RU2144565C1 - Recombinant plasmid dna pq-f35 encoding fused polypeptide f35 showing antigenic and immunogenic properties of marburg virus protein vp 35, method of its preparing and strain of bacterium escherichia coli - superproducer of recombinant polypeptide f35 - Google Patents

Abstract

FIELD: molecular biology, biotechnology, genetic engineering, medicine. SUBSTANCE: invention relates to medicinal virology and immunology and can be used for carrying out immunodiagnosis of hemorrhagic Marburg fever and for preparing specific antibodies to Marburg virus and genetic engineering vaccines against Marburg virus. Recombinant plasmid DNA pQ-f35 encoding polypeptide f35 showing antigenic properties of Marburg virus protein VP 35 and recombinant strain of bacterium E. coli JM 103 [pQ-f35] are constructed. This strain provides biosynthesis of this polypeptide at expression level 70 mg of recombinant protein/l of cultural fluid at culturing on liquid nutrient media. Plasmid pQ-f35 has optimized promoter sequence consisting of phage T5 promoter recognized by E. coli RNA-polymerase and two lac-operator sites showing high level of lac-repressor binding and effective expression of the strong phage promoter. EFFECT: enhanced specificity and sensitivity of known methods of immunodiagnosis. 3 cl, 5 dwg, 4 ex

Description

 The invention relates to biotechnology, in particular to genetic engineering and medicine, specifically to medical virology and immunology, and can be used to carry out immunodiagnostics of Marburg hemorrhagic fever, as well as to obtain specific antibodies to Marburg virus and genetically engineered vaccines against Marburg virus.

 Marburg virus (VM), a member of the filovirus family, causes hemorrhagic fever in humans and apes with high mortality rates [3, 4, 5, 6]. The natural reservoir of filoviruses is not currently known [3]. There are no effective means of specific prophylaxis and treatment of this kind of infection [3, 4, 5]. Diagnosis of filovirus fevers is currently carried out mainly in the late stages of the disease using the methods of clinical medicine, electron microscopy, serodiagnosis, which have a number of significant drawbacks associated with purely technical difficulties in organizing this kind of work with highly pathogenic viruses, as well as with some molecular biological features these viruses [3, 7, 8]. For example, the clinical picture of filovirus hemorrhagic fevers is similar to that of hemorrhagic diseases caused by arenaviruses; with electron microscopy, Marburg virus is morphologically poorly distinguishable from Ebola virus, and the specific reactivity of positive sera when diagnosed by enzyme-linked immunosorbent assay or immunofluorescence requires additional confirmation by radioimmunoprecipitation or immunoblotting [3]. Therefore, for the implementation of anti-epidemic and preventive measures, it is urgent to develop diagnostic test systems that can detect both people infected with these viruses and possible natural carriers of these viruses [3].

 Methods of immunodiagnostics of the Ebola and Marburg viruses are mainly based on the determination of antibodies to viral proteins in blood serum (serodiagnosis) [3, 8, 9]. In this case, common immunological methods are used, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence, immunoblotting and radioimmunoprecipitation [3, 8, 9, 10].

 One of the most promising ways to improve these diagnostic methods is the use of recombinant viral proteins synthesized in various expression systems. The use of recombinant proteins preserving the antigenic determinants of viral proteins as an antibody-binding substrate instead of inactivated viral antigens increases the specificity and sensitivity of these methods, makes them safe to use and reduces their cost [11].

 Currently, literature describes methods for producing recombinant prokaryotic proteins based on the glycoprotein (GP) and nucleoprotein (NP) genes of the Marburg virus [1, 2]. The BM glycoprotein gene was expressed using the QIAExpress QIAExpress expression system, but the authors do not provide data on the level of expression, purification of the recombinant protein and its use for the production of immune sera [1]. The VM nucleoprotein gene was expressed as a protein fused with β-galactosidase, however, the expression level of the recombinant nucleoprotein is not evaluated in this work either, it is not purified, and the immune antisera obtained using it are used in immunological reactions with (relatively low) working titers from 1:40 - 1: 300, which may be explained by such well-known reasons in the literature as poor solubility of recombinant proteins fused to β-galactosidase, low concentration of specific protein in m materials under applicable to the immunization, represented in this case slurry polyacrylamide gel used for electrophoretic separation of cell lysate protein-producing strains [2]. In addition, from the data presented in these studies, it can be concluded that the recombinant glycoprotein and nucleoprotein in E. coli cells undergo intensive proteolysis, which can lead to the destruction of the antigenic structure of the recombinant protein.

 The use of the Marburg virus VP35 protein in the immunodiagnostics of filovirus infections, as well as the preparation of recombinant proteins based on it, has not been previously described in the literature. Prototypes of the claimed technical solution are currently not available in the literature.

 An object of the invention is to obtain a recombinant polypeptide, an integral part of which is the amino acid sequence of the full-sized protein Mar35 virus VP35 and the creation of a bacterial strain Escherichia coli - a producer of a recombinant protein with antigenic and immunogenic properties of the virus protein VP35.

 The problem is solved by constructing recombinant plasmid DNA pQ_f35 that encodes a f35 polypeptide with antigenic properties of viral VP35, and Escherichia coli strain JM103 [pQ_f35], which ensures biosynthesis of this polypeptide with culture expression on liquid nutrient media with expression level not lower than 70 mg of recombinant protein liquids. A high inducible level of biosynthesis of the target polypeptide is ensured by the fact that plasmid pQ_f35 contains an optimized promoter operator sequence consisting of a T5 phage promoter recognized by E. coli RNA polymerase and two lac operator regions capable of a high level of lac repressor binding and efficient repression of a strong phage promoter.

The resulting plasmid pQ_f35 is characterized by the following features:
has a molecular weight of 2.99 megadaltons (4530 bp);
encodes the amino acid sequence of the VP35 VM gene;
comprises:
- a fragment of the vector plasmid pQE31 [12], including the promoter of phage T5; lac operator sequences; lambda phage and rrnB gene transcription terminators of E. coli; β-lactamase gene and replication initiation ori site;
- an SspI-HpaI fragment of a DNA copy (cDNA) of the VM genomic RNA (Popp strain) from 2879 to 3945 nucleotides (according to GENBANK Accession N Z29337 sequence), including the full-size VP35 gene of Marburg virus;
contains:
- promoter of phage T5;
- lac-operator sequences to enhance binding of the lac repressor;
- lambda phage transcription terminators (t ₀ ) and E. coli rrnB gene (t ₁ );
- as a genetic marker, the β-lactamase gene (amp ^R ), which determines the resistance of E. coli cells transformed with plasmid pQ_f35 to penicillin antibiotics;
- a nucleotide sequence encoding a block of six consecutive histidine amino acid residues, which is part of the fusion sequence of the recombinant protein f35 and allows you to select the recombinant polypeptide by metal chelate chromatography;
- unique restriction endonucleases sites having the following coordinates:
XhoI - 2
EcoRI - 89
BamHI - 148
EcoRV - 486
BglII - 736
NsiI - 991
Bsp120 - 1212
EspI - 1268
XbaI - 2233
TthlllI - 2393
PvuI - 3909
AatII - 4461
The method for constructing the pQ_f35 plasmid consists in embedding into the plasmid pQE31 [12] linearized at the SmaI site of the SspI-HpaI fragment of the cDNA of the genomic RNA of the BM (Popp strain) from 2879 to 3945 nucleotides (GENBANK Accession N Z29337). The SspI-HpaI fragment of plasmid DNA pMBG226 / 825 obtained by cloning a fragment of BglII-PstI of plasmid pMBG825 into plasmid pMBG226 [13] at BglII-Zsp2I sites is used as a cDNA fragment of the BM genome.

To create a producer strain of the recombinant f35 polypeptide, competent bacteria cells of Escherichia coli JM103 are transformed with the constructed plasmid DNA pQ_f35. The resulting strain JM103 [pQ_f35] is characterized by the following features:
Morphological signs. The cells are small, thickened, rod-shaped, gram-negative, non-spore.

 Cultural signs. When growing on the surface of an agarized nutrient medium, LB colonies are round, smooth, shiny, gray-cream, the edge is uneven, the texture is pasty. When growing on liquid nutrient media (LB, YT, 2xYT) they form an intense, even haze.

Physico-biochemical characteristics. Cells grow at a temperature of from 5 to 37 ^o C at an optimum pH of from 6.5 to 7.0. Organic compounds in the form of peptone, tryptone, yeast extract are used as a nitrogen source. Amino acids and carbohydrates are used as a carbon source.

 Antibiotic resistance. Cells show resistance to ampicillin at a concentration of up to 300 μg / ml, due to the presence of the β-lactamase gene in plasmid DNA.

 Strain JM103 [pQ_f35] provides inducible IPTG (isopropyl-β-D-thiogalactoside) synthesis of recombinant protein f35, with the expression level when growing on liquid nutrient media up to 70 mg of the target protein per liter of culture fluid.

 The essence of the invention lies in the fact that the BglII-PstI fragment of the plasmid pMBG825 [13] is cloned into the plasmid pMBG226 [13] at the BglII-Zsp2I sites, and an intermediate plasmid pMBG226 / 825 carrying the cDNA fragment of the BM genomic RNA from 2374 to 3965 nucleotides is obtained; then, the SspI-HpaI fragment of plasmid pMBG226 / 825 is inserted into plasmid pQE31 [12] at the SmaI site, and as a result, plasmid pQ_ f35 encoding f35 protein including the sequence of full-length VP35 BM is obtained.

 The resulting recombinant f35 polypeptide has the antigenic and immunogenic properties of the internal structural protein VP35 of the Marburg virus, having the ability to bind antibodies to the native VP35 protein of this virus and to induce the formation of specific antibodies after its administration to laboratory animals.

 The expression of recombinant protein f35 is carried out in E. coli JM103 cells using IPTG as an inducer. Indication of expression is carried out using protein gel electrophoresis and immunoblotting. Recombinant protein is recognized by the reference serum of a person who has had Marburg hemorrhagic fever [14]. The expression level of f35 when cultured in liquid culture media is about 70 mg / l of culture fluid. A single-stage purification of recombinant f35 to a purity of 85-90% and a concentration of 1.5-2 mg / ml is carried out using a Ni-NTA sorbent (QIAGEN). High-tonnage (titer up to 1: 650,000) immune antisera specific for the VP35 viral protein are obtained by standard methods using a fraction of soluble non-denaturing cell proteins of the producer strain JM103 [pQ_f35].

 Thus, plasmid DNA and a producer strain were obtained for the first time, providing expression of a recombinant protein including the amino acid sequence of the full-size VP35 gene of the Marburg virus and suitable for immunodiagnosis of antibodies to BM, as well as the production of highly specific antibodies to the internal protein VP35 of the virus.

The list of graphic materials:
FIG. 1. Physical map of the recombinant plasmid pQ_f35.

- обозначены жирным шрифтом и подчеркнуты функциональные районы плазмидной ДНК pQ_f35, обозначенные в тексте.Legend:
XhoI (2) - unique restriction endonucleases, the coordinates of their sites are indicated in parentheses,

- indicated in bold and underlined the functional regions of plasmid DNA pQ_f35, indicated in the text.

 FIG. 2. Scheme for the construction of plasmids pQ_f35.

- эндонуклеазы рестрикции, использованные при клонировании;
(2879) - координаты сайтов эндонуклеаз рестрикции, фрагментов кДНК, а также отдельных нуклеотидов по геномной РНК вируса Марбург штамм Popp (GENBANK Accession N Z29337);

- обозначены в рамке кленовые нуклеотидные отличия кДНК-фрагмента геномной РНК ВМ плазмиды pMBG226 от опубликованной ранее последовательности GENBANK Z29337 (вверху нуклеотид и его положение (в скобках) по GENBANK Z29337, внизу нуклеотид по pMBG226);

- показаны жирным шрифтом и подчеркнуты функциональные районы плазмидной ДНК pQ_f35, обозначенные в тексте;
tet^R - ген устойчивости к тетрациклину.Legend:
SspI - restriction endonucleases sites;

- restriction endonucleases used in cloning;
(2879) - coordinates of restriction endonucleases sites, cDNA fragments, as well as individual nucleotides for the genomic RNA of the Marburg virus Popp strain (GENBANK Accession N Z29337);

- maple nucleotide differences in the cDNA fragment of the genomic RNA of the VM plasmid pMBG226 from the previously published GENBANK Z29337 sequence (the top of the nucleotide and its position (in brackets) according to GENBANK Z29337, the bottom the nucleotide according to pMBG226) are indicated;

- shown in bold and underlined the functional areas of plasmid DNA pQ_f35, indicated in the text;
tet ^R is the tetracycline resistance gene.

 FIG. 3. Amino acid sequence of the recombinant f35 polypeptide.

и т.д. вверху - аминокислотная последовательность белка f35 (fusion-последовательность рекомбинантного белка подчеркнута);

- - показана жирным шрифтом (слева вверху) нумерация по аминокислотной последовательности белка f35;

- показан курсивом блок из шести последовательно расположенных гистидиновых аминокислотных остатков;
ТААСТ

AGA GGA и т.д. внизу - нуклеотидная последовательность фрагмента плазмидной ДНК pQ_ f35, включающего открытую рамку считывания гена белка f35 (курсивом показана нуклеотидная последовательность, кодирующая fusion-часть рекомбинантного белка f35);
82- - нумерация по нуклеотидной последовательности плазмидной ДНК pQ_f35 (слева внизу);

- показаны жирным шрифтом и подчеркнуты инициирующие кодоны рекомбинантного белка f35 и белка VP35 вируса Марбург и кодон терминации трансляции указанных белков;
EcoRI (89) - под нуклеотидной последовательностью показаны уникальные эндонуклеазы рестрикции, в скобках указаны координаты их сайтов.Legend:

etc. above - the amino acid sequence of the f35 protein (the fusion sequence of the recombinant protein is underlined);

- - shown in bold (upper left) numbering according to the amino acid sequence of protein f35;

- shown in italics is a block of six sequentially located histidine amino acid residues;
TAAST

AGA GGA etc. below is the nucleotide sequence of the plasmid DNA fragment pQ_ f35, including the open reading frame of the f35 protein gene (italics show the nucleotide sequence encoding the fusion part of the recombinant f35 protein);
82- - numbering by the nucleotide sequence of plasmid DNA pQ_f35 (bottom left);

- shown in bold and underlined the initiation codons of the recombinant protein f35 and protein VP35 of the Marburg virus and the termination codon of translation of these proteins;
EcoRI (89) - unique restriction endonucleases are shown under the nucleotide sequence, the coordinates of their sites are indicated in parentheses.

 FIG. 4. Electrophoregram. Analysis of proteins synthesized under the control of the recombinant plasmid pQ_f35 in E. coli cells of the producer strain JM103 [pQ_ f35].

Tracks:
1 - cell lysate of bacteria E. coli JM103 [pQE31], transformed with the vector plasmid pQE31;
2 - cell lysate of bacteria E. coli JM103 [pQ_f35] transformed with the recombinant plasmid pQ_f35;
3 - preparation of purified recombinant protein f35, which is marked by an arrow;
4 - markers of molecular weight of proteins (66, 45, 36, 29, 24 kD);
5 - preparation of inactivated Marburg virus (the arrow marks VP35 BM protein);
6 - molecular weight markers of Sigma proteins: 205, 116, 97, 66, 45, 29 kDa, respectively.

FIG. 5:
A. Identification by immunoblotting of the target protein in the preparations of the recombinant protein f35 for antibodies of reference serum to the Marburg virus.

Tracks:
1 - cell lysate of E. coli strain JM103 [pQE31], negative control;
2 - cell lysate of E. coli strain JM103 [pQ_f35], producer of the recombinant protein f35;
3 - purified recombinant protein f35;
Sigma protein molecular weight markers: 45, 36, and 29 kDa are indicated on the left.

 B. Immunoblot analysis of protein-specificity of polyclonal antibodies from animal sera immunized with f35 recombinant protein preparation.

Tracks:
1 - preparation of purified, inactivated Marburg virus;
Sigma protein molecular weight markers: 45, 36, and 29 kDa are indicated on the left.

 For a better understanding of the invention, the following are examples of its implementation.

 Example 1. A method of constructing recombinant plasmid DNA pQ_f35.

 Procedures for processing plasmid DNA with restriction endonucleases, modification enzymes, electrophoretic separation of DNA hydrolysates, isolation of DNA fragments from an agarose gel, ligase reactions, production and transformation of competent E. coli JM103 cells, production and analysis of recombinant clones, isolation of plasmid DNA, restriction mapping and sequencing according to the methods of Maxam-Gilbert and Sanger was carried out in accordance with the methodological manual [15].

 1.1. Construction of intermediate recombinant plasmid DNA pMBG226 / 825 carrying a full-sized DNA copy of the VP35 VM gene.

 To create the full-sized VP35 gene cDNA, plasmids pMBG226 and pMBG825 were used with insertions of cDNA fragments of the genomic RNA of the BM (Popp strain) flanked by poly-G (C) tracts and cloned by the PBI site of plasmid DNA pBR322 [13].

 15 μg of plasmid DNA pMBG825 is treated with restriction enzymes BglII and PstI; the resulting hydrolyzate is separated by electrophoresis in 1% agarose gel; a 543 bp fragment is isolated

 2 μg of plasmid DNA pMBG226 is treated sequentially with restriction enzymes BglII and Zsp2I; the resulting hydrolyzate is separated by electrophoresis in 1% agarose gel; 5567 bp of vector plasmid DNA is isolated.

 Aliquots of a fragment of plasmid pMBG825 and the vector part of plasmid pMBG226 are used to carry out a ligase reaction in a volume of 15 μl. 6 μl of ligase mixture is used to transform competent E. coli JM103 cells. Transformants are plated on LB agar containing 100 μg / ml ampicillin. The grown clones are used to generate and isolate recombinant plasmid DNA, which is analyzed by gel electrophoresis in 1% agarose gel; previously selected, on the basis of agarose gel electrophoretic mobility, plasmid DNA is then subjected to restriction mapping and clones carrying the target plasmid, designated as pMBG226 / 825, are used for its generation, isolation, detailed restriction mapping and sequencing according to the Maxam-Gilbert and Sanger methods.

 1.2. Construction of plasmid pQ_ f35 for prokaryotic expression of the VP35 VM gene.

 15 μg of plasmid DNA pMBG226 / 825 is treated with restriction enzymes SspI and HpaI; the resulting hydrolyzate is separated by electrophoresis in 1% agarose gel; a 1066 bp fragment is selected, which is a cDNA fragment of the genome of the Popp BM strain (from position 2879 to 3945 nucleotide, according to GENBANK Z29337 sequence), including the full-length coding part of the VP35 VM gene.

 2 μg of plasmid DNA pQE31 (QIAGEN) [12] is treated with restriction enzyme SmaI; the resulting DNA hydrolyzate is then subjected to dephosphorylation using alkaline phosphatase; 3464 bp vector plasmid DNA is isolated.

 Aliquots of the obtained fragment of plasmid pMBG226 / 825 and vector plasmid pQE31 are used for ligase reaction in a volume of 15 μl. 6 μl of ligase reaction mixture was used to transform competent E. coli JM103 cells. Transformants are plated on LB agar containing 100 μg / ml ampicillin. The grown clones are used to generate and isolate recombinant plasmid DNA, which is analyzed by gel electrophoresis in 1% agarose gel; and then subjected to restriction mapping; clones carrying the target plasmid, designated as pQ_f35, are used for its production, isolation, detailed restriction mapping and sequencing according to the Maxam-Gilbert and Sanger methods. The target plasmid pQ_f35 has a positive orientation of the cloned fragment, i.e. contains an open reading frame of the gene for the recombinant protein f35 formed by (i) the sequence of the vector part of plasmid DNA, part of which is the nucleotide sequence encoding a block of six consecutive amino acid residues of histidine, and (ii) the open reading frame of the VP35 VM gene.

 Scheme of plasmid DNA pQ_f35 and a method for constructing it are shown in FIG. 1 and 2.

 Thus, plasmid DNA pQ_f35 was obtained for prokaryotic expression of the full-length VP35 VM gene. Plasmid pQ_f35 contains an open reading frame encoding a recombinant f35 polypeptide with a length of 370 amino acid residues (calculated MW = 40.983 kDa), including the full amino acid sequence of VP35 BM protein (329 amino acids long, calculated MW = 36,200 kDa), as well as a fusion sequence in N- the terminal part of the protein, of which the polyhistidine block (6xHis-tag) is an integral part, which enables the isolation and purification of the f35 polypeptide by Ni-chelate chromatography. The amino acid sequence of the recombinant protein f35, determined on the basis of the nucleotide sequence of the cloned DNA fragment, is presented in figure 3.

 Example 2. Expression and purification of recombinant protein f35.

 2.1. Expression of recombinant f35 protein.

Strain E. coli JM103 (endA1, hsdR, supE, sbcB15, thi-1, strA, Δ (lac-proAB), [F ', traD36, proAB, lacl ^q ZΔM15), transformed with plasmid DNA pQ_ f35 and designated as JM103 [ pQ_f35], cultured in 100 ml of liquid LB nutrient medium with the addition of ampicillin to a concentration of 100 μg / ml, at 37 ^o C with vigorous stirring. When the optical density of the culture fluid OD ₆₀₀ = 0.8, the synthesis of the recombinant protein is induced by adding IPTG to a final concentration of 0.2 mM and cultivation is continued for 3 hours at 30 ^o C.

The culture fluid is centrifuged for 10 min at 1300 g, at 4 ^o C. The bacterial sediment is resuspended in 5 ml of phosphate buffer B (50 mM NaH ₂ PO ₄ , 300 mM NaCl, pH 7.8), the suspension is subjected to a single freeze (-196 ^o C) - thawing (+37 ^o C) and treated with ultrasound (4 times for 15 s at 300 W, with minute pauses between treatments; at 0 ^o C). The cell lysate is subjected to weak periodic stirring for 1 hour, centrifuged (A) at 1300g for 15 minutes (at 4 ^o C); the "non-clarified" supernatant A obtained at this stage is centrifuged again (B) at 26000g rpm for 15 minutes (at 4 ^° C) and a "clarified" supernatant B, designated f35B, which is a protein fraction of the cell lysate soluble in phosphate buffer B, is obtained producer strain.

Precipitation containing fragments of bacterial cell walls and insoluble inclusion bodies obtained in the centrifugation stages (A) and (B) are resuspended in 15 and 5 ml of buffer A (8 M urea, 50 mM NaH ₂ PO ₄ , pH 8.0), respectively . The resulting suspensions are combined, subjected to non-intensive periodic stirring for 1 hour at 4 ^° C, centrifuged at 26000g for 20 minutes, and the supernatant, designated f35A, which is a denaturing cell fraction of the producer strain lysate, is used to purify the recombinant protein f35 by metal chelate affinity chromatography [12].

 The concentration of total protein in the f35A, f35B lysates, as well as in the purified protein preparation f35, is determined by measuring the optical density according to the Bio-rad-proteinassay spectrophotometer SF-26 using a calibration curve constructed for the preparations of purified ovalbumin (mol. Mass 45 kDa) s known concentrations.

 2.2. One-step purification of recombinant f35 protein using the Ni-NTA-agarose sorbent (QIAGEN).

0.2 ml of Ni-NTA-agarose sorbent (QIAGEN (Germany); 50% suspension; sorption capacity of up to 10 mg / ml of sorbent), previously equilibrated in buffer A, is added to 20 ml of f35A lysate. In the centrifuge glasses, the recombinant protein is sorbed. for 1 hour at 20 ^o C, with occasional gentle stirring. The suspension is centrifuged at 600g for 5 minutes. The supernatant is carefully decanted. To remove impurity and non-specifically bound proteins, the sorbent is washed as follows: twice with buffer A, 10 ml each, then once with 10 ml of buffer C1 (8 M urea, 50 mM NaH ₂ PO ₄ , 4 pH 7.0) and 10 ml of buffer C2 (8M urea, 50 mM NaH ₂ PO ₄ , pH 6.5). Recombinant f35 protein was eluted with 0.8 ml of C3 buffer (8 M urea, 50 mM NaH ₂ PO ₄ , 0.1 M EDTA, pH 7.0). The concentration of f35 protein in the purified preparation is 1.7 mg / ml, with a purity of 85-90%.

 The obtained cell lysates of the producer strain and purified recombinant protein f35 were analyzed by the method of protein gel electrophoresis according to Laemmli in 12% polyacrylamide gel [15]. As markers of molecular masses of proteins using a commercial set of proteins from Sigma. As controls, a similarly obtained cell lysate of strain JM103 containing the vector plasmid pQE31, as well as a concentrated, inactivated preparation of the Marburg virus, are used [16].

 A comparative analysis of the protein composition of cell lysates shows that strain JM103 [pQ_ f35] expresses a recombinant polypeptide with a molecular weight of 40-41 kDa, which can be purified in a single step by metal chelate chromatography (Fig. 4).

 Example 3. Determination of antigenic properties of the recombinant protein f35.

 Control of the antigenic activity of f35 protein is carried out by immunoblotting [15]. To do this, carry out the procedure of protein gel electrophoresis (as described in example 2), then carry out the transfer of electrophoretically separated proteins to the nitrocellulose membrane using the apparatus for electroblotting for 1 hour at 24 V, 400 mA.

 To control the quality of the transfer, as well as to control the molecular weight of the proteins of cell lysates, a part of the nitrocellulose membrane is stained with a solution of 0.2% Ponso S in 3% TCA for 5 minutes at room temperature. The paint is washed twice for 30 minutes with TBS buffer (0.15 M NaCI, 20 mM Tris-HCl, pH 7.4) with 0.05% Tween-20 at room temperature.

Next, block the unpainted portion of the nitrocellulose membrane with the immobilized proteins with a solution of 5% casein prepared in 0.1 M Na-phosphate buffer, pH 7.4, for 1 hour at room temperature. After blocking, the membrane is incubated for 1 hour at 37 ^o C with a diluted 1: 500 preparation of human serum, who had Marburg hemorrhagic fever, at a dilution of 1: 500. Then, the membrane was washed three times for 30 minutes with TBS with 0.05% Tween-20 at room temperature. The membrane is then incubated with an antispecies peroxidase conjugate at 37 ^° C. for 1 hour. Repeat the procedure three times washing. The reaction is checked by adding a freshly prepared solution (100 μl of 30% hydrogen peroxide plus 50 ml of 4-chloro-1-naphthol solution (20 mg in 0.1 M Tris-HCl, pH 8.0)). Analysis shows that the recombinant f35 polypeptide binds to antibodies to the Marburg virus (Fig. 5A).

 Example 4. Obtaining specific antibodies to the recombinant protein f35.

 In order to obtain specific antibodies, laboratory animals (3-month-old BALB \ c mice) are immunized with preparations of the recombinant f35 protein. Immunization is carried out intraperitoneally with an interval of 1 week; at the first immunization - with Freund's complete adjuvant; in the following two immunizations - with incomplete Freund's adjuvant; and without adjuvant at the last immunization before blood sampling for analysis. The total immunizing dose when using the drug f35B, obtained as described in example 2, is 202.5 μg of total protein per laboratory animal. Two weeks after the last immunization, animals were bled and serum was obtained using standard methods [15]. The detection of antibodies in the blood serum of immunized animals is carried out using an enzyme-linked immunosorbent assay using purified, inactivated Marburg virus as antigen (100 ng of antigen in 0.05 M diode phosphate buffer pH 8.0 per well of a polystyrene plate) [15]. The titer of specific antiviral antibodies in serum obtained by immunization with the f35B preparation of the recombinant f35 protein is from 1: 220,000 to 1: 650,000.

 Analysis of the protein specificity of the obtained sera is carried out using immunoblotting. A purified, inactivated Marburg virus is used as an antigen [16]. Fractionation of a viral antigen by polyacrylamide gel electrophoresis, electrotransfer of proteins to a nitrocellulose membrane, blocking of membranes and their processing by animal sera immunized with f35 recombinant protein preparation, detection of specific antibodies using an antispecies conjugate; washing is carried out as described in example 3.

 The analysis (Fig. 5B) shows that the sera of animals immunized with the recombinant protein f35 preparation contain antibodies that specifically interact with the Marburg virus VP35 protein (polypeptide with a molar mass of 35 kDa).

 Thus, the recombinant protein f35 can be used to carry out immunodiagnostics of Marburg hemorrhagic fever, to obtain specific antibodies to the Marburg virus, as well as to create genetically engineered vaccines against the Marburg virus.

Literature
1. Becker S., Huppertz S., Klenk H.-D., Feidmann H. The nucleoprotein of Marburg virus is phosphorylated.// J Gen Virol 1994 Apr; 75 (Pt 4): 809-818.

 2. Will C., Muhlberger E., Linder D., Slenczka W., Klenk H. D., Feidmann H. Marburg virus gene 4 encodes the virion membrane protein, a type I transmembrane glycoprotein. // J Virol 1993 Mar; 67 (3): 1203-1210.

 3. Peters C. J., Sanchez A., Rollin P. E., et al. Filoviridae: Marburg and Ebola viruses. // "Fields Virology" / 3rd ed. - Lippincott-Raven Press. - Philadelphia. - Vol. 1. - pp. 1161-1176.

 4. Martini G.A. and Siegert R., eds // Marburg virus disease. 1971. P. 1-230. Berlin: Springer Verlag, New York.

 5. Gear JS, Cassel GA, Gear AJ, Trappler B., Clausen L., Meyers AM, Kew MC, Bothwell TH, Sher R., Miller GB, Schneider J., Koornhof HJ, Gomperts ED, Isaacson M., Gear Jh Outbreake of Marburg virus disease in Johannesburg. // Br Med. J. 1975 Nov 29; 4 (5995): 489-493.

 6. Johnson E.D., Johnson B.K., Silverstein D., Tukei P., Geisbert T.W., Sanchez A.N., Jahrling P.B. Characterization of a new Marburg virus isolated from a 1987 fatal case in Kenya. // Arch Virol Suppl 1996; 11: 101-114.

 7. Ksiazek T.G. Laboratory diagnosis of filovirus infections in nonhuman primates. // Lab Anim 1991, 20: 34-46.

 8. Johnson K. M., Elliott L.H., Heymann D.L. Preparation of polyvalent viral immunofluorescent intracellular antigens and use in human serosurveys. // J Clin Microbiol 1981 Nov; 14 (5): 527-529.

 9. Vladyko A.S., Chepurnov A.A., Bystrova S.I., Lemeshko N.N., Lukashevich I.S. Detection of the Marburg virus antigen by enzyme-linked immunosorbent assay // Vopr. virusol. 1991, 36 (5): 419-421.

 10. Wulff N., Conrad J.L. Marburg virus disease. In: Kurstak E., ed. Comparative diagnosis of viral diseases. New York: Academic; 1977: 3-33.

 11. Dead men N.P., Beklemishev A.B., Savich I.M. Modern approaches to the design of molecular vaccines. // "Science", 1987.

 12. "The QIAexpressionist" (QIAexpress Protocols) // 3rd Ed. - March 1997. - QIAGEN.

 13. Bukreyev A.A., Volchkov V.E., Blinov V.M. and Netesov S.V. The complete nucleotide sequence of the Popp (1967) strain of Marburg virus: A comparison with the Musoke (1980) strain. // Arch. Virol. - 1995. - Vol. 140. - pp. 1589-1600.

 14. Nikiforov V.V., Turovsky Yu.I., Kalinin P.P. et al. Case of laboratory infection with Marburg fever. // Journal. microbiol. epidemiol. immunobiol., 1994, N 3, p. 104-106.

 15. Sambrook J., Fritsch E.F. and Maniatis T. Molecular cloning: a laboratory manual. // 2nd ed. - 1989. - Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

 16. Bukreev A.A., Skripchenko A.A., Gusev Yu.M. and others. A promising method of preparative production and purification of the Marburg virus. // Q. virusol., 1995, N 4, p. 161-165.

Claims (3)

 1. Recombinant plasmid DNA pQ_f35 encoding a f35 fusion polypeptide with antigenic and immunogenic properties of the Marburg virus VP 35 protein, mol.m. 2.99 MDa and a size of 4530 bp, containing a fragment of the linear plasmid DNA pQE 31, 3464 bp, sized by the SmaI site; fragment SspI-HpaI (length 1066 bp) cDNA of the genomic RNA of the virus Marburg strain Porr from 2879 to 3945 nucleotide; unique restriction sites (and their coordinates): XhoI (2), EcoRI (89), BamHI (148), EcoRV (486), BglII (736), NsiI (991), Bsp 1201 (1212), EspI (1286), XbaI (2233), TthlllI (2393), PvuI (3909), AatII (4461); T5 phage promoter, lac operator sequences for enhancing lac repressor binding, f35 hybrid polypeptide gene (coordinates 115-1124 bp), which includes the full-length coding part of the Marburg virus VP 35 gene gene (coordinates 238 - 1224 p. o.), lambda phage and rrnB gene transcription terminators of E. coli, genetic marker: β-lactamase gene, which determines penicillin antibiotic resistance.  2. The method of constructing recombinant plasmid DNA PQ_f35, which consists in the fact that the fragment BglII - PstI (543 bp in length) of the plasmid pMB G 825 is cloned into the plasmid pMBG 226 at the BglII - Zsp 21 sites, and the intermediate plasmid pMBG 226/825 is obtained, carrying the cDNA fragment of the genomic RNA of the BM from 2374 to 3965 nucleotides, then the SspI-HpaI fragment (1066 bp in length) of the pMBG 226/825 plasmid is inserted into the pQE 31 plasmid at the SmaI site and as a result, the pQ_f35 plasmid encoding the f35 protein including the f35 protein is obtained the sequence of full-sized protein VP 35 Marburg virus.  3. The bacterial strain Escherichia coli JM103 [pQ_f35] is a superproducer of the recombinant protein f35 with antigenic and immunogenic properties of the internal structural protein VP 35 of the Marburg virus.

Images