KR20050028063A - Circumsporozoite protein of malaria expressed in plants and diagnostic kit comprising the same - Google Patents

Abstract

A circumsporozoite protein of malaria expressed in plants and a diagnostic kit comprising the same protein are provided, which circumsporozoite(CS) protein of malaria is cheaply mass produced using plants, and induces the identical antigen-antibody reaction to natural circumsporozoite(CS) protein, so that the circumsporozoite(CS) protein is useful for diagnosis of malaria. A polynucleotide encoding the circumsporozoite(CS) protein expressed in plants has the nucleotide sequence set forth in SEQ ID NO:1. A recombinant plant expression vector of CS protein comprises (i) the polynucleotide of SEQ ID NO:1; (ii) a promoter operately linked to the polynucleotide and forming an RNA molecule in plant cells; and (iii) 3'-non-translated region which results in polyadenylation of 3'-terminal of the RNA molecule in plant cells. The method for producing the CS protein in plants comprises the steps of: (a) introducing the recombinant plant expression vector of the CS protein into plant cells; (b) selecting the transformed plant cells; (c) regenerating a plant from the transformed plant cells; and (d) producing the CS protein in the regenerated plants.

Description

Circumsporozoite Protein of Malaria Expressed in Plants and Diagnostic Kit Comprising the Same}

The present invention relates to a malaria diagnostic kit comprising malaria CS (Circumsporozoite) protein antigen expressed in a plant, and more particularly, a novel polynucleotide encoding a CS protein, a vector comprising the same, a plant into which the vector is introduced. It relates to a method for producing CS protein using the plant and the diagnosis of malaria using CS protein.

Malaria is a blood parasitic disease called Plasmodia, which is transmitted through vampires by cancer mosquitoes. In humans, four kinds of malaria are present. In Korea, trifluid malaria, which is currently prevalent, is mainly present. In addition, there are silos, tropical fever, and oval malaria, respectively, Plasmodium vivax and Plasmodium malaria ( P. malariae ), Plasmodium Falciparum ( P. falciparum ) and Plasmodium Ovale ( P. ovale ).

Malaria was the first to record Huan del Vego's treatment with guina ficus in Peru and Ecuador in 1640. In 1880, French physician Laveran identified the causative agent for the first time in a patient in Algeria. In 1897, British doctor Sir Sir Ross Ross revealed that it was a mosquito that mediates malaria in India, and malaria research began to progress.

In World War II, the efficacy of chloroquine was discovered and used by the German doctor Resochin.Primaquine was discovered by Elderfield, an American doctor in 1950, and mefloquin ( Mefloquine) has been used to date.

Malaria is transmitted by vampires of female mosquitoes belonging to Anopheles spp., Which is transmitted by Chinese spotted mosquitoes ( An. Sinensis ) in Korea. Malaria is divided into asexual life (schizophrenia, Schizogony), a life history in humans, and sexual reproduction (sporogony), a life history in mosquitoes.When the first mosquito bites, the malaria sporozoite moves along the blood vessels to the liver. do. Proliferation in the hepatocytes or dormant state, and after a certain period of time (8-9 days), the blood comes out and full-scale proliferation. In the blood, red blood cells enter the nutrient chain, go through the proliferative and dividing process, and when the schistot releases dozens of merozoite, it enters each red blood cell and repeats the same cycle. Some of them exist in the form of germ cells, and when a mosquito has sucked a person in this state, this causes the mosquitoes to proliferate.

Malaria has a global distribution, but in some areas it has ceased or decreased since the 1960s due to effective control. However, due to the recent rise in abnormal temperature, such as El Niño, drug resistant strains, and increased resistance to insecticides, it is spreading all over the world. Malaria is divided into America, Africa, and Asia, centered on Central and South America, and Asia, centered on India and Southeast Asia. Each type shows differences in geographic distribution, and also differs greatly in species characteristics and genetic inclinations.

Korea became very popular after the June 25th incident, but it almost disappeared after the late 1970s, but it began to reappear near the truce line in northern Gyeonggi since 1993 and 1,724 cases occurred in 1997. The current recurrent trend is thought to be caused by the introduction of new protozoa from the outside, rather than by the long-awaited prevalence of malaria. There is little possibility of foreign malaria patients or foreign workers as a source of external protozoa, and it is estimated that they came from North Korea. Due to the economic crisis and the food shortage, large numbers of malaria patients occurred, and the number of mosquitoes with protozoa increased rapidly due to the increase of mosquitoes due to extreme weather. Some of these mosquitoes crossed the truce line and settled north of Gyeonggi-do. By 1998, 6142 Korean patients had developed.

Malaria is one of the most threatening diseases on the planet today, causing 200 to 300 million infections and 2.5 million to 3 million deaths each year, with more than 100 countries, 40 percent of the world suffering. In Korea, three-day fever malaria has increased exponentially, and since malaria-infected areas are banned from blood donation for three years and blood donated is discarded, more than 70% of the total blood donation depends on group blood donation. In reality, there is a big obstacle in blood supply.

The diagnosis of malaria is the most widely used blood smear to observe the malaria infected blood cells stained under a microscope, but it takes a long time, there is a problem that requires a professional inspector to determine the infected blood cells. In order to solve this problem, reagents for detecting antigens of malaria have been used, but these reagents are also used with the blood smear method because of their low sensitivity. PCR, another diagnostic method, has high sensitivity but has not been put to practical use. In addition, such a diagnostic method is particularly difficult to diagnose Korean malaria having a long incubation period and a low number of protozoa in the blood due to infection by the three-day fever malaria protozoa.

 Therefore, the development of a diagnostic method that can be quickly diagnosed with high sensitivity and specificity for malaria infection is required.

WO 0161032 discloses a method for immunologically diagnosing malaria by detecting malaria-specific antibodies in the blood using an antigen of malaria protozoa, and WO 9724141 describes a single cell antibody against plasmid-specific lactate dehydrogenase (LDH). A method of detecting the presence of plasmidum using the present invention is disclosed. Meanwhile, Korean Patent Application No. 2000-14123 discloses a malaria antibody diagnostic reagent composition comprising a merozite surface protein (MSP).

Throughout this specification, numerous citations and patent documents are referenced and their citations are indicated. The disclosures of cited documents and patents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

As a result of intensive research efforts to solve the above-mentioned needs of the art, the present inventors have successfully introduced a novel polynucleotide encoding the CS (Circumsporozoite) protein having the highest antigenicity among all the genes of the triplet malaria. The present invention was completed by confirming that the CS protein isolated and purified from the present invention exhibited the same immunological antigenicity as that of the malaria protozoa, and that it could be used for diagnosis of malaria.

Accordingly, an object of the present invention is to provide a novel polynucleotide encoding a CS (Circumsporozoite) protein.

Another object of the present invention to provide a plant expression vector of the recombinant CS protein.

Still another object of the present invention is to provide a method for preparing a transient transfected plant that expresses recombinant CS protein temporarily.

Another object of the present invention is to provide a transient transfected plant that expresses recombinant CS protein temporarily.

Another object of the present invention to provide a method for producing a recombinant CS protein.

Another object of the present invention is to provide a method for producing a transgenic plant stably expressing a recombinant CS protein.

Another object of the present invention is to provide a transgenic plant stably expressing a recombinant CS protein.

Another object of the present invention is to provide a recombinant CS protein produced by the method of the present invention.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a polynucleotide encoding a CS (Circumsporozoite) protein comprising the nucleotide sequence set forth in SEQ ID NO: 1.

CS (Circumsporozoite) protein is a protein that covers the body surface of the sporozoite of malaria protozoa and serves as a major surface antigen. Therefore, antibodies to the CS protein can be detected in a person who has been exposed to an infected mosquito, and blood tests can be performed by determining whether or not the malaria protozoa has been exposed before the onset of the patient according to the intensity of the antibody response.

The novel polynucleotides of the present invention, which encode CS proteins, have nucleotide sequences modified to optimize expression in plants without amino acid substitutions.

The design of the polynucleotides of the invention is carried out by the following criteria: (i) to make the GC content at least 50%, (ii) to have the plant's preferred codons, and (i) to remove intron-like sequences. will be. Such plant-compatible sequences can increase the translation rate of genes (Kusnadi et al ., Biotechnol. Prog. 14: 149-155 (1998)). In addition, the intron-like sequence in the foreign gene to be introduced is removed in order to rule out the possibility that the arrangement of the transgene is recognized as an intron in the host plant, resulting in cleavage in the plant nucleus and a failure to produce the desired protein.

The novel polynucleotides of the present invention include the nucleotide sequences described in SEQ ID NO: 1, as well as some sequences thereof. That is, when the amino acid sequence encoded by the above partial sequence shows antigenicity, it is included in the polynucleotide of the present invention. More specifically, when the protein encoded by the above-mentioned partial sequence reacts (antigen-antibody reaction) with respect to the antibody against the protein encoded by the nucleotide sequence described in SEQ ID NO: 1, the present invention also includes some sequences. It is included in the scope of the invention.

In addition, the novel polynucleotides of the present invention are to be interpreted to include not only the nucleotide sequence described in SEQ ID NO: 1, but also a nucleotide sequence showing substantial identity to the nucleotide sequence described above. The substantial identity is that at least 90% of the phases when the nucleotide sequence of the present invention and any other sequence are arranged to correspond as closely as possible and the sequence is analyzed using algorithms commonly used in the art. By nucleotide sequence, which shows homology, more preferably 95% homology, most preferably 98% homology.

According to another aspect of the present invention, the present invention is (i) the polynucleotide of claim 1; (Ii) a promoter operably linked to the polynucleotide of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-terminal polyadenylation of the RNA molecule, thereby providing a plant expression vector of a recombinant CS (Circumsporozoite) protein.

According to a preferred embodiment of the present invention, a promoter suitable for the present invention may be any one commonly used in the art for gene introduction of a plant, for example, the Coliflora Mosaic Virus (CaMV) 35S promoter , Nopaline synthase (nos) promoter, pigwarm mosaic virus 35S promoter, sugacran basilisform virus promoter, commelina yellow mottle virus promoter, ribulose-1,5-bis-phosphate carboxylase small sub Photoinducible promoters of ssRUBISCO, rice cytosol triosphosphate isomerase (TPI) promoters, adenine phosphoribosyltransferase (APRT) promoters of Arabidopsis and octopine synthase promoters.

According to a preferred embodiment of the present invention, the 3'-non-detoxification site causing the polyadenylation suitable for the present invention is derived from the nopalin synthase gene of Agrobacterium turmeration (nos 3 'end) ( Bevan et al., Nucleic Acids Research , 11 (2): 369-385 (1983)), derived from the Octopine synthase gene of Agrobacterium tuberculosis, of the protease inhibitor I or II gene of tomato or potato 3 'terminal portion and CaMV 35S terminator.

Optionally, the vector additionally carries a gene encoding a reporter molecule (eg, luciferase and β-glucuronidase). In addition, the vector of the present invention is a selection marker antibiotics (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.) resistant gene (e.g., neomycin phospho-transferase dehydratase (nptⅡ), high thereof Mycin phosphotransferase ( hpt ), and the like).

According to the present invention, the production of plants in which the gene encoding the CS protein is introduced is largely achieved by two methods: a transient transfected plant and a transgenic plant.

As used herein, the term "transient transfected plant" means that the introduced gene is not transferred to the next generation as the plant into which the foreign gene has been introduced. In transiently transfected plants, foreign genes are generally present separately from the chromosome of the host cell. In contrast, the term “transgenic plant” refers to a plant having a foreign gene that is transmitted to the next generation. In transgenic plants, foreign genes are generally integrated into the chromosome of the host cell, becoming the genetic repertoire of the host cell, and reliably delivered to the next generation.

Therefore, according to another aspect of the present invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention into the plant cells of the plant; And (b) provides a method for producing a transient transfected plant to temporarily express a recombinant CS (Circumsporozoite) protein comprising the step of confirming whether the gene has been introduced into the plant cell.

According to another aspect of the present invention, the present invention provides a transient transfected plant which temporarily expresses a recombinant CS (Circumsporozoite) protein produced by the above method.

According to another aspect of the present invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention into plant cells; (b) confirming whether the gene has been introduced into the plant cell; And (c) a method for producing a recombinant CS (Circumsporozoite) protein comprising a recombinant CS (Circumsporozoite) protein from a plant including a plant cell into which the gene is introduced.

In the method of the present invention, transient transfection of plant cells can be carried out according to conventional methods known in the art (Rainer Fisher et al., Biotechnol. Appl. Biochem. , 30: 113-116 (1999) ). Transient gene expression in plants is faster than in transformants with stable expression, and the result of protein expression can be confirmed as soon as possible. Therefore, whether the target protein obtained in plants prior to large-scale production using stable transgenic plants is functioning normally. Suitable for checking

Thus, the present inventors expressed, isolated and purified CS (Circumsporozoite) protein using a transient gene expression method in plants for efficient mass production of biologically active CS protein.

Transient gene expression methods are suitable approaches to test the functionality and stable expression of target genes before manufacturing plant transformants for mass production, which has the advantage of saving time and money. In particular, plant transformants produced by stable transformation show a chromosomal positional effect that is affected by the position at which the foreign gene is inserted. Transient transfection can rule out this effect. It is well suited for the efficient expression and isolation of foreign genes.

In transient transfection, there are three types of methods for delivering foreign genes to plant cells: particle bombardment, which coats and introduces naked DNA onto particles (Christou, P., Trends Plant Sci). 1: 423-431 (1996)), vacuum infiltration (vacuum infiltration) Agrobacterium-fill trafficking Orientation method (agroinfiltration containing the expression vector is introduced into plant tissue by Agrobacterium in the like) (Kapila et al, plant Sci. ., 122: 101-108 (1996)), and a modified plant virus virus vector method using a vector (viral vectors) (Scholthof, H. et al, Annu Rev. Phytopathol 34:... 299-323 (1996) ).

The transformation efficiencies of these three methods vary, so that in the case of DNA introduction by particle guns, genes are generally introduced into only a small number of cells and function only when DNA reaches the cell nucleus for transcription. This method can be used to test the stability of recombinant proteins prior to stable transformation, but is not suitable for mass expression of recombinant proteins.

Agroinfiltration allows genes to be introduced into more cells than the particle gun, whereby T-DNA containing the target gene is actively introduced into the nucleus with the help of several bacterial proteins. In this method, the introduced T-DNA is not integrated into the host cell's chromosome and continuously expressed, but is present in the nucleus independently and expresses the transient expression of the gene of interest. It is suitable for obtaining protein quickly.

In addition, the method using a viral vector introduces a foreign gene into the genome of a viral plant pathogene to be controlled by a strong promoter, and when the recombinant viral vector including the foreign gene is infected with a plant, the foreign gene introduced is During plant virus replication, they are amplified with high efficiency and then expressed to produce foreign proteins. However, when using a viral vector, the size of the protein to be produced is limited to less than 30 kDa, so the size of the gene to be introduced is difficult to use.

Therefore, in the present invention, the agroin fill method is most preferable. The agroin filtration method is generally carried out with leaves.

When the present invention is practiced in agrofiltration, more preferably, the Agrobacterium tumefaciens -binary vector system is used.

In a method using the Agrobacterium system, one specific embodiment includes the following steps: (a ') Agrobacterium tumefaciens containing a vector having a sequence of Infiltrating tissue: a nucleotide sequence set forth in SEQ ID NO: 1; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts in plant cells to cause 3'-end polyadenylation of the RNA molecule; (b ') confirming the expression of the protein in the infiltrated leaves.

In one specific embodiment described above, suitable leaves include any tissue derived from the germinated seed, preferably the leaves produced before the peduncle rises, most preferably the leaves are too young or not mature. The younger the leaves, the higher the expression level, but the tissues are more susceptible to yellowing or dying. Seed germination is carried out under suitable dark / light conditions using a suitable medium.

Introduction of genes into plant cells is carried out with Agrobacterium trimers containing Ti plasmids (Depicker, A. et al., In Genetic Engineering of Plants, Plenum Press, New York (1983)). More preferably, binary vector systems such as pBin19, pRD400, pRD320 and pHS737 are used (An, G. et al., “Binary vectors” In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986); RK Jain, et al., Biochem. Soc. Trans. 28: 958-961 (2000).

Gene introduction into the plant cells in the leaves by Agrobacterium tufasion includes methods known in the art. Most preferably, the gene introduction process includes infiltration of a culture of Agrobacterium tumeration into the tissues of the leaves.

The infiltration process may be performed by a vacuum method or a syringe method. One specific embodiment when using a vacuum is as follows. The plant leaves are placed in a culture of Agrobacterium trimmers and vacuum is applied for a short time. Quickly release the vacuum, disinfect the leaves with sterile water, and place them on a damp piece of paper with the top of the leaves facing down. The leaves are then stored at about 22 ° C. under light conditions of about 16 hours. Then, after about 2-3 hours, the target protein expressed is identified.

On the other hand, a specific embodiment when using a syringe is as follows. The back of the plant leaves is infused with a syringe of Agrobacterium turmeration using a syringe, the plant is incubated for about 3-5 days, and then the expressed target protein is identified.

Agrobacterium tuberation cultures preferably include acetosyringone, to aid in the penetration of Agrobacterium into plants.

Plants transgenic according to the present invention are confirmed the expression of the protein of interest by methods known in the art. For example, PCR can be performed using DNA samples obtained from tissues of transgenic plants to identify foreign genes present in plant cells. Alternatively, Northern or Southern blotting can be performed to confirm gene introduction (Maniatis et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). On the other hand, if the vector used to introduce the gene has a β-glucuronidase gene, a part of the leaf tissue into which the gene is introduced is selected from X-gluc (5-Bromo-4-Chloro-3-Indole-β-D- By visually observing the color development by immersion in a substrate solution such as Glucuronic acid) it can be easily confirmed whether the gene introduced (Jefferson, RA, Plant Mol. Biol. Rep. , 5: 387 (1987)).

The method of the invention is applied to a variety of plants, but preferably to tobacco, melons, cucumbers, watermelons and rapeseeds.

The CS protein can be obtained from the tissue of the transgenic plant of the present invention, ie, the transiently transfected plant (eg, leaves), and the extract obtained from the tissue can be obtained through a conventional purification process. In the present invention, the purification step may be carried out by employing a purification method commonly used in the art. For example, solubility fractionation using ammonium sulfate or PEG, ultrafiltration separation according to molecular weight, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Various methods may be used and are usually purified using a combination of the above methods.

In a specific embodiment of the present invention, when there is 6 x His at the N-terminus of the recombinant CS protein of the present invention, a Ni-NTA His-binding resin column is used to quickly and easily produce the desired recombinant GM-CSF protein. Can be isolated and purified.

According to the method of the present invention, not only can the recombinant CS protein be produced in a short time and at low cost by using a transient transfected plant, but the CS protein produced induces the same antigen-antibody response as a natural-occurring CS protein. can do.

Another approach for preparing plants in which genes encoding CS proteins have been introduced is to use transgenic plants that stably express CS proteins.

Therefore, according to another aspect of the present invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention to plant cells; (b) selecting the transformed plant cells; And (c) regenerating the plant from the transformed plant cells to obtain a transformed plant, thereby providing a method for producing a transformed plant stably expressing the recombinant CS protein.

According to another aspect of the present invention, the present invention provides a transgenic plant stably expressing the recombinant CS protein produced by the method of the present invention described above.

According to another aspect of the invention, the present invention comprises the steps of (a) introducing the plant expression vector of the present invention to plant cells; (b) selecting the transformed plant cells; (c) regenerating the plant from the transformed plant cells to obtain a transformed plant; And (d) obtaining a recombinant CS protein from the transgenic plant.

According to another aspect of the present invention, the present invention provides a recombinant CS protein produced by the above method.

In the method of the present invention, the transformation of plant cells can be carried out according to a conventional method known in the art, which is electroporation (Neumann, E. et al., EMBO J. , 1: 841 (1982) And Saul, M. et al., Plant Molecular Biology Manual , Vol. A1, Kluwer Academic Publishers, Dordrecht (1988)), particle gun method (Yang et al., Proc. Natl. Acad. Sci. , 87: 9568- 9572 (1990) and Christou, P., Trends Plant Sci. 1: 423-431 (1996)) and Agrobacterium-mediated transformation (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011). Of these, Agrobacterium-mediated transformation is most preferred. Agrobacterium-mediated transformation is generally carried out with leaf discs and other tissues (cotyledon and hypocotyls). Agrobacterium-mediated transformation is most efficient in dicotyledonous plants.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent (eg metabolic inhibitors, antibiotics and herbicides). Plant cells that are transformed and stably contain marker genes that confer selective resistance are grown and divided in the cultures described above. Exemplary labels include, but are not limited to, glycophosphate resistance genes and neomycin phosphotransferase (nptII) systems.

Methods for the development or regeneration of plants from plant protoplasts or various exploits are well known in the art (Bhojwani, SS et al., Plant Tissue Culture: Theory and Practice , Elsevier Science, New York, (1983); and Lindsey , K., Ed., Plant Tissue Culture Manual , Kluwer Academic Publishers, Dordrecht, The Netherlands, (1991)). The transformed root shoots formed are imbedded in a suitable plant growth medium. Development or regeneration of plants comprising foreign genes introduced by Agrobacterium can be accomplished according to methods known in the art (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011).

On the other hand, the present inventors have made extensive research efforts to develop novel transgenic plants (eg, tobacco, melons, cucumbers, watermelons and rapeseeds), and as a result, have established the most efficient method for the transformation of specific plants. Such a method is disclosed in PCT / KR02 / 01461, PCT / KR02 / 01462 and PCT / KR02 / 01463, which patent documents are incorporated herein by reference.

The method of the invention is applied to a variety of plants, but preferably to tobacco, melons, cucumbers, watermelons and rapeseeds.

In the present invention, the preferred transformation method is carried out using the Agrobacterium system, more preferably using the Agrobacterium tumefaciens -binary vector system.

In a method using the Agrobacterium system, one specific embodiment includes the following steps: (a ') an Agrobacterium tumer embedded in a genome DNA of a plant cell and having a vector having the sequence Infecting the plant plant with Agrobacterium tumefaciens : (i) the nucleotide sequence encoding the CS protein set forth in SEQ ID NO: 1; (Ii) a promoter operably linked to the nucleotide sequence of (iii) and acting on plant cells to form RNA molecules; (Iii) a 3'-non-detoxification site that acts on plant cells to cause 3'-end polyadenylation of the RNA molecule; (b ') re-differentiating the infected implant in a regeneration medium to obtain a redifferentiated shoot; And (c ') culturing the re-differentiated shoots in a rooting medium to obtain a transformed plant.

In one specific embodiment described above, suitable implants for transformation include any tissue derived from the germinated seed, preferably cotyledon and hypocotyl, most preferably cotyledon. Seed germination is carried out under suitable dark / light conditions using a suitable medium. Transformation of plant cells is carried out with Agrobacterium trimers comprising Ti plasmids (Depicker, A. et al., Plant cell transformation by Agrobacterium plasmids.In Genetic Engineering of Plants, Plenum Press, New York (1983) ).

More preferably, binary vector systems such as pBin19, pRD400 and pRD320 are used (An, G. et al., Binary vectors "In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986)). Binary vectors suitable for include (i) a promoter operating in a plant, (ii) a structural gene operably linked to said promoter, and (iii) a polyadenylation signal sequence Optionally, said vector comprises a reporter molecule (eg : Additionally carries genes encoding luciferase and glucuronidase) Examples of promoters used in binary vectors include the CaMV 35S promoter, 1 promoter, 2 promoter and nopaline synthase (nos) promoter. It is not limited to this.

Infection of implants with Agrobacterium trimmers includes methods known in the art. Most preferably, the infection process comprises coculture by impregnating cotyledon in a culture of Agrobacterium tumerification. As a result, Agrobacterium trimmers are infected into plants.

Explants transformed with Agrobacterium trimerization are re-differentiated in regeneration medium, which successfully leads to regeneration of shoots. Finally, transgenic plants are produced by the rooting of shoots that have been regenerated in the rooting medium.

Plants transformed according to the present invention is confirmed whether or not transformed by methods known in the art. For example, PCR can be performed using DNA samples obtained from tissues of transformed plants to identify foreign genes inserted into the genome of the transformed plants. Alternatively, Northern or Southern blotting can be performed to confirm transformation (Maniatis et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)).

The CS protein expressed in the transformant can be obtained from tissues derived from various organs (eg, stems, leaves, roots, fruits, seeds, etc.) of the transformed plant, and the extract obtained from the tissues is subjected to conventional purification. You can get it. In the present invention, the purification step may be carried out by employing a purification method commonly used in the art. For example, solubility fractionation using ammonium sulfate or PEG, ultrafiltration separation according to molecular weight, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. Various methods may be used and are usually purified using a combination of the above methods.

According to the method of the present invention, not only can the CS protein be produced in large quantities at low cost, but the produced CS protein induces an antigen-antibody response in the same manner as a natural-occurring CS protein. Therefore, the CS protein of the present invention can be used for the diagnosis of malaria.

According to another aspect of the present invention, the present invention provides a method for producing a test fluid, comprising the steps of: (a) obtaining a testing fluid from a subject under test; (b) inducing an antigen-antibody reaction with the test solution using the CS (Circumsporozoite) protein of the present invention as an antigen; And (c) it provides a method for measuring the antibody of malaria comprising the step of measuring the occurrence (occurrence) of the antigen-antibody reaction in step (b).

In the present invention, it can be applied to various biological fluids (eg, blood, serum, urine, sweat, etc.), but is preferably blood, and more preferably serum.

The method of the present invention can be used for all immunoassays based on antigen-antibody binding. Most preferably, it is performed according to an enzyme-linked immunosorbent assy (ELISA) method. More specifically, (i) adsorbing an anti-human antibody to the well plate; (Ii) reacting and washing the sample by adding serum of the sample to the well plate; (Iii) adding and reacting the CS protein; (Iii) adding and reacting a monoclonal antibody against the CS protein; (Iii) washing the well plate and reacting by adding a secondary antibody bound to a chromophore or a fluorescent substance; And (iii) determining whether or not the secondary antibody is bound.

The color development enzymes include, but are not limited to, alkaline phosphatase, β-galactosidase, horse radish peroxidase, and the like. In addition, when using alkaline phosphatase, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), ECF substrate, etc. are used as a substrate, and when using a horse radish peroxidase, Chloronaphthol, aminoethylcarbazole, diaminobenzidine, luminol, ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]) and the like are used as the substrate.

According to another aspect of the present invention, the present invention provides a kit for detecting an antibody of malaria comprising the CS protein of the present invention as an antigen.

The CS protein included in the kit of the present invention is a recombinant protein expressed in plants, which is highly infectious in humans and animals, and has very high specificity and sensitivity to the triplet malaria antibody, which is demonstrated in the following examples. have.

When the kit of the present invention is basically developed for ELISA, it may include a plate (or a plate surface-coated with CS antigen protein), a buffer, a secondary antibody labeled with a chromophore or a fluorescent substance, a chromogenic substrate, and the like. have.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited to these examples according to the gist of the present invention.

Example 1 Synthesis of New Gene of CS Protein

While encoding 37 amino acids (see SEQ ID NO: 2 sequences) identically to the genes of native CS proteins, the optimal nucleotide sequences designed by plants were designed, unlike the nucleotide sequences encoding conventional CS proteins. The design criteria for the new gene is first, to make the GC content above 50%, to have the plant's preferred codons, and to remove intron-like sequences. The nucleotide sequences thus designed were chemically synthesized at the Plant Biotechnology Institute (hereinafter referred to as "PBI") and the National Research Center (SK, Canada). The DNA encoding the synthesized CS protein is described in SEQ ID NO: 1.

Example 2: Construction of Plant Expression Vector of CS Protein Gene

The synthetic gene encoding the CS protein is composed of 111 bp and is synthesized to have a recognition site of Bam HI at the 5'-end and Xba I at the 3'-end.

PHS737 (PBI, National Research Center, Canada), a plant expression binary vector, was digested with restriction enzymes Xba I and Bam HI enzymes, digested, and isolated and purified. The 111 bp synthetic gene and the cleaved pHS737 vector were mixed and added to the ligation buffer (KOSCO, Korea) and T4 ligase (KOSCO, Korea) and reacted at 16 ° C. for at least 1 hour. The reaction was then transformed into competent cells E. coli strain DH5α (Promega, USA) treated with CaCl ₂ and antibiotic resistant strains were selected in LB medium containing the antibiotic kanamycin (100 mg / ml).

Plasmid DNA was isolated from the selected clones using an alkaline method and polymerized using forward primer 5'-AAT CTA GAA TCT AGA AAT GAG-3 'and reverse primer 5'-AAG GAT CCC GGA TCC CCT TAA-3'. Enzyme chain reaction (PCR) was performed. The enzyme used in the PCR amplification was Taq polymerase (Solgent, Korea), and the temperature conditions were set as follows: predenature for 2 minutes at 96 ° C, denature for 1 minute at 94 ° C, and connect for 1 minute at 55 ° C. The reaction was repeated 35 times at 72 ° C. for 2 minutes, and further extended at 72 ° C. for 10 minutes.

The amplification product was then analyzed on a 1.0% agarose gel to confirm that the desired insert sequence was in the plasmid.

Example 3: CS Protein Gene Introduction into Plants

Example 3-1 Preparation of Agrobacterium tumefaciens GV3101 Infected Culture

Recombinant pHS737 / CSP vector prepared in Example 2 was introduced into Agrobacterium tamperation GV3101 (Mp90) by conjugation ( Plant-cell-rep. , 15 (11): 799-803 (1996)). . Escherichia coli S17-1 with recombinant pHS737 / CSP vector and Agrobacterium tubertion GV3101 (Mp90) were each cultured in liquid LB medium to log phase. Each of these cells were mixed together 1: 1 in an Eppendorf tube and then centrifuged for 30 seconds to concentrate the cells, followed by stationary culture at 1-2C for 1-2 days. The Eppendorf tube containing the cells over time was gently shaken to float the concentrated cells, and then plated in LB solid medium to which 50 mg / L kanamycin and 30 mg / L gentamicin were added. Colonies were selected after incubation for days. The strain into which the vector was introduced was named Agrobacterium turmeration GV3101-pHS737 / CSP (Alt-Morbe et al., J. Bacteriol. 178: 4248-4257 (1996)).

Agrobacterium trimmers GV3101-pHS737 / CSP with pHS737 / CSP were inoculated in liquid LB medium and incubated at 28 ° C. for 2 days to be used as seed culture. The fully cultured spawn was added to 1/50 of the final culture volume and antibiotics (50 mg / L Kanamycin) in liquid LB medium and incubated for about 18 hours until the absorbance reached 1.5 at 600 nm. The culture solution was centrifuged at 3500 rpm for 15 minutes at room temperature to remove the supernatant, and then the cells were suspended in a 10 mM MgCl ₂ solution (containing 0.2 ppm of BA, containing 150 μM of acetosyringone) and allowed to acclimate at room temperature for at least 3 hours. It was used for infection of plants.

Example 3-2 Preparation and Agroinfiltration of Plant Cells

Seeds of the first sterilized tobacco ( Nicotina bentamiana ) were sown and incubated for at least 3 weeks and then purified in a greenhouse. Agrobacterium tuberization GV3101 (Mp90) having a pHS737 vector (pHS737 / CSP) having a CS protein gene on the leaves of the purified plant was prepared by the method described in Example 3-1, and then a needle was prepared. It was inserted into the removed 1 ml syringe (Note Green Cross) and injected until it spreads evenly on the back of the leaf. If the fungus is injected into the whole leaf, the leaf is heavy and the plant itself may fall down or the leaf disease may break. Plants infused with Agrobacterium return to their original condition after 10 minutes to 1 hour. Plants injected with Agrobacterium were harvested within 5 days after 3 days of culture and analyzed using the method of Example 4.

Example 4 Label Factor Analysis for Confirming Transgene Expression in Transgenic Plants

Confirmation of gene expression of the plant obtained in Example 3 was carried out as follows: beta-glucuronidase (β-glucuronidase, GUS) reporter gene (leaf) was collected to confirm the expression of the reporter gene (reporter gene) , 200 μl of GUS assay solution (X-gluc (5-Bromo-4-Chloro-3-Indole-β-D-Glucuronic Acid), 1 mg / ml) were placed in a container with leaves and vacuum was placed. Maintain vacuum until wet completely. After that, the appearance of color was visually observed to confirm the introduction and expression of the gene. When the gene of the leaf to which the gene is introduced is expressed, the beta-glucuronidase, a reporter gene, is produced, and the enzyme beta-glucuronidase is produced. When the X-gluc solution is added, the beta-glucuronidase is added. As the X-gluc is broken down, pigments are produced, causing the cells to appear blue. In the present invention, the leaf into which the gene was introduced was expressed by the reporter gene beta-glucuronidase expressed in blue to confirm that the gene was introduced and expressed.

Example 5: Isolation and Purification of CS Proteins from Transgenic Plants

In a mortar containing an appropriate amount of liquid nitrogen, 1 g of the leaves of the transformed plant were cut and pulverized. Finely ground was added 2 ml of protease inhibitor-containing extract buffer (250 mM sucrose, 1 M Hepes, 1 mM MgCl ₂ , 1 mM DTT) per 0.5 g of plant weight. The extract was centrifuged at 12,000 rpm and 4 ° C. for at least 30 minutes, then the supernatant was removed and transferred to a new tube. The lysed cell solution was added to a Probond column (Quiagen GmbH, Germany) containing nickel resin and gently shaken at room temperature for 10 minutes to allow CS protein in the cell solution to adsorb well to the nickel resin.

CS resin-adsorbed resin was washed twice with the same amount of pH 7.8 adsorption buffer solution (20 mM sodium phosphate, 500 mM sodium chloride) to remove plant cell-derived proteins adsorbed on the resin. An equal amount of pH 6.0 wash buffer solution (20 mM sodium phosphate, 500 mM sodium chloride) was added to the washed resin, and the mixture was gently shaken at room temperature to wash the resin again, and the same amount of pH 4.0 elution buffer solution (20 mM sodium) was added. Phosphate, 500 mM sodium chloride) was added to elute the CS protein.

CS proteins expressed in transgenic plants have six histidine residues at the amino terminus and are easily isolated by binding to a cationically nickel resin. Through this process, only CS protein from which all plant cell proteins were removed was purified. The eluted protein was concentrated using Centricon (Amicon Co., U.S.A.).

As shown in FIG. 1, CS proteins were electrophoresed on an SDS-PAGE gel and stained with Coomassie blue, and 10.1 kD of CS protein, which was not detected in wild-type tobacco, was detected in the cell eluate, especially eluted with a nickel resin column. In one case, it was confirmed that pure CS protein from which all plant cell proteins were removed was detected.

The stained protein (Protein assay kit, Bio-Rad) by the Bradford method to the CS protein purified by the above method was measured by absorbance at 595 nm with a UV-spectrophotometer and the BSA (Bovine Serum Albumin) standard Protein quantification was performed in comparison. The extracted protein was adjusted to the same amount and each sample was subjected to 8% polyacrylamide gel electrophoresis, and Western blot analysis was performed (see Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine , 108-121, CRC press). Western blot analysis was performed as follows. The protein in the SDS-PAGE gel was transferred to PVDF membrane using Semi-Dry Transfer Units (Hoefer, USA). After confirming that all proteins in the gel had migrated to the PVDF membrane, the PVDF membrane was dried. This PVDF membrane was reacted with 0.5% BSA / TBS (Tris-Buffered Saline) solution to which the monoclonal antibody (Centers of disease control, Georgia, USA), which specifically responds to CS protein antigen, was reacted for 1 hour, followed by TBS solution. The PVDF membrane was washed three times for 5 minutes. Then, after reacting with peroxidase labeled secondary antibody (peroxidase labeled-anti human IgG, KPL, USA) for 1 hour, the PVDF membrane was washed three times for 10 minutes with TBS solution. Finally, the PVDF membrane was added to the left buffer solution to which the ABTS coloring substrate solution (KPL, USA) was added, and the resultant was reacted for 30 minutes at room temperature and the result was read (see FIG. 2). As can be seen in Figure 2, Western blot analysis was carried out as a result of the CS protein of 10.1 kD was confirmed in the same manner.

Example 6: Diagnosis of Malaria Using 96-well Plate Adsorbed with CS Protein

Example 6-1 Determination of Antibody Titers on CS Proteins

In order to confirm that the CS protein isolated and purified from the transgenic plant can be used for diagnosis of malaria, the antigen-antibody response of the CS protein was measured using a 96-well microtiter plate.

Purified CS protein was serially diluted in phosphate buffer solution, dispensed 100 μl per well, and left at 4 ° C. for at least 8 hours to fix the antigen in the well. Protein-adsorbed plates were washed three times with wash buffer (0.05% Tween 20, 0.01 M phosphate, pH 7.6) to remove antigens that did not adsorb on the plate, and the monoclonal antibodies to CS protein antigens (Centers of disease). control, Georgia, USA) was diluted in a dilution (0.1% BSA, 0.05% Tween 20, 20 mM Trisma base, 150 mM NaCl) with a predetermined dilution factor and dispensed 100 μl per well and reacted at room temperature for at least 1 hour. Induced antibody response. Then, each well was washed three times with washing buffer, and then the blocking buffer was dispensed into each well, and reacted at room temperature for 1 hour, followed by washing in the same manner as above.

Peroxidase labeled secondary antibody (peroxidase labelled-anti human IgG, KPL, U.S.A.) was aliquoted and reacted and washed in the same manner as above. Finally, the color was reacted for 30 minutes at room temperature by adding ABTS color substrate (KPL, USA) to each well, and the result was read at 405 nm using an ELISA reader (ELISA Reader, Titertek, USA). Table 1 shows. The horizontal axis of the table shows the dilution fold of the CS protein of purified triline malaria (amount before dilution: 62.5 ng, 20480 fold dilution: 30 pg), and the vertical axis shows the dilution fold when the initial titer of the antibody to the protein is 1 It is shown. As can be seen from Table 1, the CS protein of the present invention shows excellent antigenicity.

Figure 3 is a schematic of the antibody titers that react with 36 pg of purified CSF malaria compared to proteins isolated from wild-type plants. As shown in the figure, the antigenicity of CS protein purified from recombinant plants was excellent, whereas the protein isolated from wild type plants showed no antigenicity at all.

Dilution multiple of CS protein Antibody Dilution Factor 10 20 40 80 160 320 640 1280 2560 5120 10240 20480 100 <3.0 <3.0 <3.0 <3.0 2.73 2.31 2.21 2.18 2.11 1.92 1.71 1.51 200 <3.0 <3.0 <3.0 2.91 2.46 2.22 2.09 2.03 2.03 1.83 1.60 1.40 400 <3.0 <3.0 2.94 2.79 2.30 2.19 2.00 2.01 1.98 1.67 1.55 1.37 800 2.98 2.95 2.75 2.61 2.11 2.05 1.99 1.95 1.93 1.54 1.50 1.20 1600 2.79 2.63 2.51 2.35 1.98 1.83 1.80 1.78 1.81 1.48 1.32 1.13 3200 2.65 2.40 2.31 2.26 1.89 1.77 1.70 1.45 1.77 1.24 1.13 0.89 6400 2.37 2.32 2.19 2.15 1.73 1.61 1.45 1.19 1.64 1.15 0.89 0.67 12800 2.16 1.94 1.75 1.67 1.31 1.27 0.94 0.9 0.65 0.66 0.65 0.61

Example 6-2 Diagnosis of Malaria

In order to confirm whether the CS protein isolated and purified from the transgenic plant can be substantially used for the diagnosis of malaria, antibody titers in the serum of malaria patients were measured.

100 μl of anti-human IgM antibody (goat anti-human IgM, KPL, USA) diluted in phosphate buffer in 96-well microtiter plates was dispensed per well, and the antibody was left at 4 ° C. for at least 8 hours. Fixed to wells. The antibody-adsorbed plate was washed three times with washing buffer (0.05% Tween 20, 0.01 M Phosphate, pH 7.6) to remove the antibody that did not adsorb on the plate, and the patient serum was diluted (0.1% BSA, 0.05% Tween 20). , 20 mM Trisma base, 150 mM NaCl) was diluted with a constant dilution factor and dispensed 100 μl per well, followed by reaction at room temperature for at least 1 hour, and washed three times with the same washing solution.

100 μl of CS protein antigen dissolved in each dilution by concentration was dispensed into each well and left at room temperature for 1 hour or more to induce a second antigen-antibody reaction. After washing each well again with wash buffer, 100 μl of each monoclonal antibody (Centers of disease control, Georgia, USA) for CS antigen protein dissolved at 10 ug / ml in dilution was dispensed at each room temperature at room temperature. After reacting for 1 hour, the solution was washed three times with a washing solution.

Then, peroxidase labeled secondary antibody (peroxidase labeled-anti human IgG, KPL, U.S.A.) was dispensed and reacted and washed in the same manner as above. Finally, the color was reacted for 30 minutes at room temperature by adding ABTS color substrate (KPL, USA) to each well, and the result was read at 405 nm using an ELISA reader (ELISA Reader, Titertek, USA). Table 2 shows. The result was positive reading of dilution factor with absorbance value of 1.5 or more.

number serum Antibody titers One Patient 1 800 2 Patient 2 400 3 Patient 3 6400 4 Patient 4 6400 5 Patient 5 1600 6 Patient 6 6400 7 Patient 7 200 8 Patient 8 400 9 Patient 9 25600 10 Patient 10 6400 11 Normal 1 <100 12 Normal 2 <100 13 Normal 3 <100 14 Normal 4 <100 15 Normal 5 <100 16 Normal 6 <100 17 Normal 7 <100 18 Normal 8 <100 19 Normal 9 <100 20 Normal 10 <100

As shown in Table 2, the serum of normal persons had an antibody titer of 100 or less, whereas the serum titer of malaria patients showed an antibody titer of 200-25600, clearly distinguishing malaria patients from normal subjects. This shows that the antigen-antibody reaction using the CS protein antigen of the triplet malaria isolated from the transgenic plant occurs with high specificity, and it can confirm that malaria patients can be discriminated through this.

In addition, as shown in Figure 4 showing the ELISA results, it can be seen that the recombinant CS protein produced by the present invention reacts with the antibody in the serum of a person with malaria disease, showing a positive antigen-antibody response.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

As described in detail above, the present invention provides a novel polynucleotide encoding a CS (Circumsporozoite) protein and a plant expression vector of the CS protein comprising the same. The present invention also provides a transient transfected plant that transiently expresses a recombinant CS protein and a transformed plant that stably expresses the recombinant CS protein. On the other hand, the present invention provides a method for producing a recombinant CS protein. According to the present invention, not only can the CS protein be produced in large quantities at low cost, but the produced CS protein can induce the same antigen-antibody response as the native CS protein. Therefore, the CS protein of the present invention can be used for the diagnosis of malaria with excellent sensitivity and specificity.

1 is a photograph showing the result of electrophoresis of CS protein expressed in transgenic plants on SDS-polyacrylamide gel. M, molecular weight marker; Lane W, protein of wild tobacco; And lane T, CS protein expressed in the transgenic plant.

Figure 2 is a photograph showing the results of Western blot analysis of CS protein expressed in the transgenic plant. Lane W, wild-type tobacco; Lane T, transgenic plant.

3 is a graph showing the antigenicity of CS proteins expressed in transgenic plants. The amount of CS protein used 36 pg; Absorbance value at Y axis, 405 nm; Dilution factor of monoclonal antibody against X axis, CS protein antigen.

4 is an ELISA plate photo of antigenicity of recombinant CS protein expressed in transgenic plants. Landscape: (+), positive control expressed in E. coli ; (-), Wild type tobacco; 1-2, MSP (malaria merozoitic suface protein) expressed in transgenic tobacco; CS protein expressed in 3-4 transgenic tobacco. Length: 1-6, serum of malaria infected person; 7, normal serum; (-) PBS buffer.

<110> NEXGEN BIOTECHNOLOGIES, INC. <120> Circumsporozoite Protein of Malaria Expressed in Plants and Diagnostic Kit Comprising the Same <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 111 <212> DNA <213> Artificial Sequence <220> <223> the nucleotide sequence coding for circumsporozoite protein <220> <221> CDS (222) (1) .. (111) <400> 1 atg ggt gat gga gct gca gga caa gct gct ggt gat gga gct gca ggt 48 Met Gly Asp Gly Ala Ala Gly Gln Ala Ala Gly Asp Gly Ala Ala Gly 1 5 10 15 caa gca gct ggt gat aga gct gca gga caa cct gca gga gat aga gct 96 Gln Ala Ala Gly Asp Arg Ala Ala Gly Gln Pro Ala Gly Asp Arg Ala 20 25 30 gca ggt caa cca gct 111 Ala Gly Gln Pro Ala 35 <210> 2 <211> 37 <212> PRT <213> Artificial Sequence <400> 2 Met Gly Asp Gly Ala Ala Gly Gln Ala Ala Gly Asp Gly Ala Ala Gly 1 5 10 15 Gln Ala Ala Gly Asp Arg Ala Ala Gly Gln Pro Ala Gly Asp Arg Ala 20 25 30 Ala Gly Gln Pro Ala 35

Claims (11)

A polynucleotide encoding the CS (Circumsporozoite) protein of malaria comprising the nucleotide sequence set forth in SEQ ID NO: 1. (Iii) the polynucleotide of claim 1; (Ii) a promoter operably linked to the polynucleotide of (iii) and acting on plant cells to form RNA molecules; And (iii) a 3'-non-detoxification site that acts on plant cells to cause polyadenylation of the 3'-end of the RNA molecule, the plant expression vector of the recombinant CS (Circumsporozoite) protein. A method for preparing a transiently transfected plant that temporarily expresses a recombinant CS (Circumsporozoite) protein comprising the following steps: (a) introducing the plant expression vector of claim 2 into a plant cell of a plant; And (b) confirming whether the gene has been introduced into the plant cell. A transient transfected plant temporarily expressing a recombinant CS (Circumsporozoite) protein prepared by the method of claim 3. Method for preparing a recombinant CS (Circumsporozoite) protein comprising the following steps: (a) introducing the plant expression vector of claim 2 into a plant cell; (b) confirming whether the gene has been introduced into the plant cell; And (c) obtaining a recombinant CS (Circumsporozoite) protein from a plant including the plant cell into which the gene is introduced. Method for preparing a transgenic plant stably expressing recombinant CS (Circumsporozoite) protein comprising the following steps: (a) introducing the plant expression vector of claim 2 into a plant cell; (b) selecting the transformed plant cells; And (c) regenerating the plant from the transformed plant cells to obtain a transformed plant. Transgenic plant stably expressing the recombinant CS (Circumsporozoite) protein prepared by the method of claim 6. Method for preparing a recombinant CS (Circumsporozoite) protein comprising the following steps: (a) introducing the plant expression vector of claim 2 into a plant cell; (b) selecting the transformed plant cells; (c) regenerating the plant from the transformed plant cells to obtain a transformed plant; And (d) obtaining recombinant CS (Circumsporozoite) protein from the transgenic plant. Recombinant CS (Circumsporozoite) protein prepared by the method of claim 5 or 8. Kit for detecting the antibody of malaria comprising the CS (Circumsporozoite) protein of claim 9 as an antigen. Method for measuring antibody of malaria comprising the following steps: (a) obtaining a testing fluid from the subject under test; (b) inducing an antigen-antibody reaction with the test solution using the CS (Circumsporozoite) protein of claim 9 as an antigen; And (c) measuring the occurrence (occurrence) of the antigen-antibody response in step (b).