Chimeric protein used for lyme-borreliosis diagnostics

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

 

Lyme borreliosis (LB) is a non-communicable infectious disease caused by a spirochete calledBorrelia burgdorferiand 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 involves several species of Borrelia, currently referred to as the group burgdorferi orBorrelia burgdorferisensu lato (includingBorrelia burgdorferisensu stricto,B. gariniiandB. afzelii). These species are pathogenic for humans.

In the U.S. involved infectious type is aBorrelia burgdorferisensu stricto. In Europe this type addedB. gariniiandB. afzelii. In Asia involved species are aB. gariniiandB. afzelii.

In the USA says about 10,000 cases. 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 are�AEB few days after the tick bite 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 speciesBorrelia burgdorferithere 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 theB. gariniithe arthritis often associated withB. burgdorferisensu stricto, chronic atrophic acrodermatitis specific toB. afzelii.

The similarity of clinical symptoms between Lyme borreliosis and other related diseases, as well as the variability of 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 symptom�x, and on the detection in serum of antibodies specific for pathogenicBorrelia burgdorferioften the ELISA method (Enzyme Linked ImmunoSorbent Assay (enzyme immunosorbent test)) or EIA, IFA.

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 samplesBorrelia burgdorferisensu 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 pathogenic bacteria, particularly Treponema pallidum (the causative agent of syphilis, spirochetes, Rickettsia, ehrlichiaeorHelicobacter pyloridiagnosis of samples with a positive result in the ELISA test must be confirmed by immunoblotting. Sensitivity is also a major factor. In practice,Borrelia burgdorferisensu lato expresses different surface proteins due to adaptation to different microcred, so genetic difference and differential gene expressionBorrelia burgdorfer patients are essential for the development of serological tests LB.

Thus, it was necessary to develop a set that mitigates the above disadvantages and to a greater extent meets the required criteria of specificity and sensitivity.

Protein VlsE (surface expressed lipoprotein with Extensive antigenic Variation (expressed surface lipoprotein with extended antigenic variation)), mainly expressedin vivotransient 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 vivoirregular 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 constant regions IR1-IR6.

It is known that VlsE proteins have considerable interspecific and intraspecific heterogeneity. In 2004 Göttner et al. [1] described the degree of identity approximately from 47 to 58% at the protein level VlsE.�walking from the four strains.

To mitigate the previously mentioned problems of sensitivity and specificity of the applicants obtained chimeric protein borrelii comprising at least one sequence of the extracellular domain of the protein VlsE borrelii of the first type corresponding to a particular strain, and at least one sequence region IR6 protein VlsE borrelii of the second kind or borrelii of the first type, but the corresponding strain different from the strain of the first type, and the indicated chimeric protein comprises (or consists principally of, or consists of):

sequence of the extracellular domain of the protein VlsE borrelii of the first type, consisting of five variable regions VR1, VR2, VR3, VR4 and VR5 and six constant regions IR1, IR2, IR3, IR4, IR5 and IR6, wherein at least one specified sequence of the extracellular domain selected from the group comprising the sequences SEQ ID NO: 1, 2, 3, 4 and 5, or a variant of one of the sequences SEQ ID NO: 1, 2, 3, 4 and 5, moreover, this variant has a degree of identity of at least 50% (preferably at least 60% or at least 70% and preferentially at least 80% or at least 85%) relative to the sequences SEQ ID NO: 1, 2, 3, 4 and 5, respectively, provided that this option is capable of forming an immunological complex with antibodies produced due Ifni�of debugger Borrelia;

- at least one sequence region IR6 borrelii of the second kind or borrelii of the first type, but the corresponding strain different from the strain of the first type selected from the group comprising the sequences SEQ ID NO: 6, 7 and 8, or a variant of one of the sequences SEQ ID NO: 6, 7 and 8, and this variant has a degree of identity of at least 80% (preferably at least 85% and preferentially at least 90%) relative to the sequences SEQ ID NO: 6, 7 and 8, respectively, provided that a variant of this sequence are capable of forming an immunological complex with antibodies produced as a consequence of infection with Borrelia.

The previously described chimeric protein can include, in addition, a variable sequence VR6 kind borrelii, and this sequence identified in SEQ ID NO: 9 in sequence listing.

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

sequence SEQ ID NO: 1 or variant of the sequence SEQ ID NO: 1, wherein this embodiment has a degree of identity of at least 50% (preferably at least 60% or at least 70% and preferentially at least 80% or at least 85%) relative to SEQ ID NO: 1;

sequence SEQ ID NO: 6 or variant of the sequence SEQID NO: 6, moreover, this variant has a degree of identity of at least 80% (preferably at least 85% and preferentially at least 90%) relative to SEQ ID NO: 6;

sequence SEQ ID NO: 7 or a variant sequence of SEQ ID NO: 7, and this variant has a degree of identity of at least 80% (preferably at least 85% and preferentially at least 90%) relative to SEQ ID NO: 7;

sequence SEQ ID NO: 8, or variant of the sequence SEQ ID NO: 8, and this variant has a degree of identity of at least 80% (preferably at least 85% and preferentially at least 90%) relative to SEQ ID NO: 8;

- and optionally, a variable sequence VR6 identified in SEQ ID NO: 9.

Thus, one of the chimeric proteins of the present invention comprises (or consists principally of, or consists of) the sequence SEQ ID NO: 1, the sequence SEQ ID NO: 6, the sequence of SEQ ID NO: 7 and the sequence SEQ ID NO: 8, in addition, optionally, the sequence of SEQ ID NO: 9.

Preferred chimeric proteins of the present invention are identified in particular as proteins comprising (or containing mostly or containing) a sequence selected from SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 23; and the most preferred protein is a protein comprising or �terjadi sequence identified in SEQ ID NO: 20 in the sequence listing.

SEQ ID NO: 1 corresponds to the sequence of the extracellular domain of VlsEB. garinii(strain pBi), shortened in its signal sequence (aa 1-19) and C-terminal region of the Mature protein, after the IR6 domain, that is, this extracellular domain includes a region IR1 VR1, IR2, VR2, IR3, VR3, IR4, VR4, IR5, VR5 and IR6B. garinii(strain pBi).

SEQ ID NO: 2 corresponds to the sequence of the extracellular domain of VlsEB. garinii(strain pBr), shortened in its signal sequence and C-terminal region of the Mature protein, after the IR6 domain, that is, this extracellular domain includes a region IR1 VR1, IR2, VR2, IR3, VR3, IR4, VR4, IR5, VR5 and IR6B. garinii(strain pBr).

SEQ ID NO: 3 corresponds to the sequence of the extracellular domain of VlsEB. garinii(strain pLi), shortened in its signal sequence and C-terminal region of the Mature protein, after the IR6 domain, that is, this extracellular domain includes a region IR1 VR1, IR2, VR2, IR3, VR3, IR4, VR4, IR5, VR5 and IR6B. garinii(strain pLi).

SEQ ID NO: 4 corresponds to the sequence of the extracellular domain of VlsEB. afzelii(strain pKo), shortened in its signal sequence and C-terminal region of the Mature protein, after the IR6 domain, that is, this extracellular domain includes a region IR1 VR1, IR2, VR2, IR3, VR3, IR4, VR4, IR5, VR5 and IR6B. afzelii(strain pKo).

SEQ ID NO: 5 �correspond to the sequence of the extracellular domain of VlsE B. burgdorferisensu stricto (strain B31), shortened in its signal sequence and C-terminal region of the Mature protein, after the IR6 domain, that is, this extracellular domain includes a region IR1 VR1, IR2, VR2, IR3, VR3, IR4, VR4, IR5, VR5 and IR6B. burgdorferisensu stricto (strain B31).

SEQ ID NO: 6 corresponds to the sequence domain IR6B. burgdorferisensu stricto (strain B31).

SEQ ID NO: 7 corresponds to the sequence domain IR6B. afzelii(strain ACA-1).

SEQ ID NO: 8 corresponds to the sequence domain IR6B. garinii(strain Ip90).

SEQ ID NO: 9 corresponds to the sequence variable regions VR6B. burgdorferisensu stricto (strain B31). This sequence can be introduced into the structure as a shoulder of the spacer between domains IR6.

A sequence of at least 6 histidinemia residues (polyhistidine chain identified in SEQ ID NO: 10 encoded by any of the nucleic acid sequences identified in SEQ ID NO: 11, 12 and 13 can be attached at the level of N-terminal or C-terminal segment of the protein to ensure it is cleaned up for metallogenetic resin, and also can be attached additional amino acids represented in SEQ ID NO: 14 and is encoded by the sequence SEQ ID NO: 15 in the early polyhistidine chain. In this configuration, the protein comprises or consists of a sequence identified as SEQ ID NO: 21. Al�alternative way a sequence of 8 histidinemia residues provided in SEQ ID NO: 16 and encoded by the sequence SEQ ID NO: 17, can be introduced in the N-terminal section of the protein sequence instead of 6 histidinemia residues, which helps to stabilize the recombinant protein adhesion on metallogenetic the resin and to improve the conditions of purification, and can also be introduced additional amino acids represented in SEQ ID NO: 18 encoded by the sequence SEQ ID NO: 19. In this configuration, the protein comprises or consists of a sequence identified as SEQ ID NO: 23.

Preferred proteins of the present invention is a protein identified as SEQ ID NO: 21 and 23 respectively encoded by the sequences SEQ ID NO: 22 and 24.

The present invention also relates to DNA sequences encoding previously identified proteins, in particular, to sequences identified as SEQ ID NO: 22 and 24.

The present invention also relates to an expression cassette which is functional in a cell derived from a prokaryotic (example:Escherichia coli) or eukaryotic organism, such as yeast (example:Pichia,Schizosaccharomyces) and to allow the expression of the previously described nucleic acids (DNA) when it is under the control of the elements permitting its expression, and that�to the same vector, incorporating such tape.

The protein of the present invention is preferably acceptable for the diagnosis of infection with Borrelia. Thus, the present invention relates to a method of diagnosis ofinvitroLyme borreliosis in a biological sample (e.g., serum sample, 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 ISM�rhenium resistance; groups detected by optical methods such as diffraction, resonance of the surface plasmon, the change in contact angle or by physical methods such as spectroscopy nuclear interactions, quantum tunneling, etc.; radioactive molecules containing32P,35S or125I. Protein is preferably immobilized on a solid support, which may represent a cone device Vidas®that plate wells for micrometrology, individual particles, gel, etc.

In one embodiment of the present invention, the sample, in addition, are brought into contact with at least one fused chimeric protein selected from proteins described later, such as:

(a) protein, amino acid sequence which includes (or consists of) the sequence SEQ ID NO: 25 and a sequence of SEQ ID NO: 26 or a sequence having a degree of identity of at least 40% relative to SEQ ID NO: 25 or a sequence having a degree of identity of at least 50% relative to SEQ ID NO: 26;

(b) protein, amino acid sequence which includes (or consists of) the sequence SEQ ID NO: 27 and the sequence of SEQ ID NO: 28 or a sequence having a degree of identity of at least 40% relative to SEQ ID NO: 27, and a sequence having a degree of identity p� least 50% relative to SEQ ID NO: 28;

(c) protein, amino acid sequence which includes (or consists of) a sequence selected from sequences like:

(i) the sequence SEQ ID NO: 29 and the sequence SEQ ID NO: 31 or a sequence having a degree of identity of at least 40% relative to SEQ ID NO: 29, and a sequence having a degree of identity of at least 50% relative to SEQ ID NO: 31;

(ii) sequence SEQ ID NO: 30 and the sequence SEQ ID NO: 31 or a sequence having a degree of identity of at least 40% relative to SEQ ID NO: 30, and a sequence having a degree of identity of at least 50% relative to SEQ ID NO: 31;

(iii) sequence SEQ ID NO: 29, a sequence of SEQ ID NO: 30 and the sequence SEQ ID NO: 31 or a sequence having a degree of identity of at least 40% relative to SEQ ID NO: 29, a sequence having a degree of identity of at least 40% relative to SEQ ID NO: 30, and a sequence having a degree of identity of at least 50% relative to SEQ ID NO: 31;

(d) a protein amino acid sequence which includes (or consists of) a sequence selected from the sequences SEQ ID NO: 32, 34, 36, or a sequence selected from the sequences SEQ ID NO: 33, 35, 37 and 38, described in more detail later.

Each of proteins, �identifitsirovannykh earlier includes at least one sequence of the extracellular domain of the protein DbpA kind borrelii selected fromB. afzelii(SEQ ID NO: 25),B. burgdorferisensu stricto (SEQ ID NO: 27) andB. garinii(group III: SEQ ID NO: 29) (group IV: SEQ ID NO: 30) or a sequence having a degree of identity of at least 40% relative to these sequences, and at least one sequence of the protein OspCB. afzelii(SEQ ID NO: 26),B. burgdorferisensu stricto (SEQ ID NO: 28) andB. garinii(SEQ ID NO: 31 or a sequence having a degree of identity of at least 50% relative to these sequences. Preferably one or more DbpA sequences are N-terminal section of the recombinant protein, and the sequence of OspC is located at the C-terminal section of the recombinant protein.

Accordingly, the previously described sequence of at least 6 histidinemia residues can be attached at the level of N-terminal 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: 10, is preferably at the N-terminal section of the structure. 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 allows for facilitating�AMB cloning in the desired sequence expressing a plasmid, for example, the link "MRGS" (SEQ ID NO: 14) encoded by the sequence ATGAGGGGATCC (SEQ ID NO: 15).

The binding region may be inserted between each of the sequences DbpA and OspC, forming a chimeric recombinant protein. The scope of this type corresponds to the field of flexible spacer that provides the best potential availability of antibodies to each of the domains. It is, in General, 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 desired sequence encoding the shoulder of DNA (or linker) to facilitate communication between the sequences encoding two required protein. For example, this may be the link "GSGG" (SEQ ID NO: 46) encoded by the sequence GGTTCCGGGGGT (SEQ ID NO: 47) and acting as a shoulder of binding between proteins DbpA group IV and OspCB. garinii.

Examples of such proteins are represented by sequences SEQ ID NO: 33, 35, 37 and 38 in the sequence listing.

Proteins, previously described and identified as SEQ ID NO: 32-38 in the sequence listing, respectively, are encoded by corresponding DNA sequences identified in SEQ ID NO: 39-45.

The present invention relates also to a kit for the diagnosis ofin vitroLyme borreliosis, containing less�her least one chimeric protein VlsE, as described previously, and optionally at least one fused chimeric protein DbpA/OspC defined previously, and preferably containing at least one kind of antiimmunoglobulin person labeled by any acceptable token corresponding to the definitions given earlier.

Examples

Example 1. Obtaining plasmid constructs encoding chimeric recombinant VlsE proteins

DNA sequences that encode different protein sequences identified in table 1.

Table 1
The origin of sequences
The speciesB. Burgdorferi
*Isolate; **amino acids (aa); * * * item no in GenBank
ProteinB. sensu strictoB.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)

Posledovatelno�and were optimized with respect to their expression in E.Coliusing 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 protein shown in example (bLYM110, bLYM125) described in table 2.

Table 2
The corresponding plasmid constructs and protein
Characteristics of recombinant proteinsCharacteristics of plasmid constructs
NameN-end tagThe sequenceB. burgdorferiParental vectorThe insertion site in the vector sequence insertion
bLYM110 SEQ ID NO: 216 × Hi 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: 238 × HispET-3d5'NcoI/3'BamHI

Example 2. Expression of recombinant proteins of example 1 and purification

For transformation of the bacteriaE. coli(strain BL21) according to a conventional method known to those skilled in the art, used a plasmid construct described in example 1. 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 2xYT medium (tripton, 16 g/l; yeast extract, 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 2xYT medium 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 p�after the addition of 0.4 mm isopropyl-β-D-thiogalactopyranoside (IPTG), and harvested by centrifugation at 6000 g for 30 min. The sediment cells were 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 centrifugally 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 dialysis buffer solution. The conditions of purification on the gel of Ni-NTA as described in table 3.

Table 3
Purification of recombinant proteins
ProteinbLYM110
SEQ ID NO: 21
bLYM125
SEQ ID NO: 23
Buffer solution for lysis and washingBuffer solution A1Buffer solution A 1+ 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 culture8,717
1Sodium phosphate, 50 mm, imidazole, 30 μm, NaCl, 500 mm, Tween 20, 0.1% glycerol, 5%, pH=7,8
2Sodium phosphate, 50 mm, imidazole, 325 mm NaCl, 500 mm, glycerol 5%, pH=7,5

Samples were analyzed on a NuPAGE gel®Novex®, 4-12%, in the circulating buffer NuPAGE®MES-SDS in accordance with the guidelines of the manufacturer (Invitrogen™). Proteins were stained with brilliant blue, Kumasi or electrop�reticence 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 3. Detection of IgG and human IgM with recombinant chimeric protein bLYM110 of example 2 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 pH=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 at pH=7.5 for 1 hour at 37°C. immunoblotting Procedures were carried out at room temperature.�tively 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 detected in �spinnomozgovoi 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 following table 4.

Table 4
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)
1720519 96
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, a highly sensitive against 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 samples of Wi�ODI with EM 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 of Lyme disease are not specific. 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 4. Obtaining plasmid constructs encoding chimeric recombinant proteins DpbA-OspC

DNA sequences encoding the various described sequence DpbA and OspC identified in table 5. DNA sequences were optimized to facilitate expression inE.Coliusing a set of GeneOptimizer™ and synthesized according to the method GenScript corporation (Scotch plains, NJ, USA) or GeneArt GmbH (Regensburg, Germany).

�table 5
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 contains at least one epitope region, corresponding to the extracellular domain of DbpA sequenceBorrelia burgdorferisensu stricto orB. afzeliiorB. gariniiand at least one epitope region, corresponding to the extracellular domain sequence of OspCBorrelia burgdorferisensu stricto orB. afzeliiorB. 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-Lin�EPA carried out by 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 protein shown in example (bLYM114, bLYM120 and bLYM121) described in table 6. 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.

The sequence of poly-His (6 histidinemia residues) 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, which further facilitates purification stage chimeric recombinant protein. This peptide HHHHHH (SEQ ID NO: 10) is encoded by the nucleotide sequences CATCATCATCATCATCAT (SEQ ID NO: 11) or CATCATCATCATCATCAC (SEQ ID NO: 12), or CATCATCACCACCATCAT (SE ID NO: 13), or any other sequence that encodes the sequence of SEQ ID NO: 10.

Should indicate that 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 6
The corresponding plasmid constructs and protein
Characteristics of recombinant proteinsCharacteristics of plasmid constructs
NameN-end tagThe sequenceB. burgdorferiNameParental vectorThe insertion site in the vector sequence insertion
bLYM114 SEQ ID NO: 336 × His - B. afzeliistrain PKo
DbpA aa 2-150 + OspC aa. 2-212 runs
pOL114pMR78*5'BamHI/3'EcoRI
bLYM120 SEQ ID NO: 356 × His - B. sensu stricto W�AMM B31
DbpA aa 28-192 + OspC aa 26-210
pOL120pMR78*5'BamHI/3'HindIII
bLYM121 SEQ ID NO: 386 × His - B. garinii
DbpA III aa 25-187 strain 40 + DbpA IV aa 24-176 strain PBi + OspC aa 32-208 strain PEi
pOL121pMR78*5'BamHI/3'HindIII
● [2]

Example 5. Expression of recombinant proteins bLYM114, bLYM120 and bLYM121 of example 4 and clean

For transformation of the bacteriaE.coli(strain BL21) according to a conventional method known to those skilled in the art, was used for plasmid construction, in which the expression vector (pMR) was inserted sequence SEQ ID NO: 40, 42 or 45. 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 2xYT medium (tripton, 16 g/l; yeast extract, 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 2xYT medium containing 2% glucose and 100 μg/ml ampicillin. �annoy culture was 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 centrifugally 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 on a column volume of 8 ml with gel of Ni-NTA, air-conditioned buffer solution A (see table 7). 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 performed by flow of buffer solution B, respectively, described in table 7, 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 RA�down. The conditions of purification on the gel of Ni-NTA as described in table 7.

Table 7
Purification of recombinant proteins
ProteinbLYM114bLYM120bLYM121
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 2100% 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 culture 80122245
1Sodium phosphate, 50 mm, imidazole, 30 μm, NaCl, 500 mm, Tween 20, 0.1% glycerol, 5%, pH=7,8
2Sodium phosphate, 50 mm, imidazole, 325 mm NaCl, 500 mm, glycerol 5%, pH=7,5

Samples were analyzed on a NuPAGE gel®Novex®, 4-12% NuPAGE buffer solution®MES-SDS in accordance with the guidelines of the manufacturer (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 6. Detection of IgG and human IgM with recombinant chimeric protein 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 � pH=7,2 was set equal to 1 mg/ml and diluted with PBS with pH=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 at 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, then 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 macroderma�item [ACA] and 10 with benign lymphocytes skin [LCB]). Antimorality IgG were found 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 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 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
Reactivity with linear immunoblotting of serum samples of patients with Lyme borreliosis with 3 chimeric recombinant proteins
Recombinant proteinIgGIgM
Stage IStage IIStage IIIStage IStage 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)
bLYM11451007122772
bLYM1206708 601172
bLYM1212105980772
Σ bLYM 114 + 120 + 1219135181721172

Samples of serum c-positive, % 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 positive results, in % and the intensity of reaction 66,7%
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 ofBorrelia afzeliiB. sensu stricto andB. gariniiaccordingly, 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

Chimeric 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 immunoblotting [4] and a commercial kit Borrelia IgM (r-biopharm®), not detected a larger number of serum samples with positive results on IgM. In addition, 3 samples of serum, which by immunoblotting set of Borrelia IgM (r-biopharm ®) is defined as negative, defined as positive by three chimeric proteins described in example (3/3), or by three proteins described in 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 7. Evaluation and validation of chimeric recombinant proteins bLYM114, bLYM120, bLYM121 and bLYM125 by VIDAS test®(bioMérieux)

This validation was performed through test VIDAS®using:

- 1) chimeric recombinant proteins bLYM114, bLYM120 and bLYM121 obtained according to examples 4 and 5, for the detection of IgM;

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

The principle of the VIDAS test®is as follows: the 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, remaining unbound, are removed by washing. Antibody antimonopo�of Ulanov 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®is proportional to the content 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 the Vidas system®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 bLYM1251 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 the formation of complexes of antigens-anti�La". 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 conditionally negative, which corresponds to a sensitivity of 96.5%.

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

The list of references

1. Chimeric protein borrelii for the diagnosis of Lyme borreliosis comprising at least one sequence of the extracellular domain of the protein VlsE borrelii of the first type corresponding to a particular strain, and at least one sequence region IR6 protein VlsE borrelii of the second kind or borrelii of the first type, but the corresponding strain different from the strain of the first type, and the indicated chimeric protein includes:
sequence of the extracellular domain of the protein VlsE borrelii of the first type, consisting of five variable regions VR1, VR2, VR3, VR4 and VR5 and six constant regions IR1, IR2, IR3, IR4, IR5 and IR6, wherein at least one specified sequence of the extracellular domain selected from the group comprising the sequences SEQ ID NO.:1, 2, 3, 4 � 5;
- the specified at least one sequence region IR6 borrelii of the second kind or borrelii of the first type, but the corresponding strain different from the strain of the first type selected from the group comprising the sequences SEQ ID NO:6, 7 and 8
moreover borrelii selected from Borrelia stricto-sensu, Borrelia afzelii and Borrelia garinii.

2. Protein according to claim 1, comprising a variable sequence VR6 kind borrelii identified in SEQ ID NO:9.

3. Protein according to claim 1, including:
sequence SEQ ID NO:1;
sequence SEQ ID NO:6;
sequence SEQ ID NO:7;
sequence SEQ ID NO:8.

4. Protein according to claim 3, additionally comprising the sequence of SEQ ID NO:9.

5. Protein according to any one of claims. 1-4, comprising a sequence selected from SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:23.

6. Nucleic acid that encodes a protein as defined in any one of claims. 1-5.

7. 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 6 and under the control of elements necessary for its expression.

8. The vector for expression of the nucleic acid according to claim 6, comprising expressing cassette according to claim 7.

9. An in vitro method for diagnosing Lyme borreliosis in a biological sample, according to which the biological specimen is lead in to�tact with at least one protein according to any one of claims. 1-5 and identify possible formation of an immunological complex between said protein and antibodies of the biological sample.

10. A method according to claim 9, in which the antibodies of the biological sample are IgG and/or IgM.

11. A method according to claim 9, in which the formation of the immunological complex is determined by adding at least one antiimmunoglobulin person labeled by any acceptable token.

12. A method according to any one of claims. 9-11, in which the protein is immobilized on a solid substrate.

13. Kit for in vitro diagnosis of Lyme borreliosis comprising at least one chimeric protein borrelii, characterized in that the protein is a protein according to any one of claims. 1-5.

14. The kit according to claim 13, comprising at least one antiimmunoglobulin person labeled by any acceptable marker.



 

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Biomarkers // 2545757

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18 cl, 2 ex, 9 dwg, 7 tbl

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33 cl, 3 dwg, 1 tbl, 2 ex

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17 cl, 53 dwg, 6 tbl, 10 ex

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FIELD: medicine, pharmaceutics.

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73 cl, 57 dwg, 4 tbl, 33 ex

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15 cl, 3 tbl, 3 ex

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4 dwg, 3 ex

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29 cl, 19 dwg, 4 tbl, 20 ex

FIELD: medicine.

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4 cl, 6 dwg, 2 tbl, 5 ex

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