KR20020024958A - DBP gene of Plasmodium viviax and the detection method of malaria using that - Google Patents

Abstract

PURPOSE: A duffy blood type binding surface protein(DBP) gene of plasmodium viviax and a detection method of malaria using the same are provided, thereby rapidly and exactly diagnosing the infection of plasmodium vivax. CONSTITUTION: The DBP gene of plasmodium vivax has the nucleotide sequence of SEQ ID NO: 1. The DBP of plasmodium vivax has the amino acid sequence of SEQ ID NO: 2. A recombinant vector KPV DBP-1 containing the DBP gene of plasmodium vivax is produced by inserting the DBP gene of plasmodium vivax into a plasmid pQE30. The transformants are produced by transforming host cells JM109 and M15 with the recombinant vector KPV DBP-1. The method for detecting malaria comprises the steps of: centrifuging the transformants capable of producing the DBP of plasmodium vivax; recovering the supernatant; subjecting the supernatant to a column chromatography; reacting the purified DBP with bovine serum albumin; reacting the resulting solution with a conjugation solution of the goat anti-human IgG and horse radish peroxidase; adding TMB into a resulting solution; and measuring its absorbency.

Description

Samil fever malaria duffy blood type surface protein gene and method for detecting malaria using the same {DBP gene of Plasmodium viviax and the detection method of malaria using that}

The present invention relates to a triplet malaria duffy blood type adhesion surface protein gene (DBP) and a method for detecting malaria using the same. More specifically, the present invention is prepared by introducing a gene, an amino acid, a recombinant vector constructed by inserting the gene for the Duffy blood type adhesion surface protein expressed on the surface of Plasmodium vivax , and inserting the vector into E. coli. One transformant and a recombinant Duffy blood type adherent surface protein expressed therefrom and a method for detecting malaria accurately and quickly using the same.

Malaria is the largest human disease that has existed since ancient times and is the epidemic that is the main concern of mankind that does not disappear and weaken even today, despite many efforts since the first protozoa was discovered in human red blood cells since the 20th century. In particular, it is emerging as a health problem in developing countries, and unfortunately, reappearance has been reported in areas where malaria has disappeared, including Korea since the 1960s.

Malaria erythrocytes and malaria as a parasite occurs in humans as causing disease belonging to the genus Plasmodium of Cryptosporidium River (class Sporozoa) in hepatocytes falciparum malaria (Plasmodium falciparum), vivax malaria (Plasmodium vivax), four days malaria (Plasmodium malariae) And four types of malaria ( Plasmodium ovale ), two of which are responsible for more than 95% of malaria incidences worldwide. Malaria is the triplet malaria that has the largest geographical distribution in temperate, subtropical and tropical regions.

In Korea, the incidence of indigenous trifluid malaria, which has disappeared for more than 10 years, continues to increase since the first patient in 1993. Malaria is transmitted by mosquitoes as a host, and the human body as an intermediate host. When mosquitoes bite humans, it is most common to inject spores from the mosquito's salivary glands and infect them. Symptoms include fever, anemia, and spleen symptoms of three symptoms, such as chills, fever, fever and fever features. Blood stocks are low and often have thrombocytopenia. The incubation period from the time of infection until the first clinical manifestations is on average 14 days long, particularly trichophytes, which remain in the hepatic tissue for months to 2-3 years, causing relapses and causing recurrence. If you have not received or have received insufficient treatment, you may be infected with mosquitoes for up to 1-3 years. Due to this long incubation period, the disease may move from the epidemic region to the distant region, and the low number of parasites in the blood may make diagnosis difficult. In order to prevent and treat malaria, drugs such as chloroquine and primaquine are being administered. However, these drugs only increase resistance to malaria, which may increase the disease further, so it is necessary to relapse an effective vaccine against malaria.

The ecology of malaria is specific, and after the body is naturally infected with malaria, the body produces antibodies against malaria and exhibits neutralizing ability, resulting in humoral immunity, but its neutralization ability persists as addictive immunity. It does not work as a protective immunity such as allowing an infection many times. In other words, humoral immunity is induced, but defense mechanisms by cellular immunity do not occur. The reason for this is that the protein of the protozoa does not have an antigen suitable for the development of a protective response, or the protozoa has developed a mechanism to escape the host's immune system.

The diagnosis of an existing malaria infection consists in the detection of protozoa in the blood through blood smear testing. Malaria should be tested for blood in all fever patients in endemic areas and should be suspected of having fever, even if only a few hours pass through the tropics. If malaria is suspected as a clinical symptom and there is no facility to perform blood smear testing or the results are not immediately available, medication may be given temporarily. However, even in this case, a blood smear should be obtained before treatment to confirm the infection. If malaria medications do not control fever, other causes of fever should be considered.In Africa, malaria is suspected of being a clinical symptom in malaria endemic areas. This is a case of low levels of protozoa in the blood due to low infection.

In addition, the Indirect Fluorescent Antibody Test (IFAT) for malaria has been developed. This method is used for epidemiological investigation as a test method that can confirm the small number of infections with malaria infection in the past. It is also used for screening malaria in the blood. Homogeneous antigens used in IFAT are obtained from red blood cell schistant obtained from humans, monkeys, and experimental blood cultures. First, the antigen, anti-human globulin and fluorescent isothiocyanate are present on the IFAT test slide, and the diluted sample is reacted to confirm the result of the antibody reaction by fluorescence microscope. This test confirms the results of malaria's immune response, not necessarily malaria parasites. A titer of 1: 200 or greater is suspected of a recent infection, and a titer of greater than 1:20 is considered positive.

Malaria detection method (IFAT) using the above antibody of the patient has the following problems. The peripheral blood smear test is a method of performing peripheral blood smears on a patient, staining them, and then examining them under a microscope. They require expensive fluorescence microscopy, require a lot of testing time, and have no objective criteria for reading. There is the hassle that must be confirmed by old doctor and is not suitable for large quantity of examination. In addition, it is difficult to confirm the case of less malaria protozoa, and it is not possible to confirm the past infection of malaria. Therefore, it is not possible to know the hypnotic invasive infection after malaria infection. In addition, there is a problem in that it is difficult to distinguish these species from each other because of high morphological similarity between malaria species, and when the antigen is produced from infected blood of a non-monkey, the false positive rate has a problem in accuracy of the test.

In view of the above facts, if the genotype of the surface protein of Korean malaria is revealed, this gene can be used to produce recombinant protein using genetic engineering techniques, which is a diagnostic reagent for the prevention of malaria, In particular, it may be a source antigen for enzyme immunoassay, which is a convenient method. In addition, this antigen can be used to develop a malaria vaccine, which can play a crucial role in the simple diagnosis, prevention and epidemiological studies of malaria. The production of diagnostic reagents and vaccines using this developed malaria antigen requires a protein expression process based on this gene.

The present invention relates to a gene encoding a duffy blood type adhesion protein (DBP), which performs the most important function when malaria invades the human body and invades adjacent red blood cells. Proteins play an important role in forming the body's immune function against malaria. Therefore, using this protein as an antigen, an immunoenzyme reagent for detecting malaria and a malaria vaccine can be developed.

In addition, the unique gene of the Korean trifluid malaria of the present invention is different from foreign trifluid malaria genes, so that the effect of producing a diagnostic reagent or vaccine based on this gene is higher in sensitivity and effect than that of a reagent imported from a foreign country. May help prevent malaria

Accordingly, it is an object of the present invention to provide a base sequence and an amino acid sequence of a gene encoding the Duffy blood type adhesion surface protein (DBP) of Korean trigeminal malaria in view of the above facts. Another object of the present invention is to introduce a recombinant vector KPV DBP-1 prepared by inserting the gene into the pQE30 plasmid and a transformant prepared by introducing a recombinant vector into E. coli and a recombinant Duffy blood type adhesion surface protein (DBP) expressed therefrom. To provide. Another object of the present invention is to provide a more accurate, sensitive and rapid triplet malaria detection method using a recombinant Duffy blood type adhesion surface protein encoded by the recombinant Duffy blood type adhesion surface protein gene of the transformant.

The object of the present invention is to perform blood smear test to extract the blood of malaria patients confirmed the presence of protozoa in the blood, extract the DNA of the protozoa, construct a recombinant primer for amplification of the extracted DNA and polymerase chain reaction ( PCR), followed by electrophoresis of the amplified product from the polymerase chain reaction to confirm the base band, and after reading the DNA and amino acid sequencing results, the base sequence of the Duffy blood type surface protein (DBP) gene and amino acid was derived. It was achieved by producing a recombinant Duffy blood type adherent surface protein of and using it to easily detect malaria.

Hereinafter, the configuration of the present invention will be described in detail.

Figure 1 shows the results of electrophoresis of the gene encoding the Duffy Blood Group Adhesion Surface Protein (DBP) of the present invention ileum malaria ( plasmodium vivax ) and reacted with the sample.

Figure 2 shows the nucleotide sequence of the gene encoding the Duffy blood type adhesion surface protein (DBP) of the present invention ternary malaria.

Figure 3 shows the amino acid sequence inferred from the gene encoding the Duffy Blood Group Attachment Surface Protein (DBP) of the present invention triplet malaria.

Figure 4 shows a cleavage map of the recombinant vector KPV DBP-1 constructed by inserting the gene encoding the Duffy Blood Group Adhesion Surface Protein (DBP) of the present invention in quinine fever malaria into the pQE30 vector.

The present invention comprises the steps of extracting the blood of the patient confirmed the presence of protozoa in the blood by setting the patient group showing the clinical symptoms of malaria as a target group and blood smear; Measuring the morphological diagnosis and blood concentration of malaria protozoa in the subject group and examining the characteristics of the subject group; Extracting DNA from the whole blood collected from the infected patient by centrifugation, vortex, heat block, etc. to extract DNA of the malaria protozoa in the blood; Constructing a recombinant primer to amplify the extracted DNA, performing a polymerase chain reaction (PCR), and electrophoresis of the PCR resultant to identify base bands; The amplified DNA was amplified again using an ABI PRISM (TM) Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer) and a heat cycler, and then separated. The result of sequencing of genes and amino acids using ABI PRISM ^TM 377 DNA Sequencer was obtained. Reading a; Cutting and ligating the DBP gene and pQE30 plasmid with BamHI and HindIII restriction enzymes to construct a recombinant vector KPV DBP-1 into which the DBP gene was introduced; Preparing and selecting transformants by introducing the recombinant vector KPV DBP-1 into JM109 and M15 host cells; Confirming the recombinant Duffy blood type adherent surface protein expressed from the transformed strain by electrophoresis and staining and measuring the molecular weight; Pulverizing the recombinant DBP producing transformant with an ultrasonic mill and centrifuging to recover the supernatant and passing through a column to purify the recombinant DBP; Western blotting is performed to react the blood plasma of the DBP-specific patient with the recombinant DBP, and then react with HRP-conjugated Anti-human secondary Ab and add color by adding 4-Chloro-1-naphthol, which is a substrate for HRP. ; In the purified recombinant DBP using bovine serum albumin as a standard protein, the sample is reacted with goat anti-human IgG and horse radish peroxidase conjugate solution, followed by color development by adding TMB, followed by absorbance to detect malaria. It is composed.

Hereinafter, specific examples of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1: Selection and epidemiological investigation of the subject group of the present invention

First step. Selection and clinical diagnosis of subjects of the present invention

During the period from 1996 to 1997, blood smear was performed on 50 patients who had visited the Armed Forces Capital Hospital and Korea University Department of Clinical Pathology and requested a blood smear to confirm the presence of protozoa. The individual interviews were followed by a history of symptoms, date of invention, place of work, fever patterns and intervals, and overseas travel. The epidemiological situation of outbreak patients was investigated.

Second step. Morphological diagnosis of malaria protozoa and blood concentration calculation of protozoa

Morphological diagnosis of malaria was performed using a vacuum blood vessel containing EDTA (Becton-Dickinson, USA), which collected 4-5 ml of venous blood from the forearm vein, followed by a thin blood smear on a glass slide. Thick blood smears were prepared at the same time and peripheral blood smears were performed using Wright-Giemsa staining and Giemsa staining. The presence of protozoa was indicated by the presence or absence of malaria according to the stage of protozoa, and was converted into the number of malaria protozoans compared to the number of leukocytes counted at the time of microscopy using an autologous hemocytometer (Celldyne 3000, U.S.A). The detailed calculation method is as follows.

Malaria Blood Protozoal Concentration (Parasite / μl)

= Number of protozoans per 100 leukocytes × leukocytes per unit volume (/ μl) / 100

Third step. Characteristics of the subject group of the present invention

From the beginning of August 1996 to the end of 1997, we investigated the characteristics of 27 subjects who were found to have malaria protozoa in their blood.

The regional distribution was 12 in Paju (48%) and 9 in Yeoncheon (36%). By gender, 24 of 25 patients (96%) and 1 of 25 patients (4%) were female.

There were 20 active soldiers (80%), three military officers (12%), and one driver and one student. The age was mainly in their twenties and the mean age was 23 years (12-45).

The test period was 23 for 96 years and 2 for 97 years. The most common monthly incidence was 8 (32%) in June, followed by September (28%) and 4 in October (12). %)

All 27 confirmed patients had never traveled abroad and had no history of blood transfusion, so they may be infected with domestically developed Plasmodium vivax. The concentrations of protozoa in the blood of the subjects ranged from a minimum of 23 / ul to a maximum of 15,330 / ul (2,040 / ul) (Table 1).

Characteristics of Patients with Triplet Malaria Infection Patient number Gender / age PCR results Malaria count / μl residence One F / 12 + 12,000 Paju-si 2 M / 21 + 2,600 Ganghwa-gun 3 M / 21 + 15,330 Paju-si 4 M / 21 + 1,941 Ganghwa-gun 5 M / 22 - 56 Paju-si 6 M / 22 - 6,840 Yeoncheon-gun 7 M / 22 W ⁺ 520 Paju-si 8 M / 23 - 340 Paju-si 9 M / 23 - 983 Yeoncheon-gun 10 M / 22 + 329 Goyang 11 M / 22 - 127 Yeoncheon-gun 12 M / 23 W ⁺ 23 Pocheon-gun 13 M / 25 - 525 Yeoncheon-gun 14 M / 22 - 217 Yeoncheon-gun 15 M / 22 + 1,000 Yeoncheon-gun 16 M / 20 + 166 Cheorwon-gun 17 M / 23 W ⁺ 32 Yeoncheon-gun 18 M / 23 + 1,320 Paju-si 19 M / 23 + 1,320 Yeoncheon-gun 20 M / 22 - 341 Paju-si 21 M / 22 + 70 Yeoncheon-gun 22 M / 23 W ⁺ 250 Cheorwon-gun 23 M / 25 + 3,400 Paju-si 24 M / 24 + 2,750 Paju-si 25 M / 24 + 970 Paju-si 26 M / 24 W ⁺ 300 Paju-si 27 M / 45 - 1,320 Cheorwon-gun Average 23 2,040 Highest 45 15,330 Lowest 12 23 Note: In the PCR results, W ⁺ shows a weak positive reaction.

Example 2: Extraction of the target group malaria protozoal DNA and determination of nucleotide sequence

First step. Extraction of Malaria Protozoal DNA

The DNA of malaria protozoa was extracted using a DNA extraction kit (InstaGene ^™ Matrix) from whole blood collected from the malaria infected patient. In the DNA extraction method, 10 μl of whole blood is added to 1 ml of distilled water, left at room temperature for 30 minutes, and then centrifuged for 3 minutes at 12,000 RPM, followed by carefully leaving only 20 μl of the supernatant. 200μl of DNA extraction solution (InstaGene ^TM Matrix) was added to the remaining precipitate, and then placed in a 56 ° C heat block for 30 minutes, followed by vigorous shaking with a vortex for 10 seconds, followed by 8 minutes in a 100 ° C heat block. After vigorously shaking with vortex for 10 seconds, centrifugation was performed at 12,000 RPM for 3 minutes to precipitate DNA in the holding solution.

Second step. Amplification of the extracted DNA by polymerase chain reaction (PCR)

Amplification of the extracted DNA was performed by polymerase chain reaction (PCR) using a GeneAmp PCR system 9600 (Perkin Elmer, USA). Based on the known Salvador I strain (Gene bank M61095, PFADUFR) to construct a recombinant primer for PCR of the gene and its properties are shown in Table 2.

Primer for PCR amplification of extracted DNA Primer 1 Primer 2 Length 22 base pair 22 base pair type Nucleic acid Nucleic acid Number of chains 1 print 1 print Topology Linear Linear Type of molecule DNA DNA order TGGGACTGTA ACACTAAGAA GG TTCATTCTCA AAAGCCACCT CG

The primers in Table 2 were each devised by the inventors based on the known Salvador I strain to make a reaction product of about 7OObp each. In the polymerase chain reaction, the mixing conditions of the reaction samples were adjusted to 1 μmol / μl of primer concentration and 20 μmol / μl of MgCl ₂ , respectively.

The polymerase chain reaction was thermally denatured at 95 ° C. for 1 minute, then bound at 53 ° C. for 1 minute, and then polymerized at 72 ° C. for 3 minutes to react for 30 cycles. 10 μl was electrophoresed on a 2% agarose gel, and the gel was stained with ethidium bromide and irradiated to confirm base bands (FIG. 1).

Third step. Base sequence determination of the DNA

The nucleotide sequence of the amplified DNA was determined using ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer), 8 μl Terminator Ready Reaction Mix per 20 μl of reaction solution, 3.2 μl of 1 μM of the primer and 30 ng of the primer. Template was added and mineral oil was added to amplify 25 cycles with a thermal cycler. The conditions for each cycle of amplification were 96 ° C 30 seconds, 50 ° C 15 seconds, and 60 ° C 4 minutes, and the first cycle adopted 96 ° C 5 minutes. After completion of the amplification, the reaction solution was mixed with 2 µl of 3M sodium acetate (pH 4,6) and 50 µl of 95% ethanol, left on ice for 1 minute, centrifuged at 11000 rpm for 20 minutes, and the precipitate was washed with 250 µl of 70% ethanol. After vacuum drying, the solution was dissolved in 6 µl injection buffer [25 mM EDTA (pH 8.0) containing loading buffer, 5 µl of deionized formamide, and 1 µl of 50 mg / ml blue dextran. After denaturing at 90 ° C. for 2 minutes immediately before injection, 1.5 μL each was electrophoresed at 3000 V on a 4% polyacrylamide sequencing gel and the DNA sequence and amino acid sequence results were read with an ABI PRISM ^™ 377 DNA Sequencer to provide a bloody adherent surface of malaria. The gene of protein (DBP) was isolated and its nucleotide sequence and amino acid sequence were analyzed (FIGS. 2 and 3).

Example 4 Preparation and Purification of Recombinant Duffy Blood Type Adhesion Surface Protein Using the Triplet Malaria Duffy Blood Type Adhesion Surface Protein (DBP) Gene

First step. Extraction of Malaria DBP DNA, Preparation of Recombinant Vectors and Transformants

The malaria duffy blood type adhesion surface protein (DBP) gene was identified by electrophoresis on a 1.2% agarose gel, and then extracted from the agarose gel using a QIAEXII gel extraction kit. It was. The PCR product, Duffy Blood Group Adhesion Surface Protein gene and plasmid pQE 30 were cut at 37 ° C. using restriction enzymes BamHI and HindIII and electrophoresed on 1.2% agarose gel to confirm the band size, and then extracted from the gel. 1 μl of 10 × bacteriophage T4 DNA ligase buffer and 1 μl of T4 DNA ligase (1wU) were added to the plasmid vector, followed by adding sterile water to a final volume of 10 μl. Reaction vector KPV DBP-1 was prepared by allowing the DBP gene to be introduced into plasmid pQE30 by overnight reaction at 16 ° C. The cleavage map is shown in FIG.

Host cells JM109 and M15 (pREP4) were incubated at 37 ° C. for 16 hours, inoculated with 500 μl in 20 ml fresh medium, and then centrifuged at 2000 × g and 4 ° C. for 5 minutes, and then 1 ml in cell pellet. Add 1 x TSS [85% LB media, 10% PEG 8,000 (w / v), 5% DMSO (v / v), 50mM MgCl ₂ ], mix well and dispense 100 μl for use in transformation. Ready.

3 μl of the ligation mixture of the recombinant vector and 100 μl of the host cells JM109 and M15 competent cells were mixed and left for 30 minutes on ice, followed by a heat shock at 42 ° C. for 90 seconds. After the addition, the mixture was immediately cooled, and 900 μl of LB was added thereto, followed by incubation at 37 ° C. for 1 hour to prepare a transformant into which the recombinant vector was introduced. This was plated on an LB / Amp (100 ㎍ / ml) plate, and 5 ml of colonies obtained by incubating at 37 ° C. for 16 hours were extracted from the recombinant plasmid. Each cleavage was confirmed to contain DBP gene, and the base sequence was analyzed to examine the accuracy of ORF (Open reading frame). The sequencing was performed using a Perkin-Elmer ABI 377 automatic sequencer as described above.

The transformed strain into which the DBP gene was inserted was deposited in a microorganism deposit institution.

Second step. Expression and Identification of Recombinant Duffy Blood Group Adhesion Surface Proteins through Small-scale Cultures

Single colonies of the transformed strains were inoculated in 10 ml of LB [100 μg / ml ampicillin, 25 μg / ml kanamycin] and incubated overnight. Incubate for about 1 hour at A ₆₀₀ until it reaches 0.7-0.9, and add IPTG (final concentration 1 mM), raise it for 2 hours and 4 hours at the same temperature, and take 1 ml each of them at 8000 rpm. Centrifugation for 10 minutes at. After removing the supernatant, 30 μl of 2 × SDS-PAGE sample buffer was added, boiled at 100 ° C. for 5 minutes, and centrifuged at 14,000 rpm for 10 minutes to perform only 12% SDS-PAGE. As a non-induced control (Uninduced control) was used that did not add only IPTG under the same conditions.

To confirm expression, 1 ml of each sample was taken and suspended in 2 × sample buffer (0.125 M Tris-HCl, pH 6.2, 4% SDS, 20% glycerol, 10% mercaptoethanol) and then 10% SDS polyacrylamide gel. electrophoresis at 150 V on (polyacrylamide gel). After the electrophoresis, the gel was stained with Commassie blue to confirm that it was 27 KDa recombinant Duffy blood type adhesion surface protein (DBP) expressed from the gene.

Third step. Purification of Recombinant Proteins

The transformed E. coli expressing recombinant DBP was suspended in fusion buffer [10 mM sodium phosphate, 30 mM sodium chloride (NaCl), 10 mM mercaptoethanol, 10 mM EDTA, pH 7.2]. Lysozyme (Lysozyme, 1 mg / mL) was mixed and allowed to stand in ice for 30 minutes to dissolve E. coli. After pulverizing three times for 5 minutes at 50% of the maximum output by the ultrasonic mill, the supernatant was recovered by centrifugation at 8000 rpm for 20 minutes at 4 ℃. An equal amount of column buffer was added to the supernatant and used for purification. Fill the column volume with column buffer (10 mM sodium phosphate, 0.2 M sodium chloride, 10 mM mercaptoethanol, 10 mM EDTA, pH 7.2) after filling the 3.2 × 10.3 cm column with Amylose resin (NEB, USA). Wash in 3-fold quantities. The E. coli lysate was passed through at a rate of 1 ml / min, washed with column buffer, and eluted with a column buffer containing 10 mM maltose to collect 5 ml fractions. Proteins were identified by SDS-polyacrylamide gel electrophoresis.

Example 5 Detection of Malaria Using the Recombinant DBP

First step. Immunoblotting of Recombinant DBP

Western blotting was performed to investigate whether the recombinant DBP, whose expression was confirmed and purified, was recognized by serum malaria patients. 50 [mu] l each of three types of serum, 99-23 and 96-73, specific for DBP was used. As in the third step of Example 4, the recombinant protein induced expression at uninduced control, 2 hours, and 4 hours, respectively, was isolated through SDS-PAGE, followed by 3 hours at a constant voltage of 60 V in the PVDF membrane. After transferring, the solution was soaked in 10 ml of blocking buffer (5% non-fat dry milk in TTBS) overnight. After washing for 15 minutes with TTBS (20 mM Tris-Cl, 137 mM sodium chloride, 0.1% Tween 20, pH 7.6) three times for 5 minutes, and then for 5 minutes, 50 μl of three serums of 99-23, 96-73 5 ml of TTBS was added and allowed to react for 1 hour while shaking slowly at room temperature. After washing 15 minutes once with TTBS and three times with 5 minutes, HRP-conjugated Anti-human secondary Ab (Sigma, U.S.A) was diluted 1: 1000 and reacted in 5 ml of TTBS for 1 hour. Thereafter, after the same washing process, 4 ml of 4-chloro-1-naphthol (4-Chloro-1-naphthol), which is a substrate for HRP, was added and the color was slowly shaken.

Second step. Determination of antigen activity and analysis of recombinant DBP by ELISA

The purified DBP protein was bovine serum albumin as a standard protein, colorimetrically quantitated at 595 nm by Bradford method, and mixed in 50 mM carbonate buffer (carbonate buffer, pH 9.6) to 10 µg / ml. Was placed in a microtiter well (Nunc-Immuno Module, Denmark) and left for 16 hours at 25 ℃. After blocking for 2 hours with casein, the sample was diluted to 1/20 with phosphate-casein buffer, 100 μl of the sample was reacted at 37 ° C. for 1 hour. After washing 5 times with phosphate buffer, goat anti-human IgG and horse radish peroxidase conjugate solution were reacted at 37 ° C. for 30 minutes. After washing, the mixture was developed by adding trimethylbenzidine (TMB) and stopped with 1.6 N sulfuric acid solution. After the absorbance was measured at 420 nm using a 96 well plate autoreader. As a result, 57% (15/27) of the samples used in the reaction were positive with a significant difference (2SD) from the OD of normal serum.

Twenty-four (85%) of 27 patients showed positive results in polymerase chain reaction using Duffy blood type adhesion surface protein (DBP), and electrophoresis in the second step of Example 2 resulted in DNA near 750 bp. Fractions could be confirmed (FIG. 1).

Peripheral blood protozoa concentrations of positive malaria polymerase reactions ranged from 23 / μl to 15,330 / μl and averaged 2,630 / μl. In peripheral smear, there was a wide distribution ranging from 56 / μl to 1,320 / μl in the case of positive in malaria polymerase reaction, but the mean protozoa concentration was significantly lower than positive.

As is apparent from the above examples, the present invention isolates the duffy blood type adhesion surface protein (DBP) of plasmodium vivax , which performs the most important function when malaria proliferates and invades human red blood cells. Analyze the nucleotide sequence of the gene and amino acid, construct and manufacture recombinant vectors and transformants incorporating the gene, isolate and purify the recombinant protein derived therefrom, and use the triplet malaria accurately, easily and in sensitivity. It is possible to provide a method for high detection, and furthermore, the gene can be used to prepare a recombinant Duffy type surface adhesion protein, and the antigen can be used to develop diagnostic reagents and vaccines for the prevention of malaria. Very useful foot for national health promotion and pharmaceutical manufacturing It is a person.

Claims (6)

The following gene sequence encoding the Duffy blood type adhesion surface protein (DBP) of ileum malaria ( Plasmodium vivax ). The following amino acid sequence making up the Duffy Blood Group Attachment Surface Protein (DBP) of Plasmodium vivax . Recombinant vector KPV DBP-1 constructed by introducing the triplet malaria duffy blood type adhesion surface protein gene according to claim 1 into pQE30. Recombinant triplet malaria duffy blood type adherent surface protein-producing transformant, wherein the recombinant vector KPV DBP-1 according to claim 3 containing the triplet malaria duffy blood type adherent surface protein gene was introduced into JM109 and M15 host cells. . Recombinant triplet malaria duffy blood type adherent surface protein production according to claim 4 A recombinant duffy blood type adherent surface protein expressed from a transformant. Recombinant tri-row malaria duffy blood type adherent surface protein production according to claim 4 The transformed Duffy blood type adherent surface protein purified by centrifugation after centrifugation of the transformant and column chromatography was purified by bovine serum albumin as a standard protein. A method for detecting malaria, comprising reacting goat anti-human IgG with horse radish peroxidase conjugate solution, and then developing color by adding TMB, followed by absorbance to detect malaria.