JPH02502337A - Plasmodium falciparum rod body antigen - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Plasmodium falciparum rod body antigen The present invention is directed to Plasmodium falciparum rod body antigen. Can inhibit the growth of Plasmodium falciparua. Asexual blood stage Plasmodium falciparum that can generate immune responses and antibodies Identification of this antigen and its use in immunization, diagnostic and therapeutic methods It concerns the use of antibodies against.

Rod body antigens are known for their accessibility to host immune mechanisms and their invasion of red blood cells. It is of particular interest in research because it is believed to be involved in stomach. A large number of rod body antigens have been detected in asexual blood in vivo or in vitro. It is known that P. falciparum can induce an immune response that inhibits the growth of P. falciparum. There is (1.2).

According to one aspect of the invention, Special Table Sen2-502337 (2) (i) has an apparent molecular weight of approximately 55kI)a; (ii)! with listic acid glycoprotein; (iii) restricted in merozoites corresponding to the location of rod bodies; (iv) the parasite antigen is present in a surfactant, e.g. When separated by phase partitioning with Riton be able to A rod body antigen of Plasmodium falciparum in the asexual blood stage, characterized by , or antigenic fragments thereof.

The antigen is a polypeptide or polypeptide having a primary structure comprising the amino acid sequence shown in FIG. is preferably an antigenic fragment thereof.

The invention also provides for administering an antigen of the invention or an antigenic fragment thereof to a host. to actively immunize the host against Plasmodium falciparum, which consists of The present invention provides a method of t.

The present invention provides an antigen of the present invention or an antigen fragment thereof, a pharmaceutically acceptable carrier, or a diluent, and optionally an adjuvant. It is something that

Other features of the invention will be apparent from the detailed description of the examples below. In this example, to enrich the putative cell membrane proteins of P. falciparum In this study, an interface was created due to temperature-dependent phase separation of an aqueous solution of Triton X-114, a surfactant. Selective partitioning of intracellular membrane proteins into the surfactant phase was used. Next, Papua After plotting human antibodies from a patient from Guinea on nitrocellulose. , which were affinity purified against these antigens. This purified human antibody , which expresses polypeptide fragments corresponding to these antigens. Identification of recombinant clones of Escherichia coli It was used to

P used in this study, Falciparum JllI1 body FCQ27/PNG (Fe The origin of 12) was described separately (2). The synchronized cells were treated with sorbitol. The organisms were treated with 0.25% hemacrite (blood red blood) along with a parasite surface in the range of 2-7%. The cells were cultured at a sphere volume ratio). Wash all samples, centrifuge to remove medium, and recalibrate human tonicity. The solution was replaced with acid buffered saline (HTPBS). Cells were then pelleted and I The RQ-encapsulated cells were snap frozen and stored at -70°C until further processing.

Solubilization and phase separation with Triton dierX4), and added the following improvements to Triton Solubilization and separation of hydrophobic, hydrophilic and insoluble fractions were carried out. to Liton It was concentrated. The previously encapsulated parasitized cells 111Q are gently injected at 10 minute intervals. 9 on water in 0.5% Toridoa'X-114' (15z12) with stirring. Solubilized for 0 minutes. The entire lle was taken as a sample and rapidly frozen.

Next, the remaining 15xI2 was centrifuged at 10.000xg for 15 minutes at 4°C to remove the insoluble I took the substance. The supernatant was removed and this step was repeated. Next, the precipitated material ( The insoluble pellet) was washed three more times in 0.5% Triton X-114, then washed with H It was quickly frozen in TPBSlzff. The surfactant soluble material was then added to the cold 6% seaweed. Sucrose cushion 10j11 of sucrose, 0.06% Triton X-114 Carefully stack on top of the 2L, put in a 37 °C water bath for 5 minutes, then 500X at 37 °C. Centrifugation was performed for 5 minutes at s+. After centrifugation, collect the upper layer 15j112 from which the surfactant has been removed. and cooled on ice. Discard the sucrose cushion 10xff and use the surfactant enriched Cold HTPBSIO of rich beret (1-2112) on the water! resuspended at 12 . The resuspended surfactant-rich phase was again layered onto the seacloth cushion and 3 The mixture was incubated at 7°C for 5 minutes and re-pelleted by centrifugation.

After this second precipitation, the surfactant-rich belay) was resuspended in HTPBS5xff. , it froze quickly. Upper layer from which surfactant was removed by sucrose cushion separation on water, add IMI2 of 11.4% Triton X-114, stir to make a solution, Incubate at 37°C for 5 minutes, then centrifuge and discard the Triton X-114 pellet. This further removed hydrophobic proteins. This series of processes was repeated three times. . Next, the remaining aqueous solution (aqueous phase) from which the surfactant was removed was rapidly frozen. All samples were stored at -70°C until assay.

Electrophoresis and immunoblotting Sodium dodecyl sulfate polyacrylamide gel electrophoresis (S’DS-PAGE) ) samples were prepared under reducing conditions and electrophoresed on a 10% slab gel. Ta Stain the gel to be assayed for protein with Coomassie Brilliant Blue. It was colored. Samples to be assayed by immunoblotting were added to a 10% slab gel. fractionated on a nitrocellulose sheet and electrophoretically transferred to a nitrocellulose sheet. After transferring, nit cellulose in 5% (W/V) skim milk powder (B1otto) in HTPBS. Blocked and diluted serum appropriately in Biotto or affinity purified Probed with human antibodies. Bound antibodies were probed with 1161-Protein A. and then detected by autoradiography. Protein A (Pharmacia Fine Chemicals, Uppsala, Sweden) acia Fine Chemicals, Uppsala, Sweden) ) was iodinated to a specific activity of 40 uC5/ltg-' by the chloramine T method.

Affinity purification of polyclonal monospecific human antibodies As in previous literature (5) and in this example, electrophoresis was performed on nitrocellulose as described below. Dynamically transferred Triton X-114 soluble antigen to generate antibodies from human serum or plasma. Affinity purified. A reduced sample of membrane antigen extracted with Triton X-114 was electrolyzed. Aerophoretically separated and transferred to nitrocellulose. to determine the area you want to investigate , a radioactive I'C high molecular weight marker (Amersham). used. A strip of nitrocellulose corresponding to the area of interest is exposed to malaria. The cells were incubated for 8 hours at 4°C with serum or plasma from treated humans.

Remove the serum and remove the nitrocellulose strips with B1otto 6 times in HTPBS. Wash vigorously for at least 2 hours with 3 changes of Syn, 0.15M sodium chloride, pH 2,6) for 15 minutes (10 minutes while).

The eluted antibody was immediately neutralized with 2M) Lis-HCff pH 8.0 and then diluted with 0.05% Sodium diide was added and stored at 4°C. This affinity purified antibody is B Immunoplot and λAll1p3 cDNA with 1=2 dilution in 1otto A to probe library filters or for immunofluorescence assays. Centricon Micro Concentrator (Centricon m1cro) concentrator (Amicon).

It was concentrated using The antibody was further coated on nitrocellulose with a lawn. were directly eluted from immunopositive λAmp3 clones grown in the same manner.

Filters with degraded lawns were pre-eluted with boric acid and glycine buffer. and incubate with serum and elute monospecific antibodies as above. Ta. This second method of affinity purification requires removal of anti-E. coli antibodies. This was done on sonicated and control λAmp3Am-n lawns.

Details of its amplification and lytic expression in E. coli are described in (6). There is. Detection of antigen-expressing clones by in-system colony immunoassay using human antibodies , (6). Positive clones were cultured in liquid culture as described in (7). The cells were grown and induced and extracted with sample buffer for electrophoresis. 10% sample Electrophoresed on a 5DS-PAGE slab gel and stained for proteins or were immunoblofted and assayed with affinity-purified antibodies to detect β-galactosidase. The presence or absence of a stable fusion polypeptide with was detected.

Antibody exhaustion (depletion) 1xj2 cultures of immunopositive clones were induced, pelleted by centrifugation, and cultured in liquid culture. removed the ground. Freeze and thaw the pellet three times and lyse the lysed cells in HTPBS. Resuspended in lxff. Add 20 μa of PNG serum and incubate with this lysate at 4°C. Incubation was carried out for 3 hours. Next, antibodies attributable to the resulting fusion polypeptide are detected. To examine the extent of depletion, centrifuge the cell debris at 10,000xy and use the supernatant. Triton X-114 extracted and immunoblotted parasite membrane protein anti- probed the original.

Subcloning of immunopositive λAs3 clones λAmp3 from immunopositive clones Isolate Am-di and insert cDNA The bodies were extracted by EcoRI digestion as described. Refined Tsuru insert, support M13 for single-stranded sequences determined by Nger's dideoxy method (8,9) Vector, and Gary Coburn (Biotech) pBTA224 kindly provided by Knology Australia Proprietor. (modification of PUR-290). Plasmid vector pBTA2 24 is an expression vector in E. coli cells, and clones were subjected to colony immunoassay. The cells were rescreened for immunoreactivity by assay.

indirect immunofluorescence Thin blood films of parasitized red blood cells from asynchronous cultures of P. falciparum Air dried and fixed in 100% acetone at -20°C for 20 minutes. Dip the slide into Fluorescein as a second antibody - purified monospecific human antibody The ink was incubated with Condiegate Shitahitsushi anti-human 1g antiserum. Parasitic Body nuclei were counterstained with ethidium bromide.

Explanation for drawings Triton X-114 extract of P. falciparum infected red blood cells was added to 10% 5DS Fractionated by PAGE and electrophoretically transferred to nitrocellulose, (a) collected adult P N G serum, (b) affinity purified polyclonal human antibody, or (C ) probed with affinity-purified monospecific human antibodies.

In (a) and (b), the lanes are (1) uninfected erythrocyte control, (2) whole non- Fractionated substance, (3) aqueous phase, (4))! (5) an insoluble substance.

In (e), Ag512. Human PNG serum was collected on a filter lawn. (1) Triton X-114 phase antigen Control strips were probed with collected serum and (2) affinity purified anti-Ag5. 12 were probed against duplicate filter strips.

Sato2Σ of At512 using the predicted amino acid sequence of a single open reading frame. Partial nucleotide sequence.

! DanΣ localized to affini bodies used in indirect immunofluorescence assays on asynchronous blood films A mature schizont with a speckled pattern of antigen-specific fluorescence is depicted.

Distribution of a subset of P. falciparum antigens to Triton X-114 triton using serum from humans repeatedly infected with P. falciparum. Immunoplot of P, falciparum antigen fractionated by X-114 medium phase separation. probing identifies several putative intracellular membrane protein antigens. child The dominant antigen for most of them was determined by 5DS-PAGE analysis and was 21,000.3 5,000.42.000, 50,000 and 55,000 relative molecules jl ( Mr. This antigen extract was combined with a large number of cloned P. falciparum Probe with antiserum raised against parm antigen or affinity purify When the molecular weight 21,000 molecular weight Triton X-114 soluble antigen was It is known that it has a noic acid hydrophobic sequence, which is a protein-associated antigen (CRA). (10), confirming that this method selects for such molecules. Other Triton X-114 soluble antigen is derived from numerous previously characterized P. It seems that it does not correspond to any of the rum antigens.

In order to obtain purified antibodies specific for a series of proteins with a molecular weight of 35-55,000, Triton X-114 extract of P. falciparum was electrophoretically fractionated and nitro Transferred to cellulose. Then cut a suitable area of the nitrocellulose filter and as an adsorbent for affinity purification of the corresponding ° antibodies from human serum. Used (Materials and Methods). The purified antibodies were purified using fractionated P, falcipa Specificity was tested by reacting with rum protein.

These purified antibodies almost exclusively partition selectively to the Triton X-114 phase. The fact that these molecules react with antigens with a molecular weight of 35-55,000 is shown in Figure 1b. It is clear that

To isolate clones expressing these antigens, P. falciparum A library of λAmp3Ammon expressing the cDNA sequence was Screening was carried out using purified antibodies. Positive lysogeny detected by this method Clones were replated to obtain single colonies.

Selected colonies corresponding to Triton X-114 soluble antigen;- Antibodies affinity-purified on a clone array were subjected to colony immunoassay on 10 clone arrays. and used to probe immunoplots of parasite antigens.

Seven clones encoded an antigen with a molecular weight of 55,000 from the Triton X-114 phase. It turned out that they were brothers. Typical of the most immunoreactive clones I selected it as a loan and named it Ag512.

Clear by 5DS-PAGE followed by protein staining or immunoblotting. When clones were assayed, all seven sibling clones of Ag512 were found to be β-galactin. It was found to produce an unstable fusion protein with tosidase. Immune serum, partial immunoreactive cDNA clones, combined with sonication of Ag512, and then Probing the immunoplot of Triton X-114 extract of P. falciparum All detectable antibody reactivity against parasite antigens was removed. Like this, This cDNA clone clearly represents the dominant natural immunogenic epitope on this antigen. Coded taupe.

Room 凰dansu mutual charm p farmhouse roof Period-specific parasite specimens were extracted with Triton X-114 and analyzed by 5DS-PAGE. and immunoplots were probed with PNG serum. Compatible with Ag512 Antigens were present at all stages of the parasite's life cycle, but only in the asexual ring. and least abundant in the trophozoite stage and most abundant in late schizont specimens. Late Schizo At the merozoite and merozoite stages, the antigen is completely absorbed by the surfactant Triton X-114. In the ring and trophozoite stages, it was very soluble in Triton 14 partitioned into both phase and insoluble pellets. Triton X-114 extract , prepared from five strains of P. falciparum obtained by culture intertone. The antigen is Strain-associated strains that are present in all strains and are evident in antigen size or immunoreactivity. There were no differences.

Ag5] 2 nucleotide and amino acid sequence nucleotide sequence of Ag512 The columns and expected translations are provided in Figure 2. A cDNA clone is a complete code code array is not coded. This is within the framework of β-galactosidase (inflationary The clone has one open reading frame located in This results in a fusion polypeptide that is unstable in the cell. The sequence is 839 base pairs long; Does not have long repeating elements. The sequence contains a stretch of hydrophobic amino acids. do not have. The encoded polypeptide has a predicted molecular weight of 33,534 Daltons. ing.

Ag512 has affinity for Ag512, which corresponds to a putative rod body antigen. Purified human antibodies were prepared as described, concentrated and plated on non-synchronized blood films. used in the immunofluorescence assay. The observed fluorescence pattern is the rod body organ of merozoites. This corresponds to the location of the antigen in the body.

Discuss 1 This example demonstrates the temperature-dependent triggering of the intracellular membrane protein antigen of P. falciparum. Figure 3 shows Ton X-114 surfactant separation. partitioned into Triton X-114 phase One such antigen is CRA, a protein with a known primary structure typical of intracellular membrane proteins. It was identified as a well-characterized antigen. Others distributed to Triton X-114 The major antigens of did not correspond to the antigen cloned in After transferring to nitrocellulose, this Affinity polyclonal monospecific human antibodies using these proteins Ag512 and Ag512, which correspond to merozoite rod body proteins, were purified and used. The named clones were isolated from the λAmp3 cDNA expression library.

Antibodies against Ag512 were present in all strains of P. falciparum screened, and none Triton X-114 with a molecular weight of 55.000, present in all stages of the sexual life cycle Indirect immunofluorescence microscopy using two of these antibodies reacted with soluble proteins. The grape-like pattern of ``double dots'' is strongly dyed during the ripening stage. Antibodies against rod body antigens gave zoites a distinctive pattern. Ag512 related antigen Part of the primary structure was discussed.

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Claims (10)

[Claims] 1. (i) has an apparent molecular weight of approximately 55 kDa; (ii) is accompanied by myristic acid; (iii) control of merozoites corresponding to the location of rod bodies; (iv) by phase partitioning of parasite antigens with surfactants; When separated, it is extracted into the surfactant phase. A rod body antigen of Plasmodium falciparum in the asexual blood stage, characterized by , or antigenic fragments thereof. 2. Claim 1 is the antigen Ag512 or an antigen fragment thereof as described herein. Antigens mentioned. 3. The antigen according to claim 1, which has a primary structure comprising the amino acid sequence shown in FIG. is its antigen fragment. 4. The antigen or antigen fragment thereof according to any one of claims 1 to 3, and and a pharmaceutically acceptable carrier or diluent. 5. 5. The composition of claim 4 further comprising an adjuvant. 6. For actively immunizing the host against Plasmodium falciparum A method comprising administering the antigen or antigen fragment thereof according to any one of claims 1 to 3 to a host. or the vaccine composition according to claim 4 or claim 5. A method consisting of. 7. An antigen of any one of claims 1 to 3 or an antigen fragment thereof. all or part of a nucleotide sequence that can be expressed as a polypeptide with or a recombinant clone containing the recombinant DNA molecule. Loning vehicle or vector or host cell. 8. Recombinant DN according to claim 7, wherein the nucleotide sequence is the sequence shown in FIG. A molecule, recombinant cloning vehicle or vector or host cell. 9. Expression of all or part of the nucleotide sequence according to claim 7 or claim 8 A synthetic polypeptide produced by. 10. A vaccine composition containing the synthetic polypeptide according to claim 9.