical potential as a natural biological response modifier " .Cancer Invest (1984) 39～49) that significantly improves the body cell immunologic function.
In order to improve body produces immune response, raising body at antigen immunity opposing level effectively, reach the purpose of enhancing immunity resistance against diseases, two kinds of different immunostimulant small peptides are linked together, bring into play the active function that they promote T lymphocyte and bone-marrow-derived lymphocyte.According to design of the present invention with stimulin TKPR as X " first function fragment ", with the plain KHG-NH of capsule
2As Y " second function fragment ", (＞the Linker that K-) constitutes is with the TKPR of multiple copied and the KHG-NH of single copy by Methionin
2Do connection, as (TKPR) 4＞K2＞KHG-NH
2Or (TKPR) 8＞K4＞K2＞KHG-NH
2Make full use of stimulin TKPR and KHG-NH
2The coordinative role that the effect of the raising immunologic function that is possessed separately and they produce comprehensively improves the immunological competence of body, and the biological action that makes synthetic product is than independent arbitrary functional section TKPR or KHG-NH
2Activity significantly improved, product can be prepared into immunostimulant.
The present invention is to two kinds of biological activity differences but the polypeptide of tool dependency or design that the oligopeptides function fragment is taked are " first function fragment " of X representative can be polypeptide or the oligopeptides fragment that any class has immune enhancing function, as capsule element, stimulin and their derivative or the like; Also can be that a class can excite bone-marrow-derived lymphocyte or T lymphocyte to produce the polypeptide or the oligopeptides fragment of immunne response effect, as polypeptide antigen fragment (can be tumour antigen, virus antigen, bacterial antigens, fungal antigen or other derive from the related polypeptide antigen fragment of pathogenic microorganism or pathogenic differential protein); Also can be polypeptide or the oligopeptides function fragment that to bring into play other effect, be not limited to this.
" second function fragment " of Y representative can be polypeptide or the oligopeptides fragment that any class has immune enhancing function, as capsule element, stimulin or the like; It can be the part fragment that any class can combine with cell surface receptor; Can be any class can with the polypeptide or the oligopeptides fragment of cell surface molecule developmental biology reaction; Can be any class can with the polypeptide or the oligopeptides fragment of cell interior molecule developmental biology reaction, as endoplasmic reticulum retention fragment KDEL or RDEL etc., also can be polypeptide or the oligopeptides fragment that any class can be brought into play other biological action.
The present invention with two kinds of function differences but relevant polypeptide of tool biology or oligopeptides fragment with the form of Xn-Linker-Y connect can design form as: (polypeptide antigen fragment)
8＞K
4＞K
2＞K-(immunostimulant fragment), or (polypeptide antigen fragment)
8＞K
4＞K
2＞K-(the part fragment of cell surface receptor), or (immunostimulant fragment)
8＞K
4＞K
2＞K-(another kind of immunostimulant fragment), design is not limited to this polypeptide active product.Immunostimulant fragment in the product has and lymphocytic cell surface molecule bonded ability, can stimulate the immune cell activation, promote in the product the multi-copy polypeptide antigen fragment with lymphocytic near with react, so the advantage of design is to have improved immune response to polypeptide antigen in the segmental help lower body of immunostimulant immunity system, and the synergistic effect of generation is beneficial to the preparation that the polypeptide antigen of preparing better effects if is used for vaccine.
Can adopt any 2 kinds of biological function differences but the polypeptide of tool biology dependency or oligopeptides produced in fragments become molecular form is the product of Xn-Linker-Y according to design of the present invention, utilize the relevant biologic activity of the related polypeptide of product or the segmental activity excitation body of oligopeptides, Zhi Bei product can be prepared into biological function or activity can surmount the segmental product of wherein single copy function in this manner.So the product that produces can be prepared as medicine, immunostimulant (immunological adjuvant) or polypeptide vaccine.
Above-mentioned explanation only is the general introduction of technical solution of the present invention, for can clearer understanding technique means of the present invention, and can be implemented according to the content of specification sheets, below with the detailed explanation of running business into particular one of embodiments of the invention.
Embodiment
Reach technique means and the effect that predetermined goal of the invention is taked for further setting forth the present invention, below in conjunction with preferred embodiment, design and concrete application implementation mode, feature and effect thereof to polypeptide product that foundation the present invention proposes are done following explanation.
U.S. scientist R.B.Merrifield invention in 1963 has been founded the amino acid whose carboxyl terminal of the carboxyl terminal of purpose peptide (C end) has been fixed on the insoluble resin, and the amino acid whose aminoterminal of bonded on the resin (N end) carries out the solid-phase synthesis that condensation reaction reaches the prolongation peptide chain with amino acid whose carboxyl terminal to be connected is arranged.The polypeptide synthetic is that the carboxyl terminal (C end) from polypeptide begins one by one amino acid condensation and connects and extend one by one to aminoterminal (N end) direction of peptide section in proper order.When carry out amino acid whose carboxyl will be when engaging reaction with it amino and side-chain radical protect and avoid reacting; at present commonly used have tertbutyloxycarbonyl (Boc) protection method and fluorenylmethyloxycarbonyl (Fmoc) protection method, and therefore the last amino acid of every connection will experience once has amino acid whose activated carboxyl generation condensation reaction to be connected to prolong peptide chain with the amino acid that is combined on the solid phase carrier as the deprotection base of amino and with the excessive next one.By such step repeated multiple times go on, promptly reach condensation-＞washing-＞go the protection-＞neutralization and the washing-＞next round condensation (connecting an amino acid again), until reaching required synthetic peptide chain length.Polypeptide is synthetic to get off the cracking from the solid-phase resin of synthetic polypeptide product after finishing again, finally obtains synthetic product through the purifying of high performance liquid chromatography.
Based on above principle, polypeptide is synthetic can be that manual operations is synthetic, also can adopt Peptide synthesizer synthetic through input composition sequence and programming automation.Nowadays solid phase method has become a common method in polypeptide and the protein synthesis.Here the synthesis mode that need to prove the Xn-Linker-Y polypeptide product is by at first synthetic Y polypeptide or oligopeptides fragment, and then the synthetic Linker of continuation, but promptly connect one have the amino Methionin of the amino acid whose activation of 2 condensations (＞K-), again simultaneously carry out amino acid whose condensation reaction on 2 the activation amino thereon, and obtain simultaneously extending in the amino acid addition condensation reaction afterwards and reach the purpose of synthetic multiple copied X function fragment simultaneously.In the process of synthetic straight-chain polypeptide because what use is that (K-), another amino on it does not participate in condensation reaction, so what down extend is linear peptides for the Methionin that has only activation amino.When we need synthesize multiple copied peptide section, the Methionin of selecting for use two amino all to be activated can to carry out condensation reaction (＞K-) (have the commodity raw material to provide) thus Methionin (＞K-) just can condensation connect 2 amino acid, if what be connected is that (＞k2＞K-) can continue down to connect 4 amino acid again to 2 Methionins, if what be connected is that (＞K4＞k2＞K-) can connect 8 amino acid again down proceeds the amino acid addition condensation reaction and just can obtain being connected with 8 segmental polypeptide products of copy X 4 Methionins successively.
Peptide molecule is the molecule of a class biologically active, their aminoacid sequence and structures shape their biological action.The present invention relates to be the polypeptide of two kinds of differences of representative but tool biology dependency or oligopeptides function fragment with X and Y (＞the Linker that K-) constitutes is connected be connected with the X fragment of the multiple copied biologically active substance of generation of the Y fragment of preparing single copy by Methionin.The polypeptide synthetic is at first from the carboxyl terminal of peptide in proper order, at first synthetic Y fragment, by have the amino Methionin of 2 activation connecting thereafter (＞K) carry out the Linker that follow-up condensation reaction produces can be＞K-Y,＞K2＞K-Y or＞K4＞K2＞K-Y, or the Methionin that utilizes the segmental N-terminal of Y to have does multiple copied and connects, thus can be on the segmental back of Y connects the X fragment of 2,4 or 8 copies.Adopt the synthetic mode that two kinds of different function fragments of multiple copied X and single copy Y are linked together, the active result that is prepared from can improve X and two kinds of function fragments of Y synergy each other, has improved the biological action of two kinds of function fragments.Polypeptide synthetic commerce services can provide synthetic product according to requirement of client at present, in this structure design and application of polypeptide product emphatically.About polypeptide synthetic detail and principle we this no longer tired chatting, see also by containing tree and advocate to compile, " present age of polypeptide hormone is theoretical and use " book that scientific and technical literature press (1998) publishes.
Embodiment 1
Mode (Fmoc) the synthetic Y fragment that adopts solid phase synthesis is the plain KHG-NH of 3 peptide capsules
2, connecting multiple copied X is the formed biologically active substances of 4 peptide stimulin TKPR.Plain molecule (Methionin-Histidine-G-NH2, Lys-His-Gly-Amide, or KHG-NH of synthetic capsule at first
2) carboxyl terminal be G-NH2, so it is synthetic that the aminoresin that adopts " G-NH2 " to link to each other with resin starts, prolong peptide chain in conjunction with amino acid one by one then and (condensation of＞K-) dual-active amino is prepared the molecule of connection multiple copied stimulin TKPR, can prepare product (TKPR) 2＞KHG-NH of 2 copies respectively by Methionin
2, the product of 4 copies (TKPR) 4＞K2＞KHG-NH
2Product (TKPR) 8＞K4＞K2＞KHG-NH with 8 copies
2, their molecular weight is respectively 1320,2542 and 4983 dalton.Synthetic product can be obtained the synthetic product of purity＞98.0% through the purifying repeatedly of high performance liquid chromatography.Use newcastle disease inactivated vaccine and synthetic peptide prod and unite use, be inoculated into no-special pathogen (Specefic pathogen Free, SPF) chick detects hemagglutination inhibition test (HI test) (HI), and whether observe synthetic peptide contrast stimulin and capsule element has immuno-potentiation to chick.
Avian pneumo-encephalitis virus (Newcastle Disease Virus, NDV) can with chicken red blood cell generation agglutination phenomenon, this is a species specific antibody neutralization reaction, but its principle is the hemagglutinin aggegation red corpuscle according to virus, but if earlier with specific antibody and virus function, add red corpuscle again, red cell agglutination does not appear then, be called hemagglutination-inhibition test (HI), the highest multiple that detects used antiserum(antisera) dilution promptly is the titre of antibody.The effect of the high more explanation immunity of the titre of antibody to be measured is good more.The HI method has the following advantages: 1. susceptibility is strong, can detect the antibody of trace, and the result is also comparatively accurate, is one of more sensitive serological reaction; 2. high specificity, viral aggegation red corpuscle can only be suppressed by special antibody; 3. detection speed is fast, and 1 HI test only needs to get final product result of determination about 2h; 4. HI test is not high to environmental requirement, simple to operate, also can detect a large amount of samples 1 time.Therefore cell agglutination inhibition test (HI) has become a kind of detection method that is usually used in detecting the poultry serum antibody, and detail sees also the chief editor by Guo Xin, " the animal immunology experiment study course " that the China Agricultyre University Press published in 2007.
Materials and methods
Vaccine: newcastle disease inactivated vaccine (La Sota strain)
There are not specific pathogeny (Specefic pathogen Free, SPF) chicken: about 1 monthly age.
Immunity grouping (every group of 10 chickens):
Control group: not vaccination.
Routine immunization group: inoculation conventional vaccine 0.3ml.
The stimulin group: inoculation conventional vaccine 0.3ml is added with TKPR 4 peptides (20 micrograms/0.1ml/ only).
The plain group of capsule: inoculation conventional vaccine 0.3ml is added with the plain KHG-NH of capsule
2(20 micrograms/0.1ml/ only).
2 sets of copies: inoculation conventional vaccine 0.3ml is added with synthetic peptide (TKPR) 2-KHG-NH of 2 copies
2(only containing 20 micrograms/0.1ml/).
4 sets of copies: inoculation conventional vaccine 0.3ml is added with synthetic peptide (TKPR) 4-K2-KHG-NH of 4 copies
2(only containing 20 micrograms/0.1ml/).
8 sets of copies: inoculation conventional vaccine 0.3ml is added with synthetic peptide (TKPR) 8-K4-K2-KHG-NH of 8 copies
2(only containing 20 micrograms/0.1ml/).
Raise: raise simultaneously and in shield retaining, freely get food drinking-water.
Blood sampling: before the inoculation, inoculate back 21 days and blood sampling respectively in 28 days, separation of serum carries out erythrocyte agglutination inhibition test (HI test).
The result:
Detected result shows that the HI antibody of 2 sets of copies, 4 sets of copies and 8 sets of copies is than high about 1 titre of antibody geometric mean titer (CMT) of normal immune group, the plain group of capsule and stimulin group, and concrete outcome sees Table as follows:
| Time | Group | HI antibody result | Average |
| ??21d | Control group | 0，0，0，0，0，0，0，0，0，0(n＝10) | ??0 |
| ??21d | The routine immunization group | 4，5，5，5，5，6，6，6，7，7，(n＝10) | ??5.6 |
| ??21d | The tuftsin group | 4，5，5，6，6，6，7，7，7，7(n＝10) | ??6.0 |
| ??21d | The plain group of capsule | 4，5，5，6，6，6，6，7，7，8(n＝10) | ??6.0 |
| ??21d | 2 sets of copies | 5，6，6，6，7，7，7，8，8，8(n＝10) | ??6.8 |
| ??21d | 4 sets of copies | 6，6，6，7，7，7，8，8，8，8(n＝10) | ??7.1 |
| ??21d | 8 sets of copies | 6，6，7，7，7，7，8，8，9，9(n＝10) | ??7.4 |
| Time | Group | HI antibody result | Average |
| Time | Group | HI antibody result | Average |
| ??28d | Control group | ??0，0，0，0，0，0，0，0，0，0(n＝10) | ??0 |
| ??28d | The routine immunization group | ??5，6，6，6，6，7，7，7，8，8(n＝10) | ??6.6 |
| ??28d | The tuftsin group | ??6，6，7，7，7，7，8，8，9，9(n＝10) | ??7.4 |
| ??28d | The plain group of capsule | ??6，6，7，7，7，7，7，8，9，9(n＝10) | ??7.3 |
| ??28d | 2 sets of copies | ??6，7，7，7，7，8，8，9，9，10(n＝10) | ??7,8 |
| ??28d | 4 sets of copies | ??6，7，7，8，8，8，9，9，9，10(n＝10) | ??8.1 |
| ??28d | 8 sets of copies | ??7，7，7，8，8，9，9，9，9，10(n＝10) | ??8.3 |
Above result shows to have the fragment " capsule element " that improves immunologic function and the product that " stimulin " obtains according to multiple copied ways of connecting (TKPR) n-Linker-KHG-NH2 has its bioactive effect of raising with two kinds, synthetic product has the effect that improves along with the increase immune effect with plain " stimulin " copy number that is connected of capsule, they and vaccine coupling can be significantly improved the antibody response of inoculation animal to vaccine, can reach the enhancing immunity ability of expection, improve the effect of immunity.
Test-results has directly proved can reach the purpose that enhancing and enhance immunity are replied level after the another kind of immune fragment stimulin of the X representative of and 2,4 or 8 copies plain when a kind of immune fragment capsule of Y representative is done multiple copied and is connected.The product that the structure design mode of this peptide species function fragment is produced can be used as immunological adjuvant and vaccine coupling to improve the immune effect of vaccine.They also can be used as stimulates the medicine of human body immune function to use separately, and this type of medicine can be used for antitumor, antiviral, anti-infective or raising human or animal's immunity of organism resistivity in treatment of diseases or the prevention.
Embodiment 2
The propagation of virus disease causes serious illness and financial loss to the human and animal, and it is pathogenic and dead that duck hepatitis virus wherein causes for the meat duck in the bird aquaculture, brings enormous economic loss to aquaculture.Use the polypeptide vaccine of polypeptide technology preparation virus, be desirably in and do not relate to or do not contact under the prerequisite of Causative virus preparation polypeptide vaccine is fast done disease for the bird (duck) that does not infect immunoprophylaxis.
We choose duck hepatitis virus and (comprise Strain A66, Strain ZJ, Strain R and Strain SH) shared polypeptide fragment YLKDELRKKEKIKE (coat protein 1944-1957,14 peptides), the mode by synthetic is with multiple copied YLKDELRKKEKIKE fragment (X) and the fragment with immunostimulant " stimulin " TKPR (Y) is connected and is prepared into a polypeptide antigen product with high immunne response effect.Synthetic product obtains the product of purity＞98% by the purifying of high performance liquid chromatograph.The stimulin TKPR fragment (Immunol.Lett (1982): Vol:4 that can react with the FC acceptor on immunocyte surface, 215), stimulate and improve the function of immunocyte, improve the immune response that body produces coupled multiple copied antigen fragment.Use TKPR and carry the antigen fragment of (connection) multiple copied as carrier, the coordinative role that TKPR and antigen fragment play body endolymph cell can stimulate and improve body to the segmental immunne response level of antigen.
Synthesize following product according to aforesaid preparation method:
Control group (8 copy antigen fragment):
(YLKDELRKKEKIKE)8＞K4＞K2＞K
Molecular weight: 15322.5 dalton
Test group (8 copy antigen fragments and are connected with stimulin):
(YLKDELRKKEKIKE)8＞K4＞K2＞K-TKPR
Molecular weight: 15804.8 dalton
Test objective:
Selection contains the bioactive molecule of the 8 polypeptide antigen produced in fragments that copy does immune effect relatively with the polypeptide antigen fragments that contain 8 copies merely.Do not adopt in the test that single copy YLKDELRKKEKIKE polypeptide antigen fragment is done the former of contrast because: so since the too for a short time immune response that is difficult to excitating organism of the molecule of the YLKDELRKKEKIKE polypeptide fragment of single copy can not be separately as antigen use (the polypeptide antigen fragment of single copy must with uncorrelated macromole coupling after could use as antigen).And the polypeptide antigen fragment of inciting somebody to action single copy is done the connection of multiple copied, or the product molecule that is connected to form with immunostimulant fragment multiple copied enough can produce immune response by excitating organism, therefore can remove from uncorrelated macromole and do link coupled complex operations process.
Materials and methods:
Experimental animal:
New Zealand's large ear rabbit male and female are not limit, 6-12 monthly age, body weight 1.5-2.0kg/, 5 every group.
Immunization ways:
, mix and emulsification with physiological saline solution and be mixed with 0.5mg/0.5ml/ usage quantity only through freeze dried synthetic product with the full adjuvant of Fu Shi (Sigma company) with volume.Do subcutaneous multiple spot immunity (about 100ul/ point) at the back of animal.The immunity second time is carried out in after the immunity first time the 28th day, and mode is the same but use Freund's incomplete adjuvant (Sigma company), later gives booster immunization once every 7 days in the sole meat pad to rabbit, dosage such as preceding but do not have adjuvant, immune 4 times altogether.
The result measures:
Got blood 2ml from every rabbit ear edge vein respectively in the 7th day before animal immune and after the 4th immunity, room temperature leaves standstill centrifuging and taking serum after 1 hour.
Adopt enzyme linked immunosorbent detection method (ELISA) to measure the antibody titer that is produced at the YLKDELRKKEKIKE polypeptide fragment in the serum.The YLKDELRKKEKIKE polypeptide is made into the concentration of 5mg/L, gets 100ul/ hole bag by elisa plate, each extent of dilution 2 hole.Control group and test group are the immunity back serum of getting the 4th time, and serum is done from the dilution of 1: 4000,1: 8000,1: 16000,1: 32000 scope, get the 100ul/ hole and do blank with preimmune serum.Detect the reading of A450nm with the goat anti-rabbit igg of HRP mark in conjunction with the rabbit reaginic antibody and with microplate reader, positive with A450＞0.23.The concrete operations of ELISA are referring to by Bao Jianfang, and Shen Jiangen chief editor's " the immunological experiment technology " of being published in 2006 by press of Zhejiang University is this no longer tired chatting.
The ELISA detected result:
| Group | 4 immunity back ELISA A450nm values |
| Blank group (preimmune serum) | ?1∶1000.176±0.03 |
| Control group (YLKDELRKKEKIKE) 8＞K4＞K2＞K | ?1∶160001.31±0.02 |
| Test group (YLKDELRKKEKIKE) 8＞K4＞K2＞K-TKPR | ?1∶320001.28±0.03 |
Evidence has following advantage with the multi-copy polypeptide antigen fragment with the polypeptide antigen that single copy immunostimulant fragment is connected preparation:
1. because the molecular weight ratio of single copy polypeptide antigen fragment YLKDELRKKEKIKE is less, be not enough to excitating organism as immunogen and produce antibody, small peptide fragment and macromolecular carrier albumen such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) are done coupling as needing as antigen.Because antigen product (YLKDELRKKEKIKE) 8＞K4＞K2＞K of preparation and (YLKDELRKKEKIKE) 8＞K4＞K2＞K-TKPR molecular weight be enough to excite immune reaction, therefore removed from and done molecule link coupled step.
Since immunostimulant fragment and polypeptide antigen fragment interconnect and make the more accessible immunocyte of antigen fragment, immune vigor at the segmental effect lower body of immunostimulant increases, and makes body obtain corresponding raising to the segmental immunne response level of polypeptide antigen.
According to this design, " first function fragment " of X representative can be the polypeptide antigen fragment that any class can the reaction of excitating organism immune cell responses, as exciting bone-marrow-derived lymphocyte to produce antibody, excite the T lymphocyte to produce the polypeptide antigen fragment of cytotoxicity, they can be the polypeptide antigen fragments in tomour specific albumen or tumor correlated albumen source; It can be the polypeptide antigen fragment that exogenous pathogenic microorganism comprises differential protein sources such as bacterium, fungi, virus; It can be the specific polypeptides antigen fragment in endogenous mutant or pathogenic associated protein source.
" second function fragment " of Y representative can be polypeptide or the oligopeptides fragment that any class has immune enhancing function, as stimulin Tuftsin (TKPR or TKLK), and the plain Busin of capsule (KHG-NH2) etc.; Can be any class can with cell surface receptor bonded polypeptide or oligopeptides part fragment; Can be any class surface of cell membrane polypeptide or oligopeptides signal peptide fragment, wear membrane-bound fragment; Can be polypeptide or the oligopeptides fragment that any class can combine or rise biologically with subcellular organelle in the cell, as ER retention signal peptide (KDEL or RDEL); Can be that function fragment etc. in any type cytokines and other have the polypeptide or the oligopeptides fragment of special biological.
Above evidence the polypeptide active molecule be to design according to artificial mode, use peptide synthesis technology with the multiple copied " first function fragment " of X representative by Methionin (＞K-) for the Linker of bridge and Y be representative singly copy the polypeptide structure product that " second function fragment " is joined together to form Xn-Linker-Y, so can by the synthetic mode with two kinds of biological function differences but the polypeptide fragment of tool dependency link together and generate expeditiously.Synthetic product can be brought into play the mutual synergy that produces between two kinds of function fragments effectively and strengthen or improve the biologic activity effect, the polypeptide antigen of synthetic be can prepare in this way, treatment and prevention that polypeptide vaccine is used for relative disease such as antitumor, antiviral prepared.
Set forth in the literary composition with two kinds of function differences but the polypeptide that mode of connection produced of the peptide class fragment of tool biology dependency by Xn-Linker-Y can be widely used in the design of polypeptide drugs design, polypeptide vaccine, its product can become medicine or vaccine is applied in treatment of diseases and the prevention area.
Claims (10)
1, a kind of multi-copy polypeptide is characterized by, and its molecular structure is: Xn-Linker-Y; Wherein X is first function fragment, n represents the copy number of X, Y is second function fragment, X and Y represent the polypeptide or the oligopeptides fragment of the biological function of two kinds of inequality but tool dependencys respectively, the Linker representative is connected with the form of multiple copied X by the connection that Methionin (Lys, or K) forms with single copy Y.
2, multi-copy polypeptide according to claim 1, it is characterized in that described Linker for＞K-,＞K2＞K-or＞K4＞K2＞K-, K wherein is a Methionin, be have that 2 activation are amino can be for the Methionins that carry out the amino acid addition condensation reaction.
3, multi-copy polypeptide according to claim 2 is characterized in that the n among the described Xn is 2,4 or 8.
4, a kind of multi-copy polypeptide, it is characterized in that be on the amino acid side chain group in each described multi-copy polypeptide of claim 1-3, aminoterminal or carboxyl terminal carry out hydroxylation, carboxylated, carbonylation, methylate, acetylize, phosphorylation, esterification, the resulting product of glycosylation.
5, a kind of multi-copy polypeptide, it is characterized in that be on the amino acid side chain group in each described multi-copy polypeptide of claim 1-3, the aminoterminal of product or carboxyl terminal add amino acid or other any chemical elements.
6, a kind of multi-copy polypeptide is characterized in that it being to insert arbitrary amino acid or other any chemical elements in each described multi-copy polypeptide of claim 1-3 in the Linker that is made up of Methionin.
7, a kind of peptide medicament is characterized in that it comprises each described multi-copy polypeptide of claim 1-5.
8, a kind of immunostimulant is characterized in that it comprises each described multi-copy polypeptide of claim 1-5.
9, a kind of vaccine is characterized in that it comprises each described multi-copy polypeptide of claim 1-5.
10, each described multi-copy polypeptide of claim 1-5 is as the application of peptide medicament, immunostimulant or polypeptide vaccine.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012119326A1 (en) * | 2011-03-10 | 2012-09-13 | 北京中天康泰生物科技有限公司 | Method for preparing branched polypepdides |
| CN103145854A (en) * | 2013-03-05 | 2013-06-12 | 河南科技大学 | Recombined Tbeta 4-BP5 fusion peptide, gene, engineering bacteria and application |
| CN102120759B (en) * | 2010-01-07 | 2015-08-12 | 北京英诺泰生物技术有限公司 | One peptide species and derivative thereof and application |
| WO2017101786A1 (en) * | 2015-12-14 | 2017-06-22 | 韩苏 | Polypeptide compound, preparation method therefor and use thereof |
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| CN105085637A (en) * | 2014-05-07 | 2015-11-25 | 北京英诺泰生物技术有限公司 | Polypeptide compound and application thereof |
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| CN100339396C (en) * | 2005-04-18 | 2007-09-26 | 中国科学院微生物研究所 | Multi-helix protein for inhibiting membrane virus infection, its coding gene and use |
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| CN1974601A (en) * | 2005-11-28 | 2007-06-06 | 上海新生源医药研究有限公司 | New-type Fc fusion protein and its production process |
| CN1994465B (en) * | 2006-10-13 | 2010-06-09 | 中国人民解放军第四军医大学 | CCR5 autogenous polypeptide vaccine and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102120759B (en) * | 2010-01-07 | 2015-08-12 | 北京英诺泰生物技术有限公司 | One peptide species and derivative thereof and application |
| WO2012119326A1 (en) * | 2011-03-10 | 2012-09-13 | 北京中天康泰生物科技有限公司 | Method for preparing branched polypepdides |
| CN102675410A (en) * | 2011-03-10 | 2012-09-19 | 北京中天康泰生物科技有限公司 | Method for preparing branch polypeptide |
| CN103145854A (en) * | 2013-03-05 | 2013-06-12 | 河南科技大学 | Recombined Tbeta 4-BP5 fusion peptide, gene, engineering bacteria and application |
| CN103145854B (en) * | 2013-03-05 | 2014-11-26 | 河南科技大学 | Recombined Tbeta 4-BP5 fusion peptide, gene, engineering bacteria and application |
| WO2017101786A1 (en) * | 2015-12-14 | 2017-06-22 | 韩苏 | Polypeptide compound, preparation method therefor and use thereof |
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