KR930010768B1 - Dna for rickettsia tsutsugamushi - Google Patents

Abstract

A method for the determination of specific DNA sequence of Rickettsia tsutsugamushi and diagnosis of Rickettsia tsutsugamushi disease comprises (a) cloning the specific DNA sequence in the Rickettsia tsutsugamushi by genetic engineering process, (b) analysing the DNA sequence by chain method, (c) preparing primers and reverse primers with 20 base size, and (d) amplifying objective gene in the Rickettsia tsutsugamushi by polymerase chain reaction. The rageat is used for diagosis of Rickettsia tsutsugamushi disease.

Description

Species-Specific DNA Sequences of Schizobacteria

Fig. 1 shows the positions of the synthetic oligonucleotide primers and the nucleotide sequence of the primers in the inserted DNA of pKT12.

2 is a nucleotide sequence of a target DNA of the DNA polymerase chain reaction.

FIG. 3 shows the results of agarose (1.4%) gel electrophoresis after amplification of pKT12 plasmid 35 times by PCR using pc4 and pc5 primers.

Fig. 4, Results of Squatting Cells by Amplifying Polymerase Chain Reaction of L929 Cells Infected with Various Rickettsia

Figure 5, Enzymatic Amplification Results of Target DNA in Samples from Mice Infected with Kato

The present invention relates to a species-specific DNA sequence of the causative agent of the disease, and a method for diagnosing the disease. More specifically, the present invention relates to a method for diagnosing a DNA polymerase chain reaction (POLYMERASE CHAIN REACTION) by specifying a target DNA sequence in the protein antigen gene of the mutant bacterium, which is the causative agent of tsuzugamushi disease.

Squatting disease occurs when the larva of the mite belonging to the genus LEPTOTROMBIDIUM attaches to the animal to suck the animal's tissue fluid in order to turn it into a pupa. do.

Squatting disease is an infectious disease caused by Rickettsia and spread to humans. It is particularly common in Asia-Pacific countries. The clinical symptoms of the disease are known as fever, headache, rash, crust, lymph node swelling, etc., but the clinical diagnosis is not yet familiar and the incidence of Zuzugamushi disease, which presents atypical symptoms, increases, leading to eruption and rash. Diagnosis of the disease in patients who do not exhibit characteristic features of the disease is very difficult. The prognosis of Zhuzigamushi disease varies greatly depending on the characteristics of Zuzugamushi disease isolated in the region, and Zhuzigamushi disease is a disease that can be treated easily when the initial diagnosis is made. 30-40% (WHO Working Group, 60: 157, 1982) or 1-60% (Muray, E.S: Microbiology, Harper and Raw press, pp772,1980) have been reported.

In the severity and prognosis of Squaw's disease, the characteristics of Rickettsia tsutsugamushi are very important. At present, the strains are classified into prototypical strains of Karp, Kato, and Gilliam according to their antigenicity (Am. J. Trop. Med. Hyg. 24: 145,1975). (Am. J. Trop. Med. Hyg. 31 (2); 395-402. 1982)

In addition, there are many possibilities for the coexistence of several types of squash bacteria in Korea. Currently, it has been found that there are 4-6 antigenic proteins on the cell surface of squash cells, and the treatment surface trypsin enzymes lose their ability to penetrate mouse L-cells. It is estimated that there is a close relationship between the pathogenic pathogens of the disease. Many studies are being carried out to clone and produce the gene of E. coli in E. coli and to analyze it for use in diagnosing and producing vaccines. 55; 1156,1987)

At present, the most widely used methods of diagnosing Zhuzi's disease include indirect immunofluorescent antibodies and indirect immunoassays. However, these methods have the disadvantage that they cannot be used in the early stages of the disease before the antibodies are produced, just like any other serologic test for the diagnosis of the disease. In order to solve these drawbacks, the present invention has been completed in order to solve these disadvantages, since the blood is collected two times or more, and the antibody titers are compared and measured.

That is, the present invention clones the species-specific DNA sequence present only in the squash bacteria by genetic engineering method and analyzes the nucleotide sequence of the specific DNA, and reveals the 20-base primer (PRIMER) with reference to the DNA base sequence thus found. ) And a reverse primer (REVERSE PRIMER) to amplify the target sequence of 388 base pairs in the 120-kiloton ton S. mutant protein antigen gene and DNA sequence of the fragment.

DNA polymerase chain reaction, which has recently been used to detect disease-causing bacteria, is not only highly sensitive and specific (J. Inf. Dis. 158; 1177, 1988, LANCET, 20; 428; 1988). This easy and automated diagnostic method can provide data for the treatment of patients, especially the early identification of disease-causing organisms and antibiotic susceptibility.

The method of diagnosing Schizophrenia by the DNA polymerase chain reaction method is based on the target DNA, the primer prepared by the present invention and the reverse primer 1 μL 10 μl 10 × Taq polymerase buffer and 200 d each of the four dNTPs. After the addition, distilled water was added to make 100 μl of reaction solution, followed by heat treatment at 95 ° C. for 5 minutes to denature the target DNA, and then added 4 units of Taq polymerase (International Biotechnology Inc.) to provide Intelligent Heat Block ( Hybaid) is used for the chain reaction.The reaction conditions are 10 seconds at 95 ° C for 10 seconds at the denaturation step, 10 seconds at 55 ° C for the annealing step, and 15 seconds at 72 ° C for the polymerization step. After 10 μl of the reaction solution, 1.4% agarose electrophoresis was performed to confirm the target DNA amplification and diagnosis.

Example 1

Cultivation and Purification of Strains

Cell culture dish containing 150 ml of EMEM (10 x 10 L929-cells) using a medium containing 5% of bovine serum in a minimun essential medium containing ear balanced salt (hereinafter referred to as EMEM). , Falcon Plastic 3025) and shake well to spread the cells on the bottom of the cell culture plate, and then monolayer culture in the presence of 5% CO ₂ at 370 ° C. When mouse L-cells proliferate and reach the late log phase, Inoculated with rickettsia tsutsugamushi proto Kato niigata from the National Institute of Health, and changed the medium to Dulbecco's Modified Eagle's Media (hereinafter referred to as DMEM) containing 2% fetal bovine serum, followed by 34 ℃, 5% CO ₂ Incubation was continued in the presence. Infected L-cells are harvested when about 8-15 days after incubation occupy about 70% of the total culture dish surface.

This was put into a Potter-Elvejhem homogenizer, and the mouse L-cells were crushed by reciprocating the pestle by hand about 10 times, and the mouse cell membranes were removed by centrifugation at 1,000 g for 10 minutes at 4 ° C. After floating the percoll in the supernatant from which the cell membranes were removed, the final concentration was 40%, and the suspension was placed in a SW 41 sec centrifuge tube and subjected to equal density centrifugation at 12,000 g for 60 minutes to isolate and purify the squash.

Example 2

Purification of DNA

Purified squash bacteria from 20 150mm cell culture dishes were added to 1 ml of lysis buffer containing SDS and proteinase K at 1% and 10 ㎍ / ml, respectively, and reacted for about 2 hours, followed by 3 times treatment with the same amount of phenol. After removing the protein, and again treated with the same amount of chloroform three times and precipitated with ethanol to prepare DNA.

Eppendorf test tube 5 was prepared by adding 40 μl of this squamous cell DNA solution (DNA concentration 100 μg / ml) to an Eppendorf test tube previously cooled on ice, adding 5 μl of 10X Sau 3A buffer solution, and mixing and cooling the same. Dogs were aliquoted with 9 μl each. Dilute each Eppendorf test tube with 1X Sau 3A buffer solution, add 1 μl of each restriction enzyme solution containing 2, 1, 0.5, 0.25, and 0.125 units of Sua 3A restriction enzyme, and mix well. After 1 hour of reaction, DNA was cleaved and EDTA was added to a final concentration of 10 mM to block the action of restriction enzymes.

The DNA solution was reacted at 70 ° C. for 10 minutes to destroy restriction enzymes, followed by electrophoresis on 0.8% agar gel to select a test tube cut to a size of about 0.5-3 Kb to prepare an insert DNA. 1 μl of DNA solution cut to about 0.5-3 Kb, 1 ml of 10X lambda ZAPII DNA solution (concentration 1 mg / ml), 1 μl of ligation buffer, 6 μl of distilled water, and 1 μl of DNA ligase (4 unit / μl) were placed in an Eppendorf test tube. The mixture was mixed well and reacted at 14 ° C. for 12 hours to bind the inserted DNA and the Voctor DNA, and 2 μl of this solution was packaged using a lambda packaging system to prepare a gene bank.

Example 3

Production of Antibodies Against Schizobacteria

About 100 mouse L-cells infected with the Kazutozocillus strain Kato were injected into Balb C mice and observed to confirm that the disease occurred, and immunization was prevented by injection with tetracycline. When the mouse completely recovered from the disease, blood was collected from the tail vein, and antibody titers were measured by indirect tube microscopy to collect antiserum with an antibody titer of 1: 800 or more.

The prepared antiserum was absorbed into E. coli XL-1 Blue again and stored at 4 ° C. About 10 recombinant phages were infected with E. coli (XL-1 Blue), mixed well with 3 ml of supernatant agar, spread on 100 mm NZCYM medium and incubated for about 3 hours until plaques were observed. When plaques were observed, the dried nitrocellose membrane soaked in 10 mM IPTG was placed on it to induce the production of the cloned protein, and the incubated protein was further incubated for 4 hours to blotting the nitrocellulose membrane. The blotting nitrocellulose membrane was detached from the medium, washed three times with TBST (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20; pH 8.0) buffer and reacted with TBST solution containing 2% fetal bovine serum for 30 minutes. After saturating the non-specific protein binding site and reacted with Kato immune serum at room temperature for 3 hours, it was washed twice with TBST solution and once with 150mM NaCl solution, and buffered with glycine-saline (0.2M glycine, 150mM NaCl: pH 2.8). The antibody attached to the nitrocellulose membrane was eluted. Eluted antibody was immediately neutralized with Tris buffer (pH 7.0) and stored at 4 ° C.

Example 4

Search of gene bank

About 30,000 phage clones were mixed with 0.6 ml of E. coli XL-1 Blue (OD = 0.3) and reacted at room temperature for about 30 minutes, then mixed with 10 ml of supernatant agar solution and layered on LB medium (150 mm diameter). Incubated for about 5 hours. After incubation, when plaques were observed, incubate for another 4-6 hours to attach to nitrocellulose containing 10 mM IPTG. Remove the nitrocellulose from the medium and react with phosphate buffer solution containing 1% fetal bovine serum for 30 minutes. Nonspecific protein binding sites were blocked.

Put the prepared nitrocellulose paper into a cell culture dish (150 × 22mm, Falcon Plastic 3026) containing 20 ml of mouse anti-Squatting bacteria serum (1: 100 dilution) absorbed with E. coli XL-1 Blue. After the reaction, the resultant was washed three times with 10 mM complete phosphate solution (pH 7.2, hereinafter referred to as PBS) and reacted with the secondary antibody solution for 30 minutes at room temperature. As a secondary antibiotic solution, anti-mouse IgG (Promega) conjugated with Alkaline phosphatase was diluted 1: 4000 times with 10 mM phosphate buffer solution (pH 7.2).

Then washed five times with PBS containing 0.05% Tween (hereinafter referred to as Tween-PBS) and nitroblue-tetrazolium (Sigma, 118F-5051), Bromochloroindolyl phosphate (Sigma, 87F-5007) and levamisole (Sigma, 68F-3439) The mixed substrate solution was added and reacted at room temperature for 15 minutes and washed with 20 mM EDTA solution (pH 8.0) to stop the enzyme reaction and observed.

Example 5

Conversion of pid clone to plasmid clone

Purified squash or E. coli bacteria were dissolved in Tris buffer solution containing 2% SDS, 10% glycerol and 5% mercaptoethanol (pH 6.8), heated at 100 ℃ for 10 minutes, and then heated at 1,000g for 10 minutes. The amount of protein was quantified by removing the insoluble substance by centrifugation. A 10% acrylamide gel was prepared, and 25 μg of protein was added to each column, followed by electrophoresis at 20 mA for 6 hours.

An electrophoretic SDS-PAGE gel was added to an electrophoretic transfer apparatus (Hoeffer Scientific), followed by electrophoresis at 30V for 18 hours to transfer the electrophoretic protein fractions to nitrocellulose paper, followed by a search in the same manner as in Example 4. As a result of the immunoblotting, three clones were produced that produced 180 kilodalton, 120 kilodalton, and 64 kilodalton protein, respectively, from the Kato prototyping strain of Zhu tzu. All of them responded, indicating that all of these antigens are species common antigens.

Add 200 μl of phage gene bank stock (10 / ml) and 1 μl of R408 helped phage (1 × 10, pfu / ml, Stratagene) to 10 mM MgSO ₄ solution in XL1-Blue host cells (OD = 1.0, 600 nm) 37 After reacting for 15 minutes at 5 ℃ 2XYT batch 5ml was added and shaking culture at 37 ℃ 3-6 hours. After reacting the culture solution at 70 ° C. for 20 minutes, the solution was centrifuged at 4,000 g for 5 minutes to collect phagemid in the supernatant, and 200 μl of the phagemid solution and 200 μl of host cell XL-Blue (OD = 1.0; 600 nm) were mixed at 37 ° C. After reacting for 15 minutes, plasmid clones were obtained by culturing on LB / Ampicillin (50 µg / ml) plate medium.

Example 6

Preparation of Restriction Enzyme Maps

The plasmid clone was placed in 5 ml of LB medium and shaken at 37 ° C. for 18 hours (200 rpm, Korean Manhattan Co.). The plasmid was purified by alkaline-lysis. In other words, the strain was immersed in an Eppendorf test tube to remove the supernatant, and 50 μl glucose, 10 mM EDTA, 25 mM Tris-Cl (pH 8.0) and 100 μl of 4 mg / ml lysozyme solution were added thereto, and left at room temperature for 5 minutes, followed by 0.2N NaOH, 1%. 200 μl of SDS solution was added thereto, and the mixture was allowed to stand on ice for 5 minutes. Then, 150 μl of 3M potassium and 5M acetate solution was added thereto, mixed well, and then allowed to stand on ice for 5 minutes, followed by centrifugation at 12,000 g for 5 minutes to collect the supernatant. Add the same amount of phenol / chloroform (1: 1 v / v) solution to the supernatant, mix well, and immerse to collect the supernatant, add 2 volumes of ethyl alcohol, and centrifuge at 12,000g for 5 minutes to collect plasmid DNA. After washing with ethyl alcohol, it was dissolved in 20 μl of TE buffer solution (including 20 μg / ml RNase) and stored at 4 ° C. to use. Thus, 2 µl of the restriction enzyme reaction buffer solution and 10 units of the restriction enzyme were mixed with 3 µg of the plasmid DNA extracted in 10X, and 20 µl of the reaction solution was prepared by adding distilled water, and then reacted at 37 ° C. for 3 hours to deliver the restriction enzyme. The cleaved plasmid DNA was subjected to 1% agarose (sigma) gel electrophoresis to measure the size of the cleaved DNA, and the results were collected to prepare restriction enzyme maps. Among the three clones, a restriction enzyme map was generated by selecting the pKT12 locus, which expresses the 120 kilolodalton antigen of S. goose (Fig. 1).

Referencing the restriction map, the inserted DNA of this clone was digested with Hind III, and the 2,100 nucleotide pair Hind III fragment was fractionated by agar electrophoresis and subjected to sequencing by Sanger et al.

Of these 2,100 base pair DNA sequences, 6 primers and 6 reverse primers were selected for DNA polymerase chain reaction.

When selecting the primer or reverse primer, GC content should be about 50%, exclude the structure of repeating polypuridine or polypyrimidine, DNA sequence itself should not form two-dimensional structure, primer and reverse primer complement each other Standards such as excluding ones are applied.

The six selected primers and reverse primers were placed in a computer program (Gene Master, BioRad, USA) and tested for similarity with the DNA sequences reported so far. Based on this, we selected pc4 and pc5 having 20 basicities, respectively. DNA sequence is shown in FIG.

Then, the DNA sequence of the 3Kb DNA fragment obtained by cutting with restriction enzyme Hind III was analyzed. DNA fragmentation of 3Kb DNA was randomly cleaved by 5 times sonication for 5 seconds after self ligation using a T4 DNA ligase (International Biotechnology Inc.) at a concentration of 30µg / 300µl.

S1 nuclease (Pharmacia) and Klenow fragment (International Biotechnology Inc.) of the cleaved DNA fragments were used to process the terminal portions of the DNA fragments.

1 μl of this DNA (30 ng / μl) and 1 μl of double-stranded M13 DNA (100 ng / μl) digested with Sma I were mixed, followed by adding 6 μl of distilled water, 1 μl of 10 × ligation buffer, and 1 μl of T4 DNA ligase. The reaction was carried out for 12 hours.

The finished DNA was transformed into JM101 host cell using CaCl ₂ method. In other words, take a set of JM101 host cells cultured in M19 minimal flat medium, shake culture with 200ml of 2 × YT medium (OD = 0.3, 600nm), wash twice with 0.1M CaCl ₂ solution, and then 2ml with 0.1M CaCl ₂ solution. After floating, the cells were left in ice for 30 minutes to produce competent cells.

Mix 300 µl of competent cells with 10 µl of ligation M13 DNA, cool on ice for 30 minutes, mix with upper layer medium containing 0.7% agar, and plate LB / AIX (Apicpicin 50 µg / ml, IPTG 10 mM, X-gal 0.2%). Poured onto the medium and cultured to obtain a translucent plaque. Only white plaques were taken from these plaques and used for DNA sequencing.

Example 7

DNA sequence analysis

White plaques were taken and shaken in 2 ml of 2 × YT medium for 10 hours, followed by centrifugation for 12,000 g for 15 minutes to collect the supernatant in an eppendorf test tube. 250 µl of a 15% PEG / 2.5M NaCl solution was added to the supernatant, which was allowed to stand on ice for 30 minutes, and then 12,000 g and 15 minutes were immersed to precipitate M13 phage.

This precipitate was dissolved in 10 mM Tris (pH 7.5), 1 mM EDTA (pH 8.0), and 100 mM NaCl solution, and then mixed with an equivalent amount of phenol / chloroform (1: 1 v / v) to remove the protein component and the outer strand for DNA base sequencing. Template DNA was used. The DNA sequence analysis was performed using chain termination method. Template DNA and M13 universal primer (New England Biolab) were mixed and heat-treated at 70 ° C. for 5 minutes, then slowly cooled to induce annealing between template DNA and primer. Thereafter, dCTP, dGTP, dTTP, Alpha-35S-dATP (Amersham), and Sequenase (United State Biolab) were added thereto, and the mixture was allowed to stand at room temperature for 5 minutes for polymerization of DNA. After completion of the reaction with ddATP, ddCTP, ddGTP and ddTTP, the DNA base sequence was analyzed by 50% Urea and 6% polyacrylamide gel electrophoresis. The analyzed base sequences were finally synthesized using a DNA sequencing computer program (Microgenie Beckman) to determine the base sequences.

Example 8

Selection and Production of Primer and Reverse Primer

The final GC sequence had a mean GC flow rate of less than 50%, randomly distributed nucleotide sequences, no secondary structure of DNA and no complementarity between primers, polypuridine or polypyrimidine. The primers and reverse primers (20 base pairs) were selected for exclusion. Using the Gene Bank Computer Program (Genemaster, Bio-rad), the selected nucleotide sequence of the selected primers was compared with the known DNA base sequences. Six primers and reverse primers were selected in the Computer Program (Gene Master, BioRad, USA) and tested for similarity. Based on this, PC4 and PC5 each having 20 base pairs were selected and shown in FIG. At this time, the size of the target sequence is 388 base pairs and is shown in FIG.

Example 9

Sensitivity and Specificity Measurement of DNA Polymerase Chain Reaction Using pc4 and pc5

In order to measure the sensitivity of the DNA polymerase chain reaction method using pc4 and pc5, DNA polymerase chain reaction method was carried out by varying the amount of purified pKT12 DNA. Electrophoresis showed about 400 base pairs of target DNA (Figure 3).

In Fig. 3, lanes 1, 2, 3, and 4 indicate that 1 pg, 100 fg, 10 fg, and 1 fg DNA were subjected to periodic electrophoresis, respectively.

In order to measure the specificity of the DNA polymerase chain reaction using pc4 and pc5, they were infected with Kazu, Karp, Gilliam, B119, TA1817, TA716, TT118, R.prowazekii, R.typhi and R.akari, respectively. The mouse L-cells were collected, DNA was extracted, and DNA polymerase chain reaction was performed using each extracted 300 ng of DNA. As a result of the reaction, the DNAs of Kato, Karp, Gilliam, B119, TA1817 and TA716 were positive for R. tsugamumishi, but TT118 and R. prowazekii, R. typhi, R.akari and Tick typhus Did not respond. It also did not react with the DNA of mouse L-cells used as controls (Figure 4).

In Fig. 4, lane 1 is R.akari, lane 2 is TT118, lane 3 is R.prowazekii, lane 4 is R.typhi, lane 5 is TA1817, lane 6 is TA716, lane B119, lane 8 is Gilliam, lane 9 infected Karp and lane 10 infected Kato.

Example 10

DNA Polymerase Chain Reaction in Mice Infected with

Mouse L-cells were infected with Kato, a strain of Kato, and about 70% of the total cells showed pathological changes. When the mouse L-cells were removed from the cell culture dish, about 100 mouse L-cells harvested were intraperitoneally injected to infect mice Regenerated 8 days after infection to collect peripheral blood, spleen and peritoneal cells. 0.5 ml of collected peripheral blood, 3 ml of phosphate buffer solution, washed with 0.5 ml of splenocytes and 3 ml of phosphate buffer solution, and 0.5 ml of collected peritoneal cell suspension was added to the cell lysis solution (0.32M sucrose, 10mM Tris-HCl, 5mM). MgCl ₂ and 1% Triton X-100) were dissolved in 0.5ml of DNA polymerase chain reaction. As a control group, peripheral blood and spleen fluid were used in normal mice. As a result, the target DNA sequence of about 400 base pairs was observed in all of the peripheral blood, spleen fluid, and peritoneal cell fluid of mice infected with R. squash. However, it did not react with peripheral blood and splenocyte suspension of normal mice under the same conditions.

In FIG. 5, lane 1 is a whole blood cell of peripheral blood vessel, lane 2 is a splenocyte of spleen fluid, lane 3 is a cell from a peritoneal cell suspension, lenin 4 is a whole blood cell of a healthy mouse, lane 5 is a healthy mouse. Enzymatic amplification of spleen cells is shown.

Claims (1)

Squash-gum species specific DNA base sequence having the following nucleotide sequence