The strain of the virus potato to obtain the antigen used in the production of enzyme diagnosticum

 

(57) Abstract:

Usage: vitaminology, in particular the immunodiagnostics of plant viruses. The new strain Y - virus potato. The inventive Strain isolated from naturally infected potato plants varieties Lorh. He was awarded the cipher UVC-0-1L. He deposited in the collection of the NPF Biotechnet under # 1. The titer of the antisera obtained by immunization with strains UVC-0-1-L was 1: 819200, and using previously known strains UVC 1:102400-1:409600 in the indirect version of the IFA and the sensitivity of diagnostic kits made from monochromatic antisera, respectively 3 and 7-30 ng/ml of virus. Strain UVC-0-1L is characterized by a high yield of virus when cleaning, stability during long-term storage and allows you to get highly specific antisera for the preparation of ELISA-based assays. table 1.

The invention relates to vitaminology, in particular to the immunodiagnostics of filoviruses, and relates to a new strain Y-virus potato (UCMJ).

Known strains Y potato virus with different antigenic activity, on the basis of which the received diagnostic serum for serodiagnosis of UVC.

Also known strain Y-virus for the immunization drug which obtained antisera with a titer of 1:4096 - 1: 8192 in test microprecipitation, which received the copyright certificate, as antigenantibody strain UCMJ.

The disadvantage of sera obtained through the above-mentioned strains UCMJ, is that for them to get used nizkozhirnye antigens that can not recommend them to receive antigenantibody highly purified preparations in the production of immunofermentic-based assays.

The aim of the invention is the selection of a new strain Y-virus potato to obtain highly purified immunologically active drugs with the aim of improving the quality of ELISA-based assays and the detection UCMJ.

Strain UVC isolated from naturally infected potato plants varieties Lorh. He was given the room UVC-0-1L, which he deposited in the collection of strains of potato viruses NGOs potato.

The strain is characterized by the following properties.

Morphological and physical properties. Strain UVC-0-1L has filiform, containing single-stranded RNA particles with a size of 730 11 nm. Point temperature inactivation 54 + 1oFrom that point limiting dilution 10-3. Infectious properties in the juice of the plant Nicotiana tabacum, with 2o the and virus 32000 D, floating density of virions to 1.32 g/cm3in CsCl.

The range of susceptible plants. Strain UVC-0-1L on the plants of potato varieties Lorch causes symptoms banded mosaic. When infected plants of N. tabacum cultivar Samsun NN causes systemic mottling vague type. The plant of physalis Physalys florydana causes systemic necrosis of stems and leaves. Inoculation of shares of leaves of indicator plants of the hybrid A6 (S. demissum x l'aquila) and TE-1 (S. chacoense) at a temperature of 22-24oWith and illumination 10-12 thousand Suite on the 7th day appear dark brown local necrosis rounded.

The cultivation. Strain UVC-0-1L is transmitted mechanically by using carborundum on tobacco plants Nicotiana tabacum cultivar Samsun NN, which are used for the accumulation of viral antigen. The virus is found in the leaves of infected plants drip methods agglutination and enzyme-linked immunosorbent assay (ELISA) 10-14 days after infection.

Characterization of the antigen. Virions strain UVC-0-1L in tobacco plants Nicotiana tabacum accumulate in high concentrations, limiting the breeding juice in DAS-ELISA variant ELISA reaches 1:10 and more. The output of the high-purity of the drug is 10-50 g/l of the tobacco leaf. Drugs ha is issledovanii drugs methods SDS-electrophoresis and immunoblotting, and high antigenicity (minimum detectable virus concentration in freshly isolated preparations of less than 30 ng/ml). Highly purified preparations were stored in 50% glycerol at -18oWith no noticeable decrease in antigenic properties of the virus after 12 months of storage.

Storage conditions of strain. Strain UVC-0-1L support on plants Nicotiana tabacum in vitro in sterile culture. In vitro plants periodically (after 1.5-2 months. ) division, and moved onto fresh medium.

Comparative study of strain.

Comparative pathogenicity of the strain UVC-0-1L conducted in the Department of biotechnology NGOs for potato, Russian agricultural Academy in 1985-1990.

The pathogenicity of the strain UVC-0-1L, compared with 8 other strains Y potato virus was studied on potato plants Solanum tuberosum L. tobacco Nicotiana tabacum L. glutinosa Nicotiana glutinosa L. and physalis Physalys floridana Rydb.

On the basis of the received data of the studied strains were divided into normal UVC-0 (3 strains), necrotic UVC-N (4-strain) and strahovate poloschatosti UVC-(2-strain). Strain UVC-0-1L referred us to a group of normal strains Y-virus potato. On the plants of potato varieties Lorch causes symptoms p is astaneh Nicotiana glutinosa, Physalis floridana and characteristic symptoms of systemic craptastic on leaves of Nicotiana tabacum. For necrotic strains of group is characterized by severe symptoms of systemic necrosis of veins Nicotiana tabacum, systemic mottling on the leaves of Physalis floridana and very mild symptoms of craptastic leaves on almost all varieties of potatoes. For strains strahovate poloschatosti characteristic reaction of hypersensitivity to many varieties of potatoes, as well as the typical symptoms of systemic strahovate poloschatosti or mild mosaic on leaves of Nicotiana tabacum.

Example 1. The application of strain UVC-0-1L to obtain antisera.

For accumulation of the virus with the aim of identifying viral antigen using plants tobacco Samsun NN, immune to TMV that infect infectious juice containing strain UVC-0-1L derived from the stem of the tobacco plant.

Infected plants are grown in isolated conditions in the greenhouse or poly-gauze house. For them to spend care, which is watered with tap water and soil loosening.

The maximum accumulation of the virus is observed after 14-20 days after infection. Before cutting the leaves of all the plants tested for the presence of virus and the absence of other virus is 2 min with chilled 0.05 M phosphate buffer pH 9,0, containing 0.005 M EDTA and 0.2% 2-mercaptoethanol in a ratio of 2 ml buffer per 1 g of leaves. Juice squeezed through cheesecloth bring the pH to 7.8 to 8.0 1 M KOH and centrifuged at 12000 rpm for 20 min at 5oC. the Precipitate discarded, and the aqueous phase add the Triton-X-100 to a final concentration of 0.5% is stirred in the cold for 30 min and precipitate the virus by adding PEG-6000 to a final concentration of 5% and NaCl to 0.15 M. the Mixture was incubated 2 hours in the refrigerator and centrifuged for 20 min at 12000 rpm, the Supernatant discarded, and the precipitate is shaken out three times virus 0.3 M glycine buffer pH 7.5, leaving each time the solution by low-speed centrifugation at 10,000 rpm for 15 minutes, the Total volume of the extract should be 1/10 the original volume of juice. Next, the extract is applied on a sucrose cushion (250 mg/ml sucrose in 0.05 M glycine buffer, pH 7.5, 15% of the volume of the tube) and centrifuged at 130000 g for 2.5 h at 12oWith the bucket-rotor ultracentrifuge. The final preparation is produced by extracting the virus from the material that passed through a sucrose cushion, 0.05 M glycine buffer, followed by low-speed centrifugation for 15 min at 1000 rpm In the received drugs UCMJ was determined by the concentration and purity of spectrophotomet the a and the ratio a/A 1,21 1,25 for highly purified preparations. The high purity of the obtained preparations also confirmed by electron microscopy, SDS-electrophoresis and immunoblotting. Samples were preserved with glycerol to 50% and kept at -18oC.

Immunization of rabbits was performed four strains UVC UVC-0-1L, UVC-0-B, UVC-N-B (highlighted in Belarus), UCMJ-N-D884 (highlighted in Germany) scheme consisting of three combined intradermal and subcutaneous injection at a dose of 100-200 mg of virus per injection with complete and incomplete adjuvant's adjuvant at intervals of 15-20 days. The blood was collected in 7-9 days after the last injection.

Received monstermovie antisera were characterized by specific and non-specific titers in indirect variant of ELISA. Isolated from antisera by using chromatography on DEAE-cellulose and vysalivaniya ammonium sulfate specific antibodies and derived conjugates with high purity horseradish peroxidase was also characterized by the sensitivity of the determination of purified virus in DAS-ELISA and the degree of antigenic relatedness of strains in PAS-ELISA variants ELISA (table).

The table data show that immunization of rabbits with antigen strain UVC-0-1L allowed to receive anticigarette with the greatest range of antigenic affinity, that gives the opportunity to obtain enzyme-based assays of higher quality.

Example 2. Receiving enzyme-linked immunosorbent diagnosticum on the basis of the antisera obtained for strain UVC-0-1L.

Native anticigarette obtained by immunization of rabbits strain UVC-0-1L, diluted 4 times in 0.01 M phosphate buffer pH 8.0 and applied to the activated and equilibrated with the same buffer to a column Packed with DEAE-Servicecom DE-52 in the ratio of 5 g of dry sorbent in 10 ml of native serum and suirable rezorbirovanny proteins buffer to obtain the value A less than 0.05. Rezorbirovanny fraction was besieged twice at 40% saturation with ammonium sulfate and centrifuged precipitate for 15 min at 10000 rpm protein content was Determined spectrophotometrically by absorption at 280 nm and the purity of the globulin fraction ratio A/A. For highly purified preparations of immunoglobulins, this ratio should be from 2.3 to 2.6. Next part of the immunoglobulins were frozen or freeze-dried and used as coating antibody and the other part conjugatively with high purity by the horseradish peroxidase method controllable periodic destruction of oxidation.

Tested immunodiagnostics obtained from Antis who imennyh drugs (3 ng/ml) had immunodiagnostics, obtained using antisera obtained for strain UVC-0-1L. Immunodiagnostics derived from strain UVC-0-1L cross-reacted with 8 other strains UVC related to three major strain groups with sensitivity higher than the sensitivity-based assays received to other strains, and did not give a positive reaction with the juices a healthy crop of potatoes and tobacco.

Thus, the use of strain UVC-0-1L allows to obtain highly purified preparations UVC high yield of virus is stable for a long time when stored, immunization, which allows to obtain high quality antisera suitable for preparation of ELISA-based assays of high quality.

The strain of The virus potato NPF "Biocenter" 1 for antigen used in the production of enzyme diagnosticum.

 

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