SU1711676A3 - Method of dna isolation for diagnosis of non-a,non-b- hepatitis - Google Patents

Abstract

The invention relates to a substance which has been isolated from stool and is associated with non-A, non-B hepatitis and which is isolated in a conventional way and, after electrophoresis and transfer to nitrocellulose, is present in the form of four bands with a molecular weight of about 64000, about 56000, about 51000 and about 43000, and to a DNA obtained therefrom, DNA fragments obtained after cloning of the DNA, and a DNA sequence, to the processes for obtaining these substances and to the use in diagnosis and therapy.

Description

The invention relates to medicine and can be used to diagnose the disease Hu-A, Ni-B-hepatitis.

A method of obtaining DNA is proposed, which preliminarily sterilizes a stool suspension by filtering it through a Millipore filter about 0.22 in size, precipitating with polyethylene glycol (PEG 6000) at a final concentration of 10%, separating the precipitate, dissolving it in a buffer, shaking with freon in a ratio of 1 : 1, separation of the freon phase, precipitation of PEG 6000 at a final concentration of 10%, treatment of RNA with az and DNA, fractionation in a gradient1 of cesium chloride, separation of the fraction with a density of 1.3 g / ml, dialysis, treatment of the floor; the fraction of proteinase K, extraction with 80% phenol and chloroform,

DNA treatment with Eco R1 restriction enzyme; cloning of the DNA fragment in the plasmid pBR322 or in lambda phages.

Example 1. Isolation of a substance associated with Ni-A Ni-B-Hepatitis from feces of patients.

Buffer used: Tris-HCl. pH 7.4, 0.05 n. in all stages of work.

a) Treatment: 0.5 g of feces is suspended in 10 ml of buffer, centrifuged at SOOOg and the supernatant is collected. The residue is washed again with 10 ml and then with 5 ml of buffer. The collected supernatants are centrifuged (30 minutes, 10,000 rpm) and filtered through a bacteria-impermeable filter (about 0.22 millipores). The supernatant was precipitated with PEG 6000 (final concentration 10%, respectively. 0.4

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mol / l). After 2-12 hours, the precipitate was separated by centrifugation and dissolved in 10 ml of buffer. This solution was shaken with 10 ml of freon, the phases were separated by centrifugation, and the freon phase was again washed with 5 ml of buffer. Deposition with PEG and NaCl is repeated (final concentration 10%, 0.4 mol / l, respectively). The pellet is treated with about 800 μl of buffer.

The resulting solution was digested with RNA and DHKase for 1 hour at 37 ° C and applied to cesium chloride gradients.

b) 1 ml of cesium chloride solution with a density of 1.4 g / ml and 3 ml of the solution with a density of 1.3, 1.25 and 1.2 g / ml are applied to the centrifugation tubes. All solutions are prepared on the indicated buffer. 800 μl of feces were layered on a gradient, centrifuged for 65-72 h at 10 ° С (31000 rpm). Then fractions of the lower region of the gradient (density 1.4-1.4 g / ml) are collected in approximately 200 μl, and large fractions can be selected in the upper region with a density of 1.1-1.2 g / ml. The density of each fraction is determined by measuring the refractive index. All fractions are dialyzed and 50 μl of each fraction are examined by NANB analysis for a substance associated with Hu-A, Ni-B-hepatitis. In positive fractions, the protein concentration is determined.

Identification and characterization of the substance.

c) Preparation of gradient stripes.

The dialyzed and possibly concentrated fractions of the cesium chloride gradient are mixed with 10 µl of SDS, 10 µl of glycerol and 5 µl of beta-mercaptoethanol, 5 µl of bromphelus blue per 100 µl of sample and boiled for 3 minutes on a water banana. They are then mixed with 5 µl of a 0.1% pyrone solution and applied to a gradient polyacrylamide gel. Duration 3.5 hours, voltage 160-300 V, current 40-25 A, power 20 W.

Approximately 5 minutes before the end of electrophoresis, 5 µl of pyronin was applied again and the operation was completed as soon as pyronin passed through the gel. The gel is either stained with silver or blotted overnight at 0.5 A, then 1 hour at 1 A.

d) Blot analysis.

At the end of the blot, the various bands (highlighted by pyrrhin markings) are cut out and, accompanied by a molecular weight standard, are stained with amido black. Sample strips are shaken for 24-72 hours at 1% -nrm.

gelatin solution in PBS. 1d6 fraction of the patient, respectively, Fab-2 fragments of this lgG fraction are labeled with iodine-125 (chloramine T): 0.5 µg per 100 µg

protein. Approximately 70% of the activity is incorporated. A radioactive indicator, the volume of which corresponds to about 2 µg of protein, is diluted in 20 ml of gelatin in PBS and then the strips are incubated with shaking for 12 hours. After this, the strips are washed 1 hour 3 times with gelatin-PBS, 2 times using PBS-tween 0.5% and 1 time with water. After drying, the strips are placed on a sensitive X-ray film and exposed at -70 ° C for 2-7 days.

Example 2. Method for the detection of an HNANB-associated substance in feces,

0 Polystyrene beads together with the serum of recovered patients who suffered from Hu-A, Ni-B-hepatitis, in a dilution of 1: 200 in carbonate buffer, pH 9.2, 0.01 mol / l, are incubated for 12 hours at room temperature

5, the beads are washed with buffered phosphate saline sodium chloride (PBS). Samples of feces in the form of a 10% suspension of feces are incubated for 2 hours at 37 ° C with covered with polystyrene as indicated, then washed abundantly with PBS, which contains 0.5% Tween-20, and then incubated at 37 ° C for 1 h with human IgG from serum recovered from Hu-A-Ni-B5 hepatitis, which is labeled with iodine-125. After washing with distilled water, the radioactivity bound to the beads is counted on a gamma counter. Feces samples in which radioactivity is bound

0 reaches three times the negative control, is considered positive for the presence of a substance associated with Ni-A, Hu-B-hepatitis.

It has been established that a positive reaction is found in patients with Hu-A, Ni-B-hepatitis, that the use of this analysis in a large number of patients with a mass of completely different liver diseases, which have nothing to do with Hu-A,

0 Hu-B-Hepatitis, a substance, if it is found at all, is found only in a very low percentage.

During the study of groups of patients with hepatitis of other genesis or

5 Hepatitis A or Hepatitis B, a substance associated with Hu-A, Hu-B-Hepatitis A substance is in a very low percentage.

It was established that patients with Hu-A, Ni-B-hepatitis have 30% positive results, as opposed to that in cases of suspected Hepatitis A, Hepatitis B and posthepatitis B in patients this number is much lower.

Example 3, DNA isolation, from feces and cloning of DNA sequences. . Patients with Hu-A and Ni-B-hepatitis, as described in Example 1, isolated particles associated with Ni-A and Ni-B-hepatitis, as described there, are processed and purified through cesium chloride gradients and dialyzed. . The dialyzed fractions are examined for the presence of a substance associated with NANB in the manner described. A fraction of 1.3 g / ml in gradients of cesium chloride is digested with 50 μg proteinase K, 1% SDS and EDTA at a final concentration of 10 mM for 6 hours at 37 ° C. The proteins are removed by extraction with 80% phenol (w / v) and chloroform, and then dissolved in the aqueous phase, DNA in the presence of 0.3 M sodium acetate is precipitated with two and a half times the volume of ethanol for 60 hours at 70 ° C. Then it is centrifuged and the precipitate is washed once with 70% ethanol, dried and then treated with TE buffer consisting of 10 mmol / l Tris, pH 3, and 1 mM EDTA in a volume ratio of 1 μl / 5 mg processing feces.

15 μl of this basic DNA solution is incubated for 1 hour at room temperature in a total reaction volume of 50 μl with 6 units of Klenow polymerase (DNA polymerase 1, large fragment), 1 μl of dATP solution, dTTP and dGTP, in a concentration. 1 μM, as well as 5 μl of alpha-32 P-dCJP in incubation buffer for Klenow polymerase, according to the manufacturer.

After that, 2.5 µl of 1 mM dCTP solution is added, incubated for 1 hour at room temperature, heated at 68 ° C for 10 minutes, and cooled to OC. Then the reaction mixture together with 2 units of T4-DNA ligase, 1 μg of phosphorylated EcoR-1 linker and 6 μl of 10 mM dATP solution are incubated for 16 hours at. The final mixture is then set to a final concentration of 150 mM NaCI and, together with 240 units of EcoR-1-restriction endonuclease (80 units / μl), are digested for 3 hours at 37 ° C. The reaction is stopped by the addition of 5 μl of 80% phenol and t μl of 20% SDS. Radioactively microorganized and equipped with linkers, DNA is separated from salt and undoped linkers through a column with sepharose (column volume 3 ml, length 25 cm) and precipitated 2.5 times with ethanol for t6 h at -7b ° C

The precipitated DNA is centrifuged for 10 min at 14000 d, the precipitate is washed with 150 μl of 70% ethanol, dried and treated with 10 μl of TE buffer.

Example 4: Cloning of isolated DNA in lambda phages and transfection with bacteria.

For ligation with the DNA vector, 2 µg of lambda-phage 1149 DNA together with 2 EcoR-1 units (4 units / µl) in a total reaction volume of 6 µl / manufacturer's incubation buffer are incubated for 1 hour at 37 ° s The enzyme is then inactivated by heating for 40 minutes at 68 ° C. This solution is ligated with 3 μl of labeled DNA, which is isolated as described in Example 3, 1 μl of 5 mM ATP and 1 unit. T4 DNA ligase at room temperature. 4 μl of this reaction mixture is packaged in lambda phage casings. These packaged phages infect Escherlchla coli strain NM514. For screening for the presence of inserted DNA, approximately 105 phages were plated onto screening plates 22x22 cm in size and incubated overnight at 37 ° C. The DNA phages are transferred to a filter from nitrocellulose by means of a print, the filter is hybridized with 1 µl of the described radioactive basic DNA solution, washed and autoradiographed for 6 hours at -70 ° C.

Out of 70 positive hybridization signals, 17 subordinate phage colonies are selected at a density of approximately 5 platelets per cm2. After purification of the platelets, 16 positive clones are prepared, which contain DNA inserts from 0.3 kb in length. to about 1.5 kb and hybridize with a sample of the original basic DNA solution.

Example 5. Characterization of a DNA fragment with a length of 0.45 kb and subcloning of this fragment into plasmid pVCl9 in Escherichia coli.

The DNA fragment is removed from the phage DNA by digesting with the endonuclease EcoR1 under the conditions described. The fragment is separated on agarose gel from lambda-phage DNA, and the corresponding DNA strips are cut out by 5 gels and eluted. The isolated DNA insert was introduced by ligation into the pVC19 vector DNA and then transfected into Escherichia coll. Strain H 1. After selection of the bacterial strains, cultivation of the strain is carried out.

The insert is labeled radioactively and tested by a Southern analysis for hybridization with the starting material, extraction from the feces of control persons, the hepatitis virus and the plasmid PBR 322.

Example 6. Detection associated with Ni-A, Ne-E-DNA in serum.

2-5 ml of serum are centrifuged at 150000 g for 2 h. The precipitate is digested at 37 ° C for 1 h in 200 µl of proteinase K solution (0.5 mg / ml, 10 mM EDTA), to the solution 165 µl mountain A saturated solution of sodium iodide (2.5 g sodium iodide / ml HiO, 75 ° C) to a final concentration of 12.5 M is heated for 1.0 min at 90 ° C and filtered directly through a nitrocellulose filter under vacuum. After filtration, nitrocellulose. the film is washed 3 times with 70% ethanol and then 10 minutes at room temperature: incubated in 100 ml of acetic anhydride (100 ml, 0.1 M triethanolamine, 250 μl of acetic anhydride). After incubation, the film is calcined in vacuum at 80 ° C for 1-2 hours. The dried film is introduced into the prehybridization solution, incubated for 3 hours at 65 ° C (6xSSC), 1 x Denhardt, 0.5% SDS, where 20 x SSC (standard salt citrate) corresponds to ZM NaCI, 1.5 M sodium citrate.

SDS is sodium dodecyl sulfate,

100 x Denhardt - solution corresponds to 2% Ficoll, 2% polyvinylpyrrolidone, 2% bovine serum albumin), 20 µg / ml denatured, heated sperm DNA), incubated with a 32P-labeled probe overnight under prehybridization conditions. The nitrocellulose film is then washed 3 times in solution (1 x 6 SSSc, Ix Denhardt, 0.5% SDS, 20 μg / ml DNA sperm, 1 x SSC, 0.5% SDS, 1 x Denhardt, 0.1 x SSCt.-0 , 05% SDS). After drying, the film is applied to the X-ray film for a period of 6 hours to 2 days at 70 ° C.

Example 7. Detection of Hu-A, No-B-DNA in serum.

200 μl of syvorki are incubated with 75 μl of the proteinase K solution (4 mg / ml) and 25 μl of 0.5 M EDTA at 37 ° C for 1 h, then added 100 μl of 1N. NaOH, incubated for 10 min at room temperature and neutralized with 250 μl of 2M ammonium acetate. 500 µl of the mixture (respectively, 181 µl of serum) is applied to nitrocellulose, neutralized with 250 µl of 2M ammonium acetate, and dried in vacuo. At 80 ° C for 45 minutes. The filters were rehybridized in 6 x SSC, 0.5% SDS, 1 X Denhardt solution and 20 μl / ml denatured (5 min, 100 ° C) sperm DNA at 65 ° C for 3 h.

For hybridization, which is carried out overnight under prehybridization conditions, a 32 P labeled Nick broadcast is used.

insertion of phage clones (specific activity of about 1-2 x 10 cpt (µg of DNA). Filters are then, under the circumstances, washed again for 20 min at 65 ° C with 6 x SSC, 1 x Denhardt solution, 0.5% SDS: 20 µg / ml of denatured Hering sperm DNA, then 1 x SSC, 0.5% SDS, 1 x Denhardt solution and 0.1 x SSC, 0.05% SDS and dried at 80 ° C. Autoradiography performed with an x-ray film and an amplifying film with an autoradiography time of 6-48 h at -70 ° C.

Example 8. Sample preparation for analysis.

1 ml of serum with 350 μl of proteinase K solution (3 mg / ml proteinase K, 10 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5% SDS) and 125 μl 0.5 M EDTA are incubated for 30 minutes at 37 ° C. The solution is then diluted with 8 ml of 2 M TCA (pH 7.0), 3.2 ml of 2M ammonium acetate, 20 µl of RNA (10 mg / ml) and 5.3 ml of NgO to 16 ml. DNA is precipitated with 10 ml of isopropanol at room temperature for 30 minutes. The DNA precipitate is separated by centrifugation (15 minutes at 8000 rpm), washed with 2 ml of 70% ethanol, dissolved in 600 µl of 10 mM Tris, pH 8.4, and 1 mM EDTA, extracted 1 time with phenol, 1 time chloroform and precipitated again. Al and quota portions of the precipitated DNA are used for Dot blot analysis or for restriction analysis.

The production of particle-associated DNA from samples of the feces is carried out in the same manner. Stool suspensions are pre-sterile filtered and precipitated with PEG.

Example 9. If there is relatively little substance associated with Hu-A, Ni-B, then it is advisable to detect the DNA by amplification.

Detection of Ni-A, Hu-B-DNA in serum is carried out by hybridization with a radioactively labeled cloned Hu-A, Ne-B-DNA sample after preliminary specific enzymatic DNA amplification.

25 μl of serum, 50 μl of Nal (saturated solution) are mixed, incubated for 2 minutes at 37 ° C, and then incubated for 10 minutes at 0 ° C. Incubate is dialyzed on a polycarbonate membrane against TE buffer (10 mM Tris, 1 mM EDTA) for 20 minutes. 5 μl of dialysate are selected for specific farm-specific DNA amplification using 0.5 μg of oligoprimer (A and B pairs, respectively, C and D). Oligomer pairs are prepared from known sequencing data using a DNA synthesizer. After amplification, the reaction mixture is separated by electrophoresis on a 2% agarose gel, applied to a nylon membrane - and hybridized with the tagged with 32 P, cloned Hu-A, Hu-B-DNA sample. After autoradiography, positive results are identified by reference to the standard and markers. Accompanying controls serve to evaluate the efficiency, amplification, using both HBV-specific primers, as well as using specific to Ni-A, Hu-B primers. Evidence of identity in infectious DNA loading with DNA from feces of patients with Ni-A, Ni-B-hepatitis speaks about the identification of DNA included in Hu-A, Ni-B-hepatitis.

Investigation of further 57 stool specimens from patients with sporadic and post-hemotransfusion Hu-A, Ni-B using radioimmunoassay, as described in the previous examples, and Dot-blotting, gives a remarkable correlation of both research methods with respect to detectable associated with Ni-A, Ni-B-substance (R 1 A) and detectable DNA (Ho-blot, see Example 7).

DNA shows the relationship with. HBV-DNA. If a cloned 0.45 kV fragment or purified material has been labeled with P and hybridized. In a Southern analysis to .HBV-DNA, both samples give a signal, albeit about 1000 times weaker than it is by itself. Plasmid pBR 322 and lmbda-phages do not give any signal. In preliminary experiments, a cleaned stool sample, without performing denaturation, can be very well labeled with a small fragment of E coll polymerase.

The processed polymerase Klenow stool sample migrates distinctly slowly in the agarose gel than the untreated sample. This suggests that the investigated DNA, as well as HBV-DNA, is partially double-stranded. The results and processing of restriction enzymes and the extra-primer proves the circular DNA structure.

The obtained DNA, in particular, the genes located in these DNAs, can be used for incorporation into appropriate expression vectors like cells and bacteria.

(e.g. E.coll and yeast, such as Saccharomyces cerevislae, as well as cell cultures) and for the production of viral antigens. Thus, it is possible to diagnose and receive immunoreagents and vaccines. AT

In the case of diagnosis, it is necessary to mention in particular the study on infectivity (the study of canned blood) and the diagnosis of an acute, chronic or past infection. Cooked

In cell cultures, antigens, as well as the corresponding, synthesized by this virus-DNA, in vivo and in vitro proteins can be used to establish an immunological diagnosis. The DNA sequence can be used to identify potential viral proteins (proteins) and produce them synthetically, therefore, obtain synthetic antigenic peptides.

Claims (1)

Invention Formula The method of obtaining DNA for the diagnosis of Ni-A, Hu-B-hepatitis, consisting in pre-sterilizing a stool suspension by filtering it through a 0.22 µm millipore filter with a pore size of 0.22 µm deposited with polyethylene glycol 6000 at a final concentration of 10%, separating the precipitate, dissolving it in a buffer, shaking with freon 1: 1, separating the freon phase, precipitating polyethylene glycol 6000 at a final concentration of 10%, treatment with RNA and DNA-az, fractionation in cesium chloride gradient, separation of the fraction with a density of 1.3 g / ml, dialysis, treatment of the obtained fraction with proteinase K, extraction with 80% phenol and chloroform, processing of DNA with EcoR1 restriction enzyme, cloning of the DNA fragment in the plasmid pBR 322 or in lambda phages.