CN1082053A - Synthetic polypeptide - Google Patents
Synthetic polypeptide Download PDFInfo
- Publication number
- CN1082053A CN1082053A CN93105907A CN93105907A CN1082053A CN 1082053 A CN1082053 A CN 1082053A CN 93105907 A CN93105907 A CN 93105907A CN 93105907 A CN93105907 A CN 93105907A CN 1082053 A CN1082053 A CN 1082053A
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- CN
- China
- Prior art keywords
- hiv
- leu
- gln
- polypeptide
- synthetic polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A61K39/21—Retroviridae, e.g. equine infectious anemia virus
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Abstract
A kind of synthetic polypeptide with at least a antigen attribute of at least one strain HIV (human immunodeficiency virus) (HIV) envelope protein, described polypeptide is made up of the aminoacid sequence of formula (I) substantially:
X-R
1-Leu-R
2-Leu-Thr-Val-Trp-Gly
-R
3-lys-Y (I)
These peptides are similar to some epitope of HIV envelope protein.
Description
The present invention relates to synthetic polypeptide, specifically, relate to tertiary structure and/or electrostatic surface and/or other physics of imitating virus enveloped protein matter specific region, the synthetic polypeptide of chemistry and structure attribute.It for the known relevant vaccine of HIV (human immunodeficiency virus) (IHV) as acquired immunodeficiency syndrome (AIDS) pathogenic agent, the immunocompetence therapeutical agent, the diagnosis and other design medical or the scientific research medicament special significance is arranged.
In 10 years of past, the AIDS serious medical problem that becomes international, and for as research, diagnosis treats and/or prevents by the pathogenic agent of HIV(AIDS) there are general urgent needs in the material that infects.Along with the protein amino acid sequence that produces by HIV I and HIV II virus (see, for example, Ratne, L.et al., Nature 313,277(1985); Meusing, M.A.et al., Nature 313,450, (1985); Wain-Hobson, S.et al., Cell 40,9(1985)) availability, design imitates the synthetic polypeptide of virus enveloped protein matter antigen attribute to become possibility.
The objective of the invention is to develop the antibody that can guide HIV virus, and the synthetic polypeptide that produces of neutralizing antibody preferably, this antibody is by passive or active immunity, and prevention HIV virus infection and/or restriction HIV virus spread.The passive immunization that is brought by this antibody can be set up treatment AIDS VICTIMS's effective ways, propagates in vivo and between individuality thereby control this virus, and slows down thus or stop the development of this disease.
Our invention provides the synthetic polypeptide of at least a antigen attribute with at least one strain HIV (human immunodeficiency virus) (HIV) envelope protein matter, and said polypeptide is made up of the aminoacid sequence of formula I substantially:
X-R
1-Leu-R
2-Leu-Thr-Val-Trp-Gly-R
3-Lys-Y （Ⅰ）
R wherein
1Be selected from Gln-Gln-R
4-R
5, Gln-R
4-R
5, R
4-R
5, R
5Or R
1Disappearance;
R
2For being selected from Gln, Lys, the amino-acid residue of Glu or Arg;
R
3For being selected from Ile, the amino-acid residue of Thr or Ala;
R
4Be His or Glu;
R
5Be Leu or Met; And
X and Y can lack independently of one another or be one or more amino-acid residues independently.
Usually, work as R
1Be Gln-GlnGln-R
4-R
5And X and Y and natural envelope protein sequence homology not when X and Y exist.More particularly, as X and Y existence and R
1When as above defining, X and Y do not provide or constitute the part of the envelope protein matter antigen attribute of at least one strain human immunity shortage virus.
That yes is useful for the peptide that exists according to the no X of above-mentioned formula I and Y, for example, and when producing the antibody of anti-HIV.When combining with carrier molecule, such peptide can be effective especially.Yet when X or Y existed, they can be any length, but preferably are less than 20 amino acid, more preferably are less than 10, for example 3 to 6.Obviously, can constitute a kind of protein according to the sequence of formula I, it with X and Y as this proteinic major portion with antigen sequence, for example, the part of exposed circle on the globular preteins.
If R
1There is R
5Be that Leu or Met are preferred, more preferably Leu, and R
4If exist, be His or Glu, more preferably His.R
2Gln preferably, Lys or Glu, more preferably Gln and R
3Ile preferably.
As in the sequence of formula I preferably, R
1Be Gln-Gln-R
4-R
5Or do not have, if when existing, R
4Preferably His and R
5Be Leu.R
2Be Leu and R
3For Gln also is preferred.
Form by following sequence as the preferred form of the polypeptide of formula I of the present invention:
(I a) for X-Gln-Gln-His-Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Y; With
X-Leu-Gln-Leu-Leu-Thr-Val-Trp-Gly-Ile-Lys-Y （Ⅰb）
Wherein X and Y as above define, though if X or Y exist, they are preferred for short relatively sequence.Preferably X lacks, and Y is 2 or 3 residue length, for example, and Gly-Cys or Gly-Cys-Ala.Consider above-mentioned I a and I b sequence, preferably lack X and in I a sequence Y be Gly-Cys and in I b sequence, be Gly-Gys or Gly-Cys-Ala; This C does not hold to extend to combine with carrier provides another site.
Simulated some surface portion of HIV1 envelope protein matter suc as formula the polypeptide of I.
Another kind of preferred form as polypeptide of the present invention is made up of the aminoacid sequence of formula II basically:
X-R
1-Leu-Arg-Leu-Thr-Val-Trp-Gly-R
3-Lys-Y （Ⅱ）
R wherein
1Be selected from Gln-Gln-Glu-R
5, Gln-Glu-R
5, Glu-R
5, lack R
5Or R
1
R
3Be Thr or Ala;
R
5Be Met or Leu; And
X and Y as above define.
Preferably, R
5If, have, be Met and R
3Thr preferably.R
1For Gln-Gln-Glu-Met or to lack equally also be preferred.
Preferred form according to the polypeptide of formula II is made up of following sequence:
(II a) for X-Gln-Gln-Glu-Met-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Y; With
X-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Y （Ⅱb）。
Preferably, lacking X and Y is 2 or 3 residue length, for example, and Gly-Cys or Gly-Cys-Ala.
According to the polypeptide of formula II some surface similar in appearance to the envelope protein matter of HIV2.
Preferred peptide sequence is that surfac topography similarity according to one or more antigenic determinants of they and HIV envelope protein chooses according to the present invention.For example, become a kind of resemblance also can demonstrate surface gram shape similarity with one or more other zones of HIV envelope protein for specific propolypeptide the design of antigen decision, this may be because genetic duplicating, or because this polypeptide is a kind of resemblance of discontinuous determiner, or because this polypeptide is designed to polyvalent.Discontinuous epitope can be regarded as by epitope closely relative sequence, itself can tool antigen significance and constitutes, and a multivalence polypeptide can contain 2 or a plurality of (continuous or discontinuous) determiner resemblance on single polypeptide chain, therefore provide a kind of method to be guided out the generation of antibody scope simultaneously, this antibody will be recognized 2 or a plurality of determiner on the HIV envelope protein.
Peptide of the present invention can be synthesized, for example adopt standard 9-fluorenyl-methoxycarbonyl (F-Moc) chemical method (see, for example, Atherton, E.and Sheppard, R.C.(1985) J.Chem.Soc.Chem.Comm.165) or fourth oxycarboxylic acid ester (T-Boc) chemical method of standard.For exactness and common 85% the purity level that surpasses, do conscientious checking to structure.Can adopt various stratographic analyses, comprise for example high performance liquid chromatography, and mass spectroscopy.
All sequences herein adopts the I.U.P.A.C. trigram code abbreviation of standard to set forth.Amino-acid residue is defined as follows: Gly-glycine, Ala-L-Ala, Val-Xie Ansuan, Leu-leucine, the Ile-Isoleucine, Ser-Serine, Thr-Threonine, the Asp-aspartic acid, Glu-L-glutamic acid, Asn-l-asparagine, the Gln-glutamine, Lys-Methionin, His-Histidine, the Arg-arginine, Phe-phenylalanine, Tyr-tyrosine, Trp-tryptophane, Cys-halfcystine, Met-methionine(Met) and Pro-proline(Pro).
Use as treatment, according to polypeptide of the present invention or its antibody can be separately or with other medicament as different levels by duplicating of viral interference genetic stew work 3 '-azido--3 '-deoxythymidine (AZT) (zidovudie) and/or check the hiv protease inhibitor administration together of the viral necessary enzyme of formation.
According to polypeptide of the present invention can as produce can with by the HIV1 of broad range and or the structured surface/surfac topography/static attribute of the interactional polypeptide of envelope protein that produces of HIV2 strain, make they be guided out very reliably can with the interactional production of antibodies of HIV envelope protein that derives from several strains or many strains, further advantage may become a big polypeptide and produces from making up several different polypeptide.This peptide species can have logical formula III:
[La-F]m-[Lb-G-]n-Lc （Ⅲ）
Wherein F and G can be any polypeptide according to formula I and II b independently of each other, and L is a catenation sequence, a, b and c be independently of one another 0 or 1 and m and n respectively be positive number, for example comprise between 1 and 10.The preferably short structure of L flexibly the polypeptide chain part as, for example and be not limited to Gly-Gly-Gly-Gly-Gly, Gly-Pro-Gly-Pro-Gly-Pro or Gly-Ser-Ala-Gly-Ser-Gly-Ala.Should be understood that to duplicate at every turn and all can have the different variants that also can not have according to polypeptide of the present invention.
Be known as false same multivalence as multivalence determiner resemblance by the definition of formula III.The varient of identical determiner resemblance duplicates in single polypeptide chain basically.In addition, simply with the multivalence polypeptide immunogen, it contains the various replisome of with good grounds formula I to the identical varient of one of any determiner resemblance of II b, also expects effectively, and is also included within the scope of the present invention.
Expect that vacation can be valuable especially as vaccine with the former polypeptide of multivalent immunogen.As vaccine, their can induce (neutralization) antibody of a scope to produce with similar but uncertain specificity, and their are understood and derive between the envelope protein of HIV strain of relative broad range and interact, and can be thus invest on the protective immunity more effective.On the different multivalence polypeptide of formation, advantage is arranged also.This different multivalence polypeptide contains the replisome (with any order) of one or more one of polypeptide according to the present invention and one or more other polypeptide resemblances of determiner resemblance.These have logical formula IV by polypeptide provided by the invention:
Ld-[G-L]m-F-[L-G]n-Le （Ⅳ）
Wherein F is according to any one the polypeptide of formula I to II b, and G is the formula I to any one polypeptide of II b or other sequence, and G is the formula I to any one polypeptide of II b or other sequence, m and n respectively are positive number, for example, comprise between 1 to 10, and d and e are 0 or 1 independently of one another.The structure that " L " preferably lacks is the polypeptide chain part flexibly, as, such as but not limited to Gly-Gly-Gly-Gly-Gly, Gly-Pro-Gly-Pro-Gly-Pro or Gly-Ser-Ala-Gly-Ser-Gly-Ala.
Should be appreciated that the subfragment of antigen meaning of peptide sequence of the above-mentioned definition that any one keeps parent's polypeptide general formula and function and/or the varient of antigen meaning all comprise within the scope of the invention.Specifically, substitute any specific residue, comprise by rare amino acid (for example, the D-steric isomer) or synthesizing amino acid resemblance substituting, include interior by residue with comparable structure and/or physical attribute.For example, in the replacement of identical part (Set) by a residue of another residue replacement, the following definition comprises within the scope of the invention; Set1-Ala, Val, Leu, Ile, Phe, Thy, Trp and Met; Set2-Ser, Thr, Asn and Gln; Set3-Asp and Glu, Set4-Lys, His and Arg, Set5-Asn and Asp; Set6-Glu and Glu; Set7-Gly, Ala, Pro, Ser and Thr.The D-steric isomer of all amino acid types all can be replaced, for example, and D-Phe, D-Tyr and D-Trp.
In a preferred embodiment of the invention, if X and Y exist, can comprise one or more protein sequence segments that have as the t cell epitope ability independently, for example, the aminoacid sequence segment of general formula 1-2-3-4, wherein 1 is Gly or charged amino acid (for example, Lys, His, Arg, Asp or Glu), 2 is hydrophobic amino acid (for example, Ile, Leu, Val, Met, Tyr, Phe, Trp, Ala), 3 is hydrophobic amino acid (as above definition) or uncharged polare Aminosaeren (for example, Asn, Ser, Thr, Pro, Gln, Gly) and 4 be polare Aminosaeren (for example, Lys, Arg, His, Glu, Asp, Asn, Gln, Ser, Thr, Pro), demonstrating the effect of T cell epitope (Rothbard, J.B.﹠amp under some situation at least; Taylor, W.R.(1988).Sequence type is same as the T cell epitope.（The EMBO Journal 7（1）：93-100）。Similar segment can be sequence 1 '-2 '-3 '-4 '-5 ', wherein 1 ' to be same as 1,2 of aforementioned definitions ' be same as 2,3 ' and 4 ' be same as 3,5 ' be same as 4(the same).These two kinds of forms all comprise within the scope of the present invention and one or more t cell epitope (preferably being less than 5) can be incorporated into according to the formula I to any polypeptide of II b.Therefore each epi-position can be that type maybe can be other structure and can be separated and comprise by the separation segment of any length or composition (preferred length is less than 5 amino-acid residues) and for example be selected from Gly as defined above, Ala, Pro, Asn, Thr, Ser residue or multi-functional connexon such as non-alpha amino acid.C or N-terminal connexon also may be represented complete protein, have therefore avoided being incorporated into may needing of carrier proteins.
Derivative according to formula I polypeptide is also included within the scope of the present invention.X or Y are or comprise " retro-inverso " amino acid in the formula I,, have the difunctional acid amides corresponding to the aminoacid functional group that is.For example according to resemblance of the present invention, the amino acid that contains retro-inverso has formula:
Wherein R is arbitrary functional group, for example, the glycine side chain, and A1 and A2 preferably respectively for the replisome (but not being must be identical) of one of its N or resemblance of the definition herein that links to each other of C-terminal.Can comprise also and can not comprise the T cell epitope, as previously discussed.
The retro-inverso of peptide modifies the reverse of one or several peptide bond more can resist the resemblance that enzyme is degenerated to produce than original molecule, and provide one easily approach go to produce branch's immunogen, this antigen compares the epi-position that medium contains high density with big immunogen.These compounds have very big potentiality in the extensive solution synthetic of the retro-inverso of short chain biologically active peptides resemblance is used.
Should be mentioned that the resemblance with retro-inverso bonded amino acid derivative can not adopt the recombinant DNA system directly to make.Yet basic resemblance is passable, and they can be purified and adopt standard peptide/organic chemistry forensic chemistry ground to be connected in retro-inverso amino acid then.As the reality of solid phase synthesis on the multiamide type resin of retro-inverso peptide and existing recently description [Gazerro, H., Pinori, M.﹠amp of novel method easily; Verdini, A.S.(1990).It is the new general method as the solid phase synthesis of retro-inverso peptide.See " Innovation and Perspectives in Solid Phase Synthesis " Ed.Roger Epton.SPCC(UK) Ltd, Birmingham, UK].
This polypeptide also can be connected on the carrier molecule, both can be by the chemical group in the polypeptide itself, also can be by being added in the additional amino acid of C or N-terminal, and, for keeping their optimum immunologic functions, can from polypeptide, isolate itself, also can be surrounded by one or more additional amino acid.Suitable be connected with many and comprise and for example use Tyr, the side chain of Cys and Lys residue.The carrier that is fit to comprises, for example, the purified protein derivative of tuberculin (PPD), tetanolysin toxoid, Toxins,exo-, cholera and B subunit thereof, Protalbinic acid, bovine serum albumin, Trypsin inhibitor SBTI, Muramyl dipeptide and their resemblance, with Braun lipoprotein, other carrier that is fit to is conspicuous to those of skill in the art.For example, can adopt the multiple antigenic peptide that comprises many lysyl nuclears such as those, for example, seven Methionins produce the reaction N-terminal.Can react with N-terminal according to polypeptide antigen of the present invention, or synthesize on N-terminal, and different polypeptide antigens can react with identical nuclear or carrier.
When adopting PPD as according to the carrier of polypeptide of the present invention the time, if the acceptor of polypeptide-PPD binding substances has been the tuberculin sensitivity, for example, the effectiveness of the BCG inoculation by early can obtain higher antibody titers.Inoculated people under the situation of cowpox, and what deserves to be mentioned is, in Britain and many other countries, people accept inoculation as usual, thereby are PPD sensitivities very.Therefore expect that PPD can be the preferred carrier that uses in these countries.
The mode of polypeptide and carrier coupling depends on by the Substance Properties of coupling.For example, the lysine residue on carrier can pass through N-maleimide butyryl oxygen-succinimide to be handled, terminal or other cysteine residues coupling (Kitagawa, T.﹠amp with the C-on the polypeptide; Ackawa, T(1976) J.Biochem.79,233).In this document, also other coupled reaction reagent is described.
Polypeptide, or it is independent or be connected in carrier molecule, can be (for example parenteral by any approach, nasal cavity, per os, rectum, intravaginal) administration, use or need not conventional auxiliary agent (as aluminium hydroxide or, when not being when imposing on human body, the complete or imperfect auxiliary agent of Freund) and/or the medicament of other immunizing potency.The present invention also comprises the preparation according to the slowly-releasing form of polypeptide of the present invention.Comprise such as subcutaneous implantation or storage, for example, liposome (Allison, A.C.﹠amp; Gregoriadis, G.(1974) Nature(London) 252,252) or by the biodegradable micro-capsule (Gresser of the copolymer of lactic acid and oxyacetic acid, J.D.and Sanderson J.E.(1984) in " Biopolymer Controlled Release Systems " pp.1278-138, Ed.D.L.Wise).
Being appreciated that can be synthetic by any ordinary method according to polypeptide of the present invention, or adopt artificial as mentioned above or the automatic peptide synthetic technology directly synthetic, or by RNA or DNA is synthetic and molecular biology and engineered routine techniques between be bonded into.Adopt these technology to produce and contain the hybridization protein that one or more polypeptide insert other peptide sequence.
Therefore, another aspect of the present invention provides at least a dna molecular according to synthetic polypeptide of the present invention of encoding, preferably with microorganism or Mammals, insect, plant, the reproducible suitable expression vector combination in fungi or other cell.DNA also can be the part as the DNA sequence of longer product (for example, also can be other protein portion polypeptide expressed).This peptide species inserts in this protein by genetically engineered.Sambrook, J., Fritsch, E.F. and Maniatis, T.(the 2nd edition 1989) " Molecular cloning:a Laboratory manual " book be a kind of practice guideline of this technology.
Can use separately or be connected on the suitable carrier according to polypeptide of the present invention and use, as:
(a) polypeptide vaccine is infected by a strain or many strains HIV as preventing;
(b) measuring, for example, in the serum of HIV positive patient as ligand;
(c) the test, for example, for polypeptide antibody exceed in conjunction with level as the quality control agent;
(d) as immunity generation mono-clonal or the polyclonal antibody of immuning agent by suitable animal, these antibody are used as the scientific research of (ⅰ) HIV virus, (ⅱ) as diagnostic reagent, for example, as histological chemistry's agent part, (ⅲ) as HIV patient's passive immunization, both can be used as a kind of therapeutic dose and be used for AIDS separately, also can with for example AZT and/or the hiv protease inhibitor combination of other medicament, (ⅳ) make other medicines (for example as a kind of, AZT or hiv protease inhibitor) hit the means of target, such medicine or linked to each other by covalency or be connected to the liposome neutralization that for example contains this medicine and mix with the antibody that any antigenic peptide causes in other mode in the HIV cells infected of its surface expression HIV envelope protein.The present invention adopts the technology of describing in the document, and genetically engineered form or the subunit antibody because of the human form of the antibody of anti-this polypeptide generation further is provided.V particularly
HThe zone; (e) treatment HIV infects, or is incorporated into the HIV virus of human or animal's cell by conversion, or by confuse in the body should virus three levels of organization, and the scientific research of the HIV virus that helps to exsomatize.
Aspect HIV or anti-HIV detection of antibodies and judgement, those of skill in the art can expect various the known immunoassaies of specialty, especially, and sandwich assay (Sandwich assay), the use of competition and non-competing mensuration and direct and non-direct demarcation.
Another aspect of the present invention provides a cover as the HIV that detects or the medicine box of anti-HIV antibody, and it comprises at least a according to synthetic polypeptide of the present invention.
Polyclone or monoclonal antibody, the human form of this antibody (see, for example, Thompson K.M.et al(1986) Immunology 58,157-160), single domain antibodies (see, for example, Ward, E.S., Gussow, D., Griffiths, A.D., Jones, P.and Winter, G.(1989) Nature 341,544-546) and can pass hemato encephalic barrier, be attached to antibody on the synthetic polypeptide of the present invention specifically, can adopt ordinary method preparation and such antibody to be considered to form part of the present invention.Antibody according to the present invention is especially, to be used in the antibody in the method for diagnosing Mammals HIV infection.This method comprises with the antibody as described in this article of significant quantity cultivates and measures the effect generation that whether reacts to each other between described sample and the described antibody with the sample of mammiferous tissue or body fluid, and also can measure degree and/or the ratio that reacts to each other if desired.The Sickledex that contains at least a described antibody also constitutes part of the present invention.
Another aspect of the present invention provides and has been used for the treatment of or prevents Mammals HIV to infect and/or stimulate mammiferous immunity system and/or the synthetic polypeptide of the cell receptor of blocking-up HIV virus and the synthetic polypeptide that is used to prepare the medicament that is suitable for such purposes.Also comprise and containing, at least a as described herein polypeptide or peptide carrier binding substances and one or more medicinal auxiliary agents, the medicinal compositions that carrier and/or vehicle interrelate as activeconstituents.These compositions can be mixed with oral, rectum, nasal cavity or particularly parenterai administration (comprising administration in the central nervous system).
The present invention also provides treatment or prevention Mammals HIV to infect and/or has stimulated immune system and/or the method for blocking-up HIV virocyte acceptor.It comprises, or separately or with other as medicament such as the AZT of treatment AIDS and/or hiv protease inhibitor combined administration significant quantity as polypeptide that preamble limited.
The following example is intended to illustrate the present invention but not limits by any way.
Embodiment 1
The solid phase F-moc method of employing standard (Atherton, E.and Sheppard, R.C., 1985; J.Chem.Soc, Comm.165-166) C-terminal of the peptide I b of the composition sequence Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Gly-Gys-Ald form that stretches.Introduce C-terminal Gly-Cys-Ala and provide another coupling site for carrier.In the presence of trifluoroacetic acid, this peptide is downcut from resin, and by gel-filtration, ion-exchange chromatography and this peptide of RPLC subsequent purificn.The purity of the peptide of gained surpasses 85%.The C-terminal L-Ala is included to support combination.Peptide is dissolved in phosphate-buffered saline (PBS; 5mg/ml) and adding glutaraldehyde reach 0.1%(W/V) ultimate density before mix with the Protalbinic acid (5mg/ml) of equivalent.With the emulsification of Freund auxiliary agent before, allow this binding mixture leave standstill 30 minutes.Every sheep (5 every group) is used the 250 μ g peptide immunity in the complete auxiliary agent of Freund (FCA) of equivalent.In 2 whens week,, every animal was given the injection with booster dose, and the peptide immunity in the incomplete auxiliary agent of Freund (FIA) with equivalent in 5-6 week again.7-10 days blood samplings after the last booster dose injection, and measure in conjunction with on the HIV gp160 envelope protein.
Adopt two strain HIV1 in the research.One strain is fully special, the HIV1 strain that identification is well also extensively adopted, name and be GB8, can be from Dr G.Farrarr, Center for Applied Microbiology and Research(CAMR), Porton Down, Salisbary, UK, obtain and at AIDS1:147-150(1987) in discussed.Another strain is to be used for the RF strain that synplasm suppress to be measured HIV1, and the RF strain of HIV1 can be from Science 224:497-500(1984) learn.From the sequence of 2 or more HIV1 strain isolated of single individuality obtained and comparative measurement tester suppress the growth of these virus strain and the ability of duplicating.Obtain to name to be GB8A, the strain isolated of GB8B and GB8D from Dr.G.Farrarr.When the HIV1 " experiment strain " with GB8 or RF infected, known clone H9, C8166 and JM supported duplicating of synplasm formation or show.
(a) mensuration that the anti-peptide antiserum(antisera) of HIV1 suppresses of living
Adopt the determination step of three kinds of standards to measure the HIV1 that lives by the sero-fast inhibition of anti-peptide (ⅰ) neutralization mensuration
This mensuration is measured anti-peptide antiserum(antisera) and is prevented that sensitive cells is by the acellular viral combination of living and the ability of infection.This measurement is determined by the synplasm counting.
To test antiserum(antisera) at 56 ℃ of following 30 minutes inactivations of heating, suitably dilution and under room temperature (RT), cultivating 30 minutes in RPMI1640 with the isopyknic HIV1 of the liquid that contains known titre.Will concentration known and to HIV1GB8 strain infect extremely sensitive CD4+ clone (JM) and be exposed in the HIV1 anti-serum mixture, placed 2 hours at 37 ℃, flushing is suspended in the cell culture medium, cultivates and carries out the synplasm quantitative measurement in the specific time.Close active in sero-fast by measuring with the minimizing of comparing synplasm quantity.
The contrast that comprises in every BT(batch testing) is that the known positive or positive serum substitute the known positive or positive serum test sera.
(ⅱ) suppress to measure
This mensuration is determined to suppress ability and measurement extracellular and the interior antigenic level of HIV1P24 of cell that HIV1 duplicates behind the anti-peptide antiserum(antisera) cell infection.In addition, carry out plasmodial detection and counting.
In known multiple infection, extremely sensitive CD4+ clone is exposed to HIV1 and cultivated 2 hours at 37 ℃.Then with PRMI1640 with this cell flushing 3 times and add the test antiserum(antisera) of hot purifying of the suitable dilution of equivalent.The cell serum suspension that infects was cultivated 1 hour microscopy and at specific subsequently time site (at 3 and 5 days) pair cell substratum sampled measurements P24 antigen at 37 ℃.Measure P24 antigen levels in the cell in same time site after adopting suitable molten born of the same parents' damping fluid dissolved cell.Storage is measured the antigenic sample of HIV1p24 until measuring with ELISA.Measured synplasm quantity at the 5th day.
Contrast as every BT(batch testing) comprises that known feminine gender and positive serum substitute the test antiserum(antisera) in other authentication step.
(ⅲ) synplasm is measured
This mensuration determine anti-peptide antiserum(antisera) prevent the HIV1 that lives from the cellular invasion that infects to the cell that does not infect with prevent that iuntercellular from passing through the ability of the reaction fusion viral glycoprotein (gp160) and the CD4 molecule.This mensuration detects by synplasm and counting carries out.
The HIV1 that the support live virus of concentration known is duplicated (the H9 cell infects with the HIV1RF strain lentamente) produces (CD4+) clone flushing 3 times, and mixes at the antiserum(antisera) of specific extent of dilution with test usefulness.37 ℃ cultivate 30 minutes after, with these cells with specific ratio with induce extremely sensitive indicator CD4+ clone (C8166) to mix to HIV1 infection and synplasm.The synplasm of observing these cells every day forms.
Contrast as every BT(batch testing) comprises that the known positive and negative serum substitute the test sera in other authentication step.
The result
Neutralization is measured
The cultivation serum of some peptide I b demonstrates the stripped neutralization activity of anti-HIV1GB8 strain.Table 1 shows that the activity of serum 17 anti-all GB8 strain isolateds subsequently is bigger.Mensuration shows in this serum and acellular virus.
Table 1: anti-peptide sheep blood serum shows the neutralization activity of the anti-HIV1 GB8 strain of exsomatizing
*Plasmodial mean number (reducing %) in plasmodial mean number/contrast in the=test
(-)=reduce≤50%
Duplicate and suppress to measure
As indicated in the table 2, just duplicate the minimizing that suppresses to measure the 5th day synplasm quantity, serum 17 and 37 demonstrates comparable activity.Moreover, as shown in the P24 antigen measuring, duplicate and be prevented from.Should be mentioned that the serum that is used in this mensuration is not diluted.
Table 2: with anti-peptide sheep blood serum external test HIV1(GB8 strain) duplicate inhibition
P9=positive human serum N SS=normal sheep serum
Synplasm suppresses to measure
Table 3 has provided the data of serum 17 in synplasm suppresses to measure.1: 100 extent of dilution reduces the inhibition activity of serum to being less than 50% slightly.What kind of horizontal dilute serum of this test prompts can prevent the diffusion that cell is separated and closed and prevent live virus thus.
Table 3: the external synplasm anti-HIV1(H9-RF strain of anti-peptide) suppresses active
*Synplasm mean number in synplasm mean number/contrast in the=test (reducing %)
Embodiment 2
HIV-2 positive human serum with reactive according to the ELISA of the polypeptide of formula II a and II b as in an embodiment synthetic the and purifying of described method have sequence
Gln-Gln-Glu-Met-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Gly-Cys （2A）
The formula II the C-terminal extension peptide and have a sequence
The C-terminal extension peptide of the formula II b of Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Gly-Cys (2B) is used from ELISA with these peptides and HIV positive human serum one then.Peptide " 10A " (being disclosed among we the unsettled embodiment 1 with the disclosed application of WO91/00903) is known be the immunity district peptide relevant with the HIV envelope protein and known be to discern the individuality that all HIV-1 infect substantially.This peptide is used to study the suitability of reacting to each other property and definite method.
The ELISA method
1. with 100 μ l parcel damping fluid (coating buffer), contain 20 μ g/ml peptides, be added in each hole of elisa plate (Dynetech), and with this plate 4 ℃ of incubated overnight.
2. with the Tris buffered saline plate is washed once and finish-drying.
3. 200 μ l blocking-up damping fluid is added each hole, cultivated 1 hour with plate shake 10 seconds and at 37 ℃.
4. the serum that is used to test dilutes 1/100 or 1/200 with dilution buffer liquid.Add 0.1ml in each hole, and plate was cultivated 1 hour at 37 ℃.Each sample triplicate.
5. with dcq buffer liquid plate is washed three times, continue 2 minutes.
6. 100 μ l alkaline phosphatase bonded goats are resisted dilution buffer liquid into IgG(1/2000) add each hole and plate was cultivated 1 hour at 37 ℃.
7. as flushing hole in (5).
8. 50 μ l alkaline phosphatase damping fluids are added each hole, then add 50 μ l substrates (10mg/ml water) and plate was cultivated 15 minutes at 37 ℃.
9. read optical density at 414nm at once.
The human serum sample of test is as follows:
20 had before shown with HIV-1 serum and had not presented interactional known HIV-2 positive serum.All 20 patients are from the Ivory Coast.
5 previous demonstrations with HIV-1 serum do not present the known HIV-2 positive serum that reacts to each other.All 5 patients from Africa but the non-Ivory Coast.
7 have shown with HIV-2 serum and have not presented the known HIV-1 positive serum that reacts to each other.
10 known HIV negative serum samples that match from age and sex and theme.
Contrast does not contain serum, but cumulative volume keeps by the dilution buffer liquid that adds 0.1ml.
Result in the table 4 clearly illustrates that:
1) do not observe reacting to each other property between HIV-1 and the HIV-2 peptide, in other words HIV-2 serum nonrecognition HIV-1 peptide and HIV-1 serum nonrecognition HIV-2 peptide;
2) in the HIV negative sample, do not find the specific activity of peptide;
3) from reactivity, having any different between Ivorian patient's serum and the serum from the patient of the other parts of the African continent; With
4) the HIV-1+ serum sample has the HIV-1(10A that can survey) antibody.
Table 4:HIV positive serum and peptide 2A, the reactivity of 2B and 10A
As expected, the no positive findings of contrast produces
These results show that the HIV-2+ serum from some patients contains and the antibody that specific reaction is arranged as the HIV-2 polypeptide of formula II a of the present invention and II b.
Embodiment 3
Give the mouse immunity with the peptide that is incorporated into carrier proteins suc as formula I a
As the method described in the embodiment 1, synthetic and purifying has sequence
Gln-Gln-His-Leu-Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Gly-Cys （ⅠA）
The peptide that stretches of the C-terminal suc as formula I a.
In conjunction with
Adopt glutaraldehyde or MBS(M-maleimide benzoyl-N-hydroxyl-succinimide ester) as coupler peptide is attached on the Protalbinic acid.
Glutaraldehyde: peptide is dissolved in PBS(5mg/ml) and add Protalbinic acid and provide 50 amino acid whose mol ratios of carrier of per 1 mole of peptide.Cumulative volume transfers to 2ml with PBS.Join the glutaraldehyde PBS solution of 2ml0.2% in the mixture that is stirring lentamente and allow solution at room temperature stir 1 hour.This mixture spends the night with a large amount of PBS dialysis and stores under-20 ℃ and was no more than for 3 weeks.
MBS: join the MBS solution (25mg/ml PBS) of 0.1ml in the Protalbinic acid solution (10mg/ml PBS) that is stirring and stirred reaction mixture 30 minutes at room temperature.By gel-filtration on usefulness PBS equilibrated Sephadex G-25 post the activatory Protalbinic acid is separated from free MBS then.Peptide is dissolved in PBS and adds in the activatory Protalbinic acid.So just obtain the ultimate density of per 50 carrier amino acid/11 mole peptides.PH is transferred to 7-7.5 and at room temperature allow reactant stir three hours.Mixture is spent the night with a large amount of PBS dialysis and under-20 ℃, store and be no more than for 3 weeks.
Be to determine the ratio of peptide/carrier proteins, can adopt the step that Habeeb describes (Habeeb, A.F.S.A., Anal, Biochem., 14,328(1966)).In this step, total free aminoacids is by the borate buffer solution with PH9.0, and trinitro-benzene-sulfonic acid (TNBS) method is measured.Read the optical density of several solution and the percentage of the free amine group acid groups that calculating is modified at 335nm.Free mercaptan is measured as also can be used for estimating combination rate (Anderson, W.L.and Wetlanfer, D.B., Anal.Biochem.67,493(1975)) by Ellman is described.
Immunity
The blood drawing in 2-3 days before initial inoculation of the black mouse of C57 is with the serum separation and-20 ℃ of storages.The negative control mice serum of this serum.Every mouse is used in the 40 μ g peptides immunity among the FCA and is used in 20 μ g peptide immunity among the FIA after 2 to 3 weeks subsequently.The inoculation volume is that 0.1ml and all injections are subcutaneous injection under every kind of situation.Just before the booster dose injection, draw blood, then 2 weeks and the back blood drawing of 4 weeks to animal.
ELISA
The ELISA method is described in embodiment 2, and except serum is diluted damping fluid dilution 1/50 and 1/100, and used antibody is and goat anti-mouse IgG bonded alkaline phosphatase.
The result
1. compared with the control, animal 2/24(8%) is to responding with the peptide of glutaraldehyde bonded suc as formula I a after drawing blood for the 3rd time.Therefore, may work as when being combined on the carrier nonspecificly, produce the antibody of anti-this peptide.
2. be incorporated into by special connection after the peptide immunization once of formula I a of Protalbinic acid with MBS, mouse 14/24(58%) just has high-level antibody.This is a kind of strong and special reaction.
The result shows, suc as formula the polypeptide of I a, when being incorporated into Protalbinic acid with MBS, is a kind of strong immunogen, and it is only once just inducing the antibody of high level after the immunity, and both to have made this peptide be the non-Protalbinic acid that is attached to specifically, also can produce antibody.
Claims (29)
1, a kind of synthetic polypeptide with at least a antigen attribute of at least one strain HIV (human immunodeficiency virus) (HIV) envelope protein, said polypeptide is made up of the aminoacid sequence of formula I substantially:
X-R
1-Leu-R
2-Leu-Thr-Val-Trp-Gly-R
3-Lys-Y(Ⅰ)
R wherein
1Be selected from Gln-Gln-R
4-R
5, Gln-R
4-R
5, R
4-R
5, disappearance R
5Or R
1
R
2For being selected from Gln, Lys, the amino-acid residue of Glu or Arg;
R
3For being selected from Ile, the amino-acid residue of Thr or Ala;
R
4Be His or Glu;
R
5Be Leu or Met; And
X and Y can lack independently of one another or be one or more amino-acid residues independently.
2, according to desired synthetic polypeptide, wherein R in the claim 1
2Be selected from Gln, Lys and Glu, and R
3Be Ile.
3, according to the desired sequence that contains in claim 1 or the claim 2
(I a) for X-Gln-Gln-His-Leu-Leu-Gln-Leu-Thr-Val-Trp-Gly-Ile-Lys-Y; With
The synthetic polypeptide of X-Leu-Gln-Leu-Leu-Thr-Val-Trp-Gly-Ile-Lys-Y (I b),
Wherein X and Y are as defined in claim 1.
4, according to the desired synthetic polypeptide that contains the aminoacid sequence of formula II substantially in the claim 1:
X-R
1-Leu-Arg-Leu-Thr-Val-Trp-Gly-R
3-Lys-Y （Ⅱ）
R wherein
1Be selected from Gln-Gln-Glu-R
5, Gln-Glu-R
5, Glu-R
5, disappearance R
5Or R
1
R
3Be Thr or Ala;
R
5Be Met or Leu; And
X and Y are as defined in claim 1.
5, contain sequence according to desired synthetic polypeptide in the claim 4:
(II a) for X-Gln-Gln-Glu-Met-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Y; Or
X-Leu-Arg-Leu-Thr-Val-Trp-Gly-Thr-Lys-Y （Ⅱb）
6, the desired any a kind of synthetic polypeptide of claim as described above, wherein lacking X and Y is Gly-Cys or Gly-Cys-Ala.
7, the synthetic polypeptide of logical formula III
[La-F]m-[Lb-G]-Lc （Ⅲ）
Wherein F and G can be that L is a catenation sequence suc as formula I any one polypeptide to the II b independently of one another, and a, b and c are 0 or 1 and m and the m positive number of respectively doing for oneself independently of one another, and be for example included between 1 and 10.
8, the synthetic polypeptide of logical formula IV:
La-[G-L]m-F-[L-G]n-Lc （Ⅳ）
Wherein F is suc as formula any polypeptide of I to II b, G be suc as formula I to any of II b or other polypeptide of sequence, m and the n positive number of respectively doing for oneself, for example included between 1 and 10, and d and e are 0 or 1 independently of one another.
9, a kind of synthetic polypeptide, it comprises as the subfragment of any one antigen meaning in the desired peptide sequence of claim 1 to 8 and/or the varient of antigen meaning.
10, also comprise a kind of T cell epitope as claim 1 to 5 or 7 to 9 desired synthetic polypeptide.
11, comprise a kind of retro-inverso amino acid as desired synthetic polypeptide in claim 1 to 5 or 7 to 10.
12, claim requires any a kind of synthetic polypeptide to be connected in a kind of carrier as described above.
13, a kind of dna molecular, its coding is at least a as the desired any a kind of synthetic polypeptide of claim 1 to 10.
14, a kind of vaccine comprises at least aly as desired any one the synthetic polypeptide of claim 1 to 12, promotes prevention HIV to infect effectively.
15, a kind of medicine box that is used to detect HIV or anti-HIV antibody, it comprises at least a as desired any one the synthetic polypeptide of claim 1 to 12.
16, a kind of medicinal compositions, it contains as effective constituent, and at least a as desired any one the synthetic polypeptide of claim 1 to 12, with one or more pharmaceutically acceptable auxiliary agents, carrier and/or vehicle mix mutually.
17, a kind of according to desired medicinal compositions in the claim 16, also comprise AZT and/or a kind of hiv protease inhibitor.
18, a kind of HIV or anti-HIV antibody or disconnected method of its antigen binding fragment of detecting, it comprises cultivates sample with desired one of any peptide species of at least a as claim 1 to 12.
19, a kind of antibody or its antigen binding fragment are disconnected, and it is combined in specifically as on the desired any a kind of synthetic polypeptide of claim 1 to 12.
20, a kind of as the judgement instrument that detects HIV or anti-HIV antibody, it contains at least a as desired antibody in the claim 19 or its binding fragment.
21, a kind of HIV or anti-HIV antibody or disconnected method of its antigen binding fragment of detecting, it comprises cultivates with at least a sample as desired antibody in the claim 19 or its binding fragment.
22, a kind of medicinal compositions, it contains, and disconnected as desired antibody in effective constituent such as the claim 19 or its antigen binding fragment, with one or more medicinal auxiliary agents, carrier and/or vehicle mix mutually.
23,, also comprise AZT and/or a kind of hiv protease inhibitor as desired composition in the claim 22.
24, a kind of method of diagnosing HIV to infect, it comprises the sample of mammiferous tissue or body fluid cultivating and measuring whether to have to interact between described sample and described antibody as desired a kind of antibody or its binding fragment in the claim 19 and take place with significant quantity, as needs, measure interactional degree and/or ratio.
25,, a kind of at antibody or the living anti-idiotype antibody of antigen binding fragment stopping pregnancy as desired in the claim 19.
26, be used for the treatment of or preventive treatment Mammals HIV infects and/or application when being used to stimulate the medicine of cell receptor of mammiferous immunity system and/or blocking-up HIV virus in preparation as desired any a kind of synthetic polypeptide in the claim 1 to 12.
27, a kind of treatment or prevention Mammals HIV infect and/or are used to stimulate the method for immune system and/or blocking-up HIV virocyte acceptor, it comprise use significant quantity as desired any a kind of synthetic polypeptide in the claim 1 to 12.
28, a kind of preparation has the method for synthetic polypeptide of at least a antigen attribute of at least one strain HIV (human immunodeficiency virus) (HIV) envelope protein, and described polypeptide is made up of the aminoacid sequence of formula I substantially:
X-R
1-Leu-R
2-Leu-Thr-Val-Trp-Gly-R
3-Lys-Y （Ⅰ）
R wherein
1Be selected from Gln-Gln-R
4-R
5, Gln-R
4-R
5, R
4-R
5, no R
5Or R
1;
R
2For being selected from Gln, Lys, the amino-acid residue of Glu or Arg;
R
3For being selected from Ile, the amino-acid residue of Thr or Ala;
R
4Be His or Glu;
R
5Be Leu or Met; And
X and Y can lack independently of one another or be one or more amino-acid residues independently,
This method comprises the known chemistry of employing itself, biology or recombinant technology coupling residue and isolated polypeptide.
29, a kind of preparation is combined in specifically and has at least one strain HIV (human immunodeficiency virus), (HIV) method of the antibody on the synthetic polypeptide of at least a antigen attribute of envelope protein, and said polypeptide is made up of the aminoacid sequence of formula I substantially:
X-R
1-Leu-R
2-Leu-Thr-Val-Trp-Gly-R
3-Lys-Y （Ⅰ）
R wherein
1Be selected from Gln-Gln-R
4-R
5, Gln-R
4-R
5, R
4-R
5, no R
5Or R
1;
R
2For being selected from Gln, Lys, the amino-acid residue of Glu or Arg;
R
3For being selected from Ile, the amino-acid residue of Thr or Ala;
R
4Be His or Glu;
R
5Be Leu or Met; And
X and Y can not have independently of one another or be one or more amino-acid residues independently, and this method comprises with described polypeptide gives inhuman mammalian immune and separation antibody.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB929208428A GB9208428D0 (en) | 1992-04-16 | 1992-04-16 | Synthetic polypeptides |
| GB9208428.4 | 1992-04-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1082053A true CN1082053A (en) | 1994-02-16 |
Family
ID=10714175
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN93105907A Pending CN1082053A (en) | 1992-04-16 | 1993-04-16 | Synthetic polypeptide |
Country Status (9)
| Country | Link |
|---|---|
| EP (1) | EP0636145A1 (en) |
| JP (1) | JPH07505878A (en) |
| KR (1) | KR950700933A (en) |
| CN (1) | CN1082053A (en) |
| AU (1) | AU3960493A (en) |
| CA (1) | CA2118033A1 (en) |
| GB (1) | GB9208428D0 (en) |
| WO (1) | WO1993021218A1 (en) |
| ZA (1) | ZA932648B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100366633C (en) * | 2006-04-18 | 2008-02-06 | 河北师范大学 | Antiviral polypeptide from Zongdian Tuanwa frog and its application in pharmacy |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5128319A (en) | 1987-08-28 | 1992-07-07 | Board Of Regents, The University Of Texas System | Prophylaxis and therapy of acquired immunodeficiency syndrome |
| US6210873B1 (en) | 1987-08-28 | 2001-04-03 | Board Of Regents, The University Of Texas System | Methods and compositions for the priming of specific cytotoxic T-lymphocyte response |
| ATE258188T1 (en) | 1992-08-27 | 2004-02-15 | Deakin Res Ltd | RETRO, INVERSO, AND RETRO-INVERSO SYNTHETIC PEPTIDE ANALOGS |
| US5603933A (en) * | 1993-08-31 | 1997-02-18 | Board Of Regents, The University Of Texas | CD4 peptides for binding to viral envelope proteins |
| AUPM411994A0 (en) * | 1994-02-25 | 1994-03-24 | Deakin Research Limited | Epitopes |
| FR2717081B1 (en) * | 1994-03-14 | 1996-06-21 | Centre Nat Rech Scient | Retropeptides, antibodies to the latter, and their uses for vaccination and in vitro diagnosis. |
| US5705522A (en) * | 1995-09-15 | 1998-01-06 | Compagnie De Developpement Aguettant S.A. | Compounds having anti-inflammatory and anti-viral activity, compositions of these, alone and in combination with reverse transcriptase inhibitors |
| JP2008173077A (en) * | 2007-01-22 | 2008-07-31 | National Institute Of Advanced Industrial & Technology | Monitor protein for analyzing expression of membrane protein |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8714802D0 (en) * | 1987-06-24 | 1987-07-29 | Proteus Biotech Ltd | Synthetic polypeptides |
| EP0330359A3 (en) * | 1988-02-25 | 1991-06-05 | Bio-Rad Laboratories, Inc. | Composition useful in the diagnosis and treating of hiv-1 infection |
| AU3557889A (en) * | 1988-04-20 | 1989-11-24 | Trustees Of The University Of Pennsylvania, The | Protective peptides derived from human immunodeficiency virus-1 gp160 |
| CA2003383A1 (en) * | 1988-11-23 | 1990-05-23 | Sushil G. Devare | Synthetic dna derived recombinant hiv antigens |
| GB9005829D0 (en) * | 1990-03-15 | 1990-05-09 | Proteus Biotech Ltd | Synthetic polypeptides |
-
1992
- 1992-04-16 GB GB929208428A patent/GB9208428D0/en active Pending
-
1993
- 1993-04-15 ZA ZA932648A patent/ZA932648B/en unknown
- 1993-04-16 JP JP5518145A patent/JPH07505878A/en active Pending
- 1993-04-16 WO PCT/GB1993/000808 patent/WO1993021218A1/en not_active Application Discontinuation
- 1993-04-16 EP EP93909057A patent/EP0636145A1/en not_active Withdrawn
- 1993-04-16 CA CA002118033A patent/CA2118033A1/en not_active Abandoned
- 1993-04-16 CN CN93105907A patent/CN1082053A/en active Pending
- 1993-04-16 AU AU39604/93A patent/AU3960493A/en not_active Abandoned
-
1994
- 1994-10-14 KR KR1019940703649A patent/KR950700933A/en not_active Application Discontinuation
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100366633C (en) * | 2006-04-18 | 2008-02-06 | 河北师范大学 | Antiviral polypeptide from Zongdian Tuanwa frog and its application in pharmacy |
Also Published As
| Publication number | Publication date |
|---|---|
| GB9208428D0 (en) | 1992-06-03 |
| WO1993021218A1 (en) | 1993-10-28 |
| ZA932648B (en) | 1994-10-15 |
| JPH07505878A (en) | 1995-06-29 |
| KR950700933A (en) | 1995-02-20 |
| AU3960493A (en) | 1993-11-18 |
| CA2118033A1 (en) | 1993-10-28 |
| EP0636145A1 (en) | 1995-02-01 |
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