WO2002059319A1 - Malaria plasmodium antigen polypeptide se36, method of purifying the same and vaccine and diagnostic with the use of the thus obtained antigen - Google Patents
Malaria plasmodium antigen polypeptide se36, method of purifying the same and vaccine and diagnostic with the use of the thus obtained antigen Download PDFInfo
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- WO2002059319A1 WO2002059319A1 PCT/JP2002/000506 JP0200506W WO02059319A1 WO 2002059319 A1 WO2002059319 A1 WO 2002059319A1 JP 0200506 W JP0200506 W JP 0200506W WO 02059319 A1 WO02059319 A1 WO 02059319A1
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- amino acid
- polypeptide
- acid sequence
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- malaria
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
- C07K14/445—Plasmodium
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
- A61P33/06—Antimalarials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to an antigen polypeptide derived from Pseudomonas malaria parasite, Plasmodium falciparum SERA (serine-repeatgen), a method for purifying the same, and a purified antigen obtained therefrom as an active ingredient.
- the present invention relates to a malaria vaccine and a diagnostic agent used as a vaccine.
- BACKGROUND ART Malaria is caused by the infection of one or more malaria parasites (Plasmo di um), and includes the following four species, Plasmo di um fa I ciparum; pf J), vivax malaria (P. vivax), malaria vivax (P. malariae) and oval malaria (P. ovale) are known.
- the protozoan invades and infects the human body in the state of sp0r0z0ite from its salivary gland by the female bite of the genus Anopheles (Anopheles).
- Anopheles Anopheles
- SE RA serine-repeatantigen
- SE RA protein antigen with a total molecular weight of 989 amino acids and a molecular weight of 115 kd expressed by the Pf gene in the inner stage of the erythrocyte, and its structure is from N-terminal to C-terminal. In order, there are three domains of 47 kd-50 kd-18 kd.
- SERA as a precursor of these domains is processed during the release of mesozoite in the inner stage of erythrocytes after expression by four exons dispersed on SERA gene DNA consisting of a total of 5868 bases, and the above three domains (Molecular and Biochemica IP arasitology, 86, pp. 249-254, 1997; and Experimental P arasitology, 85, pp. 12 1 —
- the N-terminal region of SERA (hereinafter referred to as “47 kd domain J”) consists of a total of 382 amino acids. According to a homology search between Pf strains related to the sequence, amino acid deletions and additions were found. Scattered amino acid mutations (non-synonymous substitutions), etc. at about 20 sites, which are diverse (Molecular and Bioc 0 Malaria is the world's second most common infection following acute lower respiratory tract infections, AIDS, and diarrhea, and WHO (WorlD H).
- Pf-derived single antigens such as well-known vaccine candidate antigens such as MSP-1 and AMA-1, have a narrow antigenic spectrum and are not necessarily required to prevent infection of any Pf strain. Not effective; and (c) denatured during the purification process, as is known for vaccine candidate antigens such as MSP-1 and AMA-1 above, resulting in disruption of steric structure or epitope, The antigenicity decreases or disappears.
- the present invention relates to a protein antigen (polypeptide SE36) derived from SERA of P. falciparum P f, a synthetic polynucleotide encoding the same, a method for purifying polypeptide SE36, and a method for purifying the same.
- the above problems are solved by providing a malaria vaccine and a diagnostic agent using the obtained purified antigen as an active ingredient.
- the present invention is based on the following findings and findings corresponding to the above-mentioned problems (a) to (c): (a) I against the SE36 antigen according to the present invention; Surprising discovery that gG3 antibody titers are almost completely correlated with acquired immunity to malaria in malaria-endemic populations, and that IgG3 antibodies in the sera of such populations prevent Pf proliferation in erythrocytes Findings (inhibition of P f proliferation prevents fever and red blood cell destruction due to malaria, and thus prevention of death due to cerebral malaria);
- This SE36 antigen has a broad antigenic spectrum and is a representative type of polymorphism found in the SERA47 kd domain, FCR3, Honduras-1 and K1. Strain (Molecularand Biochemical Parasito Iogy, ibid.), And the IgG3 antibody inhibits the growth of all these types of Pf parasites by a neutralization reaction. And (c) the discovery of a purification method that does not destroy or impair the antigenicity or epitope of the SE36 antigen during mass production.
- the present invention provides the following inventions (1) to (15) based on the findings and findings described above.
- Polypeptide SE 36 consisting of the entire amino acid sequence of SEQ ID NO: 4
- No. 1 85 Pro is A I a
- the polypeptide SE36 of (1) above which has at least one of the following. (3) an oligonucleotide comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 added between the 18th GIy and the 19th GIy in the amino acid sequence of SEQ ID NO: 4; Or the polypeptide SE36 of (2). (4) The method according to (1), wherein an oligonucleotide having the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 is added between No.42 AIa and No.43 Ser in the amino acid sequence of SEQ ID NO: 4. Or the polypeptide SE36 of (2).
- an oligopeptide consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 is added between the 18th GIy and the 19th GIy in the amino acid sequence of SEQ ID NO: 4, and (1) or (2) above, wherein an oligonucleotide consisting of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8 is added between No. 42 AIa and No. 43 Ser in the amino acid sequence of No. 4 Polypeptide SE36.
- the polypeptide has the antigenicity crossing with the polypeptide SE36 of (1), and the number of polymerized serine residues in the serine repeat region detected by amino acid homology search is from 0 to 1.
- At least one polypeptide selected from the group consisting of the polypeptide SE36 of the above (1) to (9) and the polypeptide of the above (10) is contained. Characterized malaria vaccine.
- At least one polypeptide selected from the group consisting of the polypeptides SE36 of (1) to (9) and the polypeptide of (10) is contained.
- a malaria diagnostic agent characterized by the following.
- FIG. I shows the amino acid sequence (amino acids 16-382) of the 47-kd SERA domain of Plasmodium um falciparum Honduras-1 and the full-length amino acid sequence of polypeptide SE36. Between ⁇ 34 amino acids (M ⁇ E) Show the differences. Described from left to right in the direction from N to C-terminus by single letter symbols of amino acids.
- Hnd-1 is Hnduras-1 strain, ... is the same sequence,-is deletion, and [number] is the amino acid number of SE36.
- FIG. 2 shows the full-length amino acid sequence as the basic structure of the polypeptide SE36 molecule.
- FIG. 3 shows the difference from that of the other P. faIc ⁇ parum strains based on the H 0 nduras—1 strain 47 kd amino acid sequence described in FIG. [Number] means the amino acid number of SE36.
- column 1 shows the molecular weight marker
- column 2 shows the whole protein of Escherichia coli that does not express SE36
- column 3 shows the electrophoresis of the whole protein of Escherichia coli expressing SE36 by adding IPTG. image.
- FIG. 5 is an SDS-polyacrylamide electrophoresis image of the SE36 sterile preparation (stock vaccine solution) prepared in Example 2. Indicates that the presence of contaminants other than SE36 is not visually detected.
- FIG. 6 is a graph showing the antibody titer of chimpanzees immunized with the SE36 protein for each IgG subclass.
- FIG. 7 is a graph showing the inhibition of malaria parasite growth by chimpanzees immunized with the SE36 protein and human IgG that has acquired malaria immunity.
- FIG. 8 shows that there is a positive correlation between the inhibition of Pf parasite growth by sera of malaria endemic areas and anti-SE36 IgG antibody titers.
- Figure 9 shows that Pf protozoan growth inhibition by sera from malaria endemic areas was significantly inhibited by the coexistence of the SE36 sterile standard (vaccine stock solution) compared to the control. Sum).
- ⁇ indicates the full-length SE36 protein having a natural three-dimensional structure, indicates the SE36 protein, and ⁇ indicates the SE50A protein without vaccine effect.
- the polypeptide SE36 (hereinafter abbreviated as "SE36J") according to the present invention comprises the SERA domain 47 kd (hereinafter referred to as "47 kd") of the aforementioned Onduras 21 strain of P. falciparum 7 protozoan Pf. SE 4 7 'antigen (V accine, 14, p. P. 106-107 6, 1996; hereinafter abbreviated as "SE 47-”) based on J) And consists of a basic structure in which the full length or a part of the 47 'kd serine repeat region has been removed.
- Figure 1 shows the difference in amino acid sequence between the basic structure of SE36 molecule and 47'kd (from Honduras-1 strain), and the full-length amino acid sequence of the basic structure of SE36 molecule.
- Figure 2 shows each. In these figures, the amino acid sequence is described from left to right in the direction from N to C terminal using amino acid one-letter symbols.
- the H 0 nduras-1 strain is abbreviated as Hond-1, and the deletion region detected by homology search between this strain and another P f strain is ---.
- the identical amino acid sequences are represented by.
- the basic structure of SE36 is ordinal numbered in the direction of the C-terminal, starting from the N-terminal methionine of a total of 382 amino acids that constitutes 47 kd of Hond-1 (the first amino acid).
- the codon of amino acid 16 (asparagine) was replaced with an initiation codon (methionine), and a translation stop codon was inserted adjacent to amino acid 382 (glutamic acid).
- This is a polypeptide consisting of a total of 334 amino acids, from which a total of 33 polymerized serine residues (from 193rd scent to 225th serine) have been excised.
- Sequence List to be compared with FIGS.
- [SEQ ID NO: 1] is the full-length nucleotide sequence of the SERA domain 47 kd gene DNA of Honduras-1 and 4 7 shows the 7 kd full length amino acid sequence.
- [SEQ ID NO: 2] shows the full length amino acid sequence of 47 kd described in SEQ ID NO: 1. This sequence is composed of a total of 382 amino acids, and the 16th to 382nd amino acids are the same as those of each H 0 nd-1 shown in FIG. 1 and FIG. 3 described later.
- [SEQ ID NO: 3] shows the full-length nucleotide sequence of the synthetic DNA after conversion of the SE36 gene to Escherichia coli codons, and the full-length amino acid sequence encoded thereby.
- the first amino acid Met is in the form of the 47 kd No. 16 amino acid Asn substituted with the start codon Met.
- the nucleotide sequence has been converted from almost all P f codons to E. coli codons. Such conversion to E. coli codons will be described later.
- [SEQ ID NO: 4] shows the full-length amino acid sequence as the basic structure of the SE36 molecule described in SEQ ID NO: 3. This sequence is the same as SE 36 described in FIG. 1 and each amino acid sequence described in FIG.
- FIG. 1 The mutation of SE36 is exemplified in FIG. This figure uses the amino acid sequence of the H 0 nduras-1 strain (Hond-1) shown in Fig. 1 as a reference, and assigns amino acid numbers with the same convention as in Fig. 1; , And the same sequence is indicated by ising
- the basic structure of the SE36 molecule is a polypeptide consisting of a total of 334 amino acids, as shown in Fig. 2 (SEQ ID NO: 4). Modified products, mutants, and the like can be obtained. These include non-synonymous substitution of amino acids based on the description in FIG. 3, addition or deletion of a lysine peptide, and limitation of the number of polymerized serine residues occupying the serine-repeat region. 1st 1st 1st 1st. 1st 1st 1st 1st 1st
- amino acid sequence of SE36 described in SEQ ID NO: 4 at least one amino acid substitution selected from the following amino acid substitutions (a) and (V) is possible:
- the number of serine residues polymerized by a peptide bond between No. 175 Asp and No. 178 Glu is 0 to 30, Preferably, it is in the range of 0 to 20, more preferably not more than 10, that is, in the range of 0 to 10.
- the restriction on the number of serine residues is based on the inventor's experience that SE47 ', which has 35 polymerized serine residues, was difficult to purify and did not reach practical use (V accine, supra).
- the number of polymerized serine residues in the serine repeat region detected by homology search for amino acids having antigenicity crossing with SE36 is, for example, in the range of 0 to 10.
- a polypeptide can be prepared.
- the crossover of antigenicity is detected by an antigen-antibody reaction, and the serine repeat region is the amino acid from the 157th GIy to the 183rd Sn of SE36 (SEQ ID NO: 4). It can be detected by homology search of amino acids using the sequence as a reference.
- the amino acid sequence of SE36 described in SEQ ID NO: 4 has various molecular structures as long as the original antigenicity and immunogenicity of SE36 and their spectra are not impaired. It can be modified, deformed or processed. That is, the polypeptide S.E36 according to the present invention is not limited to the basic structure of SE36 described in SEQ ID NO: 4, but may be obtained by addition, excision or addition of the amino acid or oligopeptide described in (1) to (4) above. It is a generic term for all of these, including derivatives or derivatives, variants, modifications, etc. of the above structure by substitution or the like.
- SE36 can be used individually as an active ingredient in vaccines and diagnostics, and can be used alone as well as at least two of them to broaden the spectrum of antigenicity and immunogenicity. Can be used. Furthermore, it should be noted that in the production of SE 36, the natural world
- SE36 is carried out by converting all of the natural P f codons encoding the amino acid sequence into E. coli codons, and obtaining a DNA fragment encoding the full-length SE36 (hereinafter, referred to as “E.
- the DNA base sequence of the SE36 gene on the desktop of P f and the amino acid sequence it encodes are: It is available on the Internet from publicly known gene database publication organizations, for example, DD BJ, Gen Bank, EB, and the like. "
- DNA synthesis is performed using a commercially available DNA synthesizer, such as a bNA / RNA synthesizer [App I ied Biosystem em Med I 392: manufactured by PE (USA)], an ASM-102 UDNA synthesizer [BIOSSET (USA)] can be used.
- a commercially available DNA synthesizer such as a bNA / RNA synthesizer [App I ied Biosystem em Med I 392: manufactured by PE (USA)], an ASM-102 UDNA synthesizer [BIOSSET (USA)] can be used.
- a bNA / RNA synthesizer App I ied Biosystem em Med I 392: manufactured by PE (USA)]
- BIOSSET ASM-102 UDNA synthesizer
- a known or commercially available host for cloning of Escherichia coli (“Cloning Vectors: AL aboratoryanual ', I—1 to l—D—i” 8, P. H. PouWels, et al., Published in EI sevier 1 988)
- Escherichia coli a known or commercially available host for cloning of Escherichia coli
- a combination of plasmid pBluescript II SK10 and E. coli XL1—BIue [S
- a restriction enzyme fragment of the above-described DNA fragment is inserted into a restriction enzyme site of a vector cleaved with the same enzyme to construct a clone.
- the clone as a transformant is obtained by transferring the obtained vector into a host, and then the clones of the double-stranded DNA fragment are amplified by culturing the above transformant, and then each of these bases is amplified.
- Arrays can be sequenced using the chaininterferor method (dide 0 Xy method) or Beam -. Determined by Giruba Bok method It should be noted that this, commercial DNA sequencer, for example, can be used ABIPRIS 3 700 [PE Co. (USA)], based on the result, 3 £ 36 gene 0 Approximately 5 to 10 clones are selected for a double-stranded DNA fragment covering the entire length of the DNA fragment.
- Cloning of the full-length SE36 gene DNA is performed by ligation of the double-stranded DNA fragment clone. For example, when 8 clones (8 pairs) are selected in the above, these full-length DNA fragments can be obtained by sequentially ligating these double-stranded DNA fragments. The full-length DNA is then cloned as described above. At the time of the above ligation, it is desired to introduce a restriction enzyme cohesiVe site for ligation at both ends of each fragment of the double-stranded DNA. However, for such introduction, it is necessary to match the base sequence as a codon so that the original amino acid sequence of P f does not change. Construction of expression system of S E 36 gene:
- a known or commercially available host for the expression vector of Escherichia coli (“Cloning Vectors: AL aboratory Manual"), which has been introduced variously as before. ), For example, a combination of plasmid p ET — 3 & with E. coli 81 (DL 3) p Lys S or E. coli BL 21 (DL 3) p Lys E [Stratagene (USA)] ] Can be used.
- Such a preparation is carried out, for example, by cutting out a restriction enzyme fragment containing the full-length SE36 gene DNA from the cloning vector, and inserting and linking the restriction enzyme site of the vector cleaved with the same enzyme.
- p ET — SE 47 ′ V ac c i n e, same as above.
- the entire or partial region encoding the serine repeat region is excised from the pET-SE47 'or the SE47' synthetic gene carried by the restriction enzyme with a restriction enzyme, thereby preparing the same as above. can do.
- a transformant is obtained by transferring the prepared expression vector into a host. From such a transformant, a suitable expression system for the SE36 gene can be selected from the viewpoint of industrial use, using mass production of the polypeptide SE36 as an index. Production of polypeptide SE36:
- Mass production of SE36 is performed by culturing the above-mentioned Escherichia coli transformant.
- a culture system may be an inducer such as IPTG (isopropyl-1-thio-—D—galactopyranosi de)), avoidance of catabolite repression, medium composition, culture temperature and time, removal of proteases in host cells, etc., can be improved or improved. Further, it can be carried out by changing the promoter or the host strain.
- the culture solution of the above-described forma transformant culture is used as a starting material for SE36 extraction. If the cells accumulate in the cells, for example, the above-mentioned SE36 is extracted from the cells collected and collected from the culture by centrifugation, filtration, or the like.
- digestion by enzymes, disruption by osmotic pressure, rapid pressure reduction and decompression, ultrasonic waves, various homogenizers, and the like can be used.
- ammonium sulfate The saturation of ammonium sulfate (hereinafter abbreviated as "ammonium sulfate”) in water at 4 ° C is set to a lower limit of 20% and an upper limit of 50%, and desirably a lower limit of 30% and an upper limit of 35%. Fractionation by salting out with addition and mixing of ammonium sulfate;
- the detection and size confirmation of the SE 36 molecule can be performed, for example, by measuring the sedimentation coefficient, molecular sieve, SDS-polyacrylamide electrophoresis, or the like.
- the antigenicity of the SE36 molecule can be confirmed by antigen-antibody reaction using ELISA, agglutination, immunofluorescence, radioimmunoassay, etc., using polyclonal or monoclonal antibodies against 47 kd of SERA. .
- the immunogenicity of the SE36 polypeptide and the ability of anti-SE36 antibody to inhibit P f The antigen-antibody reaction described above using serum of a Laria patient or an experimental small animal immunized with the polypeptide, for example, serum of a rat or mouse, etc., and the inhibition of Pfmer 0z0ite proliferation in erythrocytes (so-called medium Sum reaction), and can be confirmed by measurement of the blood Pf count of the carrier of the SE36 antibody.
- the purified polypeptide SE36 is used as an antigen, and the antigen is suspended in a solvent, for example, isotonic PBS (phosphatatebuufferrsaline) to prepare a vaccine stock solution.
- a solvent for example, isotonic PBS (phosphatatebuufferrsaline) to prepare a vaccine stock solution.
- the three-dimensional structure of the vaccine antigen can be stabilized by immobilizing it with a conventional inactivating agent.
- a conventional inactivating agent for example, formalin, phenol, daltardialdehyde, monopropiolactone, etc.
- formalin for example, formalin, phenol, daltardialdehyde, monopropiolactone, etc.
- its addition amount is about 0.005-0.1% (V / V)
- the inactivation temperature is about 4-38 ° C
- the inactivation time is about 5-180 days.
- such relaxation can be achieved by reducing the amount of inactivating agent, adding neutral amino acids or basic amino acids, etc. Can be achieved by lowering the inactivation temperature.
- Free formaldehyde remaining in the inactivation step can be neutralized by adding an equal amount of sodium bisulfite, or removed by dialysis, if necessary.
- the SE36 antigen can be processed or modified for the purpose of inducing mucosal or local immunity by oral or nasal inoculation of the vaccine.
- a technique relating to DDS (drug delivery system) using liposomes, emulsions, microcapsules, microspheres, polylactic acid, polyglycolic acid, etc. can be applied.
- the preparation thus obtained is used as the vaccine stock solution for the next step.
- the vaccine stock solution is diluted with, for example, the PBS, and the amount of the antigen in the vaccine is adjusted to an amount necessary for inducing antibody production and producing immunity.
- a stabilizer for enhancing the heat resistance of the vaccine and an adjuvant as an adjuvant for enhancing the immunogenicity can be added and mixed.
- sugars and amino acids as adjuvants and adjuvants can be used as the solution.
- an appropriate amount for example, about 1 to 20 ml, is dispensed into a vial, sealed, sealed and then used as a vaccine.
- Such vaccines are not only in liquid form, but also by lyophilization after dispensing. It can be used as a dry preparation.
- Vaccine assay :
- a dose of about 0.25 to 0.5 ml is subcutaneously administered. This inoculation should be performed 1 to 3 times at intervals of about 2 to 4 weeks.
- the vaccine usage is not limited to the above examples.
- the SE36 polypeptide can be provided as an antigen for diagnosis of malaria, for example, an antigen such as a precipitation reaction, an agglutination reaction, a neutralization reaction, a fluorescent antibody method, an enzyme immunoassay, and a radioimmunoassay.
- the polypeptide can be inoculated intraperitoneally, subcutaneously, intramuscularly, or the like of an animal, for example, a rabbit, a guinea pig, a mouse, or the like, and an antibody can be produced from the serum of the animal that has produced an antibody.
- Such antibodies can be provided, for example, for detecting antigens in the various diagnostic methods described above.
- the antigen or antibody for diagnosis according to the present invention is diluted with a solvent, for example, the above-mentioned PBS, and adjusted for use so that the content in these diagnostic agents becomes an amount necessary for exhibiting an antigen-antibody reaction. .
- the DNA base sequence of the full-length SE36 gene which was converted from P f codons to E. coli codons on the desktop, was divided into eight, and the single-stranded DNA fragments of the sense (+) strand and antisense (single) strand of each split fragment totaled 6 (8 pairs) After synthesizing and annealing to prepare a total of 8 pairs of double-stranded DNAs, these were ligated to each other to create a full-length SE36 gene, and its expression vector was constructed. The basic operation of cloning and ligation of a synthetic DNA fragment was performed according to the method of Sambr0ok et al. (Molecular CI oning: AL aboratory Manual, 2nd edition, Old Spring Laboratory Press 1989). went.
- Each of the single-stranded DNA fragments described above was a DN AZR NA synthesizer "A pp I i
- These fragments were synthesized using 10% (W / V) polyacrylamide (50 mM Tris monoborate, pB H8.3, 1m EDTA, and 8M urea) were purified by electrophoresis and then mixed with 10 to 10 complementary strands of 20p moles of purified fragment DNA. The mixture was kept at 205 ° C. for 5 minutes in 20 1 of 20 mM Tris-HCI, pH 7.0, 50 mM NaC, and 2 mM MgCI 2 ).
- coli XL1-BI The clone was amplified and cloned with ue, the nucleotide sequence of the above DNA fragment of each clone was determined by the dideoxy method, and eight clones that supported the full-length SE36 gene were selected. By ligating the synthetic double-stranded DNA fragments of these eight clones (8 pairs), double-stranded DNA of the full-length SE36 gene was prepared. At this time, a restriction enzyme site for ligation was introduced into both ends of each pair of DNAs using a base sequence that did not change the original amino acid sequence of P f.
- Example 2 coli B LZS E 36 obtained in Example 1 was replaced with LB medium containing ampicillin SOigZmI (B acto-trypton 1% (W / V), B acto-yeast extract 0.5 % (W / V), and 37% in NaCI 1% (WZV)]. C. for 18 hours to prepare seeds. Next, 5 L of the above fresh LB medium was inoculated with 50 ml of the above seed, and cultured at 37 ° C.
- ampicillin SOigZmI B acto-trypton 1% (W / V)
- B acto-yeast extract 0.5 % (W / V)
- WZV NaCI 1%
- I PTG - a isopropy I j8- D- thiogalactopyranos ⁇ de
- the cells were cultured at 37 ° C for 3 hours. After completion of the culture, the cells were collected by centrifugation (5,000 rpm, 10 minutes) to obtain 3.2 g of cell paste. After suspending the paste in 9.6 ml of ice-cold lysis (Iys ⁇ s) buffer (50 mM Tris-HC and pH 8.0, and 1 mM MEDTA), (1) The operations of (6) were performed at 4 ° C in the order described.
- Sephacry IS-300 (26) was obtained by equilibrating the above filtrate with a GF buffer [50 mM Tris-HCI (pH 8.0), 1 mM EDTA, 50 mM 2-mercaptoethanol, and 8 M urea]. / 60) After fractionation (3.5 ml fractionation) by column chromatography (flow rate: 0.3 ml / min, 4 ° C), fractions 22-43 were subjected to SDS-polyacrylamide electrophoresis. Fractions 32 to 37 containing a large amount of SE36 protein detected based on the electrophoresis image were pooled. The remaining resuspension (4.4 ml) was treated in the same manner as described above, and used in the next operation (4) together with the fraction pool. (4) Column purification II
- HIC buffer for confirmation, eluted adsorbed Ingredient by GF buffer containing no (NH 4) 2 S 0 4 .
- the unadsorbed fraction from this column is placed in a dialysis bag, and 1 L of 20 mM Tris-HCI buffer (pH 8.0) (containing 1 mM EDTA) is used as the external solution for 10 hours at 4 ° C. Dialyzed. The external solution was changed twice during this dialysis.
- the protein was precipitated by dialysis at 4 ° C for 10 hours using [20 mM Tris—HCI (pH 8.0) and 1 mM EDTA] as an external solution. This precipitation The substance was collected as a sediment by centrifugation (12,000 rpm, 10 minutes), and then suspended in 2 ml of GF buffer.
- the above suspension was kept at 60 ° C for 10 minutes and then returned to 4 ° C. This was filtered through the 0.45 m-filter, and the filtrate was flown at 0.3 ml / min.
- the S-300 column (26/60) equilibrated with [1 OmM Tris-HC I (pH 8.0), 1 mM MEDTA, 20 mM 2 monomercaptoethanol, and 8 M urea] And fractionated.
- each fraction was subjected to SDS-polyacrylamide electrophoresis, fractions of $ E36 protein were selected, and these were pooled to obtain 12 mi of the fraction.
- This fraction pool was added to the dilution buffer [1 OmM Tris—HCl (pH 8.0), 1 mM EDTA, and 2 M urea] by stirring, and the concentration of SE36 protein was 25 ⁇ gZm Diluted to be I.
- the external solution was changed twice during this dialysis.
- the inner solution after completion of the dialysis was concentrated with Centprep 30 and then sterilized by filtration through a D urapore 0.22 m filter [ ⁇ II IIpore (USA)], and 1 mgZm of SE 36 protein was obtained. 10 ml of a sterile sample containing I was obtained, stored as a stock solution of SE36 vaccine at 4 ° C, and used for the subsequent assay.
- Example 3 Amino Acid Sequence Determination
- the N-terminal amino acid sequence of the SE36 protein obtained in Example 2 was determined by Edman degradation using a protein sequencer Applied Biostems 473 A [manufactured by PE (USA)]. .
- the results are shown in FIG. 2 and SEQ ID NO: 4 in the sequence listing.
- Example 4 Assay of antigenicity and immunogenicity The antigenicity and immunogenicity of the SE36 protein in the stock solution of SE36 vaccine obtained in Example 2 were assayed as follows (1)-(3).
- the above vaccine stock solution was serially diluted 5-fold with PBS to prepare solutions each having an antigen amount (g / 0.05 ml) of 200, 40, and 8, respectively, and an equal volume (VZV) of Freund's complete adjuvant, and The mixture was mixed with Freund's complete adjuvant to prepare an emulsion, and the former was used for the first immunization, and the latter was used for the second and third immunizations.
- mice A total of 20 7-week-old female BALB / c mice [manufactured by C-EA (Japan)] (5 groups, 4 groups in total) were used. Subcutaneous inoculation was carried out three times on days 7 and 21. Group 1 mice received a vaccine with an antigen amount of 200 Mg, group 2 40 ig, group 3 8 g, group 4 mice
- mice in Groups 1 to 3 above were healthy during breeding after vaccination, as were the mice in Group 4 for comparison, and had abnormal weight loss, behavior, excretion, appearance, and No deaths were seen, confirming the safety of the vaccine.
- Honduras-I te of PI asm 0 diumfa I cipar um was used, and its culture and maintenance were carried out by the method of Trager and ensen (Sc ⁇ ence, 193, 673—675, 1976), and B This was performed by the method of anya I and Innerberg (American Journal of Tropical and Medica IH ygiene, 34, 1055—1064, 1985).
- the maintenance medium is diluted with fresh erythrocytes, and the parasite erythrocyte count is counted as the total erythrocyte count.
- mice in the first and second groups had the above inhibition rate of 90%, and the average value of the five mice in the third group was 70%.
- the mouse antiserum and the control mouse serum were each subjected to the following pretreatment before the inhibition test described above: 0.5 ml of each of these was mixed with 2 ml of a red blood cell pack. After that, incubate at 37 ° C for 2 hours, then centrifuge at room temperature (500 rpm, 5 minutes), and the supernatant was collected.
- a VecstasstainABC kit [VectorLaboraboritas Co. (USA)] was used.
- E and I SA SE 36 protein is used as an antigen, and 2, 2'-az is used as a substrate.
- P f parasitized erythrocytes were sourced from cultures of erythrocytes, at least 80% of which were mature tr0 ph0z0ite and schizont. This parasitic red blood cell
- the average values of the ELISA titers of Groups 1 to 3 were 90,000 for Group 1, 88,000 for Group 2, 42,000 for Group 3, and Four groups of comparison controls were less than 100.
- the protein reactive with each mouse antiserum in the first to third groups was 1%.
- a prototype vaccine prepared by adsorbing SE36 protein on aluminum hydroxide gel is inoculated into chimpanzees, and the blood collected at each time point is subjected to hematological and blood biochemical tests, and the serum is used for immunological response. Tested.
- SE36 protein For three chimpanzees, 450 g (male, 11 years old), 50 g (female, 10 years old), and 10 g (female, 6 years old) of SE36 protein were given at a time. The back was inoculated subcutaneously. The study schedule consisted of a first vaccination at week 0 and a booster at week 4. Blood was collected before each vaccination, and at the time of additional vaccination and over time thereafter.
- SE36 protein-specific antibody titers of the obtained sera were measured for each IgG subclass, and as a result of the inoculation at 450 times, a high increase in antibody titer was confirmed for all subclasses. In particular, it reached about 1000 times after the second inoculation. This high The antibody titer was maintained at 10 weeks (FIG. 6A).
- the same immune response was obtained with the 50 v ⁇ inoculations as with the 450 gZ vaccinations (Fig. 6B). Inoculation at 10 doses requires some time for an immune response to be obtained, but antibody titers in all subclasses increase at week 4, and about ⁇ after the second dose.
- IgG-dependent ADCs were obtained using immune sera obtained by inoculation at 450 gZ.
- the total IgG fraction concentration of the added chimpanzee was approximately 1/10 of the IgG concentration in the chimpanzee serum, and the total IgG fraction before immunization was reduced.
- the addition of the whole IgG fraction after immunization resulted in approximately 30% better inhibition of protozoan growth compared to the case of the addition, indicating that the almost complete inhibitory effect was observed in the blood of living organisms. Be expected.
- IgG3 specific to the SE36 protein is used for the measurement in human blood, it is comparable to the protozoan growth inhibitory effect of humans that have acquired immunity to malaria parasites. Considered enemy. From the above results, the effectiveness of the vaccine of the present invention was confirmed.
- the anti-SE36 IgG3 antibody titer and the growth inhibitory ability showed a positive correlation, providing an epitope of a human antibody in which the SE36 protein acts to inhibit protozoan growth. It was suggested that you. Furthermore, it was suggested that SERA gene polymorphism had no effect on the patterning effect (Fig. 8).
- Reference Example 5-Neutralizing Ability of SE36 The anti-SE36IgG3 antibody titer of the malaria highly endemic area used in Reference Example 4 in the presence of the sterilized SE36 sample obtained in Example 2 was used. The fate of the ability to inhibit Pf proliferation by a high representative serum was measured by the in vitro test for inhibiting the growth of P. falciparum described in Reference Example 4. As a control for comparison with SE36, full-length SERA protein with natural conformation produced by baculovirus vector and SE50A protein derived from the central domain of full-length SERA protein (no vaccine effect) (V accine, ibid.).
- the present invention inhibits the growth of malaria parasite (P f) ; in erythrocytes, which is the root cause of cerebral malaria, which causes malaria symptoms and death with fever.
- An extremely effective vaccine against malaria that induces antibodies is provided.
- a malaria diagnostic agent will be provided.
- the present invention contributes as an excellent and powerful tool for controlling the world's leading multiple infectious disease, malaria, and at the same time there is a concern that the area where malaria occurs due to global warming is concerned. It has a long-awaited and tremendous effect and gospel in securing global and sound activities not only for human health but also for tourism, economy and politics.
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JP2002559804A JP4145145B2 (ja) | 2001-01-24 | 2002-01-24 | マラリア・プラスモジウム抗原ポリペプチドse36、その精製方法、並びにこれより得られる抗原を用いるワクチン及び診断剤 |
AU2002228347A AU2002228347C1 (en) | 2001-01-24 | 2002-01-24 | Malaria plasmodium antigen polypeptide SE36, method of purifying the same and vaccine and diagnostic with the use of the thus obtained antigen |
EP02710343A EP1279735B1 (en) | 2001-01-24 | 2002-01-24 | Malaria plasmodium antigen polypeptide se36, method of purifying the same and vaccine and diagnostic with the use of the thus obtained antigen |
DE60233619T DE60233619D1 (de) | 2001-01-24 | 2002-01-24 | Malaria-plasmodium antigen-polypeptid se36, verfahren zu seiner reinigung und impfstoff und diagnose unter verwendung des so erhaltenen antigens |
BRPI0203949-4 BRPI0203949B8 (pt) | 2001-01-24 | 2002-01-24 | polipeptídeo se36, processo para purificação do polipeptídeo se36, vacina para malária, agente de diagnóstico para malária e fragmento de dna sintético |
US10/239,517 US20040137512A1 (en) | 2001-01-24 | 2002-01-24 | Malaria plasmodium antigen polypeptide se36, method of purifyng the same and vaccine and diagnostic with the use of the thus obtained antigen |
CA2404429A CA2404429C (en) | 2001-01-24 | 2002-01-24 | Malaria plasmodium antigen polypeptide se36, method of purifying the same and vaccine and diagnostic with the use of the thus obtained antigen |
AT02710343T ATE442440T1 (de) | 2001-01-24 | 2002-01-24 | Malaria-plasmodium antigen-polypeptid se36, verfahren zu seiner reinigung und impfstoff und diagnose unter verwendung des so erhaltenen antigens |
HK04101841.5A HK1058948A1 (en) | 2001-01-24 | 2004-03-12 | Malria plasmodium antigen polypeptide se36, method of purifying the same and vaccine and diagnostic with the use of the thus obtained antigen |
US11/517,455 US7462358B2 (en) | 2001-01-24 | 2006-09-08 | Antigenic polypeptide SE36 of malaria plasmodium, process for purification thereof, and vaccine and diagnostic agent using the antigen |
US12/292,206 US7887819B2 (en) | 2001-01-24 | 2008-11-13 | Antigenic polypeptide SE36 of malaria plasmodium, process for purification thereof, and vaccine and diagnostic agent using the antigen |
US12/458,388 US20100016572A1 (en) | 2001-01-24 | 2009-07-10 | Antigenic polypeptide SE36 of malaria plasmodium, process for purification thereof, and vaccine and diagnostic agent using the antigen |
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US10/239,517 A-371-Of-International US20040137512A1 (en) | 2001-01-24 | 2002-01-24 | Malaria plasmodium antigen polypeptide se36, method of purifyng the same and vaccine and diagnostic with the use of the thus obtained antigen |
US11/517,455 Continuation US7462358B2 (en) | 2001-01-24 | 2006-09-08 | Antigenic polypeptide SE36 of malaria plasmodium, process for purification thereof, and vaccine and diagnostic agent using the antigen |
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WO2010126151A1 (ja) * | 2009-05-01 | 2010-11-04 | 国立大学法人大阪大学 | 新規マラリアワクチン |
JP2017210441A (ja) * | 2016-05-26 | 2017-11-30 | 国立大学法人大阪大学 | マラリアワクチン |
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EP2116261A1 (en) * | 2008-05-07 | 2009-11-11 | Institut Pasteur | Sub-region of a plasmodium protein with improved vaccine potential and medical uses thereof |
WO2010036293A1 (en) * | 2008-09-24 | 2010-04-01 | The Johns Hokins University | Malaria vaccine |
US8277650B2 (en) | 2009-03-13 | 2012-10-02 | Terrasep, Llc | Methods and apparatus for centrifugal liquid chromatography |
CN102838659A (zh) * | 2011-06-22 | 2012-12-26 | 中国医学科学院基础医学研究所 | 间日疟原虫抗原的多肽、IgY抗体及其在疟疾诊断上的应用 |
WO2013082500A2 (en) * | 2011-12-02 | 2013-06-06 | Rhode Island Hospital | Vaccine for falciparum malaria |
CN103965313B (zh) * | 2013-01-28 | 2016-04-13 | 苏州偲聚生物材料有限公司 | 多肽、包含该多肽的检测器件和检测试剂盒 |
CN103965324B (zh) * | 2013-01-29 | 2016-08-10 | 苏州偲聚生物材料有限公司 | 多肽、包含该多肽的检测器件和检测试剂盒 |
CN103965327B (zh) * | 2013-01-29 | 2016-08-10 | 苏州偲聚生物材料有限公司 | 多肽、包含该多肽的检测器件和检测试剂盒 |
CN103570817B (zh) * | 2013-11-06 | 2015-07-22 | 中国疾病预防控制中心寄生虫病预防控制所 | 间日疟原虫PvMSP1重组抗原蛋白、其制备方法和用途 |
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DE3741057A1 (de) | 1987-03-18 | 1988-09-29 | Behringwerke Ag | Klonierung von malaria-spezifischen dna-sequenzen:isolierung des gens fuer das 140 kd protein |
US6333406B1 (en) | 1988-08-12 | 2001-12-25 | Joseph W. Inselburg | Gene encoding protein antigens of Plasmodium falciparum and uses therefor |
WO1990001549A2 (en) * | 1988-08-12 | 1990-02-22 | Trustees Of Dartmouth College | Gene encoding protein antigens of plasmodium falciparum |
DE4041836A1 (de) | 1990-12-24 | 1992-06-25 | Behringwerke Ag | Protektive plasmodium falciparum hybridproteine, die teilsequenzen der malaria-antigene hrpii und serp enthalten, ihre herstellung und verwendung |
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DATABASE BIOSIS [online] HORII T. ET AL.: "Antibodies reactive with the N-terminal domain of plasmodium falciparum serine repeat antigen inhibit cell proliferation by agglutinating merozoites and schizonts", XP002909448, accession no. STN Database accession no. 1999:232590 * |
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Cited By (6)
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WO2010126151A1 (ja) * | 2009-05-01 | 2010-11-04 | 国立大学法人大阪大学 | 新規マラリアワクチン |
US8808712B2 (en) | 2009-05-01 | 2014-08-19 | Osaka University | Malaria vaccine |
US9028843B2 (en) | 2009-05-01 | 2015-05-12 | Osaka University | Malaria vaccine |
JP5748658B2 (ja) * | 2009-05-01 | 2015-07-15 | 国立大学法人大阪大学 | 新規マラリアワクチン |
AU2010242352B2 (en) * | 2009-05-01 | 2016-01-07 | Osaka University | Novel malaria vaccine |
JP2017210441A (ja) * | 2016-05-26 | 2017-11-30 | 国立大学法人大阪大学 | マラリアワクチン |
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US7462358B2 (en) | 2008-12-09 |
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BR0203949A (pt) | 2003-01-28 |
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HK1058948A1 (en) | 2004-06-11 |
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BRPI0203949B1 (pt) | 2018-07-03 |
EP1279735B1 (en) | 2009-09-09 |
CA2404429A1 (en) | 2002-08-01 |
ATE442440T1 (de) | 2009-09-15 |
CA2404429C (en) | 2011-02-22 |
AU2002228347C1 (en) | 2006-05-11 |
JPWO2002059319A1 (ja) | 2004-05-27 |
US20100016572A1 (en) | 2010-01-21 |
CN100516219C (zh) | 2009-07-22 |
US7887819B2 (en) | 2011-02-15 |
EP1279735A4 (en) | 2005-05-25 |
US20090104219A1 (en) | 2009-04-23 |
US20040137512A1 (en) | 2004-07-15 |
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