Recombinant fused protein, preparation for immunotherapy based on thereof and method for immunotherapy of relapsing larynx papillomatosis
FIELD: medicine, biotechnology.
SUBSTANCE: invention relates to the development of a recombinant fused protein consisting of an oncoprotein E7 VPCH 11 type with a sequence SEQ № 2 and protein of "heat shock" Hsp 70 from M. tuberculosis cells prepared by using plasmid pQE30-E711-dnaK in E. coli cells. The preparation is used against relapsing human papillovirus comprises the recombinant fused protein in the effective amount and a pharmaceutically acceptable carrier. Method for immunotherapy of relapsing larynx papillomatosis involves intracutaneous administration of indicated preparation in a patient. The advantage of invention involves the development of a novel preparation showing improved immunological indices. Proposed preparative formulations can be used in immunotherapy of relapsing larynx papillomatosis in humans.
EFFECT: valuable medicinal properties of protein, improved method for treatment.
3 cl, 6 dwg, 7 ex
Recurrent (return) respiratory papillomatosis (RRP) is the most common tumor of the respiratory tract, occupying the first place among the causes of chronic obstruction of the larynx in children. Although the papilloma is a benign tumor in children because of frequent recurrence, rapid growth and anatomical narrow the lumen of the larynx disease clinically difficult, with symptoms of airway stenosis, which is absent timely assistance can lead to death.
Etiological factor RRS is the human papilloma virus (HPV). Currently identified more than 70 types of the virus, however, according to the literature, in the identification of HPV DNA in biopsy material most patients RRS determined mainly virus genotypes 6, 11, 16 and 18. The data presented in the literature on the relative prevalence of different subtypes of HPV in children, their relationship with disease severity and impact on the effectiveness of anti-relapse treatment is very controversial.
According to the literature, in the USA the incidence of RRP is around 2350 new cases per year in children and 3600 - adults (0.6 to 4.3 per 100000 and 1.6 to 3.8 per 100,000, respectively). In Denmark, the frequency of the RRS is 3.84 per 100,000, including children -3,62 per 100,000 adults - 3,94 per 100,000 in the population.
In the clinic, reconstructive surgery of the larynx DGKB St. Vladimir (clinical database research group SIC MMA. Sechenov) in 2002 was on treatment 182 children suffering from RRS; every year the clinic will be hospitalized 15-30 new patients. In General, in the Russian Federation annually about 4,000 new cases of laryngeal papillomatosis in children and adults.
Treatment of children with RRP is an unsolved problem. The primary method of treatment is surgical removal of papillomas, which consists in the removal of papillomas of the respiratory tract by micro tools, laser or ultrasound under the supervision of modern optics. However, the isolated use of surgical methods does not prevent the development of subsequent relapses. Moreover, re-operation often lead to the development of cicatricial stenosis of the larynx, leading to profound disability. In addition, the growth of warts on the vocal folds leads to obvious violations of speech, until aphonia, determining and many psychological and social problems, and also causing persistent against non-adaptable changes the personality of the child.
In this regard, in recent decades actively search for opportunities to improve the effectiveness of treatment RRS. Currently, the most effective one is by application, along with the surgical method, interferon (IFN). Data about the effectiveness of preparations contradictory. According to the results of various authors, the effect of IFN therapy ranges from 20% to 50%. In addition, the use of recombinant IFN has significant economic, social and health disadvantages: high cost of drugs, the need for intramuscular injection 3 times a week for 1-2 years and more, possible side effects.
The most promising appear to develop such new treatment methods RRS immunotherapy with vaccines.
Pharmaceutical company "Stressgen Biotechnology" (Stressgen Biotechnologies corp.) developed a therapeutic vaccine for the treatment of RRP. Based vaccine is a recombinant hybrid protein consisting of the oncoprotein E7 of HPV type 16 protein and "heat shock" of M.tuberculosis Hsp 65 (U.S. patent 6524825, Chu Nandal, Mizzen Lee, Woo Huacheng bill. Immune responses to HPV antigens, called tracks, which are HPV antigen and a stress protein or expression vector capable of expression of these proteins. (Chu N.Randall, Mizzen Lee, Wu Huacheng Bill. Immune responses against HPV antigens elicited by compositions comprising an HPV antigen and a stress protein or an expression vector capable of expression of these proteins). This solution can be specified as the closest analogue of the prototype.
Recently the published results of clinical trials of this vaccine. Triple vaccine is given in a dose of 500 µg allowed more than 40% increase in the interval between relapses.
However, full stop recurrence of the authors was not achieved. In our opinion, the failure of the vaccine because the vaccine composition includes oncoprotein E7 of HPV 16-th type, whereas the RRS is caused mainly HPV 11 and 6 types. Moreover, as was shown by us, special analyses, in biopsy material of patients RRS mainly detected HPV-11 type. In addition, HPV 11 type causes disease with a more aggressive course. Thus, the use of oncoprotein E7 16th-type pharmaceutical company "Stressgen Biotechnology" (Stressgen Biotechnologies corp.) it was not quite justified. Sufficient to compare the amino acid sequences of proteins E7 type 11 and 16, to verify the absence of any marked homology between them, and hence the cross-antigenic determinants. Therefore, it is difficult to expect a pronounced therapeutic effect in the treatment of RRP HPV-11 type, the vaccine is designed based on the protein type 16 E7. In our opinion, for immunotherapy RRS should be used E7 11 type that will allow you to achieve maximum effect. Moreover, a similar effect should be expected when using our vaccines in the case of therapy RRS due to HPV type 6, as between what they have expressed homology. To improve the immunogenic properties of the oncoprotein E7 company "Stressgen Biotechnology" (Stressgen Biotechnologies corp.) used protein "heat shock" Hsp 65.
The present invention is the preparation of recombinant protein drug based on it having improved immunological characteristics, as well as more effective immunotherapy.
The problem is solved a new drug created on the basis of recombinant protein consisting of oncoprotein E7 of HPV-11 type protein and "heat shock" of M.tuberculosis Hsp 70, and its use for immunotherapy of recurrent papillomatosis.
Recombinant hybrid protein is characterized by the fact that consists of oncoprotein E7 of HPV-11 type sequence SEQ No. 2 and protein "heat shock" of M.tuberculosis Hsp 70, and obtained using the plasmid pQE30-E711-dnaK in E. coli cells.
The drug contains synthesized specified recombinant hybrid protein in an effective amount and a pharmaceutically acceptable carrier. As the carrier can be any acceptable for introduction of immunogenic protein basis, in particular, can be used aluminium hydroxide, mannitol, polyoxidonium. Under the effective number should be same number, which should be sufficient for the treatment or prophylaxis. It will depend on age, sex, weight, condition of the patient. Effective the number is preferably from 100 to 2500 mcg. The drug has a high degree of standard, low reactogenicity.
There is also a method of immunotherapy of recurrent laryngeal papillomatosis, according to which the patient intradermally injected the drug against papillomavirus man in an amount of 0.1-2.5 mg protein per dose, if necessary, with the re-introduction of the drug in two weeks.
According to our data, Hsp 70 has the best immunomodulatory characteristics that do not depend on the HLA type. Developed the drug gives the best clinical results, namely: greatly increases the interval between relapses, and can be used for vaccination heterogeneous human population, as well as severe forms of the disease.
The invention can be illustrated by the following examples of its implementation.
Typing papilloma virus in patients with a diagnosis of RSD.
Postoperative material from patients diagnosed with RRS was used to determine the types of HPV. HPV DNA was able to identify all 26 children (including the primary outcome was confirmed by repeated trial, all 3 children surveyed twice). Isolated infection 11 type virus was detected in 15 patients (57,7%); isolated infection 6 type virus was detected in 7 children (26.9 per cent); the remaining 4 children (15.4 per cent) were in zirovanii and 6 and 11 HPV types simultaneously (Figure 1 and 2). Infection 16 and 18 HPV types in any case have been identified.
Figure 1 shows a photograph of agarose gels in the study warts HPV type 6. (The arrow shows the target bands. Because the primers were selected for the subsequent synthesis of the protein E7 of HPV 6 and 11 types, they gave additional bands with human genomic DNA).
Figure 2 presents a photograph of agarose gels in the study warts HPV 11 type. (The arrow shows the target bands. Because the primers were selected for subsequent protein synthesis E 7 HPV 6 and 11 types, they gave additional bands with human genomic DNA).
A comparative analysis of the amino acid sequence of the E7 oncoproteins of HPV 6, 11 and 16 types.
Alignment of E7 protein, HPV 6 and 11
E7 VPC SEQ No. 1
E7 VPC SEQ No. 2
E7 VPC SEQ No. 3 CDSNVRLWQCTETDIREVQQLLLGTLNIVCPICAPKT
E7 VPC SEQ No. 4 CDSNVRLWECTDGDIRQLQDLLLGTLNIVCPICAPKP
Multiple alignment of E7 proteins, HPV 6, 11 and 16
E7 VPC CGCDSNVRLWQCTETDIREVQQLLLGTLN1VCPICAPKT
E7 VPC CGCDSNVRLWECTDGDIRQLQDLLLGTLNIVCPICAPKP
E7 VPC CKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP
Construction of recombinant vaccine that encodes the synthesis of a hybrid protein E7 11-Hsp 70 (see figure 3).
PR is measures 4
Getting a producer of hybrid protein containing the E7 peptide HPV 11 type and DnaK (HSP70) .tuberculosis.
1. PCR amplification E7 gene of human papillomavirus 11 type.
As the matrix was used provided a preparation of genomic DNA from clinical sample that has undergone a preliminary check for the presence of DNA 11 types of human papillomavirus.
PCR amplification was performed using primers:
The initiating codon is in the same reading frame with the sequence 6HIS tag termination codon is missing.
2. Cloning of the gene sequence of E7 in the recombinant vector-producer DnaK.
Has been used previously received vector pQESO-dnaK-Y (see the diagram on figure 4), providing for the expression of protein DnaK, fused with a 6HIS sequence at N-end. PCR fragment of the E7 gene was treated restrictase BamHI and BgIII and cloned in the BamHI site of the plasmid pQE30-dnaK-Y. Recombinants having the insert in the correct orientation were identified by restriction analysis. Circuit design is provided on the attached figure 4. It should be noted that the recombinant plasmid pQE30-E-dnaK provides products hybrid protein 6HIS-E7(type 11)-DnaK.
3. Grow the s strain producer of the protein E7 of HPV type 11 and DnaK(HSP70) M.tuberculosis.
For the cultivation of the producer strain using a nutrient medium Luria-Bertani composition:
10 l take 100 g of tryptone, 50 g yeast extract, 100 g of sodium chloride and bacto agar. To the sterilized medium add ampicillin to a final concentration of 50 mg/ml Incubated with strain at 37°With during the night.
4. Products E7(11)-DnaK
Synthesis of a hybrid protein E7(11)-DnaK induced using IPTG as follows:
Night culture DLT1270/ pQE30-E711-dnaK, grown in LB-broth, diluted 1:100 and grown in LB-broth (medium Luria-Bertani) on the rocking chair to the density OD600-0.5.
Then was made 0.1 mM IPTG and continued growing for 3 hours.
Production of the protein was monitored using SDS-PAGE. Watched the fusion protein of the expected molecular weight.
5. Selection of recombinant hybrid protein consisting of the E7 oncoprotein of HPV 11-type sequence SEQ No. 2 and protein "heat shock" of M.tuberculosis Hsp 70, and obtained using the plasmid pQE30-E711-dnaK in E. coli cells.
The raw material is biomass recombinant E.coli strain.
Recombinant protein, with the growth of the strain accumulated in the cytoplasm in the form of Taurus enabled.
Receiving and washing Taurus enabled.
The cell biomass of E. coli was added to the solution A: 50 mM Tris (121.14 g/mol) 0.5 M NaCl (58.44 g/mol), 5 mM EDTA (372.2 g/mol), 1% non-ionic detergent Nonidet P-40, ImM PMSF (174.2 g/the ol). pH=7.5±0.3
Received the homogeneous solution, which was subjected to sonication on the cage B.Braun (USA).
Processed ultrasound solution suspension was centrifuged in a centrifuge Beckman J2-21, at 10,000 RPM at +4°within 10 minutes the Supernatant was discarded. To the precipitate (bullock inclusion) solution was added In: 50 mM Tris (121.14 g/mol), 0.5 M NaCl (58.44 g/mol), 4 mM EDTA (372.2 g/mol), 2M urea (60.06 g/mol), 0.5 mM PMSF (174.2 g/mol), 0.05% Nonidet P-40 (w/w), pH=7.5±0.3, homogenized him and centrifuged in the same mode.
The processing solution was repeated 2 times with the same parameters.
Then, Taurus inclusion were treated 2 times so the same solution: 50 mM Tris (121.14 g/mol), 0.5 M NaCl (58.44 g/mol), 1.5 mM EDTA (372.2 g/mol), 2 M urea (60.06 g/mol).
Dissolving the washed Taurus on and refolding of the target product.
To the resulting precipitate (bullock inclusion) solution was added D: 25 mM Tris (121.14 g/mol), 0.25 M NaCl (58.44 g/mol), 8M urea (60.06 g/mol), 10 mM DTT (154.24 g/mol), pH=8.0±0.2 subsequent gomogenizirovannom.
The resulting mixture was treated with ultrasound on the cage B.Braun (USA).
After sonically, the solution decantation, and centrifuged in a centrifuge Beckman J2-21, rotor Beckman JA-14, at 12000 RPM at +4°within 20 minutes
The precipitate was discarded, and the supernatant decantation. The solution Taurus inclusions were placed on the magnetic stirrer, was fed a solution of PBS: .1 M Na 2HPO4(142 g/mol), 1.45 M NaCl (58.44 g/mol), pH=7.5±0.2 and stirred.
The resulting solution was subjected to tangential ultrafiltration with simultaneous dialysis in PBS solution. The solution was concentrated.
Figure 5 and 6 shows the expression of a hybrid protein consisting of the E7 oncoprotein of HPV 11-type sequence SEQ No. 2 and protein "heat shock" of M.tuberculosis Hsp 70, and obtained using the plasmid pQE30-E711-dnaK in E. coli cells.
The manufacture of dosage forms of the vaccine.
Purified recombinant protein is mixed with a preparation of aluminium hydroxide (in the ratio of 1 mg of protein 0.4 mg aluminium hydroxide), thoroughly mixed under mild conditions for 20 minutes. After that, the suspension is centrifuged at 5000 revolutions/min, supernatant collected and used for measurement of residual protein. Sediment posing an aluminum hydroxide with adsorbed protein, suspendered in phosphate buffer and poured into bottles. The concentration of protein in a single dose is 0.5 mg
Purified recombinant protein at a concentration of 2.5 mg per ml of phosphate buffer overflows in the vials 0.2 ml, containing 10 mg of mannitol, and liabilitiesa. Each vial contains 0.5 mg of protein.
Method of immunotherapy of recurrent papillomatosis of the larynx.
Reaction to widediapasone drugs are extremely individual, due to the peculiarities of the organization of the main histocompatibility complex. In this regard, each case of drug requires an individual approach and objective laboratory criteria the effectiveness of therapy. As such the criteria used level antitelomerase in the serum of patients after administration of each dose and the rate of activation of the delayed-type hypersensitivity (GST) in respect of the E7 protein. This parameter was estimated using the skin test by intradermal injection of 10 μg of peptides containing antigenic determinants of the protein E7. 12 patients diagnosed with recurrent respiratory papillomatosis" vacciniavirus intradermally one dose of the drug obtained in example 1 and 4 patients drug in in example 2. Re-introduction of the drug were conducted over two weeks. All patients determined the titer of specific antibodies and assessed the level GST. In 8 patients recorded enough antitelomerase and level GST. In 8 patients, these figures were not expressed, in this regard, it was recommended re-introduction of the drug. After two vaccinations with the drug every 2 weeks performance antitelomerase and skin sample has reached the required level. Monitoring of patients during the year showed otsutstvie relapses.
1. Recombinant hybrid protein, characterized in that it consists of oncoprotein E7 of HPV-11 type sequence SEQ No. 2 and protein "heat shock" of M.tuberculosis Hsp 70, and obtained using the plasmid pQE30-E711-dnaK.
2. Drug against recurrent papillomavirus person, characterized in that it contains a recombinant hybrid protein according to claim 1 in an effective amount and a pharmaceutically acceptable carrier.
3. Method of immunotherapy of recurrent papillomatosis of the larynx, characterized in that the patient intradermally injected the drug against papillomavirus man according to claim 2 in an amount of 0.1-2.5 mg protein per dose, if necessary, with the re-introduction of the drug in two weeks.
FIELD: molecular biology, biochemistry, medicine, oncology.
SUBSTANCE: invention relates to DNA sequences found in analysis of mDNA from squamous carcinoma cellular lines of different origin wherein these DNA sequences represent transcripts from rearranged genes SCCA1 and SCCA2. Result of rearrangement is formation of fused gene consisting of exon 2-7 of gene SCCA1 and exon 8 of gene SCCA2, or exons 2-7 of gene SCCA2 and exon 8 of gene SCCA1. Prepared expressing vectors comprising above said combinations of exons of two genes provide synthesis of corresponding fused protein in host-cell. Proposed sequences of nucleic acids and genetic constructions based on thereof represent novel agents for diagnosis squamous carcinomas.
EFFECT: valuable biological and medicinal properties of transcripts.
8 cl, 9 dwg, 1 tbl, 5 ex
FIELD: genetic engineering, medicine.
SUBSTANCE: invention relates to isolated DNA encoding human peptide related to urocortin and peptide named as urocortin II. These peptides are relative with corticotropin-releasing factor and involves in mechanisms for initiation of hypophysis-suprarenal responses for the stress. Pharmaceutical composition comprising such peptide in combination with acceptable vehicle can be used in treatment of pathophysiological states, such as enhanced body temperature, appetite disorder, congestive cardiac insufficiency, stress, anxiety state and undesirable low levels of ACTH.
EFFECT: valuable medicinal properties of urocortin proteins.
33 cl, 27 dwg, 2 tbl, 16 ex
SUBSTANCE: method involves determining complex formed between HCV anticore antigen and NS3/4a-antibody capable of recognizing both HCV- antigens and antibodies available in sample when using common solid base and solid substrates usable in immunoassay.
EFFECT: high accuracy and sensitivity of hepatitis C diagnosis.
47 cl, 8 dwg, 10 tbl
FIELD: biotechnology, molecular biology, medicine.
SUBSTANCE: invention discloses amino acid sequences of human obesity polypeptide (OB) two isoforms possessing capacity for modulation of animal body mass, their signal peptide-containing precursors and analogues. Polypeptide isoforms are prepared as result of insertions, deletions and amino acid changes that retain activity typical for nonmodified forms of OB-polypeptides, and polyclonal and monoclonal antibodies interaction specifically with new agents modulating the body mass value also. Invention describes DNA sequences encoding these polypeptides and their analogues, vector structures comprising these sequences used for preparing recombinant forms of OB-polypeptides. Invention proposes using new polypeptides and their analogues as an active component in pharmaceutical compositions. Using this invention can promote to solving the problem for providing medicine, veterinary science and animal husbandry with effective agent used for decreasing the body mass value. Invention can be used in medicine for diagnosis and treatment of pathological states associated with disturbance of regulation of human body mass, and in animal husbandry and veterinary science.
EFFECT: valuable biological, medicinal and veterinary properties of polypeptide.
23 cl, 71 dwg, 12 tbl, 17 ex
FIELD: biotechnology, peptides, genetic engineering.
SUBSTANCE: invention relates to constructing nucleic acid molecule comprising functional in starch-containing plant tissues promoter, fragment encoding transit peptide for translocation of useful peptide into amyloplast, fragment encoding useful peptide, region encapsulating into starch and terminator. In insertion into plant genome DNA molecule expresses hybrid polypeptide comprising the desirable protein encapsulated into starch matrix. Prepared vegetable material can be used, for example, in manufacturing fodders for mammals, fishes and poultries. Also, invention can be used in food industry.
EFFECT: valuable properties of nucleic acid and polypeptides.
15 cl, 19 dwg, 9 tbl, 7 ex
FIELD: genetic engineering, pharmaceutical and medical-biological industry.
SUBSTANCE: invention proposes a chimeric sequence of nucleic acid encoding a fused polypeptide able to bind with the vessel endothelium growth factor (VEGF) and to inhibit its specific mitogenic effect. The fused polypeptide molecule comprises immunoglobulin-like domain 2 of VEGF-receptor Flt1, immunoglobulin-like domain 3 of VEGF-receptor Flk 1 or Flt4 and multimerizing component represented by either domain Fc IgG or heave chain IgG. By expression of the proposed chimeric sequence or its two successively joined copies in a host-cell a monomer or dimer of the fused polypeptide are prepared, respectively, that can be used for suppression of VEGF activity in mammals, in particular, in humans. New VEGF inhibitors differ from the known one by the improved pharmacokinetics.
EFFECT: improved preparing method, valuable biological properties of polypeptide.
23 cl, 67 dwg, 1 tbl, 35 ex
FIELD: biotechnology, microbiology, genetic engineering.
SUBSTANCE: invention proposes a new recombinant plasmid pR752 (5269 pair bases) comprising genetic construction under control of bacteriophage T5 promoter and encoding a module polypeptide consisting of 6 histidine residues, hemoglobin-like protein of E. coli, modified fragment of large T-antigen SV-40, translocation domain of diphtheria toxin, spacer sequence (Gly-Ser)5 and human epidermal growth factor (6 His-HMP-NLS-Dtox-(Gly-Ser)5-EGF) and designated for the directed transfer of photosensitizers into target-cell nuclei. By transformation of the strain E. coli M15 (rep 4) with plasmid pR752 the recombinant strain E. coli VKPM B-8356 as a producer of new polypeptide vector is prepared. The usage of this new strain is able to enhance the effectiveness of effect of photosensitizers by some orders. Invention can be used in medicinal-biological industry in preparing agents providing the directed transport of photosensitizing agents into tumor cell nuclei.
EFFECT: valuable biological and medicinal properties of polypeptide.
3 dwg, 4 ex
FIELD: medicine, oncology, biochemistry.
SUBSTANCE: invention relates to fused proteins, namely to the multifunctional fused protein cytokine-antibody. This fused protein involves immunoglobulin region and cytokine fused protein of the formula IL-12-X or X-IL-12 wherein interleukin-12 (IL-12) represents the first cytokine and X represents the second cytokine taken among the group comprising IL-2, IL-4 and GM-CSF bound covalently either by amino-end or carboxyl-end to subunit p35 or p40 of interleukin-12 (IL-12) in its heterodimeric or a single-chain form. Indicated fused cytokine protein is fused by either its amino-end or carboxyl-end with indicated region of immunoglobulin. Multifunctional fused protein cytokine-antibody shows an anticancer activity.
EFFECT: valuable medicinal properties of protein complexes.
13 cl, 40 dwg, 18 ex
FIELD: genetic engineering, proteins, medicine, pharmacy.
SUBSTANCE: invention relates to a method for preparing a fused protein representing immunoglobulin Fc-fragment and interferon-alpha and can be used in treatment of hepatitis. Method involves construction of a fused protein comprising immunoglobulin Fc-fragment prepared from Ig G1 or Ig G3 in direction from N-end to C-end and the end protein comprising at least one interferon-alpha. Fc-fragment and the end protein are joined directly or by a polypeptide bridge. The fused protein is used for preparing a pharmaceutical composition used in treatment of liver diseases and in a method for targeting interferon-alpha into liver tissues. Invention provides preparing the fused protein eliciting with biological activity of interferon-alpha providing its concentrating in liver and showing enhanced solubility, prolonged half-time life in serum blood and enhanced binding with specific receptors.
EFFECT: improved targeting method, valuable biological properties of fused protein.
10 cl, 5 dwg, 9 ex
FIELD: biotechnology, microbiology, medicine.
SUBSTANCE: method involves selection of signal sequence suitable for the effective expression of Leu-hirudine in E. coli cells by the polymerase chain reaction-screening method. Method involves construction of a protein as a precursor of hirudine based on the selected signal sequence of surface membrane protein from Serratia marcescens, oprF protein from Pseudomonas fluorescens or fumarate reductase from Shewanella putrifaciens by joining the Leu-hirudine amino acid sequence with C-end of selected signal sequence. Prepared precursor of Leu-hirudine is used in a method for preparing Leu-hirudine. Invention provides preparing Leu-hirudine by the direct secretion in E. coli cells with the high yield. Invention can be used in preparing the hirudine precursor.
EFFECT: improved preparing method.
4 cl, 1 dwg, 2 tbl, 12 ex
FIELD: genetic engineering, virology, medicine.
SUBSTANCE: invention relates to method for production of modified Vaccinia virus Ankara (MVA). Claimed method includes contamination of mammalian continuous cell line with Vaccinia virus Ankara (MVA) of wild type, followed by viruses cultivation and collection. Further fresh cells of the same cell line are infected with newly formed viruses. Abovementioned steps optionally are repeated. Also disclosed are strains of modified Vaccinia virus Ankara (MVA) and utilization thereof. Said strains are capable to growth in continuous cell lines.
EFFECT: strains having decreased virulence in relates to mammalians.
20 cl, 5 tbl
FIELD: organic chemistry, chemical technology, medicine.
SUBSTANCE: invention relates to acyclic nucleoside phosphonate derivatives of the formula (1): wherein means a simple or double bond; R1 means hydrogen atom; R2 and R3 mean hydrogen atom or (C1-C7)-alkyl; R7 and R8 mean hydrogen atom or (C1-C4)-alkyl; R4 and R5 mean hydrogen atom or (C1-C4)-alkyl possibly substituted with one or more halogen atoms, or -(CH2)m-OC(=O)-R6 wherein m means a whole number from 1 to 5; R6 means (C1-C7)-alkyl or 3-6-membered heterocycle comprising 1 or 2 heteroatoms taken among the group consisting of nitrogen (N) and oxygen (O) atoms; Y means -O-, -CH(Z)-, =C(Z)-, -N(Z)- wherein Z means hydrogen atom, hydroxy-group or halogen atom, or (C1-C7)-alkyl; Q (see the claim invention); its pharmaceutically acceptable salts or stereoisomers. Also, invention proposes methods for preparing compounds of the formula (1) and their using in treatment of hepatitis B or preparing a medicinal agent designated for this aim.
EFFECT: improved preparing method, valuable medicinal properties of compounds and agent.
16 cl, 10 tbl, 87 ex
SUBSTANCE: invention relates to method for production of porphyrinopeptides satisfying the formula I , wherein R1 and R2 independently from one another represent amino acids or peptides comprising 2-15 of amino acid residues, wherein α-carboxylic groups of amino acids or peptides may be modified by C1-C8-alkyl ester and side functional groups of amino acids or peptides may be protected; in particular R1 is ArgOMe; R2 is -OH (III); R1 is LeuHisOMe; R2 is -OH (IV); R1 is LeuLeuValPheOMe; R2 is -OH (V); porphyrin carboxylic group may be modified by methyl or other C1-C9-ester or pharmaceutically acceptable salt; Y- represents Cl-; Me represents Zn, Cu, Fe, Mn. Claimed method includes activation of porphyrin carboxylic group with N-oxy-5-norbornene-2,3-dicarboxyimede in molar ratio of 1:1 in presence of N,N'-dicyclohexylcarbodiinide; or with diphenylphosphorylazide (DPPA) in equimolar ratio of porphyrin/DPPA in presence of base. Then porphyrin with activated carboxylic group is brought into reaction with amino component (amino acid or peptide) in form of mineral acid salt, which is neutralized with base. Also disclosed are methods for application of compounds (I) as nucleotic agents.
EFFECT: new nucleotic agents.
4 cl, 7 ex, 1 tbl
FIELD: medicine, in particular treatment of malignant neoplasm.
SUBSTANCE: claimed method includes administration of vaccine representing complex of heat shock protein and tumor peptide, isolated from subject tumor tissue, and radioactive preparation Oncofer. Oncofer is administered both during vaccination course and after vaccination.
EFFECT: stable antitumor immunity, enhanced tumor cell apoptosis and blocked blood supply due to alteration of erythrocyte properties in tumor caused by radioactive preparation and activation of clot formation in said vessels.
2 cl, 4 ex, 2 dwg
FIELD: organic chemistry, polymers, immunochemistry of high-molecular compounds.
SUBSTANCE: invention describes a synthetic hapten based on copolymers of N-vinylpyrrolidone, crotonic acid and p-crotonoyl aminophenol comprising covalently joined 2-naphthyl amine as a hapten of the following general formula:
wherein m = 88.5 mole%; n = 6.8-10.3 mole%, and l = 1.2-4.7 mole%. Invention provides specificity of antibodies raised against 2-naphthylamin as a hapten and possibility for preparing highly specific antibodies raised against carcinogen 2-naphthylamine as a hapten by immunization of rabbits with synthetic antigen comprising a copolymer of N-vinylpyrrolidone, crotonic acid and p-crotonoyl aminophenol as a macromolecular carrier. Also, invention provides possibility for practice using these antibodies for immunological recording 2-naphthylamine as a hapten in blood of industrial workers subjected for exposition with 2-naphthylamine. The proposed compound elicits antigenic features and can be used for detection of oncological risk among workers subjecting with contact with 2-naphthylamine.
EFFECT: valuable properties of antigen.
1 tbl, 1 dwg, 6 ex
SUBSTANCE: vaccine is high molecular weight protein conjugate with angiotensine II taken in high molecular weight protein : angiotensine II proportion of 1:12-55 in % by weight. The conjugate is modified with equilibrium quantity of immunocompetent polyelectrolyte like polyoxydonium.
EFFECT: stable physiological response within prolonged period of 6-12 months.