CN1867588A

CN1867588A - Product comprising a c4bp core protein and a monomeric antigen, and its use

Info

Publication number: CN1867588A
Application number: CNA2004800299249A
Authority: CN
Inventors: 弗盖尔·希尔
Original assignee: Avidis SA
Current assignee: Avidis SA
Priority date: 2003-08-12
Filing date: 2004-08-12
Publication date: 2006-11-22
Also published as: JP2007528210A; WO2005014654A2; AU2004263387A1; CA2535517A1; WO2005014654A3

Abstract

The invention provides a product which comprises a C4bp core protein and a monomeric antigen, desirably in the form of a fusion protein. Monomeric antigens include malarial and influenza antigens. The C4bp core protein provides for assembly of multimeric complexes of the monomeric antigen, or mixtures thereof. The complexes are useful as vaccines.

Description

Comprise product of C4BP core protein and monomeric antigen and uses thereof

The application requires the right of priority of PCT/EP2003/008926, and its content is incorporated herein by reference.

Technical field

The present invention relates to the macromole assembly, fusion rotein for example, it comprises adjuvant and antigen, compares with this antigen of independent use, and this assembling physical efficiency causes at this antigenic enhanced immunne response.

Background technology

Adjuvant improves at antigenic immunne response, and is therefore available in vaccine.But have only limited several adjuvant to be licensed for human body, and because the stronger adjuvant of effect from zooscopy, know, therefore be very tangible for using safely in human body, needs with immunological adjuvant of more pretending usefulness.Several pieces of nearest summaries, ask for an interview " vaccine adjuvant progress " (" Advances in vaccine adjuvants ") (Nature Biotechnology, 1999 the 17th volumes, the 1075-1081 page or leaf) and " discovery of vaccine adjuvant and the latest developments of transmission " (" Recent Advances in the discovery and delivery ofvaccine adjuvants ") (Nature Reviews in Drug Discovery, 2003 the 2nd volumes, the 727-735 page or leaf).

Complement system is made up of a series of serum proteins, and these protein have vital role in immune the replying at exotic antigen.Complement system main ingredient therein is activated when being cut, and product causes the proteolysis cascade reaction separately or with the complement protein that other protein activates other.The activation of complement system can bring multiple replying, and comprises the activation of vascular permeability enhancing, cytophagous chemotaxis, inflammatory cell, to the direct killing effect and the tissue injury of external particulate opsonization and pair cell.The activation of complement system may be (classical pathway) that is caused by antigen-antibody complex, and perhaps normal activation slowly may be exaggerated (alternative pathway) in the presence of for example biological invasion such as bacterium and virus cell walls.Complement system and cell immune system interact by the specificity approach that relates to C3, and C3 is the key protein matter at classical pathway and alternative pathway.The activation of the proteolysis of C3 can produce big fragment (C3b), and expose can with the chemically reactive inherent thioester bond of external nucleophile (for example cell surface protein of biological invasion or external cell) covalent interaction.Consequently potential antigen combines with this albumen until C3b further is hydrolyzed to iC3b and C3d, g with C3b " mark " and maintenance.The part (CR2 is also referred to as CD21) that described latter's fragment is respectively complement receptor CR3 and CR2.Become the mechanism that target has the immune cell of these acceptors with the C3b labelled antigen like this.

This target mechanism is important for the enhancing of immunne response, this at first be experimental results show that by following, wherein the circulation C3 of mouse exhausts and with antigen (sheep red blood cell (SRBC)) immunity, the removal of C3 has weakened antibody to this antigenic (M.B.Pepys that replys, J.Exp.Med., 140,126-145,1974).Produce the effect that studies have shown that C3 (J.M.Ahearn and D.T.Fearon, Adv.Immunol., 46,183-219,1989) that the animal of upstream components (being C2 and C4) of the complement cascade reaction of C3 carries out by disappearance C3 in heredity or disappearance.Show recently, compare that model antigen combines remarkable enhancing (the 1000-10000 doubly) (P.W.Dempsey etc. that cause mouse antibodies to reply with the linearity of the mouse C3d fragment sequence of copy more than two with not modified antigen control, Science, 271,348-350,1996; WO96/17625, PCT/GB95/02851).This reinforcing effect is not using traditional adjuvant for example also can take place under the situation of Freund's complete adjuvant, and this adjuvant has toxicity and be unsuitable for human the use.To be polyvalent C3d construct combine with CR2 high-affinity on the B cell mechanism that has proved this remarkable effect, and CR2 is connected altogether with another kind of B cell membrane protein CD19 and membrane bound immunoglobulin afterwards, produces the signal to B cell nuclear.

Yet the homologous recombination protein matter that verified generation contains three C3d copies in a large number is very difficult.Subject matter is:

1) genetic instability that contains the construct of (three) tumor-necrosis factor glycoproteins;

2) folding (or dissolving and folding again) of the recombinant protein of the inclusion body that forms in the next comfortable intestinal bacteria.

Make the minimized method of genetic instability of the construct that repeats to copy that contains the C3d gene in WO99/35260 and WO01/77324, description be arranged.The technology of describing in these applications is to use the different sequences of the DNA of coding C3d repeated fragment.

Use the multimerization system of c4 binding protein (C4bp) description to be arranged at WO 91/11461.People C4b conjugated protein (C4BP) is the plasma glycoprotein with high molecular (570kDa), and it has by 7 identical α chains and 1 spider shape structure that the β chain is formed.C4bp α chain has this molecule of being responsible for and is assembled into polymeric C-terminal nucleus.According to standard model, the halfcystine of halfcystine C4bp monomer+498 and another monomer+510 forms disulfide linkage.The less important form that only contains 7 α chains is also found in human plasma.The natural function of this plasma glycoprotein is the classical pathway that suppresses complement activation.

The major part of C4bp α chain is made up of the structural domain of about 60 amino acid whose 8 arranged in series of length, is called complement control albumen (CCP) tumor-necrosis factor glycoproteins.WO91/11461 proposes ability that C4bp protein carries out multimerization can be used for preparing the fusion rotein that comprises all with portion C 4bp and purpose bioprotein.Preferably include one or more CCP tumor-necrosis factor glycoproteinss (being also referred to as SCR) in the fusion rotein of in WO91/11461, describing.

WO91/11461 also advises this fusion rotein as vaccine.Now made some concrete protein, it comprises at least one C4bp SCR zone of merging with the hepatitis B virus e antigen fragment.Employed e antigen fragment is the cAg fragment that can form the polymer structure.

Libyh M.T. etc. (1997, Blood, 90,3978-3983) propose, all CCP can both depleted (57 amino acid of only remaining C-terminal) and do not stop multimerization.This of C4bp section C-terminal zone is called as the C4bp core here.

Shinya etc. (1999, Biomed ﹠amp; Pharmacother Vol.53:471) has also set forth the polymer solubility CD4-C4bp fusion rotein that has from the body assembling, and wherein this fusion rotein is expressed in people's 293 clones.

(2000, Journal of Immunology has also described the purposes of C4bp in Vol.164:1505) at Oudin etc.

(2000, Journal of Virology Vol.74:4672) has discussed the therepic use of CD46-C4bp fusion rotein to Christiansen etc.

WO2004/020639 provides the method that obtains recombination fusion protein in prokaryotic hosts, it comprises the optional support that merges with heterologous polypeptide of C-terminal core protein of C4bp α chain, and described recombination fusion protein can form the polymer of soluble form in prokaryotic host cell.

Summary of the invention

The present invention is based on the new discovery of the concrete kind of relevant C4bp fusion rotein.Prior aries more discussed above propose, and are on the basis of inert support basically in the C4bp part, the proteinic purposes of C4bp fusion rotein delivery therapeutic interest.

The described WO91/11461 of preamble has exemplified to comprise and can form polymeric antigenic fusion rotein.

By comparison, the present invention is based on and can forms naturally that polymeric antigen is unsuitable for and the C4bp core merges, because this antigen in fact may disturb the C4bp core to be assembled into polymer.In addition, existing proof, surprisingly, the fusion of C4bp core and monomeric antigen can cause at this antigen intensive immunne response.Show that in example subsequently the fusion rotein comprise monomeric antigen causes that high titre, inhibiting antibody reply, by comparison, when antigen and freund's adjuvant are injected jointly, can cause the antibody response of low titre, non-inhibity.

Therefore, the invention provides by monomeric antigen and C4bp core protein are combined the immunogenic method of raising monomeric antigen in mixture.In a preferred method, described monomeric antigen and C4bp core protein covalent attachment.In preferred method, described monomeric antigen and C4bp core protein heredity merge.

The present invention also provides the method for inducing at the high titre of antigenic antibody, and the sero-fast purposes of high titre, and described antiserum(antisera) is by using described method to produce in the infectivity by passive immunization or malignant disease prevent and/or treat.In a preferred method, partial purification is at described antigenic high titre antibody by the immunoglobulin fraction in separation hyperimmunization blood plasma or the serum, in the best approach, the immunoglobulin fraction of described hyperimmunization blood plasma or serum is to separate from preventing or treat the same species individuality of infectivity or malignant disease with described antiserum(antisera).

Therefore the invention provides product, it has comprised:

The C4bp core protein; With

Monomeric antigen.

This first and second component can be the form of fusion rotein.Perhaps, they can be by chemical coupling, and described coupling is by any amino acid side chain of first component, or the amino acid whose side chain that is added on first component by specificity comes described second component of chemical coupling.

Described first or second component can be by tight but non-covalent combination.For example, the amino acid side chain of described first component can be modified to have additional vitamin H group, and this vitamin H can be used to and streptavidin combination (streptavidin is as second component), and perhaps the antigen with the streptavidin fusion can pass through this vitamin H and first combination of components.In other possibility, biotinylated antigen and biotinylated first component can be by the mixture that adds streptavidin matter and purifying gained tight but combine non-covalently.

For avoiding query, name " first " and " second " component does not hint or is illustrated in the concrete linear precedence in the product of these two kinds of components.These two kinds of components can anyly be linked in sequence.

Therefore working as two kinds of components all is that polypeptide and described product are made into fusion rotein, and the N-terminal of these two kinds of components and C-terminal order can exchange arbitrarily.

The present invention also provides the nucleic acid of the fusion rotein of described first and second components of encoding.The present invention also provides carrier that comprises described nucleic acid and the host cell that carries described carrier.

In another specific embodiments, the invention provides the method for preparing product, described product comprises:

The C4bp core protein; With

Polypeptide monomer antigen,

This method comprises expresses the nucleic acid of coding with these two kinds of components of fusion rotein form, and reclaims described product.

The C4bp core protein; With

It is non--polypeptide monomer antigen,

This method comprises the nucleic acid of expressing coding C4bp core protein, described core protein is connected with described antigen, and reclaims described product.

The method for preparing described product can be carried out in eucaryon or prokaryotic cell prokaryocyte.

The present invention also provides the method for inducing antigenic immunne response, and the product according to the present invention that this method comprises significant quantity gives the experimenter.

The present invention also provides the purposes of product of the present invention in the method for the method, particularly induce immune response of treatment human body or animal body.

The present invention also provides pharmaceutical composition, and it comprises the associating of product of the present invention and pharmaceutically acceptable carrier or thinner.

The present invention also provides the method for preparing the protective immunity serum that uses in the passive immunization at infectious substance, and this method comprises with product inoculation animal of the present invention, reclaims antiserum(antisera) from this animal.This antiserum(antisera) can be used for the passive immunization method to people experimenter then.This people experimenter can be the experimenter who infects or have this infectious substance risk of infection.

The accompanying drawing summary

Fig. 1 provides the comparison of C4bp core protein.

Fig. 2 is given in the proteinic result of expression in escherichia coli.

Fig. 3 is given in the proteinic contrast of electrophoretic the present invention on the reduction and the gel of non-reduced condition.

Summary of the invention

The core protein of C4bp α chain

Here refer to " C4bp core protein " or " core protein ", or " C4bp support ". These terms Can exchange. This protein can be mammiferous C4bp core protein, or it can shape Become polymer and bring into play the fragment of adjuvant effect, or it can form polymer and can bring into play adjuvant and do With synthetic or chimeric variant.

Among the present invention, the C4bp core protein or comprise described core protein C4bp α chain fragment (please Detailed description sees below) as adjuvant. The people C4bp core protein of SEQ ID NO:1 is corresponding to ability The territory is known to form the amino acid of polymeric total length C4bp protein sequence+493 to+549.

The derivative that the present invention has further comprised described C4bp core is in improve antigen immune originality Purposes. These derivatives comprise its mutant, and it may comprise amino acid deletions, add particularly half Guang The interpolation of propylhomoserin residue or replacement, formed by the fusion of the part of the different members of C4bp family assorted Close or the protein scaffolds of chimeric molecule and/or annular arrangement, all can be used for the adjuvant character that keeps above-mentioned.

The present invention can also use artificial total that comparison obtains based on different plant species C4bp core sequence The C4bp sequence. An example of this class chimeric molecule in a lot of possibilities provides (SEQ hereinafter ID:20, Fig. 1).

Therefore adjuvant may be the one or more of C4bp core and optional and described core integration SCR.

In a most preferred specific embodiments, the C4bp component of product of the present invention is C4bp α chain Core protein, the core protein that does not link to each other with any C4bp SCR sequence that namely limits here. In this specific embodiments, described C4bp core is ideally by the residue 1-57 of SEQ ID NO:1 Or the corresponding residue of its homologue forms, or by at least 47 ammonia of SEQ ID NO:1 or its homologue The fragment of base acid forms.

The C4bp core of product of the present invention can comprise the extension of N end or C end in addition as connecing flexibly Head. Common this joint length is several amino acid, is 1 to 20 such as length, for example 2 to 10 Amino acid. A kind of such joint is (Gly_m-Ser) _nJoint, wherein m and n are separate is 1 to 4. These are used for making protein domain to be connected to each other in this area. Therefore described first component can be with described the Two components link to each other by such joint.

Preferably when first component was the C4bp core, it was positioned at the C end of product.

There is the sequence of many mammal C4bp protein in this area. These comprise people C4bp core Protein (SEQ ID NO:1). This area also has the homologue of many people C4bp core proteins. Have Two class homologues: directly to homologue (orthologue) and side direction homologue (paralogue). Directly to homologue Be defined as the homologous genes in the different organisms, namely these genes have the common ancestor, and this is common Ancestors form event with the species that produce these genes and take place simultaneously. The side direction homologue is defined as identical life Deriving in the object has the homologous gene of gene, chromosome or genome duplication, namely the common ancestor of gene from Last species formation event plays appearance.

For example, search GenBank, original gene group vestige (raw genomic trace) and EST (Expressed sequence tags) database displaying comprise chimpanzee, macaque, rabbit, rat, dog, horse, The mammal C4bp core SEQ ID NO:1 homologue egg of the species of mouse, cavy, pig and Niu Nei In vain. The side direction homologue of C4bp SEQ ID NO:1 and directly be included in ratio among Fig. 1 to homologue Centering.

In a word, the comparison of SEQ ID NOs:1-19 as shown in Figure 1. Can find out all 19 orders Row have high similarity, although at the C end largely variation is arranged. And, use such as BLAST Search utility commonly used, can identify by the database of search DNA or protein sequence C4bp nuclear Heart protein matter.

If in database, do not have the C4bp protein of required mammal, can use this area The conventional cloning process that has had obtains it. Basically, this technology comprises with the existing C4bp of coding The nucleic acid of one of core protein is as the C4bp core egg in probe recovery and definite other purpose species The sequence of white matter. This is existed multiple technologies, for example with suitable genomic DNA or mRNA source (for example from the embryo or in the noble cells or tumour cell of active division) pcr amplification is also cloned this base Cause, or use and to comprise from mammal and obtain cDNA library (for example from the cDNA in one of above-mentioned source The library) method, in moderate to highly strictly spending condition (for example 0.03M sodium chloride and 0.03M lemon Acid sodium, about 50 ℃ to 60 ℃) with the known described library of C4bp nuclei acid probe, and reclaim and encode The cDNA of the sort of mammiferous all or part of C4bp protein. After having obtained part cDNA, Complete encoding sequence can be determined by the primer elongation technology.

The fragment that can form polymeric C4bp core protein can comprise at least 47 amino acid, and is excellent Select at least 50 amino acid. This fragment forms polymeric ability and can detect by the following method: according to The present invention expresses this fragment in prokaryotic host cell, but at whole 57 the amino acid C4bp nuclears of multimerization The condition of the heart reclaims described C4bp fragment, and determines whether this fragment also forms polymer. Ideally, The fragment of C4bp core includes at least residue 6-52 of SEQ ID NO:1 or the correspondence of its homologue Residue.

Also can use the variant of C4bp core and can form polymeric fragment, described variant is similar Kept and formed polymeric ability (can be such as above-mentioned mensuration for fragment). Described variant is preferably with wild Type mammal C4bp core or its polymer form fragment at least 70%, and more preferably at least 80%, Very more preferably at least 90%, at least 95% or most preferably at least 98% sequence homogeneity for example.

On the one hand, the C4bp core should be the glycine that appears at position 12 that comprises SEQ ID No:1, Appear at position 28 alanine, appear at position 29,34,36 and 41 leucine, appear at The tyrosine of position 32, appear at the lysine of position 33 and optimize present position 6 and 18 The core of two cysteine residues. Ideally, this variant will be retained in the phase between these residues To space length.

The homogeneity degree of above-mentioned appointment be with respect to arbitrary among the SEQ ID NOs:1-20 or its can form Polymeric fragment.

Most preferably the homogeneity degree of described appointment is can form poly with respect to SEQ ID NO:1 or its The fragment of body.

Sequence homogeneity degree can determine that by the GAP algorithm this algorithm is to be extensive use of in this area The part of algorithm " Wisconsin program package ", can (be Genetics Computer originally from Accelrys Group, Madison, WI) obtain. GAP uses Needlema and Wunsch algorithm with maximization Join number and compare two complete sequences with the mode that minimizes the breach number. GAP can be used to compare tool Therefore the sequence that is closely related that the weak point of similar length is arranged is suitable for determining whether a certain sequence reaches above-mentioned The homogeneity level. GAP can use under the condition of default parameters.

The synthetic variant of mammalian cell C4bp core protein comprises and has at C end or N end The variant of one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, disappearance, insertion or interpolation. Replacement is most possible. Replace and comprise conservative the replacement. The conservative example that replaces comprises for the similar ammonia that is commonly referred to the Dayhoff group Those replacements of the group of base acid. As follows:

The 1st group

D，E，N，Q

The 2nd group	I，L，V，M
The 2nd group	I，L，V，M	The 3rd group	F，Y，W
The 4th group	K，R，H	The 3rd group	F，Y，W
The 4th group	K，R，H	The 5th group	S，P，T，A，G
The 6th group	C	The 5th group	S，P，T，A，G

Can prepare and detect it and form polymer and comprise SEQ ID NOs:37 to 44 as fragment and the variant of the C4bp core protein of the ability of adjuvant, as shown in table 1:

Table 1:

A	B	C
A	B	C	37	----GCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQS------	100
38	ETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQS------	100	37	----GCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQS------	100
38	ETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQS------	100	39	----GSEQVLTGKRLMQSLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL	96
40	ETPEGCEQVLTGKRLMQCLPNPEDVKMALEIYKLTLEIEQLELQRDSARQSTLDKEL	96	39	----GSEQVLTGKRLMQSLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL	96
40	ETPEGCEQVLTGKRLMQCLPNPEDVKMALEIYKLTLEIEQLELQRDSARQSTLDKEL	96	41	ETPEGCEQVLTGKRLMQCLPNPEDVKMALEIYKLSLEIKQLELQRDSARQSTLDKEL	96
42	---EGCEQILTGKRLMQCLPDPEDVKMALEIYKLSLEIKQLELQRDRARQSTL----	91	41	ETPEGCEQVLTGKRLMQCLPNPEDVKMALEIYKLSLEIKQLELQRDSARQSTLDKEL	96
42	---EGCEQILTGKRLMQCLPDPEDVKMALEIYKLSLEIKQLELQRDRARQSTL----	91	43	ETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIKQLELQRDRARQSTLDKEL	96
44	---EGCEQILTGKRLMQCLPNPEDVKMALEIYKLSLEIEQLELQRDRARQSTLDK--	95	43	ETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIKQLELQRDRARQSTLDKEL	96

A=SEQ ID NO, the B=sequence, C=% homogeneity, reference has the SEQ ID of equal length The fragment computations of NO:1.

If make sequence deletion, the brachymemma except N end or C end preferably is limited in disappearance not Surpassing 1,2 or 3 can be continuous or discontinuous disappearance.

If insert, perhaps the N of core protein sequence end or C end extend, and number also ideally Should be restricted, be no more than 20 amino acid of wild-type sequence length with the length that guarantees core protein More than, preferably be no more than 15 amino acid, more preferably no more than 10 amino acid. Therefore at SEQ ID In the situation of NO:1, through inserting or prolonging the core protein of modifying, ideally not super on length Cross 77 amino acid.

Antigen

Antigen is can be by any molecule of antibody or TXi Baoshouti identification.Yet, be not all antigen all be immunogen.Immunogen is any material that causes immunne response.On the one hand, the present invention makes and is not that immunogenic antigen becomes immunogen, and makes as weak immunogenic antigen and become immunogen preferably.

Key character of the present invention be when with antigen by merging when producing with C4bp core heredity, monomeric antigen is extremely preferred, is assembled into oligomerization thereby form that have function because they can not hinder the C4bp core protein.

But on the other hand, when antigen by chemical process or non-covalent when being coupled to the C4bp core protein, described antigen is can right and wrong monomeric.

Therefore monomeric antigen can be divided into two main groups:

1) antigen can be to be the proteinic fragment of parent or the derivative of (polymer dimerization or higher category) of poly under the state of nature, but this antigen does not form polymer from forming in parent's protein under the polymeric condition; With

2) state of nature is monomeric antigen.

These two kinds of antigenic examples will go through hereinafter.

Monomeric antigen has identical point and is, they can be encoded on wall scroll DNA, and when this DNA also translated into protein subsequently with the DNA fusion of coding C4bp core protein, this antigen linked to each other by the unique and wall scroll C4bp core protein chain on the antigen.This antigenic simple case is the N,O-Diacetylmuramidase from egg albumen.The cDNA of coding total length N,O-Diacetylmuramidase open reading frame can not hinder the mode and the C4bp core open reading frame of the C4bp assembling partly of gained fusion rotein to merge.。

After the biosynthesizing, the wall scroll polypeptide chain that merges with the C4bp core will, for example by proteolytic enzyme, thereby processedly produce new N-terminal and C-terminal in that polypeptide chain is inner.If two of producing by proteolysis cutting or more chains interconnect by for example disulfide linkage, the C4bp fused protein can not be connected with not thinking monomeric protein usually when process finishing so.Yet, being purpose of the present invention, such protein is considered to monomeric, because they are encoded as the single fusion rotein in the single open reading frame.Such example is a proinsulin, and it is processed to have two chains that are called A and B after biosynthesizing, and these two chains link to each other by disulfide linkage.Insulinogenic fragment is called the C peptide chain, removes in the proteolysis processing back of fusion rotein precursor.

It is monomeric protein that described monomeric antigen can derive from also nonessential under its state of nature.Therefore the antigen that is in the polymer state in many natures can be modified, and is for example by the protein renovation technique, monomeric thereby they become.Three examples are arranged.As this antigenic example is a kind of proteinic antigen of influenza virus hemagglutinin that derives from.Known this antigen forms compound tripolymer structure (Wilson etc., Nature 289,366-373,1981) under its state of nature.But, might make the coiled coil of this molecule tripolymerization obtain the monomer fragment by removal.The work of Jeon and Arnon provides an object lesson (Viral Immunology 15,165-176,2002).These authors have only used the residue 96-261 of hemagglutinin, only to be comprised the fragment of described hemagglutinin bulbous region.

Another example is plasmodium sporozoite surface protein 1 (MSP1).The protein of this huge (about 200kDa) has been decorated the surface of the sporozoite in the blood stage of causing malaria infection.This protein generally is fixed on the sporozoite surface by C-terminal GPI anchor (wherein GPI is the glycosylation phosphatidylinositols).Before this GPI anchor is amino acid whose hydrophobic section.Because this anchor, the C-terminal fragment (even when sporozoite invade erythrocytic the time it is still membrane-bound) of finding total length MSP1 and be called MSP1.19 in nature is not free state.Many membranins with single hydrophobic transmembrane zone all have same situation.The present invention is preferably undertaken by deleting described hydrophobic section.Asking for an interview following Example about the fusion of MSP1.19 protein and C4bp core protein describes.

Of the present invention one preferred aspect, product of the present invention is the fusion of monomeric antigenicity fragment of plasmodium MSP1 and C4bp core protein.Described plasmodium MSP1 antigenicity fragment can comprise by about 50 to about 200, and preferred about 50 to about 150 amino acid.Described antigenicity fragment can be from any plasmodium species, as plasmodium falciparum (Plasmodium falciparum) or Plasmodium vivax (Plasmodium vivax) or Plasmodium ovale (Plasmodium ovale) or malariae (Plasmodium malariae) (these can both cause people's disease) or Plasmodium yoelii.

Although disappearance is to obtain monomer or oligomer the simplest proteinic method, one or more amino acid that suddenly change sometimes are just enough.Such example is Cpn10, and is similar to the C4bp core that is main isotype, and Cpn10 protein is heptamer protein in its native state.The sudden change of the single amino acids of Cpn10 makes it be transformed into the monomer mutant, and this makes it be fit to merge (Guidry etc., BMC Biochemistry 4,14-26,2003) with the C4bp core protein.It is the amino acid of disappearance N-terminal or C-terminal that another kind makes the method for this protein monomersization, (Llorca etc., Biochem.Biophysica Acta1337,47-56,1997; Seale and Horowitz, J.Biol.Chem.270,30268-30270,1995), thereby disappearance is responsible for the interactional zone between the subunit.

Generally speaking, for those have be assembled into the inclining strongly of oligomer structure (for example viral capsid proteins) after, thereby destroy the albumen of the assembling of the C4bp core protein that merges with them, the principle that disappearance is responsible for the residue of interactional zone of bak protein or abrupt interface can be used for obtaining monomeric protein.

Antigen can be divided into two classes, and this two class all is applicable to the present invention.The first kind is an exogenous antigen, and is included in all molecules in the infectious organism.Bacterial immune is former, parasite immunogen and virus immunity are former can prepare polymer or heteromultimeric C4bp fusion rotein as vaccine as polypeptide portion.

These immunogenic bacterial origins comprise those that cause bacterial pneumonia, meningitis, cholera, diphtheria, Whooping cough, tetanus, pulmonary tuberculosis and leprosy.

The parasite source comprises malarial parasite, as plasmodium, taper worm and leishmania kind.

Viral source comprises poxvirus, variola virus for example, vaccinia virus and orf virus; Simplexvirus, as I type and II herpes simplex virus type, B-virus, varicella zoster virus, cytomegalovirus, and Epstein-Barr virus; Adenovirus is as adenosis of breast poison (mastadenovirus); Papovavirus (papovavirus), as HPV16, and polyomavirus (polyomavirus) is as BK and JC virus as papilloma virus (papillomavirus); Microvirus (parvovirus) is as adeno associated virus; Reovirus (reovirus) is as 1,2,3 type reoviruses; Orbivirus (orbivirus) is as colorado tick fever; Rotavirus (rotavirus) is as the human rotavirus; Alphavirus is as east encephalitis and VE virus; Rubella virus (rubivirus) is as rubella (rubella); Flavivirus (flavivirus), as yellow fever virus, dengue fever virus, japanese encephalitis virus, tick biography property (Tick-borne) encephalitis, and hepatitis C virus; Coronavirus is as the people coronavirus; Paramyxovirus, for example 1,2,3,4 type parainfluenza virus and parotitiss; Measles virus (morbillivirus) is as Measles virus (measles virus); Pneumovirinae is as respiratory syncytial virus; Bubble venereal disease poison is as vesicular stomatitis virus; Rabies virus (lyssavirus) is as rabies virus (rabi virus); Orthomyxovirus is as A and Type B influenza; Bunyavirus (bunyavirus) is as lacrosse virus (laCrosse virus); Sand fly virus (phlebovirus) is as valley fever virus (Rift Valley fever virus); Na Yiluo virus (nairovirus) is as Congo hemorrhagic fever virus; Bite Hepadnaviridae (hepadnaviridae), as hepatitis B; Arenavirus (arenavirus), as 1cm virus, lock set virus (lasso virus) and Junin virus (Juninvirus); Retrovirus, HTLV I for example, HTLV II, HIV-1 and HIV-2; Enterovirus, as 1,2,3 type polioviruses, Coxsackie virus, ECHO virus, human intestine's virus, hepatitis A virus, hepatitis E virus, norwalk virus (Norwalk-virus); Rhinovirus is as the human rhinovirus; And Filoviridae (filoviridae), as Marburg virus (Marburg virus) and Ebola virus (Ebola virus).

Can be used for preparing polymer protein from the antigen in these bacteriums, virus and parasite source as vaccine.Described polymer can comprise and have different antigenic monomeric mixtures.

Antigen from these bacteriums, virus and parasite source can be considered to exogenous antigen, because they generally are not present among the host, and is not encoded in host genome.On the contrary, endogenous antigen generally is present among the host or in host genome and is encoded, perhaps both.Generation is carried described antigenic tumour to the ability of the antigenic immunne response of endogenous in treatment, perhaps neutralizes at being useful in the growth of tumor factor.The example of first kind homologous antigen is exactly HER2, and it is the target that is called the monoclonal antibody of Herceptin.The example of the endogenous antigen of the second class somatomedin class is gonadotropin releasing hormone (being called GnRH), and it is to the nutritious effect of prostatic certain cancers.

Immunogen with the present invention's preparation can be used for research or therapeutic purpose.For example, research is used and to be comprised that producing antiserum(antisera) predicts gene product in genomic sequence data.This demand is applied to the gene product of prokaryotic organism (as bacterium) and eukaryote (comprising fungi and Mammals).Antigen can be this area for vaccine any length commonly used, from small peptide to very big protein.

Non-polypeptide type immunogen may be for example sugar or nucleic acid.The polysaccharide shell of Nai Seshi kind and pneumonia streptococcus bacterial classification is the example that can be used for the polysaccharide of the object of the invention.

When non-polypeptide immunogen was portioned product of the present invention, described immunogen can be by covalently bound first component to described product of conventional synthetic method.Usually, described immunogen may be connected to the N-or the C-end of the C4bp core protein that comprises described first component, perhaps is connected to amino acid side chain group (for example sulfydryl of the epsilon-amino of Methionin or halfcystine), or their combination.Each fusion rotein can add more than one immunogen.For auxiliary coupling, cysteine residues can be added to described C4bp core protein, for example as N-or C-end.

The present invention has a lot of advantages on the generation immunne response.For example, use polymer can allow some antigens to be presented to immunity system simultaneously.Thereby can prepare the polyvalent vaccine that can cause at the immunne response of an above antigenic determinant, described antigenic determinant can exist in single or some different organisms.

Therefore, on the other hand, described monomeric antigen may be the synthetic antigen that comprises two different antigenic determinants, and described antigenic determinant is from two different organisms or from two kinds of different proteins of same organism.The latter's example is the fusions of sporozoite antigen sequence, for example two or more NANP tumor-necrosis factor glycoproteinss of the circumsporozoite protein that is connected with the MSP1 sequence.Second example of the latter described the M2e sequence that ((Nature Medicine 5,1157-1163,1999)) and monomeric influenza hemagglutinin fragment merge by Neirynck etc.

So, inoculation when vaccine formed according to the present invention can be used at more than one diseases, perhaps simultaneously target in a plurality of antigenic determinants on known pathogenic agent.These determinants may be present in the one monomeric unit or provide on the different monomeric unit of heteromultimers combining.

C4bp core fusion rotein is useful especially in immunity, because described core protein under normal circumstances is present in the serum or blood plasma of accepting the immune, and described core protein does not cause the immunne response at itself.Known C4bp protein all exists in a lot of mammalian species, and can be found by those skilled in the art by the gene clone technology of standard for the suitable homologue of mammalian species.

Nucleic acid

Product of the present invention can be by the nucleic acid construct with code for said proteins, and expressed fusion protein prepares in prokaryotic organism or Eukaryotic host cell.If antigen is polypeptide, expressed fusion protein can be used to produce product of the present invention from nucleotide sequence.

So the invention provides the nucleic acid construct of code book invention product, normally DNA or RNA.

Described construct is generally the form of replicable vector, wherein the sequence of code for said proteins operationally be suitable in the ideal host cell expressing described proteic promotor and be operably connected.

Provide replication orgin to reach the instrumentality of promotor alternatively may for described carrier.Described carrier may contain one or more a plurality of selected marker.Known in the art have a lot of such eukaryote and procaryotic expression vectors, and the present invention can utilize any carrier according to those skilled in the art's individual preference.

Multiple prokaryotic organism host cell can be used for method of the present invention.These hosts can comprise that Escherichia, Rhodopseudomonas, bacillus, lactobacillus, thermophilic Pseudomonas, salmonella, enterobacteria belong to or the bacterial strain of streptomyces.For example, if the intestinal bacteria from Escherichia are used in the methods of the invention, the preferred strain of this used bacterium comprises the derivative of BL21 (DE3), comprises C41 (DE3), C43 (DE3) or CO214 (DE3) (as described in the WO98/02559 and can get).

Very more preferably, the derivative that lacks these bacterial strains of prophage DE3 can not used when promotor is not the T7 promotor.

The prokaryotic organism carrier comprises the carrier bacterial plasmid, for example comprises ColEI, pCR1, pBR322, pMB9 and their derivative from colibacillary plasmid; The plasmid of wideer host range is RP4 for example; Phage DNA, for example various derivatives of lambda particles phage, for example NM989; And other DNA phage, for example M13 and thread single stranded DNA phage.Can handle these and other carrier with the recombinant DNA method of standard, introduce the nucleic acid of the present invention that can be operatively connected with promotor.

Described promotor may be an inducible promoter.Suitable promotor comprises T7 promotor, tac promotor, trp promotor, λ promotor P _LOr P _R, and other well known to a person skilled in the art promotor.

Also can use multiple eukaryote host cell, comprise for example yeast, insect and mammalian cell.Mammalian cell comprises CHO and mouse cell, cercopithecus aethiops cell such as COS-1 and people's cell.

A lot of eukaryote carriers of known suitable marking protein.These carriers can be designed as by karyomit(e) and mix the eukaryotic cells genome or be retained in beyond the genome, or only of short duration being retained in the eukaryotic cell.These nucleic acid can be operatively attached to suitable promotor, as strong viral promotors (comprising the CMV promotor) and SV40 T-antigen promotor or retrovirus LTR.

In order to obtain product of the present invention, carry the host cell of carrier of the present invention and can cultivate being suitable for expressing in the described proteinic environment, and reclaim described protein from the cell of described substratum.

Cell cultures

Coding can be introduced in the host cell with transformation technology commonly used according to the plasmid of fusion rotein of the present invention, and cultivates described cell helping to produce under the condition of described fusion rotein.If the use inducible promoter is cultivated described cell at the beginning when lacking no described inductor,, in a single day described then cell just adds the protein recovery that described inductor obtains maximum thereby growing into higher density.

Cell culture condition is known in this field, and described condition can be used according to same known method.

Although WO91/11461 thinks that the prokaryotic organism host cell can be used for preparing the protein based on C4bp, does not have experimental evidence for this preparation.

Recently, have been found that and protein that the C4bp core for preparing in the prokaryotic expression system merges still keeps their functionally active.This is open in WO2004/020639, and its content is introduced text as a reference.These methods can be used in the preparation of fusion rotein of the present invention.

From culture, reclaim protein

Described albumen can be produced in case described cell has grown into, just described protein can be from described cell, reclaimed.Because we are surprised to find, it is solvable that described protein keeps, and described cell is got off by centrifugation and the cracking by supersound process usually.For example in that (as one hour 15, centrifugal back protein fraction 000rpm) keeps solvable and this fraction still remains in the supernatant more at a high speed.

Fusion rotein in described supernatant protein fraction can be with incompatible being further purified of any suitable groups of the protein chromatography technology of standard.We have used ion exchange chromatography gel permeation chromatography then.Other chromatographic technique such as affinity chromatography are also available.

In a specific embodiments, we find in the centrifugal back of lysate or heat the supernatant sample after other any purification steps to help to reclaim protein.Described sample can be heated to about 70-80 ℃ about 10-30 minute.

According to described proteinic purpose purposes, described protein can be handled with more purification step (for example dialysis) or enrichment step (for example freeze-drying).

Composition and use thereof

Can prepare with the form of pharmaceutical composition according to product of the present invention.Described product can exist with one or more pharmaceutically acceptable carrier or thinner.Described composition will prepare according to the approach that gives of purpose purposes and product.Therefore the invention provides the product of the present invention that comprises the polymer form and the composition of one or more pharmaceutical acceptable carrier or thinner, and the purposes of described composition in treatment or prevention human or animal experimenter's immunotherapy method.

Medicine acceptable carrier or thinner are included in those that use in the preparaton that suitable per os, rectum, intranasal, part (comprising cheek and hypogloeeis), vagina or parenteral (comprise subcutaneous, intramuscular, intravenously, intradermal, in the sheath, epidural) give.These preparatons can exist with unit dosage form (dosage form) easily and can be used in the interior known any method of pharmaceutical field and prepare.

The pharmacy of liquid can give composition and can pass through, for example (for example by the optional pharmacy adjuvant in fusion rotein of the present invention such as dissolving, dispersion and the carrier, water, physiological saline glucose solution, glycerine, ethanol and similar substance) prepare, thus form solution or suspension.If necessary, the described composition that give also may comprise auxiliary substance such as pH buffer reagent and similar material.The practical methods for preparing this dosage form be for those skilled in the art known maybe will be conspicuous.For example see Remington ' s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 19th Edition, 1995.

Can comprise one or more adjuvant in addition according to composition of the present invention, for example mineral substance salt such as aluminium hydroxide or calcium phosphate, perhaps cytokine is as being situated between plain-12 or GM-CSF certainly.The more complete inventory of suitable adjuvant is at Singh and O ' Hagan, Nature Biotechnology, and 17,1075-1081 provides in 1999 the table 1, and its content is incorporated herein by reference.

According to product of the present invention, with the form of composition or preparaton, can pass through administration of human or this product of animal subjects or its composition ideally, be used for methods of treatment as herein described.The significant quantity that alleviates the experimenter's who receives treatment symptom will be decided according to the patient and the situation of being treated by the doctor.Can prepare and comprise scope at the activeconstituents of 0.25-95% and dosage form or the composition of forming (balancemake up) from the trim of non-toxic carrier.

Administered parenterally generally is to be feature with subcutaneous, intramuscular or intravenous injection.Injectable agent (injectable) can common type prepare, described common type is liquor or suspension, be adapted at injecting before dissolving or be suspended in solid form or milk sap in the liquid.Appropriate excipients is, for example water, physiological saline, glucose, glycerine, ethanol or similar substance.The approach of an administered parenterally that designs recently is implantation slow release or sustained release system, has so just kept the constant dosage level.See for example U.S. Patent No. 3,710,795.

The dosage of described product will be determined by described antigenic character, and can decide by give described antigenic actually operating in vaccine mixture commonly used.

Passive immunization

On the other hand, the invention provides the method with the immune serum passive immunization experimenter who comprises antibody, described immune serum obtains by inoculating product of the present invention for host experimenter.Described host experimenter can be people or inhuman Mammals.So in another aspect, the invention provides the immune serum that obtains by this method, and the purposes of this immune serum in the method for treatment human body or animal body.

Dna vaccination

On the other hand, the invention provides the eukaryote expression vector of the nucleotide sequence that comprises code book invention recombination fusion protein product, this carrier is used for the treatment of human body or animal.

This treatment can reach its result of treatment by the antigenic nucleotide sequence of introducing coding initiation immunne response.The transportation of nucleic acid can realize with plasmid vector (with the form of " exposing " or preparation) or recombinant expression vector.The summary of DNA inoculation is seen Ada G. and Ramshaw I, in Expert Opinionin Emerging Drugs 8,27-35,2003.

The virus vector that much can be used for the gene transmission comprises adenovirus, simplexvirus, vaccinia virus or RNA viruses such as retrovirus.Retroviral vector can be retroviral derivative mouse or bird.The example of retroviral retroviral vector that can insert single foreign gene is including, but not limited to Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (MuMTV), murine mammary tumour virus (MuMTV) and Rous sarcoma virus (RSV).When the experimenter is the people, can use as gibbon ape leukemia virus (gibbon ape leukaemia virus, carrier GaLV).

Described carrier can comprise transcription regulating nucleotide sequence, and being specially is enough to the synthetic promoter region that starts of guide RNA.Suitable promoter in eukaryote comprise the metallothionein(MT) I gene of mouse promotor (Hamer etc., 1982, J.Molec.Appl.Genet.1:273); The TK promotor of simplexvirus (McKnight, 1982, Cell 31:355); The SV40 early promoter (Benoist etc., 1981, Nature290:304); Rous sarcoma virus promoter (Gorman etc., 1982, Proc.Natl.Acad.Sci.USA79:6777); And cytomegalovirus promoter (Foecking etc., 1980, Gene 45:101).

Give the experimenter with the present invention's carrier in this respect as plasmid vector or as the part of virus vector, may be by a lot of different approaches influences.Plasmid DNA can be expose or do direct or indirect transportation with the preparation of positively charged ion and neutral lipid (liposome) or by micro-encapsulation.Dna sequence dna also can be contained in virus (as adenovirus, retrovirus, simplexvirus, the poxvirus) carrier, and these carriers can be used for direct or indirect transportation.Transport pathway includes but are not limited to: per os, intramuscular, intradermal (Sato, Y.et al., 1996, Science 273:352-354), suction in the intravenously, intra-arterial, sheath, in the liver,, the intravaginal (Bagarazzi etc. that instil, 1997, J Med.Primatol.26:27), in internal rectum, the tumour or intraperitoneal.

So, the present invention includes carrier described herein as pharmaceutical compositions, it can be used for described some cells of dna vector transfection, like this will the express therapeutic polypeptide and have therapeutic action, just induce antigenic immunne response.Pharmaceutical compositions according to the present invention is to prepare by the form (with solvent, carrier, haulage system, vehicle and additive or auxiliary) that construct according to the present invention is become be suitable for giving the experimenter.Often the solvent that uses comprises the water and the physiological saline (buffered or non-buffered) of sterilization.A kind of carrier comprises gold grain, and it can pass through Living system (biolistically) and transmit (under at air pressure).Other carrier or haulage system that often uses comprises cationic-liposome, volution (cochleate) and microcapsule, and these can give, be enclosed in the transportation capsule with liquor or mix food.

The another kind of preparaton that gives the gene transport agent relates to liposome.Liposome is provided the another kind of preparaton that gives polynucleotide and expression vector.Liposome by one or more double-layers of lipoid around the micro-vesica formed of water-based compartment (compartment).Usually see Bakker-Woudenberg etc., 1993, Eur.J.Clin.Microbiol.Infect.Dis.12 (Suppl.1): S61, and Kim, 1993, Drugs 46:618.Liposome is similar to the composition of cytolemma, so liposome can be given by safety and be biodegradable as a result.According to the preparation method, liposome may be individual layer or multiwalled, and liposome can be at diameter from 0.02 μ M to dimensional change greater than 10 μ M.See for example Machy etc., 1987, LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (John Libbey) and Ostro etc., 1989, described in the American J.Hosp.Phann.46:1576..

Expression vector can be wrapped in the liposome by standard technique.Various different liposome composition and synthetic method are well known to a person skilled in the art.See for example US-A-4,844,904, US-A-5,000,959, US-A-4,863,740, US-A-5,589,466, US-A-5,580,859, and US-A-4,975,282, these all are incorporated herein by reference.

Usually, the dosage that gives of the carrier of liposome will change with patient's age, body weight, height, sex, general curative state and factors such as medical history before.The dosage range of concrete preparaton can be determined by using suitable animal model.

The present invention can illustrate by following embodiment.

The fusion rotein of embodiment 1-plasmodium falciparum MSP1.19-rabbit C4bp.

This embodiment has illustrated monomeric antigen (the amino acid/11 567-1661 that comprises plasmodium falciparum MSP1) and the proteinic fusion of rabbit core C4bp.This fusion rotein is called AVD174, expresses also therefrom purifying in bacterial strain C41 (DE3).Described fusion rotein itself can not need to add the independent immune rabbit of any adjuvant.

The clone

The synthetic 294bp dna fragmentation of coding MSP1 residue of protein 1567-1661 with NdeI and BamHI digestion, and is connected to the pAVD181 that has digested with NdeI and BamHI.So forming the open reading frame of 95 amino acid whose MSP1.19 protein fragments of coding merges with 57 residues of C-terminal at the rabbit C4bp in T7 late promoter downstream α chain.Check that by dna sequencing this is called the construction of pAVD174.

The nucleotide sequence of coding AVD174 fusion rotein is:

atgttaaacatttcccagcaccagtgcgttaagaaacagtgcccgcagaa

ctctggttgtttccgtcatctggacgagcgtgaagagtgcaaatgtctgc

tgaactacaaacaggaaggtgataaatgtgttgagaacccaaacccgacc

tgtaacgaaaacaacggcggttgtgacgctgatgctaaatgcaccgagga

agacagcggttctaacggtaagaaaatcacctgcgagtgtactaaaccgg

actcctacccgctgttcgacggtatcttttgctccGGATCCGAGGTCCCG

GAAGGCTGTGAGCAGGTGCAAGCGGGTCGCCGTCTCATGCAGTGTCTCGC

AGACCCATACGAAGTGAAAATGGCCCTGGAGGTCTACAAGCTGTCTCTGG

AGATTGAACTCCTGGAACTGCAGCGCGATAAGGCACGTAAAAGCTCTGTG

CTGCGCCAGCTGTAA(SEQ?ID?NO：21)

The aminoacid sequence of the fusion rotein AVD174 of this construct coding is as follows:

MLNISQHQCV?KKQCPQNSGC?FRHLDEREEC?KCLLNYKQEG?DKCVENPNPT

CNENNGGCDA?DAKCTEEDSG?SNGKKITCEC?TKPDSYPLFD?GIFCSGSEVP

EGCEQVQAGR?RLMQCLADPY?EVKMALEVYK?LSLEIELLEL?QRDKARKSSV

LRQL(SEQ?ID?NO：22)

The residue 1-95 of SEQ ID NO:22 is corresponding to the residue 1567-1661 of plasmodium falciparum MSP1 (monomeric antigen), and the residue 98-154 of SEQ ID NO:22 is corresponding to 57 residues of rabbit C4bp core protein.The GS joint sequence appears between these two components.

Described proteinic about molecular weight is 17,319 dalton, and theoretical pI is 5.05.

Express

The plasmid pAVD174 of coding for Plasmodium-rabbit C4bp core protein is expressed in coli strain C41 (DE3).Cell transformed grows into OD600 in 37 ℃ and is approximately 0.6 in the LB substratum, add IPTG then to final concentration 0.5mM abduction delivering, and makes described culture 37 ℃ of regrowths three hours, gathers in the crops described cell by centrifugal then.

The purifying of AVD174 fusion rotein

Protein purification AVD174 from 1 liter C41 (DE3) cell.Come lysing cell by supersound process in the damping fluid of MESpH6.5 that contains 20mM and 5mM EDTA after, all fusion roteins are all found in solvable fraction.Supernatant after centrifugal is splined on the HitrapS pillar.

Cation seperation column (HiTrap S)

At 20mM MES pH6.5, balance pillar in the 20mM edta buffer liquid (buffer A).Described protein comes wash-out with the gradient (buffer A adds 0.5MNaCl) from the buffer A to the buffer B of 10 column volumes.AVD174 in the concentration of about 200mM NaCl by wash-out.

The HiTrapS fraction that comprises AVD174 concentrates with Millipore thickener (cutoff value 30K), goes up sample then to gel-filtration column.

Gel-filtration column (Superdex 200 26/60 prep grade)

With the Tris damping fluid of 20mM pH8.0,150mM NaCl balance Superdex 200 26/60 pillars are gone up the spissated AVD174 protein of sample from the HiTrapS fraction then.

Described protein at two peaks by wash-out.The protein of correct folding and assembling by wash-out, and being not correct assembling or folding at the accessory peak early of 120mls wash-out at 156mls.

Bio-physical property

The oligomer state that contains the C4bp fusion rotein of disulfide linkage ((be present in except mouse at the core protein of all core proteins except the mouse source, see Fig. 1)) can be checked by proteinic behavior described in the SDS-PAGE gel that relatively has or do not exist reductive agent β mercaptoethanol ((BME)) simply.The proteic apparent size of AVD177 is about 140KDa under the situation that does not have BME, yet it has been reduced under the situation that BME exists, and analyzes its apparent molecular weight and has just surpassed 20KDa.

Mass spectrum

AVD147 protein is detecting by the electrospray mass spectrum by the β mercapto-ethanol reduction and after with N-ethyl maleimide (NEM) alkylation.The result shows that 14 NEM molecules are added ((each 125Da)) molecular weight is confirmed as 19,072Da protein.

Level of endotoxin

Level of endotoxin in the protein purification is determined as every micrograms of protein 21EU with LAL ((king crab amoebocyte lysate)) kit form Biowhittaker.

Embodiment 2-plasmodium falciparum falciparumMSP1.19 albumen and the-proteic fusion rotein of people C4bp.

This embodiment has illustrated the fusion of monomeric antigen (the amino acid/11 567-1661 that comprises plasmodium falciparum plasmodium falciparum MSP1) with people C4bp core protein core protein.This fusion rotein is expressed also therefrom purifying in bacterial strain C41 (DE3).Described fusion rotein itself can not need to add the independent immune people of any adjuvant.

The clone

The dna fragmentation of 294 base pair bp of the synthetic of coding MSP1 residue of protein 1567-1661, the 1567-1661 section amino acid of coding MSP1 is cut with NdeI and BamHI digestive ferment, and is connected to the pAVD181 plasmid that has been cut by these two enzymes with NdeI and BamHI digestion.So the open reading frame of 95 amino acid whose MSP1.19 protein fragments of the open reading-frame (ORF) of the opening that forms coding is fused to 57 residues at the carbon C-terminal of the α chain of the C4bp of people's in T7 late promoter downstream and merges, and starts the downstream of word late period at T7.Check that by dna sequencing this construct that is called pAVD177 is called PAVD174, proves its reliability by dna sequencing.

The accounting nucleotide sequence of coding AVD177 fusion rotein is:

atgttaaacatttcccagcaccagtgcgttaagaaacagtgcccgcagaa

ctctggttgtttccgtcatctggacgagcgtgaagagtgcaaatgtctgc

tgaactacaaacaggaaggtgataaatgtgttgagaacccaaacccgacc

tgtaacgaaaacaacggcggttgtgacgctgatgctaaatgcaccgagga

agacagcggttctaacggtaagaaaatcacctgcgagtgtactaaaccgg

actcctacccgctgttcgacggtatcttttgctccGGATCCgagaccccc

gaaggctgtgaacaagtgctcacaggcaaaagactcatgcagtgtctccc

aaacccagaggatgtgaaaatggccctggaggtatataagctgtctctgg

aaattgaacaactggaactacagagagacagcgcaagacaatccactttg

gataaagaactataa(SEQ?ID?NO：23)

The aminoacid sequence of the fusion rotein AVD177 of this construct coding is as follows:

MLNISQHQCV?KKQCPQNSGC?FRHLDEREEC?KCLLNYKQEG?DKCVENPNPT

CNENNGGCDA?DAKCTEEDSG?SNGKKITCEC?TKPDSYPLFD?GIFCSGSETP

EGCEQVLTGK?RLMQCLPNPE?DVKMALEVYK?LSLEIEQLEL?QRDSARQSTL

DKEL(SEQ?ID?NO：24)

The residue 1-95 of SEQ ID NO:24 is corresponding to the residue 1567-1661 of plasmodium falciparum plasmodium falciparum MSP1 (monomeric antigen), and SEQ ID NO: the residue 98-154 correspondence amino-acid residue of sequence number 24 is with corresponding in the C4bp of people's core protein core protein 57 residues mutually.A GS connecting joint sequence appears between these two ingredient components.

This described proteinic about molecular weight is 17,261 dalton, and theoretical iso-electric point pI is 4.72.

Express

Protein expression as shown in Figure 2.Shown among this figure that AVD77 protein do not inducing (U) or inducing the back at three kinds of different bacterial strain C41 (DE3) at 37 ℃ or 30 ℃, the SDS-PAGE gel of expression among BL21 (DE3) and the C43 (DE3).Swimming lane on the described gel is as follows:

Swimming lane 1: molecular weight marker (from top to bottom 66,60,46,36,28,20,14,12,6KDa)

Before swimming lane 2:C41 (DE3) induces

Swimming lane 3:C41 (DE3) induced back three hours for 37 ℃

Swimming lane 4:C41 (DE3) induced back three hours for 30 ℃

Before swimming lane 5:BL21 (DE3) induces

Swimming lane 6:BL21 (DE3) induced back three hours for 37 ℃

Swimming lane 7:BL21 (DE3) induced back three hours for 30 ℃

Before swimming lane 8:C43 (DE3) induces

Swimming lane 9:C43 (DE3) induced back three hours for 37 ℃

Swimming lane 10:C43 (DE3) induced back three hours for 30 ℃

As can be seen, in C41 (DE3) and bacterial strain C41 (DE3), obtained good expression.On the contrary, under tested condition, in bacterial strain BL21 (DE3), do not find to express (see figure 2).Be about 0.6 in 30 ℃ and 37 ℃ grown culture in the LB substratum to optical density(OD) (OD600), add the come abduction delivering of IPTG then to final concentration 0.5mM.

The purifying of AVD177 fusion rotein

From inducing protein purification AVD177 1 liter of C41 (DE3) cell of 3 hours of back growth at 37 ℃.After the bacterial precipitation cracking, all fusion roteins all are present in the solvable fraction.In the damping fluid that comprises 20mM MES pH6.5 and 5mM EDTA, come lysing cell by supersound process.Sample is to the MonoS post on the supernatant after centrifugal.

Cation seperation column (Mono S HR 10/10)

At 20mM MES pH 6.5, balance pillar in the 20mM edta buffer liquid (buffer A).Described protein uses the gradient of 10 column volumes from the buffer A to the buffer B to come wash-out, and buffer B is that buffer A adds 0.5M NaCl.AVD177 in the concentration of about 200mM NaCl by wash-out.

The MonoS fraction that contains AVD177 concentrates with Millipore thickener (blocking 30K), goes up sample then to gel-filtration column.

Gel-filtration column (Superdex 200 26/60 prep grade)

With the Tris damping fluid (pH8.0) of 20mM pH 8.0,150mM sodium chloride nacl balance Superdex 200 26/60 pillars right concentrate the spissated AVD177 protein from the MonoS fraction of passing through that sample is gone up in the back.

Described protein wash-out has at two peaks by wash-out.The correct folding and protein of assembling assembling by wash-out, and not being correct assembling or folding at the accessory peak early of 115mls wash-out when 150mls.

Bio-physical property

The oligomer state that contains the C4bp fusion rotein of disulfide linkage (be present in core proteins all except mouse, see Fig. 1) can be checked by proteinic behavior described in the SDS-PAGE gel that relatively has or do not exist reductive agent β mercaptoethanol (BME) simply.The proteic apparent size of AVD177 is about 140KDa under the situation that does not have BME, yet it has been reduced under the situation that BME exists, and analyzes its apparent molecular weight and has just surpassed 20KDa.

Mass spectrum

AVD147 protein is detecting by the electrospray mass spectrum by the β mercapto-ethanol reduction and after with N-ethyl maleimide (NEM) alkylation.The result shows that 14 NEM molecules are added (each 125Da) molecular weight is confirmed as 19,072Da protein.

Level of endotoxin

Level of endotoxin in the protein purification is determined as every micrograms of protein 21EU with LAL (king crab amoebocyte lysate) kit form Biowhittaker.

The fusion rotein of embodiment 2-plasmodium falciparum MSP1.19-people C4bp.

This embodiment has illustrated the fusion of monomeric antigen (the amino acid/11 567-1661 that comprises plasmodium falciparum MSP1) with people C4bp core protein.This fusion rotein is expressed also therefrom purifying in bacterial strain C41 (DE3).Described fusion rotein itself can not need to add the independent immune people of any adjuvant.

The clone

The synthetic 294bp dna fragmentation of coding MSP1 residue of protein 1567-1661 with NdeI and BamHI digestion, and is connected to the pAVD181 that has digested with NdeI and BamHI.So forming the open reading frame of 95 amino acid whose MSP1.19 protein fragments of coding merges with 57 residues of C-terminal at the people C4bp in T7 late promoter downstream α chain.Check that by dna sequencing this is called the construct of pAVD177.

The nucleotide sequence of coding AVD177 fusion rotein is:

atgttaaacatttcccagcaccagtgcgttaagaaacagtgcccgcagaa

ctctggttgtttccgtcatctggacgagcgtgaagagtgcaaatgtctgc

tgaactacaaacaggaaggtgataaatgtgttgagaacccaaacccgacc

tgtaacgaaaacaacggcggttgtgacgctgatgctaaatgcaccgagga

agacagcggttctaacggtaagaaaatcacctgcgagtgtactaaaccgg

actcctacccgctgttcgacggtatcttttgctccGGATCCgagaccccc

gaaggctgtgaacaagtgctcacaggcaaaagactcatgcagtgtctccc

aaacccagaggatgtgaaaatggccctggaggtatataagctgtctctgg

aaattgaacaactggaactacagagagacagcgcaagacaatccactttg

gataaagaactataa(SEQ?ID?NO：23)

MLNISQHQCV?KKQCPQNSGC?FRHLDEREEC?KCLLNYKQEG?DKCVENPNPT

CNENNGGCDA?DAKCTEEDSG?SNGKKITCEC?TKPDSYPLFD?GIFCSGSETP

EGCEQVLTGK?RLMQCLPNPE?DVKMALEVYK?LSLEIEQLEL?QRDSARQSTL

DKEL(SEQID?NO：24)

The residue 1-95 of SEQ ID NO:24 is corresponding to the residue 1567-1661 of plasmodium falciparum MSP1 (monomeric antigen), and the residue 98-154 of SEQ ID NO:24 is corresponding to 57 residues of people C4bp core protein.The GS joint sequence appears between these two components.

Described proteinic about molecular weight is 17,261 dalton, and theoretical pI is 4.72.

Express

Before swimming lane 2:C41 (DE3) induces

Swimming lane 3:C41 (DE3) induced back three hours for 37 ℃

Swimming lane 4:C41 (DE3) induced back three hours for 30 ℃

Before swimming lane 5:BL21 (DE3) induces

Swimming lane 6:BL21 (DE3) induced back three hours for 37 ℃

Swimming lane 7:BL21 (DE3) induced back three hours for 30 ℃

Before swimming lane 8:C43 (DE3) induces

Swimming lane 9:C43 (DE3) induced back three hours for 37 ℃

Swimming lane 10:C43 (DE3) induced back three hours for 30 ℃

The purifying of AVD177 fusion rotein

Cation seperation column (Mono S HR 10/10)

Gel-filtration column (Superdex 200 26/60 prep grade)

With the Tris damping fluid of 20mM pH 8.0,150mM NaCl balance Superdex 200 26/60 pillars are gone up the spissated AVD177 protein of sample from the MonoS fraction then.

Described protein at two peaks by wash-out.The protein of correct folding and assembling by wash-out, and not being correct assembling or folding at the accessory peak early of 115mls wash-out when 150mls.

Bio-physical property

The oligomer state that comprises the C4bp fusion rotein of disulfide linkage (be present in core proteins all outside the deratization, see Fig. 1) can be easily by relatively checking in proteinic behavior described in the SDS-PAGE gel that has or do not exist reductive agent β mercaptoethanol (BME).The results are shown in shown in Figure 3ly, wherein shown the SDS-PAGE gel, its shown under reductive condition (left side ,+BME) and under the non-reduced condition (right side ,-AVD177 the albumen BME) analyzed is distinguished by molecular weight marker and (is followed successively by from top to bottom: 66,60,46,36,28,20,14,12,6kDa).From Fig. 3 as seen, do not exist under the BME situation, the proteinic apparent size of AVD177 is about 140kDa, and it has been reduced under the situation that BME exists, and analyzes its apparent molecular weight and has just surpassed 20KDa.

Mass spectrum

AVD177 albumen is reduced by BME and after N-ethyl maleinamide (NEM) alkylation, checks by the electrospray mass spectrum.The result shows, 14 NEM molecules (each 125Da) is added its molecular weight be confirmed as 19, the protein of 015Da.

Level of endotoxin

The level of endotoxin of protein purification is defined as every milligram of protein 38EU with LAL (the king crab amoebocyte lysate) detection kit of Biowhittaker.

The plasmodium falciparum MSP1.19-rabbit C4bp fusion rotein of embodiment 3-sudden change

By example, second plasmodium falciparum MSP1.19-rabbit C4bp protein has been described here.Its key distinction is to utilize differently for the tool codon of monomeric antigen gene with pAVD174 and pAVD177, and also comprises three amino acid changes (seeing Uthaipibull etc., J Mol Biol.307,1381-1394,2001 description).This fusion rotein is known as AVD178.

The nucleotides sequence of coding AVD178 fusion rotein is classified as:

atgctgaatatttcccagcaccagtgcgtaaagaaacagtgtcctcagaa

ctctggttgcttccgccatctggacgaacgcgaatattgcaaatgccgtc

tgaactacaaacaggaaggtgacaagtgcgttctgaacccgaacccaact

tgtaacgagaacaacggtggctgcgatgctgatgctaaatgcactgaaga

agacagcggttctaacggcaaaaaaatcacctgcgagtgcaccaaaccgg

acagctatccgctgttcgacggcattttttgttctggatccGAGGTCCCG

GAAGGCTGTGAGCAGGTGCAAGCGGGTCGCCGTCTCATGCAGTGTCTCGCAGACCCATACGAA

GTGAAAATGGCCCTGGAGGTCTACAAGCTGTCTCTGGAGATTGAACTCCTGGAACTGCAGCGC

GATAAGGCACGTAAAAGCTCTGTGCTGCGCCAGCTGTAA(SEQ?ID?NO：25)

The aminoacid sequence of the fusion rotein AVD178 of this construct coding is as follows:

MLNISQHQCVKKQCPQNSGCFRHLDEREYCKCRLNYKQEGDKCVLNPNPTCNENNGGCDADAK

CTEEDSGSNGKKITCECTKPDSYPLFDGIFCSGSEVPEGCEQVQAGRRLMQCLADPYEVKMAL

EVYKLSLEIELLELQRDKARKSSVLRQL(SEQ?ID?NO：26)

The amino acid of three sudden changes is runic and has drawn underscore.

The residue 1-95 of SEQ ID NO:25 is corresponding to the residue 1567-1661 with the plasmodium falciparum MSP1 (monomeric antigen) of three sudden changes, and the residue 98-154 of SEQ ID NO:24 is corresponding to 57 residues of rabbit C4bp core protein.The GS joint sequence appears between these two components.

The expression of AVD178, purifying and table exist

This process is described with AVD174 and AVD177 albumen basically.From HiTrapS post wash-out is at about 200mM.In gel-filtration, described oligomer protein when the 159mls volume by wash-out.In the mass spectrum, molecular weight is 19 when being with 14 NEM residues on each monomer, 133Da.Level of endotoxin is measured as every milligram of protein 58EU.

Embodiment 4-Plasmodium yoelii MSP1.19-mouse C4bp fusion rotein

The clone

Be prepared as follows AVD108 albumen: the synthetic DNA fragment of coding MSP1.19 and from the part MSP1.33 of Plasmodium yoelii, be cloned into 54 amino acid whose upstreams of C-terminal of mouse C4bp α chain as the NdeI-BamHI fragment.

The nucleotide sequence of encoding fusion protein AVD108 is as follows:

ATGAGATCTCACATTGCCTCTATTGCTTTGAACAACTTGAACAAGTCTGG

TTTGGTAGGAGAAGGTGAGTCTAAGAAGATTTTGGCTAAGATGCTGAACA

TGGACGGTATGGACTTGTTGGGTGTTGACCCTAAGCATGTTTGTGTTGAC

ACTAGAGACATTCCTAAGAACGCTGGATGTTTCAGAGACGACAACGGTAC

TGAAGAGTGGAGATGTTTGTTGGGTTACAAGAAGGGTGAGGGTAACACCT

GCGTTGAGAACAACAACCCTACTTGCGACATCAACAACGGTGGATGTGAC

CCAACCGCCTCTTGTCAAAACGCTGAATCTACCGAAAACTCCAAGAAGAT

TATTTGCACCTGTAAGGAACCAACCCCTAACGCCTACTACGAGGGTGTTT

TCTGTTCTTCTTCCGGATCCGAGGCCTCTGAAGACCTTAAGCCTGCGCTT

ACAGGCAACAAGACCATGCAGTATGTGCCAAATTCACACGATGTGAAAAT

GGCTCTGGAGATCTACAAGCTGACTCTGGAGGTTGAACTACTACAGCTCC

AGATACAAAAGGAGAAACACACTGAAGCACACTAA(SEQ?ID?NO：27)

The aminoacid sequence of a-protein VD108 is as follows:

MRSHIASIAL?NNLNKSGLVG?EGESKKILAK?MLNMDGMDLL?GVDPKHVCVD?TRDIPKNAGC

FRDDNGTEEW?RCLLGYKKGE?GNTCVENNNP?TCDINNGGCD?PTASCQNAES?TENSKKIICT

CKEPTPNAYY?EGVFCSSSGS?EASEDLKPAL?TGNKTMQYVP?NSHDVKMALE?IYKLTLEVEL

LQLQIQKEKH?TEAH(SEQ?ID?NO：28)

The residue 3-138 of SEQ ID NO:28 is corresponding to the residue 1619-1753 of Plasmodium yoelii MSP1 (monomeric antigen), and the residue 141-194 of SEQ ID NO:28 is corresponding to 54 residues of rabbit C4bp core protein.The GS joint sequence appears between these two components, and the sequence of short coding restriction site is positioned at before first component.

The expression of AVD108

In coli strain C41 (DE3), express AVD108 protein.Be about 0.6 37 ℃ of three liters of culture to optical density(OD) (OD600) of growing in the LB substratum, adding IPTG then is the abduction delivering that comes of 0.7mM to final concentration.Induced the described cell of centrifugal results back four hours.The described cell of cracking in buffer A (50mMTris pH 9,5mM EDTA) is by the centrifugal cell debris of removing.

The purifying of AVD108 and sign

Come purifying AVD108 albumen with four column chromatography steps.In the first step anion-exchange chromatography step, use DEAE HR16/10 post.Described protein is added in the buffer A, and in the buffer B gradient of (buffer A adds 1M NaCl) by wash-out.In the wide peak of AVD108 between 180-300mM NaCl by wash-out.

In the second step hydrophobic interaction chromatography step, will comprise from sample on the concentrated fraction of the AVD108 of DEAE post to Macro-Prep phenyl sepharose post, 1M in the salt gradient of the decline of 0M NaCl by wash-out.In the end two the step chromatographic step in, AVD108 protein at Superdex S20026/60 post by the gel-filtration purifying.At first, be that the urea of 8M comes the described protein of sex change by adding final concentration, be incubated overnight at 4 ℃.Monomer is post wash-out from then at 203mls volume place.By 4 ℃ come renaturation with respect to damping fluid H (20mM Tris pH 7.5,150mM NaCl) dialysed overnight after, at last sample before the same column, balance in damping fluid H.Now at 164mls volume place wash-out oligomer.By mass spectrum, determine that quality is 21,257Da.The level of endotoxin that LAL detection kit by Biowhittaker records is every milligram of protein 4EU.

Embodiment 5-comes immunity with plasmodium falciparum MSP1.19-rabbit C4bp fusion rotein

Immunity

AVD174 protein with preparation as described in example 1 above comes immunity three New Zealand white rabbit (NZW).Immunization protocol is as follows: every rabbit is accepted four injections (that is to say, the 0th, 14,28 and 42 days) with the interval in two weeks.Per injection is subcutaneous, and it is included in 345 micrograms (or the 20 nanomoles) protein in the buffered normal isotonic saline solution, and does not add any known adjuvant.

Abreast, the AVD172 protein with 212 micrograms (or 20 nanomoles) in the freund's adjuvant comes three NZW rabbits of immunity.AVD172 is identical with AVD174, but lacks 57 amino acid of C-terminal of rabbit C4bp.AVD172 has following aminoacid sequence:

MLNISQHQCV?KKQCPQNSGC?FRHLDEREEC?KCLLNYKQEG?DKCVENPNPT?CNENNGGCDA

DAKCTEEDSG?SNGKKITCEC?TKPDSYPLFD?GIFCS(SEQ?ID?NO：29)。

Immunization protocol is as follows: every rabbit is accepted four injections (that is to say, the 0th, 14,28 and 42 days) with the interval in two weeks.The injection complete Freund's adjuvant first time to every rabbit gives by intradermal, and gives by subcutaneous in three full freund's adjuvants that too many or too much for use subsequently.

Antibody titers

Inject the antibody titers of one week (promptly the 63rd day) anti-plasmodium falciparum sporozoite surface, back MSP1 the last time, the indirect immunofluorescence that is used on the red corpuscle smear of falciparum infection of acetone fixed is measured (as Ling etc., Vaccine 15,1562-1567,1997 is described for Plasmodium yoelii).

Be 1/81,920 with two titres the highest in the rabbit of AVD174 protein immunity.In the rabbit with the AVD172 protein immunity in the freund's adjuvant, two titres the highest are 1/20,480.

This proof can be induced higher immune titre with the identical monomeric antigen that monomeric antigen and C4bp core fusion ratio give in the freund's adjuvant.

Produce the sign of antibody

The high titre of antibody is not sufficient to prevention or treatment is infected, and the specificity of the antibody that well-known antigen immune produces is most important.Guevara Patino etc., (J.Exp.Med.186,1689-1699,1997) have described detection in detail at the dead front type of MSP1.19 and the method for inhibition type antibody.These methods are used to detect exist (described antibody is very useful, thereby MSP1.42 is processed as MSP1.33 and MSP1.19 prevention red corpuscle is infected because they can be blocked) whether inhibition type antibody is arranged.

Inhibition type antibody only is found in the antibody of AVD174 protein induce.They none in antiserum(antisera), be found with the rabbit of AVD172 protein immunity.On the contrary, the AVD172 protein in the freund's adjuvant as people's natural malaria infection, can be induced dead front type antibody and is deleteriously (to see Guevara Patino etc., as mentioned above).

Embodiment 6-comes immunity with Plasmodium yoelii MSP1.19-mouse C4bp fusion rotein

Immune mouse

AVD108 protein with preparation as described in embodiment 5 comes six BALB/c mouse of immunity.Do not use adjuvant, described protein oozes in the salts solution at buffered etc.40 micrograms (1.9 nanomole) protein is used in per injection.With interval (that is to say) every mouse of subcutaneous injection in 4 weeks three times at the 0th, 28 and 56 day.Abreast, with six BALB/c mouse of 23 micrograms (also being 1.9 nanomoles) AVD183 protein immunity, this protein is identical with AVD108, but lacks 54 amino acid (being that it only is a Plasmodium yoelii MSP1.19 protein) of the C-terminal of mouse C4bp.This protein of 23 micrograms (using identical buffering normal isotonic saline solution with AVD108) is used in per injection.Every mouse of subcutaneous injection in the 0th, 28 and 56 day three times.

Antibody titers

Inject the antibody titers of back two weeks (promptly the 70th day) anti-Plasmodium yoelii sporozoite surface MSP1 the last time, measure by the indirect immunofluorescence on the red corpuscle smear that infects at the Plasmodium yoelii of acetone fixed and (to describe as Ling etc., Vaccine 15,1562-1567,1997).

The result shows that it is that the 1/40,960, six mouse titre is 1/10,240 that 5 antibody titerss are arranged in six proteic mouse of immune AVD108.In contrast, not have an antibody that detects at MSP1 in the proteic mouse of the 1/80 immune AVD183 of dilution.This proof merges monomer M SP1.19 antigen can be made the antigenic titre that is obtained increase to the C4bp core to be up to 500 times.

Parasite is attacked

Attack two groups of each six mouse of immunity as mentioned above with the red corpuscle that 5,000 Plasmodium yoeliis of lethal dose infect.This detection was described by (as mentioned above) such as Ling.Described six mouse that cross with the immunity of AVD183 protein are attacked in back 7 days all dead parasite.On the other hand, five survivals in six mouse that cross with AVD108 immunity, and do not have parasite to have (Giemsa staining as the thin blood smear of microscope inspection is assessed) in its blood.The 6th mouse had titre 1/10,240 at the 70th day, it is in attack death in back 19 days; Infected above 70% red corpuscle at the 19th day by visible this mouse of Giemsa staining.

In a word, attack and experiment showed, that being used in the monomeric antigen that merges with the C4bp core protein when not having any known adjuvant comes immunely, can not protect and be infected by the plasmodium of lethality.Believe that at present this is that first example does not have any known adjuvant and follows the successful inoculation that resists the plasmodium infection with single albumen.

Embodiment 7-influenza hemagglutinin-C4bp fusion rotein

This embodiment has proved the core C4bp proteinic fusion of monomeric antigen (the residue 91-261 that comprises the HA1 hemagglutinin matter of influenza virus A) with people, rabbit and mouse.Under the normal circumstances, total length HA1 is assembled into tripolymer at the virosome cell surface, therefore only just can change it into monomeric antigen effectively with this peptide fragment.These are called AVD272 and express in bacterial isolates C41 (DE3) and from its purifying to the fusion rotein of AVD274.These fusion roteins are used to immune mouse and rabbit separately and do not add any adjuvant.

Among the AVD272, the HA1 segment (as Jeon and Arnon, Viral Immunology, 15,165-176,2002 is described) merge with mouse C4bp support.

The aminoacid sequence of AVD272 is as follows:

kafsncypyd?vpdyaslrsl?vassgtlefi?tegftwtgvt?qnggsnackr

gpgsgffsrl?nwltksgsty?pvlnvtmpnn?dnfdklyiwg?ihhpstnqeq

tslyvqasgr?vtvstrrsqq?tiipnigsrp?wvrglssris?iywtivkpgd

vlvinsngnl?iaprgyfkmr?GSEASEDLKP?ALTGNKTMQY?VPNSHDVKMA

LEIYKLTLEV?ELLQLQIQKE?KHTEAH(SEQ?ID?NO：30)

Among the AVD273, HA1 fragment and rabbit C4bp support merge.

The aminoacid sequence of AVD273 is as follows:

kafsncypyd?vpdyaslrsl?vassgtlefi?tegftwtgvt?qnggsnackr

gpgsgffsrl?nwltksgsty?pvlnvtmpnn?dnfdklyiwg?ihhpstnqeq

tslyvqasgr?vtvstrrsqq?tiipnigsrp?wvrglssris?iywtivkpgd

vlvinsngnl?iaprgyfkmr?GSEVPEGCEQ?VQAGRRLMQC?LADPYEVKMA

LEVYKLSLEI?ELLELQRDKA?RKSSVLRQL(SEQ?ID?NO：31)

Among the AVD274, HA1 fragment and people C4bp support merge.

The aminoacid sequence of AVD274 is as follows:

kafsncypyd?vpdyaslrsl?vassgtlefi?tegftwtgvt?qnggsnackr

gpgsgffsrl?nwltksgsty?pvlnvtmpnn?dnfdklyiwg?ihhpstnqeq

tslyvqasgr?vtvstrrsqq?tiipnigsrp?wvrglssris?iywtivkpgd

vlvinsngnl?iaprgyfkmr?GSETPEGCEQ?VLTGKRLMQC?LPNPEDVKMA

LEVYKLSLEI?EQLELQRDSA?RQSTLDKEL(SEQ?ID?NO：32)

With the AVD272 protein that does not add any adjuvant (2 nanomole) with the interval in two weeks (promptly the 0th; 14 and 28 days) subcutaneous immune mouse three times; shown considerable antibody titers (determining) as Jeon etc. is above-mentioned, and at the obvious provide protection of the deadly attack of the mouse adaptability A/PR/8/34 bacterial strain of 5LD50 dosage.

Embodiment 8-influenza M2 peptide-C4bp fusion rotein

This embodiment illustrates the core C4bp proteinic fusion of monomeric antigen (the proteinic residue 2-24 of M2 that comprises influenza virus A, born of the same parents' outside part) to people, rabbit and mouse.Under the normal circumstances, total length M2 is assembled into the tetramer in virosome and infected cells, therefore only can change it into monomeric antigen effectively with this peptide fragment.These called afters AVD275 expresses in bacterial strain C41 (DE3) and from its purifying to the fusion rotein of AVD278.These fusion roteins self are used to immune mouse and rabbit and do not add any adjuvant.

Among the AVD275, the outer M2 peptide of born of the same parents (as Nature Medicine 5 such as Neirynck, 1157-1163,1999 is described) merge with mouse C4bp support.

The aminoacid sequence of AVD275 is as follows:

SLLTEVETPI?RNEWGCRCND?SSDGSEASED?LKPALTGNKT?MQYVPNSHDV?KMALEIYKLT

LEVELLQLQI?QKEKHTEAH(SEQ?ID?NO：33)

Among the AVD276, outer M2 peptide of born of the same parents and rabbit C4bp support merge.

The aminoacid sequence of AVD276 is as follows:

SLLTEVETPI?RNEWGCRCND?SSDGSEVPEG?CEQVQAGRRL?MQCLADPYEV?KMALEVYKLS

LEIELLELQR?DKARKSSVLR?QL(SEQ?ID?NO：34)

Among the AVD277, outer M2 peptide of born of the same parents and people C4bp support merge.

The aminoacid sequence of AVID277 is as follows:

SLLTEVETPI?RNEWGCRCND?SSDGSETPEG?CEQVLTGKRL?MQCLPNPEDV?KMALEVYKLS

LEIEQLELQR?DSARQSTLDK?EL(SEQ?ID?NO：35)

Among the AVD278, wherein two the born of the same parents' variant and fusions of people C4bp support of M2 peptide outward that halfcystine is replaced by serine residue.

The aminoacid sequence of AVD278 is as follows:

SLLTEVETPI?RNEWGSRSND?SSDGSETPEG?CEQVLTGKRL?MQCLPNPEDV?KMALEVYKLS

LEIEQLELQR?DSARQSTLDK?EL(SEQ?ID?NO：36)

With the AVD275 protein that does not add any adjuvant (2 nanomole) with the interval in two weeks the (promptly the 0th; 14 and 28 days) subcutaneous immune mouse three times; shown considerable antibody titers (basic as Neirynck etc. above-mentioned definite), and at the obvious provide protection of the deadly attack of the mouse adaptability A/PR/8/34 bacterial strain of 5LD50 dosage.

Claims

1. product, it comprises:

The C4bp core protein; With

Monomeric antigen.

2. according to the product of claim 1, wherein said C4bp core is by the residue 1-57 of SEQ ID NO:1 or the corresponding residue of its homologue, and perhaps at least 47 of SEQ ID NO:1 or its homologue amino acid whose fragments are formed.

3. according to the product of claim 2, wherein said homologue is any one among the SEQ ID NO:2-20.

4. according to the product of claim 2 or 3, wherein said homologue be with SEQ ID NO:1-20 in any one has the variant of at least 70% amino acid identity.

5. each product in requiring according to aforesaid right, wherein said monomeric antigen is fused to described C4bp core protein N-or C-end.

6. according to the product of claim 5, wherein said fusion is by joint flexibly.

7. according to each product among the claim 1-6, wherein said monomeric antigen is the monomeric antigen fragment of plasmodium schizont surface protein 1.

8. according to each product among the claim 1-6, wherein said monomeric antigen is the monomeric antigen fragment of influenza virus hemagglutinin protein or influenza M2e peptide.

9. composition, it comprises among the claim 1-8 each product and pharmaceutically acceptable thinner, carrier or adjuvant.

10. according to each the product or the composition of claim 9 among the claim 1-8, it is used for the methods of treatment of human body or animal body.

11. the method for immunotherapy malaria comprises the product according to claim 7 that gives individual effective dose.

12. according to the method for claim 11, wherein said individuality is infected by malarial parasite.

13. according to the method for claim 11, it is used for preventative inoculation.

14. according to the product of claim 7, it is used for the treatment or the prevention of malaria.

15. the purposes of product in the medicine of preparation treatment or prevention of malaria according to claim 7.

16. the way of the antibody of preparation antimalarial protozoan parasite, described way comprise the product according to claim 7 is introduced non-human mammal, and from this Mammals, reclaim immune serum.

17. the method for carrying out passive immunization at experimenter's disease, described way comprises and gives the immune serum that described experimenter comprises antibody, and the described serum that comprises antibody is inoculated host experimenter according to each described product of claim 1-5 and obtained by using.

18. the method for passive immunotherapy human experimenter's malaria, described method comprise the immune serum according to claim 16 generation that gives described people's significant quantity.

19. the immune serum that the method by claim 16 obtains, it is used for the method for immunotherapy of people experimenter's malaria.

20. the method for immunotherapy influenza comprises the product according to Claim 8 that gives individual effective dose.

21. the method for claim 20, wherein said individuality is by influenza infection.

22. the method for claim 20, it is used for preventative inoculation.

23. product according to Claim 8, it is used for the treatment of or flu-prevention.

24. product according to Claim 8 preparation be used for the treatment of or the medicine of flu-prevention in purposes.

25. produce the way of anti influenza antibody, described way comprises introduces non-human mammal with product according to Claim 8, and reclaims immune serum from described Mammals.

26. the passive immunotherapy method of people experimenter's influenza, described way comprise the immune serum according to claim 25 generation that gives described people's significant quantity.

27. the immune serum that the method by claim 25 obtains, it is used for the immunotherapy method of people experimenter's influenza.

28. the method for preparing product, it comprises:

The C4bp core protein; With

Non-polypeptide monomer antigen,

Described method comprises the nucleic acid of expressing coding first component, described fusion rotein is connected on second component, and reclaims described product.

29. the method for preparing product, described product comprises

The C4bp core protein; With

The antigenic fusions of polypeptide monomer,

Described method comprises the nucleic acid of expressing the described fusions of coding, and reclaims described product.

30. the method for claim 28 or 29, wherein said nucleic acid is expressed in prokaryotic host cell.

31. according to the method for claim 30, wherein said fusion rotein reclaims with the polymer form.

32. the immunogenic method of raising monomeric antigen, described method comprise described antigen is connected on the C4bp core protein.

33. expression vector, it comprises the nucleotide sequence of coding C4bp core protein and the antigenic fusion rotein of polypeptide monomer, and described nucleotide sequence is operably connected with functional promotor in the host cell.

34. the expression vector of claim 33, each limits in wherein said C4bp core protein such as the claim 2 to 5.

35. the expression vector of claim 33 or 34, wherein said monomeric antigen such as claim 7 or 8 qualifications.

36. bacterial host cell with each expression vector conversion in the claim 33 to 35.

37. eukaryotic host cell with each carrier conversion in the claim 33 to 35.

38. the purposes of each expression vector in the method for treatment human body or animal body in the claim 33 to 35.