Mel-tci-a0201 artificial gene coding mel-tci-a0201 polyepitope immunogenic protein, recombinant plasmid dna of pmel-tci-a0201, providing expression of mel-tci-a0201 artificial gene and mel-tci-a0201 artificial immunogenic protein containing multiple ctl- and th-epitopes of melanoma antigens

FIELD: medicine.

SUBSTANCE: invention refers to biochemistry, particularly to artificial immunogenic proteins having the properties of melanoma antigens. What is declared is an artificial gene coding MEL-TCI-A0201 polyepitope immunogenic protein containing multiple cytotoxic restricted HLA-A*0201 and T-helper epitopes of NY-ESO-1, MART1, MAGE-A1, MAGE-A3, MAGE-A11, MAGE-C1 melanoma antigens, having a sequence of 1,535 base pairs presented in Fig. 3. There are also declared a recombinant plasmid DNA containing the above artificial gene, and MEL-TCI-A0201 immunogenic protein with the properties of the melanoma antigens.

EFFECT: invention enables providing higher immunogenicity of the artificial polyepitope T-cell immunogen inducing a higher level of cytotoxic T-lymphocyte response.

3 cl, 11 dwg, 3 tbl, 2 ex

 

The invention relates to artificial proteins-immunogenum having properties of melanoma antigens used to generate synthetic polyepitope immunotherapy vaccines against melanoma, and can be used in molecular biology, genetic engineering and medicine.

Currently numerous studies aimed at developing vaccines against various types of cancer. Known strategies for the creation of such vaccines based on the use of various forms of cancer antigens, lysates of cancer cells, polyepitopic antigens containing multiple T-cell epitopes that induce a cytotoxic response (CTL - cytotoxic T-lymphocyte) and/or helper (Th - T-helper) T lymphocytes. It uses a variety of approaches: the use of modified viruses or bacteria encoding the target antigen; DNA vaccination or the use of cellular immunotherapy vaccines.

Of considerable interest is the immunogenicity of melanoma cells. There were cases of spontaneous complete remission of malignant melanoma associated with spontaneous induction of both humoral and T-cell immune response [1]. The high immunogenicity of melanoma tumors suggests the possibility of creating an effective therapeutic melanoma vaccine, using p the recent advances in molecular immunology and cell technology.

The pioneer in this field work were created individual autologous vaccines: 64 patients with stage IV melanoma were immunized with preparations of autologous tumor tissue, and it has been shown that antigen-specific immune response can be induced in many patients [1]. Currently describes a number of highly immunogenic protein antigens that are specific for melanoma and some other types of cancer that are absent (or almost absent) in normal tissues of the human body [1-3]. Many of these antigens such as Melan-A/MART-1, gp100, tyrosinase, MAGE-3 and NY-ESO-1 were used to develop candidate vaccines, some of which is currently undergoing clinical trials [1,3]. The results of clinical trials indicate a slight amount of side effects of vaccination, such as hypersensitivity at the site of injection and induction of autoimmune reactions, such as vitiligo (when vaccination proteins associated with differentiation of melanocytes) [1].

It is believed that the most effective for the treatment of neoplastic diseases should be a comprehensive approach, including surgical techniques, and methods of chemotherapy along with individualized immunotherapy of malignant tumors. In the last decade have been developed principally the new approaches to the immunotherapy of neoplastic diseases, based on cellular technology is the induction of T-cell immune responseex vivo. Vaccines derived from cell technologies, has demonstrated promising results. In 2010, one of these immunotherapy vaccine Sipuleucel-T (Provenge) is a vaccine against prostate cancer, was approved for use in clinical practice [4].

However, such vaccines have significant drawbacks: as a rule, currently cellular immunotherapy vaccines are individualized drugs, brewed on the basis of the lysate of autologous tumor cells obtained surgically. Thus, these drugs in addition to tumor antigens contain a large number of proteins present in normal tissues of the body - such an approach is fraught with the development of the patient's autoimmune reactions, in addition to the target antigens of the cancer cells lysates may contain potentially pathogenic molecular factors. In addition, when using this approach it is difficult to achieve a high level of activation of cytotoxic T-lymphocytes, because the presentation of antigens captured in the result of endocytosis, as a rule, is carried out by MHC-II-dependent mechanism - using immature dendritic cells (DC), lagausie high capacity for phagocytosis. It is shown that the ability of immature DC to induce a cytotoxic response of CD8+ T lymphocytes is significantly lower than in Mature DC [5].

One of the promising approaches to the development of new effective and safe vaccines - the creation of artificial polyepitopic antigens [6,7]. Such vaccines compared with vaccines that are created on the basis of more traditional approaches have the following advantages, namely:

1) they do not contain full-sized molecular structures of pathogens, which can be pathogenicity factors and/or can inhibit the formation of protective immunity or to stimulate the development of autoimmunity;

2) polyepitope may include in its membership a large number of CTL and T-helper epitopes from cancer antigens;

3) they can be designed taking into account the prevalence of different allomorph of HLA class I molecules in the target human population of any given genetic characteristics of a particular patient;

4) polyepitope can be designed to maximize the efficiency of processing and presentation of most of the target epitopes [8-12];

5) to increase the efficiency of the induction response of T-lymphocytes target polyepitope may contain additional signal sequence (for example, N-terminal ubiquitin-like, N-terminal leader peptide IP-terminal fragment of the protein LAMP-1 person) [13-15].

Currently, there are a large number of synthetic vaccine constructs containing multiple CTL epitopes. In particular, described polyepitope design, containing seven adjacent minimum HLA-A2-restrictively CTL epitopes of HIV-1 [16], poly-CTL-epitope vaccine against human papilloma virus [17], poly-CTL-epitope vaccine against hepatitis C virus [18], polyepitope vaccines against melanoma [19, 20], etc. Immunogenicity of these constructs was confirmed by numerous studies, in particular, [19, 20].

The closest analogue is polyepitope design for immunotherapy of melanoma, containing ten HLA-A2 epitopes of the five antigens of melanoma [19, prototype]. It was shown that this artificial polyepitope vaccine design is immunogenic, while the CTL line derived from vaccinated HLA-A2 transgenic mice were able to lyse melanoma cells iv vitro. The findings suggest the applicability polyepitope vaccines for immunotherapy of melanoma.

Along with this, we can note some important points that distinguish the invention from the prototype design. First, for the design of polyepitope used ten CTL epitopes, whereas the inventive artificial gene encodes a 19 CTL epitopes. Secondly, the prototype does not contain CD4+T-helper the x epitopes, while the inventive artificial gene encodes 45 T-helper epitopes of antigens of melanoma that is required for the induction of the response of CD4+T-lymphocytes, which are known to enhance the responses of CD8+CTL [41]. Thirdly, as part of the prototype epitopes were combined sequentially at the junction interface without speyeria sequences that optimize the binding of peptides with TAR and release of peptides in the proteasome or immunoproteasome splitting, which is required for efficient induction of T-cell response [9, 22]. Fourth, when designing polyepitope was not taken into account the number of non-target epitopes, which may be formed at the joints. Fifthly, there was a search for an optimal placement of epitopes within polyepitopes design [22]. And finally, when assessing the immunogenic properties of the prototype was not used positive control, that is not compared with the immune response to a full-sized antigens of melanoma. Unlike the prototype when assessing the immunogenicity politions design MEL-TCI-A0201 were compared its immunogenic properties with the full-length protein MART1. It was shown that, despite the fact that the composition of MEL-TCI-A0201 is only four epitope MART1, its effectiveness induction of T-cell response in the ex vivo system using Mel melanoma cells Is HLA-A*0201+, MART1+) with anima with the response to the design, encoding a full-sized antigen MART1. Given that the structure of the MEL-TCI-A0201 also includes CTL-epitopes from other melanoma antigens such as NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-EN, MAGE-C1, it can be expected that the claimed immunogen will also induce an immune response against melanoma cells positive for these antigens antigens.

The technical result of the claimed invention is the use of computer design more optimized artificial polyepitope T-cell immunogen MEL-TCI-A0201, is able to induce specific T-cell immune response against antigens of melanoma by rational allocation of the target T-cell epitopes in the composition polyepitope immunogen and selection of the optimal spacer elements amino acid sequences flanking epitopes as part polyepitopes design

This technical result is achieved by a computer system design artificial protein immunogen MEL-TCI-A0201 having the amino acid sequence shown in figure 2, and containing multiple cytotoxic restrictively HLA-A*0201 and T-helper epitopes of melanoma antigens NY-ESO-1, MART1, MAGE-A1, MAGE-A3, MAGE-EN, MAGE-C1, combined with the use of spacer elements amino acid residues, and N is the end of polyepitope contains the signal peptide of the protein HER2 (the 04626), and With the end includes the last 11 amino acid residues of the protein LAMP-1 person. Design polyepitopes design aimed at increasing the efficiency of processing polyepitopes design and presentation of target epitopes, as there is convincing evidence from the prior art that the optimization processing increases the immunogenicity polyepitope vaccine design [9, 22]. This takes into account the relative locations of the epitopes in the composition polyepitopes design, is calculated optimal spacer elements sequence for each pair of epitopes and minimizes the number of non-target epitopes in polyepitopes design.

This technical result is also achieved by constructing recombinant plasmid DNA pMEL-TCI-A0201, carrying the gene of artificial polyepitope protein MEL-TCI-A0201 under control of the CMV promoter, providing the last expression in mammalian cells, which has a size 6952 P.N. and consists of the following parts (figure 4):

- NheI-HindIII vector fragment of DNA plasmids pcDNA3.1(-) size 5423 P.N.;

- NheI-HindIII fragment size 1535 BP, containing the gene MEL-TCI-A0201.

In order to explain the principle of constructing artificial polyepitope protein immunogen MEL-TCI-A0201, the following explanations are presented below.

Therefore, the search for peptides, the way the s contact with high affinity with a wide range of allelic variants of HLA molecules, important.

An important role in the processing of endogenous antigens plays a proteasome - intracellular multispherical proteolytic complex [28, 29]. It is shown that the proteasome generates the C-end of peptides binding to MHC molecules of class I [30, 31], thus, when selecting epitopes must take into account the specificity of proteasome cleavage of target antigens. Peptides resulting from cleavage of proteins by the proteasome, selectively linked heterodimeric complex TAP (transporters associated with processing) and are transported into the endoplasmic reticulum (ER), where contact with molecules MHC class I and formed complexes are transported to the cell surface [23-25,32].

As a result of the literature analysis for design polyepitopes designs intended for immunotherapy of melanoma, the target (optimal) epitopes were selected from six of the most immunogenic antigens:

1. Cancer/testis antigen 1 (CTAG1A, NY-ESO-1) (P78358size 180 amino acids.

2. MART1, Melanoma antigen recognized by T-cells 1 (Q16655), size 118 A.K.

3. MAGE-A1 (P43355size 309 A.K.

4. MAGE-A11 (CT1.11) (P43364size 429 A.K.

5. MAGE-A3 (P43357size 314 A.K.

6. MAGE-C1 (O60732size 1142 A.K.

Prediction of epitopes in these antigens was carried out using established authors previously is put together his original software TEpredict [33], allowing for (immuno)proteasome processing, the binding affinity of oligopeptides with TAP and hold a selection of epitopes with regard to frequency of occurrence allomorph of HLA molecules in the target human population.

When creating polyepitope vaccine design is necessary to provide the most efficient induction of T-cell immune response is included in its composition antigenic peptides (epitopes).

The problem is solved by choosing the optimal placement of the target T-cell epitopes in the composition polyepitope immunogen and selection of the optimal spacer elements of amino acid sequences to optimize processing of epitopes and increase immunogenity polyepitopes design [9-22], and also by selecting a special signal sequences that optimize MHC I and/or MHC II-dependent antigen presentation [8-15].

The design of artificial polyepitope T-cell immunogen MEL-TCI-A conducted using the developed by the authors earlier original software PolyCTLDesigner [34-35] (Figure 1). Analysis of cell lysate T, transfected with a plasmid pMEL-TCI-A0201, showed that artificial polyepitopic protein MEL-TCI-A0201 not only effectively synthesized, but is also exposed to the efficient processing in transfected cells (Fig.9)that the plan is fully consistent with its design.

Below is a list of graphical materials illustrating the invention.

Figure 1 presents the algorithm of the program PolyCTLDesigner.

Figure 2 shows the amino acid sequence polyepitope protein immunogen MEL-TCI-A0201.

Figure 3 presents the nucleotide and amino acid sequence of the synthetic gene encoding polyepitopic immunogen MEL-TCI-A0201.

Figure 4 shows the physical and genetic map of the recombinant plasmid pMEL-TCI-A0201.

Figure 5 shows the circuit construction of recombinant plasmid DNA pMEL-TCI-A0201.

Figure 6 presents electrophoregram in 1% agarose gel. The DNA fragments after hydrolysis of plasmids by restrictase NheI and HindIII.

Figure 7 depicts electrophoregram in 1% agarose gel. The DNA fragments after hydrolysis plasmids pMEL-TCI-A0201-restrictable BglII and PsiI.

On Fig presents immunochemical staining preparations of cells T, transfected with plasmid DNA pMEL-TCI-A0201 (pcDNA-G1).

Figure 9 shows the immunoblotting of proteins after separation in 15% SDS page with monoclonal antibodies 29F2 (marker epitope).

Figure 10 presents electrophoregram products FROM RT-PCR in 1% agarose gel.

Figure 11 presents the results of the study the immunogenicity of DNA vaccine constructs pMEL-TCI-A0201, coding polyepitopic immunogen, the content is the overall cytotoxic epitopes of antigens of melanoma, restrictively HLA-A*0201.

Design and construction of the proposed objects of the invention by the method of computer simulation.

1. Design poly-CTL-epitope immunogen fragment MEL-TCI-A0201

Using TEpredict in the selected antigens were predicted CTL epitopes. Design target polyepitopes design MEL-TCI-A0201 were selected epitopes for which the predicted value pIC50≥6.8, which corresponds to a high or medium affinity binding of the peptide to a molecule HLA-A*0201 Peptides that were predicted to have lower affinity of binding to MHC molecule (pIC50<6.8), were excluded from further analysis. In addition, predicted proteasome and immunoproteasome processing of antigens, as well as the binding affinity of peptides with conveyors associated with the processing of antigens, and only those peptides that were predicted with sufficient affinity binding to TAP, and With the end of which was predicted cleavage site, were selected for constructing polyepitope antigen. In the result, we selected 19 of the peptides listed in table 1.

Table 1

The epitopes selected for constructing poly-CTL-epitope of fragmenta target immunogen

MEL-TCI-A0201FLIIVLVMI, ALMDKSLHV, ILILSIFI, HLYIFATCL, VIWDVLSGI,
GILTVILGV, LMWITQCFL, YIFATCLGL, FLWGPRALA, LLIIVLAII,
SLAQDAPPL, VCMQLLFGI, FFSSALLSI, HLLLRKYRV, FLAMLKNTV,
IMPKAGLLI, YVLVTCLGL, AMDAIFGSL, CILESLFRA

Next, using PolyCTLDesigner (Fig. 1) was carried out design poly-CTL-epitope fragment using degenerate spacer elements of the motif [ARSP][DLIT][LGA][VKA] to optimize proteasome cleavage. When choosing the best spacers for each pair of epitopes was carried out to minimise the number of non-target epitopes formed by the docking of peptides, restrictively allaluminum option of HLA class I molecules - A*0201. The length of the resulting construction amounted to 213 ago, the share of the spacer elements of the sequence is equal to 18.31 %.

In Fig. 1 presents the algorithm of the program PolyCTLDesigner. Note: I is the prediction of the affinity of binding of peptides to TAP and add N-terminal flanking ako (if necessary); II - optimal spacer elements sequences for each pair of peptides (spacer elements sequence meets the following criteria: provides education site proteasome cleavage at the C-end of the 1st peptide, provides, in comparison with other variants of the spacer, forming the smallest number of non-target epitopes at the junction and has the smallest possible length) and the creation of a directed weighted graph, in which vertices represent the target e is atopy, and the edges represent valid ways of combining them (each edge corresponds to a set of parameters: spacer elements sequence and the vector of weights corresponding to the predicted efficiency of the cleavage site, the number of non-target epitopes and the length of the spacer); III - construction sequence polyepitope immunogen (required sequence is defined as the longest simple path in the generated graph with the smallest weight).

Thus, was designed poly-CTL-epitope fragment (213 ACO) immunogen MEL-TCI-A0201 , containing 19 CTL-epitopes (restrictively HLA-A*0201) of the six most immunogenic melanoma antigens.

In Fig. 2 shows the amino acid sequence polyepitope protein immunogen MEL-TCI-A0201. The length of the resulting construction was 502 ago, the share of the spacer elements of the sequence is equal to 18.31 %. On the N-end signal peptide is shown bold white font on a black background. After is poly-CTL-epitope fragment (213 ago to PADRE epitopeAKFVAAWTLKAAA,the selected frame). Spacer elements ako optimizing proteasome processing in bold and underlined. -The end is the C-terminal fragment of the protein LAMP1 (RKRSHAGYQTI)shown bold white font on a black background. Before him is a marker of b-cell epitope of the protein Ga HIV-1 EPFRDYVDRFYKTLR(italics and underlined) to confirm the expression of the target protein. Between PADRE and marker epitopes is poly-T-helper epitope fragment. Gray in a poly-T-helper fragment shows the amino acid spacers, optimizing lysosomal processing polyepitope antigen.

2. The design of poly-T-helper fragment immunogen MEL-TCI-A0201

For the most efficient induction of T-cell immune response is necessary to stimulate a response is not only CD8+, and CD4+ T-lymphocytes, therefore, the next task was to build a poly-Th-epitope fragment. To do this, using TEpredict was conducted predicting the composition of selected cancer antigens Th epitopes with the most broad specificity with respect to HLA class II. Using PolyCTLDesigner was selected 6 fragments ranging in length from 20 to 30 ACO, which contained the largest number of Th epitopes with the most broad specificity with respect to different allomorphs HLA class II (PL. 2). N-the ends of the selected fragments were extended for 5 ACO relative to the beginning of the first epitope and the C-ends - 5 ACO relative to the end of the last epitope.

Table 2

The sequence selected for constructing poly-T-helper fragment of the target immunogen

td align="justify"> 34
Sequence AntigenThe beginning and end of the fragmentIn allomorph HLA IIIn a foreseeable-
these epitopes
EEAAGIGILTVILGVLLLIGCWYCRRRNGYRALMDKSMAR125-614511
LSYDGLLGDNQIMPKTGFLIIVLVMIAMEGGHAPEEMAGA1177-212478
MSQNRLLILILSIIFIKGTYASEEVIWMAGC1994-1020486
SFSQDILHDKIIDLVHLLLRKYRVKGLITKAEMLGSVMAGEA11216-252408
KASSSLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLMAGA3153-189416
VSGNILTIRLTAADHRQLQLSISSCLQQLSLLMWITQCFCTG1B128-1666

In table. 2 shows the sequence of the peptide, the name of the antigen, the beginning and the end of the selected peptide, the number of interacting fragment allomorph HLA II and the number of predicted fragment Th epitopes. In addition, it was decided to include in the composition of the poly-Th-epitope constructs a universal T-helper epitope PADRE (PanDRepitope). To confirm the expression of a target polypeptide in its composition was included marker of b-cell epitope Gag protein of HIV-1 (Fig. 2). The fragments were combined with the use of the motif [KR][KR] - forming sites of cleavage for lysosomal cathepsins B and L, involved in MHC-II-dependent processing of antigens.

Thus, was designed poly-Th-epitope immunogen fragment MEL-TCI-A0201 containing 45 T-helper epitopes (restrictively not less than 48 allelic variants of HLA molecules class II) of the six most immunogenic melanoma antigens.

3. The final design polyepitopes designMEL-TCI-A0201

Using PolyCTLDesigner poly-CTL - and poly-Th-epitope fragments were combined into a single structure by means of spacer elements selected amino acid sequence. To increase the induction response of CD4+ and CD8+ T lymphocytes, in addition to N-con is polyepitope was added to the signal peptide of the protein HER2 ( P04626), a guide formed polypeptide in the endoplasmic reticulum and on the end was added for the last 11 amino acid residues of the protein LAMP-1 person to provide redirection of the polypeptide from the secretory pathway to degradation in complementary mechanism, where its peptide fragments resulting from proteolysis, may contact recirculating MHC class II molecules. The selection sequence of the signal peptide was carried out using a server SignalP 3.0 [39]. Consistency and General appearance of the final polyepitope immunogen is shown in Fig. 2.

4. Designing an artificial gene encoding the immunogen MEL-TCI-A0201

Was designed sequence synthetic gene encoding the target polyepitopic antigen MEL-TCI-A0201-optimized for expression in human cells. To design the sequence of the synthetic gene was used various programs and on-line services (DNASTER, VectorNTI, NCBI-Dlast and others), allowing to optimize the sequences of genes for their high expression in human cells. At the 5'end of the newly inserted site of the restriction enzyme BmtI, the Kozak sequence and initiating codon at the 3' - site of Hind III and three of the stop codon. The sequence of the synthetic gene has a length of 1535 BP (Fig. 3).

In Fig. 3. presents the nucleotide and amino acid p is coherence of artificial gene coding polyepitopic immunogen MEL-TCI-A0201. The sequence of the synthetic gene has a length of 1535 BP At the 5'end of the newly inserted site of the restriction enzyme BmtI (highlighted in black). In gray in the figure selected Kozak sequence. The initiating codon ATG in bold and underlined. Stop codons are shown in bold italic marked in grey. On the 3' - HindIII site (marked in black).

5. Construction of recombinant plasmids containing an artificial gene that encodes a protein-immunogenic and MEL-TCI-A0201

The gene encoding the protein MEL-TCI-A0201, was obtained by chemical synthesis and cloned into the commercial vector pAL-TA (Evrogen, Russia). To ensure the artificial gene expression in eukaryotic cells it was periglomerular in the expression vector pcDNA3.1 (Invitrogen,USA) according to the restriction sites NheI/HindIII. After transformation of cellsE. coli DH5αF'the obtained recombinant plasmid pMEL-TCI-A0201 containing target genes, was selected standard screening procedures using restriction analysis. The structure of the cloned sequence was confirmed by sequencing.

The constructed recombinant plasmid DNA pMEL-TCI-A0201 has a size 6952 gel (Fig. 4) In the composition of this plasmid target gene that encodes a synthetic polyepitope protein MEL-TCI-A0201, is under the control of the prom is the Torah CMV, ensure its expression in mammalian cells. Plasmid DNA pMEL-TCI-A0201 consists of the following fragments:

NheI-HindIII - vector DNA fragment of the plasmid pcDNA 3.1(-) [Invitrogen, USA] size 5423 gel containing the CMV promoter and the sequence of BGH poly A, providing for the expression of a gene MEL - TCI-A0201 in mammalian cells; a gene of resistance to ampicillin (bla) and pMB1 ori, providing breeding and reproduction of target plasmids in bacteriaEscherichia;

NheI-HindIII - fragment 1535 BP, containing the gene MEL-TCI-A0201 and the Kozak sequence from the initiating codon ATG, obtained by processing restrictase NheI-HindIII plasmid pAL-TA - MEL - TCI-A0201.

In addition, plasmid pcDNA-MEL-TCI-A0201 contains unique restriction sites:NruI-209,NheI-896,HindIII-2430,NarI-3792,SmaI-3605.

The molecular mass of the target recombinant plasmid DNA is equal to 4.5×103kDa.

The position of genes on a plasmid pMEL-TCI-A0201:

MEL - TCI-A0201 - the beginning (895) - the end (2429);

Neo-r - the beginning (of 3,663) - end (4457);

Bla - start (5962) - end (6822);

BGH polyA - start (2551) - end (2778);

pMB1 - the beginning (5144) - end (5817);

the eukaryotic CMV promoter pr - the beginning (209) - end (863).

In order to prove the ability of the resulting plasmid to induce the synthesis of the corresponding target polypeptide in eukaryotic cells, was carried out transfection T-tile is dedicated to plasmid DNA. The presence of a target polypeptide in transfected cells was confirmed using monoclonal antibodies to marker epitope Gag protein of HIV-1 immunoactive cells and Western blot turns, as well as on the synthesis of specific mRNAs using RT-PCR.

Thus, the designed artificial protein immunogen MEL-TCI-A0201 containing multiple cytotoxic and helper T-cell epitopes of melanoma antigens (NY-ESO-1, MART1, MAGE-A1, MAGE-A3, MAGE-A11, MAGE-C1). Based on the designed amino acid sequence carried out the design and synthesized an artificial gene that encodes polyepitopic protein immunogen MEL-TCI-A0201. The data obtained confirm that the immunochemical properties of the expression product of the gene MEL-TCI-A0201 matches the target immunogen MEL-TCI-A0201.

Created antigenic structure can be used for immunotherapy of melanoma using three different protocols: either as DNA vaccines, either to engage in individualized immunotherapy on the basis of whole cell vaccines using autologous Mature DC, transfected with the DNA vaccine design, or based on the adoptive transfer of autologous effector T-lymphocytes obtainedex vivo.

Below are examples of specific performance of the declared objects of the invention.

Example 1.With ntes artificial gene, encoding the immunogen MEL-TCI-A0201 and preparation of recombinant plasmids pMEL-TCI-A0201, the coding target polyepitopic immunogen MEL-TCI-A0201

1.1. The artificial synthesis of the gene encoding the immunogen MEL-TCI-A0201

The choice of method of synthesis was dictated by considerations of efficiency and accuracy. The most optimal method was based on the use of thermostable RNA ligase (NEB, USA) with subsequent PCR and cloning into the vector pAL-TA. The synthesis of oligonucleotides was carried out on the synthesizer ABI3900 (AppliedBiosystems, USA). In the synthesis we have used the modified Protocol, which allows to obtain a long oligonucleotides to 90 parts of a purity of more than 95 %. Trimmed and desalted oligonucleotides straight chain mineralis, then added primers-stand and held the ligation reaction. Received lligat was cloned in the vector pAL-TA. After screening were selected by three clones carrying an insert of them were isolated plasmids. The correctness of the synthesized sequence was confirmed after subsequent sequencing on an automated sequencing machine ABI3730x1.

1.2. The preparation of recombinant plasmids pMEL-TCI-A0201, the coding target polyepitopic immunogen MEL-TCI-A0201

To construct plasmids pMEL-TCI-A0201 used plasmid pcDNA3.1-mycchislacZ(-) [Invitrogen, USA]. The DNA fragment containing the target gene was obtained by hydrolysis of the restriction endonucleases NheI and Hind III plasmid pAL-TA-MEL-TCI-A0201. In Fig. 5 is a diagram of the construction of recombinant plasmid DNA pcDNA-MEL-TCI-A0201.

Next, 0.5 μg of the fragment obtained gene ligated under standard conditions with 0.1 µg plasmid pcDNA3.1, hydrolyzed with the restriction endonucleases NheI and HindIII. Ligase mixture was used to transform competent cells ofE. coliDH5F' clones, grown on medium containing ampicillin, were isolated plasmid DNA was subjected to restriction analysis with restriction endonucleases NheI, HindIII (Fig. 6) and BglII, PsiI (Fig. 7).

1.3. Confirmation of the structure of plasmid DNA restriction analysis

According to theoretically calculated nucleotide sequence by the hydrolysis of plasmid pMEL-TCI-A0201 using endonucleases NheI (site recognition GCTAGC) and HindIII (site recognition AAGCTT) should appear in the fragments (in BP) 1528 and 5329 (Fig. 7). When the hydrolysis of plasmid pMEL-TCI-A0201-restrictable BglII (site recognition AGATCT) and PsiI (site recognition ACATGT) should receive the sets of fragments (in BP) 2320, 1797, 1468, 1367 (Fig. 7). From the data shown in Fig. 6-7 shows that the mobility of the fragments of the hydrolyzate of the target and the control plasmid (pcDNA-G2) plasmids in a 1 % agarose relative to the molecular weight marker and the hydrolysate of the original plasmid pcDNA-TCI coincides with theoretically calculated.

In Fig. 6 presents electrophoregram in 1 % agarose gel. The DNA fragments after GI is rolisa plasmids by restrictase NheI and HindIII, where:

1. pcDNA3.1 - hydrolysis NheI and HindIII, the fragments 5329 and 98 BP

2. pMEL-TCI-A0201 - native

3. pMEL-TCI-A0201 - hydrolysis NheI and HindIII, the fragments 1528, 5329 P.N.

4. pcDNA-G2 - hydrolysis NheI and HindIII, the fragments 3421, 5329 P.N.

5. pcDNA-G2 - native

6. the molecular weight marker 1Kb (M12). Specified fragments 3000 BP (upper arrow) and 1000 BP (bottom arrow);

In Fig. 7. Electrophoregram in 1 % agarose gel. The DNA fragments after hydrolysis plasmids pMEL-TCI-A0201-restrictable BglII and PsiI.

1. the molecular weight marker 1Kb (M12). Specified fragments 3000 BP (upper arrow) and 1000 BP (bottom arrow);

2. pMEL-TCI-A0201, hydrolysis BglII - PsiI, fragments 2320, 1797, 1468, 1367 BP

1.4. Confirmation of the structure of the plasmid by sequencing

The structure of the gene MEL-TCI-A0201 inthe composition of the obtained recombinant plasmidspMEL-TCI-A0201in addition to the restriction analysis was also confirmed by sequencing. It is shown that the nucleotide sequence of the gene in the sample coincides with theoretically calculated sequence.

Example 2. The study of the ability of the target gene to provide a fusion protein MEL-TCI-A0201 in securitychecker cells

2.1. Transfection of cells T recombinant plasmids

Transfection of cells T the plasmid pcDNA-MEL-TCI-A0201 were performed for achieving cell proliferation to state 60-70 % of the monolayer. In the tube type Eppendorf were placed 200 μl medium DM is M, 5 ál of plasmid DNA (1 mg/ml) and 5 μl of FuGENE® HD Transfection Reagent (Roshe). The mixture was thoroughly mixed and incubated at room temperature for 15 minutes After incubation, the mixture was made to culture the cells and placed in a culture plate in a CO2incubator for 4 hours. Then to each well was added 1 ml of medium, DMEM containing 10 % fetal bovine serum, and placed in CO2incubator for 48 hours.

2.2. Immunoablative transfected T cells

After the procedure transfection T cells by plasmidpMEL-TCI-A0201 the cells were fixed with 4 % formaldehyde solution for 30 min at room temperature. Washed with PBS solution 3 times. To remove peroxidase activity was made in 200 μl of 3 % solution of sodium azide and 0.3% hydrogen peroxide solution. Incubated at room temperature for half an hour. Washed three times with PBS solution. To each well was added 200 μl of a solution of PBS and 100 µl of monoclonal antibodies 29F2 that are associated with epitope EPFRDYVDRFYKTL P24 HIV-1, part polyepitopes design. Incubated 1 hour at room temperature. Unbound antibody was removed by washing in PBS. Specifically bound peroxidase antibodies were revealed using peroxidase labeled antibodies rabbit against mouse IgG in PBS buffer with 5 % fetal serum. Commonly used breeding conjugate antibodies 1/1000. Washed with PBS buffer 3 times. Staining was performed by adding 200 μl of a solution of the following composition: 0.5 mg/ml diaminobenzidine phosphate, 0.01 % hydrogen peroxide solution, 50 mm Tris-HCl (pH 7.4), 50 mm imidazole. The coloring was performed in the dark at room temperature for 3 min, then the product was thoroughly washed with distilled water. The presence of stained cells was determined using an Olympus microscope.

When immunochemical staining preparations of transfected cells as a negative control preparations were used T, transfected with a vector plasmid pcDNA3.1, as a positive control plasmid pcDNA-TCI and pcDNA-G2, the products of expression of which contain a marker epitope EPFRDYVDRFYKTL P24 of HIV-1.

In Fig. 8 presents immunochemical staining preparations of cells T, transfected with plasmid DNApMEL-TCI-A0201 (pcDNA-G1). Vector plasmid pcDNA3.1 - negative control plasmid pcDNA-TCI - positive control. Staining with monoclonal antibodies 29F2 and conjugate of rabbit antibodies against mouse IgG with horseradish peroxidase. The data in Fig. 8 show that the staining of cells was observed only when transfection of cells T target plasmidpMEL-TCI-A0201 and plasmid pcDNA-TCI and pcDNA-G2 (positive control) and was not observed with transfection of the original vector pcDNA3.1, which confirms the presence of transferir the data cells of the products of expression of a target gene. In the case of transfection with plasmid pMEL-TCI-A0201 the number of stained cells was ≈1.6%. The low level of stained cells by transfection with plasmids pMEL-TCI-A0201 and pcDNA-G2, apparently due to rapid degradation of the target immunogens, because they were designed to provide high speed processing required for the release of CTL epitopes.

Indeed, according to the design of artificial polyepitopic protein MEL-TCI-A0201 contains a signal sequence (ER and LAMP-1) and amino acid residues that are intended to ensure the efficient processing target polyepitopes designs on the way, MHC classes I and II. Together with the processing of the target protein, probably, is the cutting of the marker epitope, which is recognized µa 29F2, thereby reducing the number of stained cells, transfected with plasmid pMEL-TCI-A0201.

2.3. The definition of products of expression of a target gene in transfected cells using Western blot turns

Lysates of 293 cells T, transfected with plasmid pMEL-TCI-A0201 were analyzed by electrophoresis in 15% polyacrylamide gel followed by transfer of the separated cellular proteins on a nitrocellulose filter and holding staining using the MAB antibodies to 29F2 and conjugate with horseradish peroxidase (Fig.9). The lysate of cells T, Tr is spellbunny the plasmid pMEL-TCI-A0201, analyzed by electrophoresis in 15% polyacrylamide gel followed by transfer of the separated cellular proteins on a nitrocellulose filter and holding staining using the MAB antibodies to 29F2 and conjugate with horseradish peroxidase. Figure 9 presents the immunoblotting of proteins after separation in 15% SDS page with monoclonal antibodies 29F2 (marker epitope). As positive control we used the lysate T cells, transfected with pcDNA-martI, and as negative control - plasmid vector without insert - pcDNA-3.1.

On the tracks:

M - molecular weight marker (14, 24, 45, and 66 KD)

1 - the lysate of cells T, transfected with pDNA plasmid-mart1

2 - the lysate of cells T, transfected with a plasmid pMEL-TCI-A0201

3 - cell lysate T, transfected with the plasmid pcDNA 3.1

Figure 9 shows that in the transfected cells are the target proteins identified µa 29F2 that confirms their synthesis, directed target DNA vaccine constructs. It should also be noted that in the case of the analysis of the lysate of cells T, transfected with the plasmid pcDNA-martI, is seen in the immunoblot only one major band corresponding in mol. the mass of the target protein MART1 (track 1). In the study of cell lysate T, transfected with a plasmid pMEL-TCI-A0201, shows a discrete set of lines of different mol mass ranging from 52 KD (which is corresponds to the calculated mol. the mass of the original protein MEL-TCI-A0201) to low molecular weight peptides. This fact suggests that artificial polyepitopic protein MEL-TCI-A0201 subjected to an effective processing in the cell, and hence the sites of processing inherent in the protein, effectively recognized by cellular proteases.

2.4. Analysis of the expression of target genes by definition synthesis of specific mRNAs using RT-PCR

The allocation of total RNA was performed from 0.8×106cells T, transfected plasmids pMEL-TCI-A0201, pcDNA-G2 (positive control)using the dial to highlight the total RNA of the company Promega (cat. NZ3100) according to the attached instructions. Method of isolation involves the stage of cell lysis, Dnesneho hydrolysis to remove impurities DNA, and the final purification of RNA on microcolony.

Obtaining cDNA was performed on RNA in the reverse transcription reaction: 10 μl of RNA was added to 40 μl of reaction mixture containing in a final volume of 50 μl buffer REV×1, 100 µg/ml corresponding to this gene direct primer for revertase, 0.5 mm each of the four deoxynucleotides and 800 u/ml of the enzyme reverse transcriptase M-MuLV (reverts) company of Sibenik. The reaction of synthesis of cDNA was carried out for 2 hours at 42°C, then the contents of the tubes warmed up for 3 minutes at 95°C.

Then received the th cDNA was used for PCR using specific primers to genes MEL-TCI-A0201, G2 (Table 3.)

Amplification was performed in 25 µl reaction mixture containing 5-10 ng of cDNA, 0.2 mm dNTPs, 200 ng each of the two primers corresponding to the gene, 1×buffer for PCR and 1 unit of Taq-DNA-polymerase firm of Sibenik.

Table 3
The primers mel-tci-a0201
GeneSequences of forward and reverse primersThe size of the amplification product (BP)The melting temperature for primers °C
MEL-TCI-A02015'-GGACAAATCCCTGCACGTGTC37560.4
5'-AGATCTGCTGCGCGAAATAGC59.3

The mixture was placed in an amplifier with a given program time-temperature cycles: 95°C 1 min, 60°C, 72°C - 1 min 20 sec; total of 30 cycles. At the end of the last cycle of PCR was performed the final synthesis at 72°C for 5 min. amplification Products were analyzed by electrophoresis in 1%agarose gel.

Figure 10 presents electrophoregram products FROM RT-PCR in 1% agarose gel, where:

1, 2 products FROM RT-PCR using the quality matrix of the original plasmids pMEL-TCI-A0201, pcDNA-G2 (positive control);

3 is a marker of molecular weights (M12 firm of Sibenik);

4, 5 - products FROM RT-PCR, obtained using as template cDNA MEL-TCI-A0201, G2 transfected T cells.

The results presented in figure 10 show that the sizes of the amplified fragments correspond to theoretically calculated amplification products (see p). These data confirm the presence of total fraction of cDNA sequences encoding proteins MEL-TCI-A and G2, and hence indicates the gene expression and the synthesis of specific mRNAs.

2.5. Example. Study the immunogenicity of DNA vaccine constructs pMEL-TCI-A0201, coding polyepitopic immunogen containing cytotoxic epitopes of antigens of melanoma, restrictionenzyme HLA-A*0201

Study the immunogenicity of DNA vaccine constructs pMEL-TCI-A0201, coding polyepitopic immunogen containing cytotoxic epitopes, restrictionenzyme HLA-A*0201, were carried out in the system of the induction of T-cell immune response ex vivo using peripheral mononuclear blood cells conditionally healthy HLA-A*0201-positive donor. The study was conducted jointly with the staff of the laboratory of molecular immunology NIIKI WITH the RAMS. Permission to conduct these studies was approved by the local ethics Committee at fsri NIIKI With the RAMS (Protocol №68 of the meeting of the ethics Committee NIIKI SORAN from 13 February 2012).

As target cells was used for cell culture human melanoma Mel Is (HLA-A*0201+, MART1+), courtesy of the Russian oncological scientific center named after N.N. Blokhin Russian Academy of medical Sciences. As a negative control were used culture of mononuclear cells (MNCs) and co-culture of MNCs and dendritic cells (DC), transfected with a control (vector) plasmid. As a positive control was used for co-culture of MNCs and DK, transfected with plasmid pcDNA-mart1 that encodes a full-sized melanoma antigen MART1. Obtaining dendritic cells and induction of maturation was carried out according to the methods described previously (Hripko et al., 2008). Transfection of dendritic cells with DNA vaccine constructs were made with the help of the magnetic transfection (MATra - Magnet-Assisted Transfection) according to the Protocol recommended by the manufacturer (Promokine, USA). The ability polyepitopes design to induce specific T-cell immune response was studied in the reaction of IFNγ-ELISpot (11).

Comparison of the ability of DC transfected with different plasmids to induce T-cell response (11) showed that in the presence of lysate of melanoma cells (Mel Is) in the joint cultures OLS and DK, transfected plasmids pMEL-TCI-A0201 (DCA.Lys) and pcDNA-mart1 (DCM.Lys - positive control)produced significantly greater quantity the STV IFNγ-producing cells, than under cultivation OLS (PBMC.Lys) (p=0.0004 and 0.0398) and DK, transfected with a control plasmid (DCC.Lys) (p=0.0107 and p=0.0864) in the presence of lysate.

The level of cells producing IFNγ in the joint culture of MNCs and DK, transfected target plasmid pMEL-TCI-A0201, and the stimulation of DC transfected with plasmid pcDNA-martI, did not differ (p=0.599). This group DCC, DCA and DCM is not statistically differ from each other, but significantly different from RVMS (p<0.005).

Group DCA.Lys and DCM.Lys (positive control) differ from the negative control group DCA and DCM (p<0.028). Statistical analysis was performed using paired test Wilcoxon signed using FDR correction.

Figure 11 shows that the target polyepitope design and a plasmid encoding the full-size protein MART1, provide the greatest expression of IFNγ. All experimental groups, which were conducted stimulation with lysate of melanoma cells demonstrate bimodality distribution - that is, apparently, the cells of some patients did not respond to stimulation with lysate. Only 15 patients out of 21 (71.4%), the number of IFNγ-producing cells in the presence of lysate exceeded 4 times the average number of cells producing IFNγ, in the negative control group RUMS in the absence of lysate.

Figure 11 shows the results of IFNγ-ELISpot for 15 patients, demonstrated the induction of T-letocnalaget, the total number of IFNγ-producing cells obtained for each patient. Target polyepitope design to the greatest extent stimulates the expression of IFNγ. However, in this case, differences between groups DCA.Lys and DCM.Lys were statistically insignificant.

Figure 11 presents the following information:

(A) the Number of spot-forming IFNγ-producing cells (100,000 cells) in the culture of mononuclear peripheral blood cells (MNCs) conditionally healthy donors and in co-culture with autologous DC (n=21).

(B) the Percentage of the maximum number of spot-forming IFNγ-producing cells (for each patient) in the culture of mononuclear peripheral blood cells (MNCs) conditionally healthy donors and in co-culture with autologous DC (n=15). Bold horizontal line indicates the average value.

RVMS (peripheral blood mononuclear cells) and control culture OOC; DCC - co-culture of MNCs and DK, transfected with a control plasmid; DCA - co-culture of MNCs and DK, transfected with plasmid specific for HLA-A0201 (pMEL-TCI-A0201); DCM - co-culture of MNCs and DK, transfected with a plasmid that encodes a full-sized protein MART1; PBMC.Lys, DCC.Lys, DCA.Lys and DCM.Lys - appropriate culture lysate of cells Mel Is

Thus, studies conducted in the induction system T-cell immunol the response ex vivo, showed that was designed using the developed models and algorithms polyepitope design MEL-TCI-A0201 has the ability to induce T-cell immune response against antigens of human melanoma and is not inferior in efficacy full-sized melanoma antigen MART1. It is quite convincing, given that the target structure contains only part of CD8+T-cell epitopes from protein MART1.

From the above results it follows that the created genetic design pMEL-TCI-A0201 containing the gene encoding the artificial polyepitopic T-cell protein immunogen, which is due to the optimal selection and placement of epitopes provides high speed processing polyepitopes design and the induction response of cytotoxic T-lymphocytes, is not inferior to the response to the full-size protein MART1, which is one of the key antigen of melanoma.

The results provided the rational design of targeted polyepitopes design, which in addition to the epitopes of the protein MART1 includes other epitopes of protein antigens of melanoma - NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-EN, MAGE-C1. Given that the optimization processing of epitopes increases the immunogenicity polyepitopes design [9], the design of the target immunogen was aimed at optimizing the processing of all the epitopes included in the price the eve of the structure. This allows data on immunogenicity obtained for some epitopes (in this case for epitopes of the protein MART1), to extrapolate to other epitopes included in polyepitope design MEE-TCI-A0201 (in this case, the protein epitopes of NY-ESO-1, MAGE-A1, MAGE-A3, MAGE-EN, MAGE-C1).

Indeed, analysis of the cell lysate T, transfected with a plasmid pMEL-TCI-A0201, showed that artificial polyepitopic protein MEL-TCI-A not only effectively synthesized, but is also exposed to the efficient processing in transfected cells (Fig.9)that the plan fully complies with its design.

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1. Artificial gene that encodes polyepitopic protein immunogen MEL-TCI-A0201 containing multiple cytotoxic restrictively HLA-A*0201, and T-helper epitopes of melanoma antigens NY-ESO-1, MART1, MAGE-A1, MAGE-A3, MAGE-A11, MAGE-C1, having a sequence length 1535 BP shown in Fig. 3.

2. Recombinant plasmid DNA pMEL-TCI-A0201, providing products of the protein immunogen MEL-TCI-A0201 in mammalian cells and has a size 6952 gel containing in accordance with the physical and genetic map shown in Fig. 4, the target gene under item 1, encoding an artificial polyepitopic protein MEL-TCI-A0201, under the control of the CMV promoter, which ensures its expression in mammalian cells, and consisting of the following fragments:
- NheI-HindIII - vector DNA fragment of the plasmid pcDNA3.1(-) size 5423 P.N.;
- NheI-HindIII - fragment 1535 BP, containing the gene MEL-TCI-A0201.

3. Artificial protein immunogen MEL-TCI-A0201 properties of melanoma antigens having the amino acid sequence shown in Fig. 2.



 

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2 cl, 4 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention concerns genetic engineering, biotechnology, immunology and medicine and can be applied in obtaining antitumoral vaccine and melanoma treatment. Vaccine compositions are based on heat shock proteins and antigen peptides for treatment of tumor disease, the said compositions containing hybrid protein consisting of heat shock HSP70 family protein and one of specific melanoma MAGE peptides (A1, A2, A3) or gp 100, as well as suitable pharmaceutical carrier. The invention also claims a method for obtaining the said hybrid proteins by gene expression in synthetic recombinant vectors in producer strains of E coli BL21 (DE3) cells.

EFFECT: improved immunising power.

3 cl, 4 ex, 1 tbl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly to genetically engineered production of human proteins, and may be used for preparing human epidermal growth factor (hEGF) in bacterial cells in the form of glutathione-3-transferase fusion protein. What is constructed is the recombinant DNA coding GST-hEGF fusion protein which consists of an amino acid sequence of glutathione-S-transferase and an amino acid sequence of human epidermal growth factor divided by a cleavage site by enterokinase, and characterised by the nucleotide sequence SEQ ID NO:1. The KpnI/XhoI fragment of the vector pET41 and the above recombinant DNA are used to create the recombinant plasmid pAS007 for expression of GST-hEGF fusion protein in E.coli cells.

EFFECT: invention enables reaching high GST-hEGF expression levels in Ecoli cells.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, gene and protein engineering and specifically to recombinant plasmid DNA pG1-Rm7, which facilitates synthesis of hybrid protein G1-Rm7 in Escherichia coli cells, which is capable of biding the tumour necrosis factor and has bioluminescence of luciferase Renilla muelleri, where said plasmid DNA includes the nucleotide sequence SEQ ID NO: 1 and can be in medicine. The invention also relates to the protein pG1-Rm7 having molecular weight of 65.4 kDa, consisting of a single-strand anti tumour necrosis factor antibody, a GGSGGS peptide and modified luciferase Renalla muelleri and characterised by SEQ ID NO: 2.

EFFECT: invention enables to obtain a highly sensitive reporter for detecting a tumour necrosis factor via bioluminescent analysis.

2 cl, 4 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to genetic engineering, specifically to creation of fibroblast growth factor receptor (FGFR) muteins, and can be used in medicine. The polypeptide of the FGFR4 receptor extracellular domain (ECD) acidic region mutein is an FGFR4 ECD chimera or a FGFR4 long acid box version and has more acid residues in the D1-D2 linker region than the wild-type FGFR4 ECD. The muteins may include a point mutation that inhibits glycosylation. The mutein is used to treat a disease associated with one or more FGFR ligands, e.g., proliferative diseases, including various types of cancer, angiogenic disorders and macular degeneration.

EFFECT: invention enables to obtain an FGFR4 ECD acidic region mutein, having low capacity to bind with tissue, by increasing the number of amino acid residues within the D1-D2 linker region.

32 cl, 22 dwg, 11 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, specifically PTH receptor agonists, and can be used in medicine. A polypeptide of formula PTH(1-X)/PTHrP(Y-36) is constructed, where denotes an integer between 11 and 18, and Y=X+1, where PTH(1-X) is an amino acid from 1 to X of the human PTH sequence (SEQ ID NO:5) and PTHrP(Y-36) is an amino acid from Y to 36 of the human PTHrP sequence (SEQ ID NO:6). The polypeptide contains one or more of the following mutations in the PTH(1-X) sequence: Ala in position 1, Ala or Aib in position 3, Gin in position 10, Arg or homoarginine in position 11, Ala in position 12, and Trp in position 14. The obtained polypeptide is used to repair fractures, treat hypoparathyroidism, hyperphosphatemia, tumoral calcinosis, osteoporosis, osteomalacia, arthritis, thrombocytopenia, as well as increase stem cell mobilisation in a subject.

EFFECT: invention enables to obtain a polypeptide having prolonged activity on the PTH receptor.

18 cl, 37 dwg, 8 tbl, 11 ex

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: inventions refer to polynucleotide sequence coding the structured pertactin protein (Prn); a vector including such a sequence and compositions containing protein or vector. The characterised polynucleotide sequence codes 300 first amino acids that are the closest to N-end of this type of natural mature Prn (PrnX300), and amino-acid sequence including 620 last amino acids that are the closest to C-end of this type of natural mature Prn (PrnY620) so that structured pertactin PrnX300-PrnY620 of Bordetella class is obtained. Structured molecules Prn include polymorphisms of different B. Pertussis strains and cause immune responses with increased protective ability and opsonophagocytic activity, which exceed the corresponding properties of the preceding vaccines.

EFFECT: protein obtained as per the presented invention can be used in medicine and veterinary as a component of antibacterial vaccines against Bordetella pertusis.

12 cl, 3 dwg, 2 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: proposed chimeric protein with SEQ ID NO:02 is fluorescent biosensor, built on the basis of HyPer protein and mutant of PH-domain of Btk tyrosine kinase.

EFFECT: proposed inventions allow performing simultaneous monitoring of product of hydrogen peroxide and phosphatidyl inositol-3,4,5-triphosphate in a living cell.

4 cl, 4 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: microorganism-producent is cultivated in a suitable nutrient medium with subsequent extraction and treatment of target protein. At the same time the producent is yeast strain Saccharomyces cerevisiae, transformed with the expression vector, which contains an area of initiation of replication of endogenic 2-mcm plasmid of yeast Saccharomyces cerevisiae, and also a yeast promotor GAL1, which controls gene expression, including a DNA sequence SEQ ID NO: 1, coding a fused protein, the composite parts of which are aminoacid sequences of the protein E7-HSP70 and the protein of ubiquitin of yeast Saccharomyces cerevisiae, occupying within the fused protein an N-end position ending with a processing site, which separates it from the sequence of the protein E7-HSP70 and recognised natural ubiquitin-specific yeast proteinases. The yeast strain Saccharomyces cerevisiae VKPM Y-3853 - producent of the protein E7-HSP70 - is produced by transformation with the expression vector pPDX3U-E7-HSP70 of the yeast strain Saccharomyces cerevisiae D702.

EFFECT: group of inventions provides for high level of biosynthesis and yield of treated protein E7-HSP70, production of a water-soluble correctly processed protein E7-HSP70, principal absence of toxic and pyrogenic bacterial LPS in preparations of the protein E7-HSP70.

2 cl, 4 dwg, 12 ex

FIELD: biotechnologies.

SUBSTANCE: fusion peptide is presented for neutralisation and destruction of organophosphorous compounds, comprising a signal peptide TAT3 with amino acid sequence SEQ ID NO: 5, presented in the description, functionally connected to the sequence of organophosphate hydrolase SEQ ID NO: 18, classified as protein EC 3.1.8. The following components are described: extracted polynucleotide, which codes the specified fusion protein; a vector containing the specified polynucleotide; and a procaryotic host cell containing the specified vector and expressing the specified fusion protein. The method is proposed to produce a ferment, which destroys organophosphorous compounds, including expression of the specified polynucleotide in the procaryotic host cell and production of the specified ferment.

EFFECT: invention makes it possible to increase expression of organophosphate hydrolase in a host cell.

13 cl, 1 tbl, 9 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: described fused protein contains at least two amino acid sequences. The first amino acid sequence, having 90% sequence identity with an amino acid sequence represented in SEQ ID NO:2, is fused with a second amino acid sequence, having at least 90% sequence identity with an amino acid sequence represented in SEQ ID NO:4.

EFFECT: invention provides immunity against various clinically vital strains of group B streptococci.

9 cl, 5 dwg, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an antibody representing a neutralising VEGFR-2/KDR antibody with its hypervariable regions being identical to the hypervariable regions of TTAC 0001 of VEGFR-2/KDR antibody fused with a binding domain of angiopoietin 2 which is Tie-2 ligand for treating cancer by angiogenesis inhibition. A DNA coding the above antibody, an expression vector containing the above DNA, and a CHO host cell transformed by the above vector for preparing the antibody are also described. What is also presented is a method for preparing the antibody involving: host cell incubation, and the antibody recovery from a culture fluid of CHO cell. What is described is a pharmaceutical composition for treating an angiogenesis-related disease, containing an effective amount of the above antibody and at least one pharmaceutically acceptable carrier.

EFFECT: invention enables preparing the VEGFR-2/KDR antibody fused with the binding domain of angiopoietin 2 which may be used for effective treatment of a disease related to excessive angiogenesis.

13 cl, 10 dwg, 8 ex

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