Borrelia hybrid protein, nucleic acid coding this protein, expressing cartridge, vector, method and kit for diagnosing lyme borreliosis, vaccine for borreliosis prevention

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented group of inventions concerns fused proteins, nucleic acids coding these proteins, an expressing cartridge providing nucleic acid expression, a vector comprising this cartridge, a diagnostic technique for in vitro borreliosis, a kit for this diagnostic technique, which use these proteins, as well as a vaccine composition for preventing borreliosis containing these proteins. The characterised fused proteins contain (i) at least one sequence of DbpA protein of the species Borrelia specified in B. afzelii, B. burgdorferi sensu stricto and B. garinii, and (ii) least one sequence of OspC protein of the species Borrelia specified in B. afzelii, B. burgdorferi sensu stricto and B. garinii.

EFFECT: presented group of inventions enables performing more sensitive and specific analyses related to the presence of certain pathogenic species Borrelia.

11 cl, 8 tbl, 7 ex

 

Lyme borreliosis (LB) is a non-communicable infectious disease caused by a spirochete called Borrelia burgdorferi, and is transmitted to humans by the bite of ticks of the family Ixodidae ticks. LB if untreated, causes various pathological disorders (dermatologic, arthritic, cardiac, neurological and sometimes ophthalmic). It represents the transmission of infectious disease, most common in the U.S. and in some countries of the Northern hemisphere temperate.

In this infection are several species of Borrelia, currently referred to as the group burgdorferi or Borrelia burgdorferi sensu lato (including Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii). These species are pathogenic for humans.

In the U.S. involved infectious type is a Borrelia burgdorferi sensu stricto. In Europe this type of added B. garinii and B. afzelii. In Asia, the participating species are B. garinii and B. afzelii.

Celebrated in the USA approximately 10,000 cases per year. In Europe the frequency of occurrence is less than 5 cases per 100,000.

Lyme borreliosis is evolving through three different stages from early infection to late. Early stage (stage I) may be asymptomatic or be expressed pseudagrion pattern. In 50-80% of cases, a few days after �locus of the mite on the skin, the appearance of inflamed rashes very specific appearance, called erythema migrans (EM). In the absence of treatment for hematogenous dissemination of Borrelia appears a few weeks later, the sudden emergence of inflammatory arthritis, neurological (neuroborreliosis) and meningeal lesions, manifestations on the skin and in the activity of the heart (stage II). After a few months or years, the disease becomes chronic atrophic form, encephalopathy, encephalomyelitis and chronic arthritis (stage III).

For each species Borrelia burgdorferi, there is a special organic tropism. If the first stage of erythema migrans unclear associated with three species, the transition in neurologic form preferably is associated with the species B. garinii, arthritis often associated with B. burgdorferi sensu stricto, chronic atrophic acrodermatitis specific for B. afzelii.

The similarity of clinical symptoms between Lyme borreliosis and other related diseases, as well as the changing manifestations complicates clinical diagnosis. If evidence of no history (tick bite or EM), the diagnosis of Lyme disease based on clinical observations may be particularly constrained. The early stage of the disease may be asymptomatic until it reaches a very advanced clinical stages.

That's why diagnosis of LB is based on clinical signs and the detection�research Institute in serum antibodies, specific for pathogenic Borrelia burgdorferi, the most common method of ELISA (Enzyme Linked ImmunoSorbent Assay (enzyme immunosorbent test)) or EIA, IFA. IgM antibodies anti-Borrelia burgdorferi appear, in General, after a few days or weeks after infection and can persist for disease development. The IgG response occurs later. The predominant proportion of patients with IgG detected approximately one month after the start of active infection, and IgG can persist for many years after the initial exposure, and symptoms are gone.

In Europe assessment of serological response is complicated because of the existence of the three pathogenic species and interspecific variability in relation to major immunodominant antigens. The antigens currently used in the standard program detect IgG and IgM LB, are ultrasonically treated cell samples of Borrelia burgdorferi sensu lato. The results of serological studies with these antigens are highly variable in terms of specificity and sensitivity. Thus, due to the lack of specificity involving cross-reactivity with antibodies associated with various pathogenic bacteria, particularly Treponema pallidium (the etiological agent of syphilis, spirochetes, Rickettsia, ehrlichiae or Helicobacter pylori, diagnosis of�samples with a positive result in the ELISA test must be confirmed by immunoblotting. Sensitivity is also a major factor. In practice, Borrelia burgdorferi sensu lato expresses different surface proteins due to adaptation to different microcred, so genetic difference and differential gene expression by Borrelia burgdorferi in patients are essential for the development of serological tests LB. Lipoprotein OspC (Outer-surface protein C (surface protein C)) and protein DbpA (Decorin-binding protein A (decorin-binding protein (A)) form part of these proteins. DbpA is expressed mainly in the mammal following infection. These proteins have high sequence variability in the species Borrelia burgdorferi and between species. Proteins DbpA are particularly variable and can be divided into four groups: a group corresponding to genocide Borrelia afzelii, the group corresponding to genocide Borrelia sensu stricto, and two groups corresponding to genocide Borrelia burgdorferi garinii. The species identity of amino acid sequences between proteins DbpA is only 40-44%. For OspC proteins is equal 54-72%.

Thus, the need to develop a set that fits the required criteria of specificity and sensitivity and, in particular, improves the detection of IgM sensitivity in the case of fresh infection.

The present invention proposes to resolve complex faults �previous level thanks to new technology recombinant chimeric proteins, which can be easily synthesized and purified, and have a strong immunoreactivity against sera of patients could be infected by one or more pathogenic species of Borrelia burgdorferi. Such fused chimeric proteins help to alleviate the problems of sensitivity and specificity associated with the presence of several pathogenic species of Borrelia burgdorferi, with a large variability of the sequences of surface antigens of Borrelia burgdorferi and the need to apply a few representative antigens of the species B. garinii, B. burgdorferi sensu stricto and B. afzelii, through the development of a diagnostic test for Lyme borreliosis, based at least on the detection of anti-OspC and anti-DbpA.

Fused chimeric proteins of the present invention allows at the same time to eliminate the difficulties arising from the expression of some antigens in recombinant form in increased amounts. In practice, despite considerable work on the design of genes for optimized their expression in E. coli, the inventors first showed that OspC proteins are poorly expressed in recombinant form in E. coli, at the same time, a completely unexpected way, it was found that DbpA proteins could be expressed in the same conditions in a soluble form in conditions that do not cause denaturation, and with more� high yield. Ease of DbpA protein expression was used to generate chimeric proteins that form DbpA and OspC, and the authors were able to show that chimeric proteins expressionlist better than isolated proteins OspC that was completely unexpected, since it was never described and never even made the assumption that proteins DbpA could possess the properties of the fusion proteins. In view of this, to improve the expression levels of the authors of the invention were constructed chimeric proteins DbpA-OspC through the use of unexpected properties of the fusion proteins DbpA to improve expression and solubility of recombinant proteins. Molecular constructs for expression of a chimeric protein of the present invention the gene encoding the protein DbpA, preferably inserted at the beginning of one or more genes encoding one or more proteins OspC. Depending on such preferred molecular structure of the chimeric protein of the present invention to the N-terminal section contains a sequence belonging to a protein sequence DbpA, and the C-terminal section contains a sequence belonging to a protein sequence of OspC. This preferred type of construction, besides the fact that it allows you to simplify and optimize the expression and solubility of the chimeric protein has, in addition, another advantage of the improved�and recognition Chimera antimorality antibodies due to better demonstrate them immunodominant region of protein OspC. An additional advantage of chimeric proteins of the present invention is to limit the number of recombinant proteins in the kit for the diagnosis of Lyme borreliosis. At the same time, the properties of the fusion protein DbpA make it an excellent candidate for expression of the chimeric vaccine proteins DbpA-OspC for the prevention of infection with Borrelia. Consequently, the chimeric proteins of the present invention are acceptable as the active agent in the prophylactic vaccine against Lyme disease.

Thus, the present invention relates to fused chimeric protein DbpA-OspC borrelii, not having a natural origin, and which are synthetic, i.e. genetic engineering (recombinant protein) or by peptide synthesis, and referred to a protein selected from the group consisting of:

(a) protein, amino acid sequence which includes (or consists of) the sequence SEQ ID NO: 1 and the sequence SEQ ID NO: 2, or a variant referred to protein, amino acid sequence which includes (or consists of) a sequence having a degree of identity of at least 40% relative to SEQ ID NO: 1, and a sequence having a degree of identity of at least 50% relative to SEQ ID NO: 2, provided that this option is capable of forming an immunological compl�COP with antibodies, produced due to the infection with Borrelia, or this variant is able to induce the production antimorality antibodies;

(b) protein, amino acid sequence which includes (or consists of) the sequence SEQ ID NO: 3 and a sequence of SEQ ID NO: 4, or a variant referred to protein, amino acid sequence which includes (or consists of) a sequence having a degree of identity of at least 40% relative to SEQ ID NO: 3, and the sequence having a degree of identity of at least 50% relative to SEQ ID NO: 4, provided that this option is capable of forming an immunological complex with antibodies produced as a consequence of infection with Borrelia or this variant is able to induce the production antimorality antibodies;

(c) protein, amino acid sequence which includes (or consists of) the sequence SEQ ID NO: 5 and a sequence of SEQ ID NO: 7, or a variant referred to protein, amino acid sequence which includes (or consists of) a sequence having a degree of identity of at least 40% relative to SEQ ID NO: 5, and the sequence having a degree of identity of at least 50% relative to SEQ ID NO: 7, provided that this option is capable of forming an immunological complex with �ntially, produced due to the infection with Borrelia, or this variant is able to induce the production antimorality antibodies;

(d) a protein amino acid sequence which includes (or consists of) the sequence SEQ ID NO: 6 and the sequence SEQ ID NO: 7, or a variant referred to protein, amino acid sequence which includes (or consists of) a sequence having a degree of identity of at least 40% relative to SEQ ID NO: 6, and the sequence having a degree of identity of at least 50% relative to SEQ ID NO: 7, provided that this option is capable of forming an immunological complex with antibodies produced as a consequence of infection with Borrelia or this variant is able to induce the production antimorality antibodies;

(e) protein, the amino acid sequence of which includes (or consists of) the sequence SEQ ID NO: 5, the sequence of SEQ ID NO: 6 and the sequence SEQ ID NO: 7, or a variant referred to protein, amino acid sequence which includes (or consists of) a sequence having a degree of identity of at least 40% relative to SEQ ID NO: 5, a sequence having a degree of identity of at least 40% relative to SEQ ID NO: 6, and the sequence having a degree of identity �ENISA least 50% relative to SEQ ID NO: 7, provided that this variant is capable of forming an immunological complex with antibodies produced as a consequence of infection with Borrelia, or this variant is able to induce the production antimorality antibodies;

(f) protein, amino acid sequence which includes (or consists of) a sequence selected from the sequences SEQ ID NO.: 8, 9, 10, 11, 12, 13 and 14.

Each of the proteins identified previously, includes at least one sequence of the extracellular domain of the protein DbpA kind borrelii selected from B. afzelii (SEQ ID NO: 1), B. burgdorferi sensu stricto (SEQ ID NO: 3) and B. garinii (group III: SEQ ID NO: 5) (group IV: SEQ ID NO: 6), or a sequence having a degree of identity of at least 40% relative to that sequence, and at least one sequence of the protein B. afzelii OspC (SEQ ID NO: 2), B. burgdorferi sensu stricto (SEQ ID NO: 4) and B. garinii (SEQ ID NO: 7) or a sequence having a degree of identity of at least 50% relative to that sequence. Preferably, one or more DbpA sequences are N-terminal section of chimeric recombinant protein, and the sequence of OspC is located at the C-terminal section of chimeric recombinant protein.

A sequence of at least 6 histidinemia residues can be attached at the level of the N-end�wow or C-terminal segment of the protein to ensure it is cleaned up for metallogenetic resin. A sequence of 6 histidinemia residue identified in SEQ ID NO: 22 is preferably at the N-terminal section of the structure. As an example of such an arrangement is illustrated by the sequences SEQ ID NO: 9, 11, 13 and 14, which contain the N-terminal section of chain poly-His(6). Chain poly-His can be encoded by any of sequences identified in SEQ ID NO: 23, 24 and 25.

Additional amino acids may be in the beginning of a chain poly-His as a result, by inserting a DNA sequence that encodes a small sequence that facilitates cloning in the desired sequence expressing the plasmid. This applies in particular to the sequences SEQ ID NO: 9, 11, 13 and 14 containing the link "MRGS" (SEQ ID NO: 26) in the beginning of a chain poly-His. Link "MRGS" encoded by a sequence ATGAGGGGATCC (SEQ ID NO: 27).

The binding region may be inserted between each of the sequences DbpA and OspC, forming a chimeric recombinant protein. The area of this type is an area of the flexible spacer that provides the best potential availability of antibodies to each of the domains. It contains a lot of amino acid residues Gly and Ser, which are amino acids, characterized as amino acids, giving flexibility in the tertiary structure of the protein. It is also possible to enter in the required to�drugshow sequence shoulder of DNA (or linker) to facilitate communication between the sequences code two required protein. This applies in particular to the sequence of SEQ ID NO: 14, containing the link "GSGG" (SEQ ID NO: 28) encoded by the sequence GGTTCCGGGGGT (SEQ ID NO: 29) and acting as a shoulder of binding between proteins DbpA group IV and B. garinii OspC.

Preferred proteins identified in SEQ ID NO: 8, 9, 10, 11, 12 13 and 14. They are respectively encoded by corresponding DNA sequences identified in SEQ ID NO: 15, 16, 17, 18, 19, 20 and 21.

The present invention also relates to DNA sequences encoding previously identified proteins, in particular to the sequences identified in SEQ ID NO: 15, 16, 17, 18, 19, 20 and 21.

The present invention also relates to an expression cassette which is functional in a cell derived from a prokaryotic (e.g. E. coli) or eukaryotic organism, such as yeast (e.g. Pichia, Schizosaccharomyces) and to allow the expression of the previously described nucleic acids (DNA) when it is under the control of elements allowing its expression and to a vector comprising a cassette.

Proteins of the present invention are preferably acceptable for the diagnosis of infection with Borrelia. Thus, the present invention relates to an in vitro method for diagnosing Lyme borreliosis in a biological sample (e.g., obra�TSE serum blood, plasma, etc.), according to which the biological sample is brought into contact with at least one previously determined protein and identify the possible formation of an immunological complex between said protein and a biological sample antibodies (IgG and/or IgM), for example by adding at least one antiimmunoglobulin person labeled by any acceptable marker. Under the understand the marker indicator capable of generating a signal. In non-restrictive list of such indicators include enzymes that produce a signal that is detected, for example, by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose-6-phosphatedehydrogenase; chromophores such as fluorescent, luminescent or dye compounds; groups having electron density, apparently detected by electron microscopy or by their electrical properties such as conductivity, ways of amperometry or voltammetry or by resistance measurement; groups detected by optical methods such as diffraction, resonance of the surface plasmon, the change in contact angle, or physical methods such as spectroscopy nuclear interactions, quantum tunneling, etc.; radioactive molecules containing32 P,35S or125I. One or more proteins, preferably immobilized on a solid support, which may represent a cone device Vidas®, plate wells for micrometrology, gel, individual particles, etc.

In one embodiment of the present invention, a biological sample, in addition, are brought into contact with at least one chimeric protein VlsE, described below.

Protein VlsE (surface expressed lipoprotein with Extensive antigenic Variation (expressed surface lipoprotein with extended antigenic variation) is mainly expressed in vivo transient and soon after infection of the host. He is very immunogenic in the infected host and induces the production of IgG and IgM. The Vls locus is linear plasmid length 28 T. p. O. (Ip28-1) available to the three genospecies of Borrelia responsible for Lyme borreliosis, and consists of the silent cassettes and expression site (VlsE). In vivo irregular recombination between expressing and silent cassettes suddenly arise in the course of infection and are the basis of antigenic variability VlsE. Protein VlsE is composed of six variable regions VR1-VR6 located on the surface of the VlsE protein and separated by intervals of regions IR1-IR6, called a constant.

Chimeric protein VlsE includes (or consists mainly of):

(i) according to møn�Shea least one sequence, selected from the sequences identified in SEQ ID NO: 30, 31, 32, 33 and 34, and the sequences having a degree of identity of at least 50%, preferably 60 or 70% and preferentially at least 80 or 85% relative to SEQ ID NO: 30, 31, 32, 33 and 34;

(ii) at least one sequence comprising the sequence of SEQ ID NO: 35 or a sequence having a degree of identity of at least 80%, preferably at least 85% and preferentially at least 90% relative to SEQ ID NO: 35, a sequence of SEQ ID NO: 36 or a sequence having a degree of identity of at least 80%, preferably at least 85% and preferentially at least 90% relative to SEQ ID NO: 36, a sequence of SEQ ID NO: 37 or a sequence, having a degree of identity of at least 80%, preferably at least 85% and preferentially at least 90% relative to SEQ ID NO: 37, and optionally the sequence SEQ ID NO: 43. Chimeric protein VlsE preferably comprises the sequence of SEQ ID NO: 43.

Accordingly, the previously described polyhistidine chain (×6) can be attached at the level of the N-terminal segment of the chimeric protein to ensure it is cleaned up for metallogenetic resin, and also can be attached additional amino acids at the beginning of a chain poly-His.

Preferred chemical�RNA protein comprises (or consists essentially of, or consists of):

(i) the sequence SEQ ID NO: 30 or a sequence having a degree of identity of at least 50%, preferably at least 60 or 70% and preferentially at least 80 or 85% relative to SEQ ID NO: 30;

(ii) a sequence comprising the sequence of SEQ ID NO: 35 or a sequence having a degree of identity of at least 80%, preferably at least 85% and preferentially at least 90% relative to SEQ ID NO: 35, a sequence of SEQ ID NO: 36 or a sequence having a degree of identity of at least 80%, preferably at least 85% and preferentially at least 90% relative to SEQ ID NO: 36, a sequence of SEQ ID NO: 37 or a sequence having a degree of identity of at least 80%, preferably at least 85% and preferentially at least 90% relative to SEQ ID NO: 37, and the sequence of SEQ ID NO: 43.

Preferred chimeric protein comprises (or consists essentially of, or consists of):

(i) the sequence SEQ ID NO: 30;

(ii) a sequence comprising the sequence of SEQ ID NO: 35, 36, 37 and 43.

Protein includes or consists of a sequence identified as SEQ ID NO: 38.

SEQ ID NO: 30 corresponds to the sequence of the extracellular domain of VlsE B. garinii (strain pBi), shortened in its signal sequence� (aa 1-19) and C-terminal region of the Mature protein, after the IR6 domain.

SEQ ID NO: 31 corresponds to the sequence of the extracellular domain of VlsE B. garinii (strain pBr), shortened in its signal sequence and C-terminal region of the Mature protein, after the IR6 domain.

SEQ ID NO: 32 corresponds to the sequence of the extracellular domain of VlsE B. garinii (strain pLi), shortened in its signal sequence and C-terminal region of the Mature protein, after the IR6 domain.

SEQ ID NO: 33 corresponds to the sequence of the extracellular domain of VlsE B. afzelii (strain pKo), shortened in its signal sequence and C-terminal region of the Mature protein, after the IR6 domain.

SEQ ID NO: 34 corresponds to the sequence of the extracellular domain of VlsE of B. burgdorferi sensu stricto (strain B31), shortened in its signal sequence and C-terminal region of the Mature protein, after the IR6 domain.

SEQ ID NO: 35 corresponds to the sequence of the IR6 domain of B. burgdorferi sensu stricto (strain B31).

SEQ ID NO: 36 corresponds to the sequence of the IR6 domain B. afzelii (strain ACA-1).

SEQ ID NO: 37 corresponds to the sequence of the IR6 domain B. garinii (strain Ip90).

SEQ ID NO: 43 corresponds to the sequence variable regions VR6 B. burgdorferi sensu stricto (strain B31).

The present invention relates also to a kit for in vitro diagnosis of borreliosis containing at least one himem�th protein DbpA-OspC, as described previously, and preferably containing at least one kind of antiimmunoglobulin person labeled by any acceptable token corresponding to the definitions given earlier. In addition, the kit can contain a chimeric protein VlsE, defined earlier.

Proteins of the present invention are also suitable for use as active ingredient to obtain the vaccine compositions for the prevention of infection with Borrelia. Thus, the present invention also relates to vaccine compositions containing at least one protein as defined previously, and pharmaceutically acceptable filler.

The following examples are given by way of explanation, and do not have any limiting nature. They allow a better understanding of the present invention. The order of sequences encoding different immunodominant epitope region of chimeric recombinant proteins, can be changed if necessary. Epitopes may also have changes compared to the sequences described in the examples, depending on the species Borrelia burgdorferi and one or more of the strains they represent. Length of binding regions can also be modified to improve flexibility between the two domains. In conclusion, the field of fixation can be inserted inside areas tie�tion.

EXAMPLES

Example 1. Obtaining plasmid constructs encoding chimeric recombinant proteins DpbA-OspC

DNA sequences encoding the various described sequence DpbA and OspC identified in table 1. DNA sequences were optimized to facilitate expression in E. coli using a set of GeneOptimizer™ and synthesized according to the method GenScript corporation (Scotch plains, NJ, USA) or GeneArt GmbH (Regensburg, Germany).

Table 1
The origin of sequences
The speciesB. Burgdorferi
*Isolate; **amino acids (aa); * * * item no in GenBank
ProteinB. sensu strictoB.afzeliiB.garinii
DbpA*B31; **aa 2-192; ***AF069269*PKo; **aa 2-150; ***AJ131967*40; **aa 2-187; ***AF441832
*PBi; **aa 2-176; ***AJ841673
OspC*B31; **aa 26-210; ***X73622*PKo; **aa. 2-212 runs; ***X62162*PEi; **aa 32-208; ***AJ749866

Each recombinant chimeric protein comprises at least od�the epitope region, corresponding to the extracellular domain of DbpA sequence of Borrelia burgdorferi sensu stricto or B. afzelii, or B. garinii, and at least one epitope region, corresponding to the extracellular domain sequence of OspC of Borrelia burgdorferi sensu stricto or B. afzelii, or B. garinii.

The Association of different nucleotide sequences, coding sequences DbpA and/or OspC, and changes in nucleotide sequences, such as deletions, addition sequence binding or adding sequence-linker was carried out by way of genetic engineering with the use of PCR techniques well known to those skilled in the art and described for example in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989.

The DNA sequence encoding the desired chimeric proteins were introduced into the expression vector pMR [2] between the restriction site BamHI at 5' and site EcoRI or HindIII at 3'. The corresponding plasmid constructs and proteins mentioned in the example (bLYM114, bLYM120 and bLYM121) described in table 2. MRGS in the N-terminal section of recombinant protein corresponding to the nucleotide sequence ATG AGG GGA TCC were introduced by the method of cloning used for expression vector pMR. In sequence is really only required the initiation codon ATG, and therefore, the amino acid Met.

Polyhistidine�th sequence (6×His) was introduced at the N-terminal section of each recombinant protein. This sequence provides purification of recombinant proteins on the column for metallogenetic affinity chromatography. It is an area of the fixation of the gel of Ni-NTA to facilitate later stage purification of chimeric recombinant protein. This peptide HHHHHH (SEQ ID NO: 22) is encoded by the nucleotide sequences CATCATCATCATCATCAT (SEQ ID NO: 23) or CATCATCATCATCATCAC (SEQ ID NO: 24), or CATCATCACCACCATCAT (SEQ ID NO: 25), or any other sequence that encodes the sequence of SEQ ID NO: 22. This area is special fixation, including histidinol sequence, provides, in particular, oriented fixation of recombinant protein on a substrate formed of silicon dioxide or metal oxides.

Table 2
The corresponding plasmid constructs and proteins
Characteristics of recombinant proteinsCharacteristics of plasmid
structures
NameN
end
label
The sequenceB. burgdorferiParental
the vector
Website insertions
in the vector
posledovatelno�and
insert
bLYM114 SEQ ID
NO: 9
6 × His - B. afzeliistrain PKo
DbpA aa 2-150 + OspC aa. 2-212 runs
pMR78*5'BamHI/3'EcoRI
bLYM120 SEQ ID
NO: 11
6 × His - B. sensu stricto strain B31
DbpA aa 28-192 + OspC aa 26-210
pMR78*5'BamHI/3'HindIII
bLYM121 SEQ ID
NO: 14
6 × His - B. garinii
DbpA III aa 25-187 strain 40 + DbpA IV aa 24-176 strain PBi + OspC aa 32-208 strain PEi
pMR78*5'BamHI/3'HindIII
* [2]

Example 2. Expression of recombinant proteins bLYM114, bLYM120 and bLYM121 of example 1 and purification

For transformation of E. coli (strain BL21) according to a conventional method known to those skilled in the art, was used for plasmid construction, corresponding to the sequence SEQ ID NO: 16, 18, or 21, is inserted in the expression vector (pMR). Transformed bacteria are selected by their resistance to ampicillin, given by the vector pMR.

Then carried out the selection of clone recombinant bacteria for inoculation of preculture in 40 ml of medium 2×YT (tripton 16 g/l; ek�tract yeast 10 g/l; NaCl 5 g/l, pH 7.0) containing 100 μg/ml ampicillin. After incubation for 15-18 hours at 30°C with stirring at 250 rpm this preculture used for the implementation of planting in 1 l of medium 2×YT containing 2% glucose and 100 μg/ml ampicillin. This culture is incubated at 30°C with stirring at 250 rpm until reaching an optical density DO at 600 nm values 1,0/1,2. The culture was allowed to stand for 3 hours 30 minutes or 4 hours at 30°C after addition of 0.4 mm isopropyl-β-D-thiogalactopyranoside (IPTG), and harvested by centrifugation at 6000 g for 30 min. the Precipitate of cells was stored at -60°C. For the purification of wet biomass was thawed and were resuspended in lysing buffer containing protease inhibitors without EDTA (Roche) and benzenetoluene (Novagen), and carried out the destruction of cells at 1.6 kbar in the destructor cells (Constant System Ltd, Daventry, UK). Then the lysate was centrifuged at 10,000 rpm for 45 min at 2-8°C. the Obtained supernatant contains soluble proteins. The supernatant was filtered through a filter of porosity 0.45 μm and was purified by affinity chromatography on metallogenetic column (matrix "Nickel-nitrinoxus acid (Ni-NTA, Qiagen)). With this purpose, the supernatant was injected (1 ml/min) at 18-25°C in the column with 8 ml of gel of Ni-NTA, air-conditioned buffer solution A see table 3). Then the column was washed with buffer A to achieve at the outlet of column with DO280 nm=0. Elution of the recombinant protein was carried out by a flow of buffer solution B, respectively, described in table 3, and the purified protein is dialyzed in a dialysis cassette with a cut-off of 10,000 or 20,000.e.m. (Slide-A-Lyser®, Pierce) against dialysis buffer solution. The conditions of purification on the gel of Ni-NTA as described in table 3.

Table 3
Purification of recombinant proteins
ProteinbLYM114
SEQ ID NO: 14
bLYM120
SEQ ID NO: 11
bLYM121
SEQ ID NO: 14
Buffer solution for lysis and washingBuffer solution A1
Elution buffer solutionBuffer solution B2
Elution stage 190% of buffer solution A + 10% buffer solution B (4CV)92% of buffer solution A + 8% of buffer solution B (4CV)100% of buffer solution B
Elution, stage 2 100% of buffer solution B100% of buffer solution Bno data
Exit cleaning, mg protein/g of wet biomass121320
The output is clean, mg of protein/l of culture80122245
1Sodium phosphate 50 mm, 30 mm imidazole, 500 mm NaCl, Tween 20 and 0.1%, glycerol 5%, pH 7.8
2Sodium phosphate 50 mm, 325 mm imidazole, 500 mm NaCl, glycerol 5%, pH 7,5

The samples were analyzed on the gel NuPAGE® Novex®, 4-12%, in buffer NuPAGE® MES SDS according to the manufacturer's instructions (Invitrogen). Proteins were stained with brilliant blue, Kumasi or electrophoresis were transferred onto nitrocellulose membrane. The membrane was blocked with 5% (wt./about.) a solution of dry milk in PBS and incubated with anti-pentamethylenebis antibody (Qiagen) in PBS containing 0,05% Tween 20. Conjugate goat anti-mouse IgG labeled with horseradish peroxidase, (Jackson Immunoresearch laboratories) in PBS/Tween was used as the secondary antibody.

Determining the concentration of proteins was carried out using the set of Bradford (Pierce Coomassie Plus, Perbio Science) with BSA as the standard protein.

Example 3. Detection of IgG and human IgM with recombinant chimeric proteins by the method of linear immunoblotting

Each recombinant protein was coated on polyvinylidenedifluoride membrane (PVDF, Immobilon, Millipore®, Bedford, mA, USA) according to the following method.

The protein concentration in PBS with a pH of 7.2 was set equal to 1 mg/ml and diluted with PBS with a pH of 7.2, supplemented with 0.03% of Tween 20 (dilution 1/200). The membrane of PVDF wetted with methanol, washed in demineralized water and applied to paper with a wet spot. Plastic ruler was immersed into the diluted protein solution and fixed on the membrane of PVDF. After applying the protein and drying, the membrane was cut in a vertical direction into strips. Before applying the strips were incubated with 5% solution of gelatin in TBS pH 7.5 for 1 hour at 37°C. immunoblotting Procedures were carried out at room temperature, respectively, described by A. G. Bretz et al. [3]. The strips were incubated for 2 hours with samples of human serum, diluted in a ratio of 1/200 in TBS with 1% gelatin, washed and incubated with the antibody anti-IgG or anti-human IgM labeled with alkaline phosphatase (Sigma, St. Louis, USA) and diluted at a ratio of 1/1000 in TBS with 1% gelatin. After washing, strips were incubated with the substrate BCIP-NBT alkaline phosphatase (KPL, Gaithersburg, MD, USA) for 30 min, W�those washed in distilled water and dried.

The group of samples tested sera

Sample human serum was collected from a typical, clinically well-characterized patients with LB, LB corresponding to different stages (22 with erythema migrans [EM], 5 with carditis, with 20 neuroborreliosis [NB], 20 with Lyme arthritis [LA], 20 with chronic atrophic acrodermatitis [ACA] and 10 with benign lymphocytes skin [LCB]). Antimorality IgG were found earlier described by immunoblotting using lysates of whole cells [4] in serum of patients with LA, ACA and carditis. EM, NB and LCB were identified clinically, but all relevant serum samples were not identified as positive by the use of standard immunoblotting [4] or by commercial kits (VIDAS® Lyme (biomérieux), Borrelia IgG (Diasorin®) and Borrelia IgM (r-biopharm®)). On the contrary, in all cases NB included in the study had antibodies detectable in the cerebrospinal fluid [LCR] (the index was in the range from 2 to 27.1 in determining the set of VIDAS® Lyme (biomérieux)). The presence of IgM was only identified in clinical cases of stages I and II, but not in chronic stages.

The negative control group consisted of 31 samples of serum, samples were previously defined in the traditional test as negative for the presence of antiparasitic antibodies. In addition, recombinant protein were testing�Ana 64 serum samples of healthy blood donors, living in a region endemic for the disease of lime (Monthey, Valais, Switzerland).

The intensity of the reaction was evaluated as follows: [+], [++], [+++], [-] or mixed results. Mixed results were taken as negative.

The results are presented in table 4.

Table 4
Reactivity with linear immunoblotting of serum samples of patients with Lyme borreliosis with 3 chimeric recombinant proteins bLYM114 (SEQ ID NO: 9), bLYM120 (SEQ ID NO: 11) and bLYM121 (SEQ ID NO: 14)
ProteinIgGIgM
Stage
I
Stage
II
Stage
III
Stage
I
Stage
II
EM (n=22)NB (n=20)Carditis (n = 5)LA (n=19)ACA (n=20)LCB (n=10)EM (n=22)NB (n=20)Carditis (n=5)
bLYM1145 1007122772
bLYM1206708601172
bLYM1212105980772
Σ bLYM 114 + 120 + 1219135181721172
Serum samples c put-to enhance results, in % and the intensity of reaction40,9%1[+++] 4[++] 4[+]59,1%8[+++] 2[++] 3[+] 100%4[+++]
1[+]
94,7%7[+++] 8[++] 3[+]85%8[+++] 5[++] 4[+]20%
1[++] 1[+]
50%1[+++] 7[++] 5[+]35%
5[++] 2[+]
40%
2[++]

Total put-found-result-ing in % and the intensity of reaction66,7%
28[+++]
20[++]
16[+]
42,5%
1[+++]
14[++]
7[+]

The specificity is 100% on the basis of 31 serum samples collected from healthy participants and is defined as a Lyme-negative by standard commercial tests.

Detection of IgG

The results show that the fused chimeric recombinant proteins represent a diagnostic tool that is sensitive in respect of IgG and IgM in all stages of infection. They reveal the complementary action of three recombinant proteins based on the sequences of Borrelia afzelii, B. sensu stricto and B. garinii, respectively, for the detection of IgG. The combined use of the three chimeric recombinant proteins allows for stage I of infection to detect IgG in 9 cases of patients with EM of 22 (or 40.9% of sensitivity).

Detection of IgM

Hemery� of antibully IgM was found in 11 cases out of 22 (50% sensitivity). Thus, in serum of patients with LB stage I such chimeric proteins can detect IgM often than IgG. In tests carried out for monitoring by the use of standard immunoblotting [4] and a commercial kit Borrelia IgM (r-biopharm®), detected a larger number of serum samples with positive results on IgM. In addition, 3 samples of serum, which by means of immunoblotting and set Borrelia IgM (r-biopharm®) is defined as negative, defined as positive by three chimeric proteins mentioned in the example (3/3), or by one of three proteins mentioned in the example (1/3). The combined use of three recombinant proteins allows for improved 13.6% sensitivity for the detection of IgM at the first stage of infection.

Example 4. Obtaining plasmid constructs encoding chimeric recombinant VlsE proteins

DNA sequences that encode different protein sequences identified in table 5.

Table 5
The origin of sequences
The speciesB. burgdorferi
*Isolate; **amino acids (aa); * * * item no in GenBank
ProteinB. sensu stricto B.afzeliiB.garinii
VlsE--*PBi; **aa 20-293; ***AJ630106 (GenScript Corp)
IR6*B31; **aa 274-305; ***U76405 (GeneArt GmbH)*ACA-1; **aa 172-188; ***U76405 (GeneArt GmbH)*Ip90; **aa 167-191; ***AAN87834 (GeneArt GmbH)

Sequences were optimized with respect to their expression in E. coli using a set of GeneOptimizer™ and synthesized according to the method GenScript corporation (Scotch plains, NJ, USA) or GeneArt GmbH (Regensburg, Germany).

Additional DNA changes, deletions or Association of different sequences implemented by the PCR method (PCR) used in genetic engineering, using PCR techniques, well known to those skilled in the art and described for example in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989. DNA sequences have been linked in the expression vector pMR [2] or pET-3d (Novagen®). The corresponding plasmid constructs and proteins mentioned in the example (bLYM110, bLYM125) described in table 6.

Table 6
The corresponding plasmid constructs and proteins
Brushless�IKI recombinant proteins Characteristics of plasmid constructs
NameN-end tagThe sequenceB. burgdorferiParental vectorThe insertion site in the vector sequence insertion
bLYM110 SEQ ID NO: 396 × His - VlsEgariniipBi aa 20-293 + 3 IR6 [sensu stricto B21aa 274-305+afzeliiACA-1 aa 172-188 +gariniiIp90 aa 167-191]pMR785'BamHI/3'HindIII
bLYM125 SEQ ID NO: 418 × HispET-3d5'NcoI/3'BamHI

Example 5. Expression of recombinant proteins of example 4 and clean

For transformation of E. coli (strain BL21) according to a conventional method known to those skilled in the art, used a plasmid construct described in example 4. Transformed bacteria are selected by their resistance to ampicillin, given by the vector pMR or pET.

Then carried out the selection of clone recombinant bacteria for inoculation of preculture in 40 ml of medium 2×YT (tripton 16 g/l; yeast extract 10 g/l; NaCl 5 g/l, pH 7.0), Sumerians�nd 100 ug/ml ampicillin. After incubation for 15-18 hours at 30°C with stirring at 250 rpm this preculture used for the implementation of planting in 1 l of medium 2×YT containing 2% glucose and 100 μg/ml ampicillin. This culture is incubated at 30°C with stirring at 250 rpm until reaching an optical density DO at 600 nm values 1,0/1,2. The culture was allowed to stand for 3 hours 30 minutes or 4 hours at 30°C after addition of 0.4 mm isopropyl-β-D-thiogalactopyranoside (IPTG), and harvested by centrifugation at 6000 g for 30 min. the Precipitate of cells was stored at -60°C. For the purification of wet biomass was resuspended in lysing buffer containing protease inhibitors without EDTA (Roche) and benzenetoluene (Novagen®), and carried out the destruction of cells at 1.6 kbar in the destructor cells (Constant System Ltd, Daventry, UK). Then the lysate was centrifuged at 10,000 rpm for 45 minutes at 2-8°C. After filtering through a filter of porosity 0.22 μm supernatant was injected into a column of Ni-NTA (Quiagen®), air-conditioned lytic buffer solution. Then the resin was washed with the same buffer solution to achieve A280 nmvalues for the baseline. Elution was carried out by eluting with a buffer solution, and the purified protein was subjected to dialysis cassette, Pierce for dialysis Slide-A-Lyser® with a cutoff of 10,000 or 20,000.e.m. against Dialin�th buffer solution. The conditions of purification on the gel of Ni-NTA as described in table 7.

Table 7
Purification of recombinant proteins
ProteinbLYM110
SEQ ID NO: 39
bLYM125
SEQ ID NO: 41
Buffer solution for lysis and washingBuffer solution A1Buffer solution A1+ urea, 2 M
Elution buffer solutionBuffer solution B2Buffer solution B2modified 600 mm imidazole
Elution stage 186% of buffer solution A + 14% of buffer solution B (4CV)92% of buffer solution A + 8% of buffer solution B (4CV)
Elution, stage 2100% of buffer solution B100% of buffer solution B
Exit cleaning, mg protein/g of wet biomass0,50,8
The output is clean, mg of protein/l of culture8,7 17
1Sodium phosphate 50 mm, 30 mm imidazole, 500 mm NaCl, Tween 20 and 0.1%, glycerol 5%, pH 7.8
2Sodium phosphate 50 mm, 325 mm imidazole, 500 mm NaCl, glycerol 5%, pH 7,5

The samples were analyzed on the gel NuPAGE® Novex®, 4-12%, in the circulating buffer NuPAGE® MES SDS according to the manufacturer's instructions (Invitrogen™). Proteins were stained with brilliant blue, Kumasi or electrophoresis were transferred onto nitrocellulose membrane. The membrane was blocked with 5% (wt./about.) a solution of dry milk in PBS and incubated with anti-pentamethylenebis antibody (Qiagen®) in PBS containing 0,05% Tween 20. Conjugate goat anti-mouse IgG labeled with horseradish peroxidase, (Jackson Immunoresearch laboratories) in PBS/Tween was used as the secondary antibody.

Determining the concentration of proteins was carried out using a set Bradford Assay Kit (Pierce Coomassie Plus, Perbio Science) with BSA as the standard protein.

Example 6. Detection of IgG and human IgM with recombinant chimeric protein bLYM110 of example 5 by the method of linear immunoblotting

Recombinant protein was coated on polyvinylidenedifluoride membrane (PVDF, Immobilon, Millipore®, Bedford, mA, USA) according to the following method.

The protein concentration in PBS with a pH of 7.2 was set equal to 1 mg/ml and diluted with PBS with a pH of 7.2, supplemented with 0.03% of Tween 20 (dilution 1/20). The membrane of PVDF wetted with methanol, washed in demineralized water and applied to paper with a wet spot. Plastic ruler was immersed into the diluted protein solution and fixed on the membrane of PVDF. After applying the protein and drying, the membrane was cut in a vertical direction into strips. Before applying the strips were incubated with 5% solution of gelatin in TBS pH 7.5 for 1 hour at 37°C. immunoblotting Procedures were carried out at room temperature, respectively, described by A. G. Bretz et al. [3]. The strips were incubated for 2 hours with samples of human serum, diluted in a ratio of 1/200 in TBS with 1% gelatin, washed and incubated with different from human IgG or IgM labeled with alkaline phosphatase (Sigma™, St. Louis, USA) and diluted at a ratio of 1/1000 in TBS with 1% gelatin. After washing, strips were incubated with the substrate BCIP-NBT (KPL, Gaithersburg, MD, USA) for 30 minutes, washed in distilled water and dried.

The group of samples tested sera

Sample human serum was collected from a typical, clinically well-characterized patients with LB, LB corresponding to different stages (22 with erythema migrans [EM], 5 with carditis, with 20 neuroborreliosis [NB], 20 with Lyme arthritis [LA], 20 with chronic atrophic acrodermatitis [ACA] and 10 with benign lymphocytes skin [LCB). Antimorality IgG were found earlier described by immunoblotting using lysates of whole cells [4] in serum of patients with LA, ACA and carditis. EM, NB and LCB were identified clinically, but all relevant serum samples were not identified as positive by immunoblotting [4] or by commercial kits (VIDAS® Lyme (biomérieux®), Borrelia IgG (Diasorin®) and Borrelia IgM (r-biopharm®)). On the contrary, in all cases NB included in the study had antibodies detectable in the cerebrospinal fluid [LCR] (the index was in the range from 2 to 27.1).

The negative control group consisted of 31 samples of serum, samples were previously defined in the traditional test as negative for the presence of antiparasitic antibodies. In addition, recombinant protein were tested 64 serum samples of healthy blood donors living in a region endemic for the disease of lime (Monthey, Valais, Switzerland). The intensity of the reaction was evaluated as follows: [+], [++], [+++], [-] or mixed results. Mixed results were taken as negative.

The results are presented in the following table 8.

Table 8
IgG
Stage IStage IIStage IIIDonors
EM (n=22)NB (n=20)Carditis (n=5)LA (n=19)ACA (n=20)The lymphocytes (n=10)(n=64)
17205192096
77,3%100%100%100%100%90%9,4%
12[+++]11[+++]4[+++]13[+++]20[+++]3[+++]6[+]
4[++] 7[++]1[++]4[++]2[++]
1[+]2[+]2[+]4[+]
Total positive reactions to IgG: 93,7%
IgM
EM
(n=22)
NB
(n=20)
Carditis (n=5)(n=64)
5421
22%20%40%1,5%
1[++]2[++]1[++]1[+]
4[+]1[+]1[+]
Total positive IgM reactions to: 23,4 %

Detection of IgG

The results show that the recombinant protein bLYM110 is a diagnostic antigen, is highly sensitive in respect to IgG at all stages of infection. At the first stage of the infection IgG were detected in 17 cases of patients with EM of 22 (or 77,3% sensitivity). Five patients with EM have negative results with a recombinant protein, the same results the post and immunoblotting with commercial kits. Seven serum samples with EM, was found positive with the recombinant protein were detected by immunoblotting, which was an improvement of the sensitivity of the recombinant protein with 31.8%. In the first stage of infection in the absence of the characteristic redness diagnosis may be difficult as other clinical manifestations bol�FDI, are not specific for Lyme. In addition, traditional tests were found only in a few patients who had EM. Thus, the protein of the present invention improved the detection of IgG antibodies to phase I of infection, ensuring the detection of more than 77% of patients who had EM.

Detection of IgM

Chimeric antibully IgM was found in 23.4% of serum samples with LB. In serum of patients with LB stages I and II protein can detect IgG more often than IgM.

Example 7. Evaluation and validation of chimeric recombinant proteins bLYM114, bLYM120, bLYM121 and bLYM125 through test VIDAS® (bioMérieux)

This validation was performed through test VIDAS®, using:

1) chimeric recombinant proteins bLYM114, bLYM120 and bLYM121 obtained according to examples 1 and 2, for the detection of IgM;

2) chimeric recombinant proteins bLYM114, bLYM120 obtained according to examples 1 and 2, and chimeric protein bLYM125 obtained according to examples 4 and 5, for the detection of IgG.

The principle of the test VIDAS® consists of the following: cone forms a solid substrate, which also serves as the feeding system of the reagents contained in the cavity of the cone. One or more recombinant proteins are fixed on the cone. After the step of diluting the sample is taken by aspiration and administered over several sessions inside the cone. This procedure allows antimorality immunoglobulins of the sample contacting the recombinant proteins. Components left�I unbound, are removed by washing. The antibody of antiimmunoglobulin person, anywhereman with alkaline phosphatase (PAL), were incubated in the cone where it attaches to antimorality immunoglobulins. At the stages of rinsing remove conjugate, remaining unattached. During the last stage of the detection substrate of alkaline phosphatase (PAL), which is a 4-methylumbelliferone, to hydrolyze 4-methylumbelliferone, the fluorescence of which is measured at 450 nm. The fluorescence intensity measured by the optical system Vidas®, proportional to the content of antimorality immunoglobulins present in the sample. The results are analyzed automatically by the system VIDAS® and expressed in RFV (Relative Fluorescent Value (relative fluorescence units)).

Thus, through a system of Vidas® were analyzed 255 positive serum samples (samples with ambiguous results + samples with a positive result) and 298 of the negative serum samples (samples with ambiguous results + samples with a negative result).

Cones Vidas® Lyme IgG were sensibilized 300 μl of a solution containing proteins bLYM114, bLYM120 and bLYM125 of the present invention with the concentration of each in General sensitising solution, equal to 1 μg/ml.

In the first stage, serum samples were incubated for 5,3 min for images�of complexes "antigen-antibody". In the second stage anti-human IgG labeled PAL, were incubated for 5,3 min.

The results were expressed as an index relative to the threshold positive value of 135 according to the RFV method.

- 255 tested positive 246 serum samples found positive and 9 false negative, which corresponds to a sensitivity of 96.5%.

- From 298 tested negative 284 serum samples found negative and 14 false positive, which corresponds to a specificity of 95.3 per cent.

The LIST of references

1. Gottner G. et al., Int. J. Microbiol. 293, Suppl. 37, 172-173 (2004).

2. Arnaud N. et al., Gene 1997; 199:149-156.

3. Bretz A. G., K. Ryffel, P. Hutter, E. Dayer and O. Peter. Specificities and sensitivities of four monoclonal antibodies for typing of Borrelia burgdorferi sensu lato isolates. Clin. Diag. Lab. Immunol. 2001 ; 8: 376-384.

4. Ryffel K., Peter O., B. Rutti and E. Dayer. Scored antibody reactivity by immunoblot suggests organotropism of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii and B. valaisiana in human.J. Clin. Microbiol. 1999; 37:4086-92.

1. Fused chimeric protein DbpA-OspC borrelii for the diagnosis of Lyme borreliosis selected from the group consisting of:
(a) protein, the amino acid sequence of which comprises at its N-end sequence of SEQ ID NO: 1 and at its C-end of the sequence SEQ ID NO: 2;
(b) protein, the amino acid sequence of which comprises at its N-end sequence of SEQ ID NO: 3 and at its C-end of the sequence SEQ ID NO: 4;
(c) protein, amino acid followers�nost of which comprises at its N-end sequence of SEQ ID NO: 5 and at its C-end of the sequence SEQ ID NO: 7;
(d) a protein comprising at its N-end sequence of SEQ ID NO: 6 and at its C-end of the sequence SEQ ID NO: 7;
(e) a protein comprising at its N-end sequence of SEQ ID NO: 5, the sequence of SEQ ID NO: 6 and at its C-end of the sequence SEQ ID NO: 7;
(f) protein, amino acid sequence which comprises a sequence selected from the sequences SEQ ID NO.: 8, 9, 10, 11, 12, 13 and 14.

2. Nucleic acid that encodes the protein according to claim 1.

3. Expressing cassette which is functional in a cell derived from a prokaryotic or eukaryotic organism, providing the expression of the nucleic acid according to claim 2 and under the control of elements necessary for its expression.

4. The vector comprising the expression cassette of claim 3.

5. An in vitro method for diagnosing Lyme borreliosis in a biological sample, according to which the biological sample is brought into contact with at least one protein according to claim 1 and identify possible formation of an immunological complex between said protein and antibodies of the biological sample.

6. A method according to claim 5, in which the antibodies of the biological sample are IgG and/or IgM.

7. A method according to claim 6, in which the formation of the immunological complex is determined by adding at least one antiimmunoglobulin man, m�Chennai by any acceptable token.

8. A method according to any one of claims.5-7, in which the protein is immobilized on a solid substrate.

9. Kit for in vitro diagnosis of Lyme borreliosis comprising at least one chimeric protein fused borrelii, characterized in that the protein is a protein according to claim 1.

10. The kit according to claim 9, comprising at least one antiimmunoglobulin person labeled by any acceptable token.

11. Vaccine composition for the prevention of infection with Borrelia, which has at least one protein according to claim 1 and pharmaceutically acceptable filler.



 

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33 cl, 31 dwg, 8 tbl, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology. Presented are variants of anti-CD20 modified antibody or its antigen-binding fragment. Each of the variants is characterised by the fact that it contains a variable light and heavy chain domain, and induces a higher apoptosis level as compared to anti-B-Ly1 chimeric antibody. There are presented: a mixture of antibodies, wherein at least 20% of oligosaccharides in Fc domain have a branched chain and are not fucosylated, as well as a pharmaceutical composition for producing a therapeutic agent for a malignant haematological or autoimmune disease by using the antibodies or the mixture of antibodies. Described are: an expression vector, a based host cell, variants of coding polynucleotides, as well as a method for producing the antibody in the cell.

EFFECT: using these inventions provides the new antibodies with the improved therapeutic properties, including with increased binding of Fc receptor, and with the increased effector function that can find application for treating the malignant haematological or autoimmune disease.

32 cl, 3 ex, 9 tbl, 26 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to field of biochemistry, in particular to method of obtaining bivalent bispecific antibody, which includes transformation of host cell by vectors, containing molecules of nucleic acids, coding first light chain and first heavy chain of bivalent bispecific antibody, and vectors, containing molecules of nucleic acids, coding second light chain and second heavy chain of bivalent bispecific antibody, cultivation of host cell under conditions, providing synthesis of molecule of bivalent bispecific antibody from said culture. Said antibody contains first light chain and first heavy chain of antibody, specifically binding with first antigen, and second light chain and second heavy chain of antibody, specifically binding with second antigen, in which variable domains VL and VH of second light chain and second heavy chain are replaced by each other and constant domains CL and CH1 of second light chain and second heavy chain are replaced by each other.

EFFECT: invention makes it possible to increase output of correct bispecific antibody by increasing the level of correct heterodimerisation of heavy chains of wild type and modification of heavy chains resulting from crossing over.

2 cl, 31 dwg, 3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.

EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.

23 cl, 74 dwg, 17 tbl, 33 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to antibodies including human antibodies and their antigen-binding portions, which specifically bind to CCR2, in particular to human CCR2, and can act as CCR2 inhibitors. Anti-CCR2 antibodies are those binding to first and/or second extra-cellular CCR2 loops. The present invention also refers to human anti-CCR2 antibodies and to their antigen-binding portions. The present invention refers to the recovered heavy and light chains of immunoglobulin initiated from human anti-CCR2 antibodies, and to nucleic acid molecules coding such immunoglobulins. The present invention also refers to methods for preparing human anti-CCR2 antibodies and their antigen-binding portions, to compositions containing such antibodies or their antigen-binding portions, and to methods for using antibodies and their antigen-binding portions, and compositions for diagnosing and treating.

EFFECT: invention refers to methods for gene therapy with the use of nucleic acid molecules coding molecules of heavy and light chains of immunoglobulin, wherein the above molecules contain anti-CCR2 antibodies and their antigen-binding portions.

25 cl, 24 dwg, 8 tbl, 17 ex

Csf-1r antibody // 2547586

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. There are presented an antibody and its antigen-binding fragment specifically binding human colony-stimulating factor-1 receptor (CSF-1R) characterised by sequences of complementary-determining regions (CDR). There are also disclosed a nucleic acid coding the antibody according to the invention or its antigen-binding fragment, a vector providing the expression of the antibody and its antigen-binding fragment, and a pharmaceutical composition applicable in treating the diseases associated with an inflammation or an autoimmunity, or cancer.

EFFECT: invention can find further application in diagnosing and therapy of the CSF-1 associated diseases.

23 cl, 18 dwg, 4 tbl

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, in particular to peptydoglycane hydrolase biosynthesis, and represents a protein with the peptydoglycane hydrolase activity, a plasmid, containing a peptydoglycane hydrolase-coding fragment, a bacterium-producer, a method of microbiological peptydoglycane hydrolase synthesis, as well as a pharmaceutical composition, containing the obtained peptydoglycane hydrolase, for the therapy of diseases, caused by Gram-negative microflora.

EFFECT: elaborated method of microbiological synthesis makes it possible to obtain bacteriophage S-394 peptydoglycane hydrolase in an effective way.

22 cl, 2 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to compositions for intensive generation of a target protein in eucariotic cells, which includes a DNA vector with an insert of target protein gene and an agonist of cell receptors. Besides, the invention relates to methods for increasing generation of a target protein coded with a transgene in eucariotic cells by using the above compositions.

EFFECT: invention allows effective increase of generation of a target protein in eucariotic cells.

28 cl, 4 dwg, 7 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: inventions relate to chimeric proteins, nucleic acid, coding such a protein, an expression cassette, providing the expression of nucleic acid, a vector, including the expression cassette, a method of diagnostics and a set for diagnostics. The characterised chimeric Borrelia protein includes at least one sequence of an extracellular domain of the VlsE Borrelia protein of the first type, corresponding to a certain strain, and at least one sequence of IR6 area of the VlsE Borrelia protein of the second type or Borrelia of the first type, but corresponding to a strain, different from the strain of the first type, with Borrelia being selected from Borrelia stricto-sensu, Borrelia afzelii and Borrelia garinii.

EFFECT: claimed inventions make it possible to carry out diagnostics of Lyme-borreliosis with an increased specificity and sensitivity.

15 cl, 8 tbl, 7 ex

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