Attenuated strain of sendai virus

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology. The subject of the invention is a new strain of the Sendai virus Sen293nsk1 adapted to effective replication in the human cell culture HEK293. The produced strain of the Sendai virus possesses lower virulence for laboratory mice and higher ability to destroy human tumour cells. The strain is supposed to be used experimentally as a therapeutic oncolytic preparation for treating malignant diseases. The invention can be used in treating oncologic diseases.

EFFECT: invention enables providing higher clinical effectiveness in oncologic diseases by using the murine Sendai virus non-pathogenic for humans, possessing an increased oncolytic activity and intensifying anti-tumour immunity.

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The invention relates to the field of immunology and medicine, particularly to Oncology, and in particular to a new strain of Sendai virus.

The level of technology

In many cases, metastatic cancer remains an incurable disease, and therefore requires the development of new anticancer therapeutic strategies. The idea of using viruses for the treatment of malignant diseases dates back to the early XX century, when it was noticed that some patients had spontaneous regression of the tumor after anti-viral vaccinations or viral diseases (Mullen, J. T., Viral Oncolysis, Oncologist 7, 2002, p.106-119). In Oncology was introduced the term "oncolytic" virus. It is believed that this virus is able to destroy specific cancer cells and leave normal intact. Such specific destruction of malignant cells can occur directly as due to preferential replication of virus in these cells and indirectly by stimulating the immune system during asymptomatic viral infection.

Therapy using oncolytic viruses has several advantages over standard methods of cancer treatment. Viruses can be directionally modified with the use of recombinant DNA technology (E. Kelly, History of oncolytic viruses: genesis to engineering, Mol. Ther. 15, 2007, p.651-659). Thus, it is possible �perative to implement the latest knowledge in the field of molecular cell biology in the process of creating oncolytic viruses. Oncolytic virus properties can also be enhanced by biological selection, aimed at the selection of strains with maximum selectivity against tumor cells and hitting with greater efficiency the maximum number of types of malignant cells (Beier R., Hermiston, T., D. Mumberg, Isolation of more potent oncolytic paramyxovirus by bioselection, Gene Ther., 23, 2012, p.13).

The present invention relates to oncolytic virus belonging to the family Paramyxoviridae. Among members of this family have been investigated as anticancer agents attenuated strains of measles and mumps, as well as harmless to humans animal pathogens such as the virus of Newcastle disease (NDV) and Sendai virus (SeV).

Attenuated strain of measles virus (MV) has demonstrated strong antitumor activity in several model systems (Galanis E., Therapeutic potential of Oncolytic Measles Virus: Promises and Challenges. E Galanis, Clinical Pharmacology & Therapeutics, Nov. 2010, Vol.88, No. 5:620-625).

Anticancer effect of mumps virus (genus Rubulavirus) have been less studied. Described three clinical trials for the treatment of cancer with the use of mumps virus (Shimizu Y., Immunotherapy of advanced gynecologic cancer patients utilizing mumps virus, cancer Detect. Prev. 12, 1988, p.487-495).

Currently used in the production of human leukocyte interferon as inductor wah�tiny strain "H" VBI - Newcastle (NDV) is an effective viral inducer (K. E. Mogensen et al., 1977; Frolova V. M. and others, 1984). Anti-cancer activity of NDV virus is being studied in a number of countries, including in Russia (patent RF 2379055 C1, publ. 20.01.2010).

In world practice anti-cancer activity of Sendai virus (SeV), also known as parainfluenza virus of mice 1 type, or hemagglutinins Japanese virus, are just beginning to explore. This paramyxovirus (family Paramyxoviridae) that belongs to the genus Respirovirus. The genome of the virus presents minus RNA chain length 15.3 kilobase. All the virus detected six genes encoding proteins. Of these, two proteins encode surface glycoproteins HN and F, three encode the protein nucleocapsid: proteins NP, P and L, and one deglycosylation internal protein M. the SeV Virus causes a highly communicable infection of the respiratory tract in mice, hamsters, Guinea pigs, rats, and sometimes in pigs. SeV is able to spread through the air or by direct contact. It can be detected in colonies of mice around the world, but it is considered highly safe for humans.

Described oncolytic strain of Sendai virus was derived from the virus that was isolated in the laboratory of V. M. Zhdanov at the Institute of Virology. D. I. Ivanovsky of medical Sciences of the USSR from mice in the early 60-ies and further studied as model Pat�gene (Zhdanov V. M., Bukrinskaya A. G., Autoradiographic study of the penetration of Sendai virus into tissue culture cells. I. Preparation of Sendai virus labelled with radioactive isotopes, 1961, Probl. Virol., v.6, pp.588-93). This virus was also sent to study in the Institute of prevention and control of viral diseases, Beijing, people's Republic of China. For this strain of the virus, called BB1, Chinese colleagues had obtained the complete nucleotide sequence of the genome (GeneBank accession number DQ219803). The origin of the strain BB1 and its location in the phylogenetic tree of other paramyxoviridae described L. Y. Shi, A new paramyxovirus, Tianjin strain, isolated from common cotton-eared marmoset: genome characterization and structural protein sequence analysis, Arch. ViroL, 2008; v.153, pp.1715-1723). Variant of the original strain of the virus Sendai, held an unknown number of passages in chicken embryos at Cancer research center of RAMS, was sequenced at Novosibirsk state University in 2012 (Tikunova N. In., Kochneva G. V., A. Y. Tikunov, Netesov S. V., Matveeva O. V., Chumakov P. M., unpublished) and has received the name "the Moscow strain of Sendai virus" (M-SeV). This strain different from the strain BB1 with just a few substitutions of nucleotides.

Known strain of parainfluenza virus 1 Sendai STB N 2339. Strain bred in the Department of interferons company "Biomed" to them. Mechnikov, typed with a strain of parainfluenza virus 1 Sendai STB N 326 and differs from it titre� infectious activity. Strain is used to create an antiviral drug for the induction of interferon Genesis. He deposited in the State collection of viruses of the Institute of Virology. Ivanovo (patent RU 2108804 C1, publ. 1998.04.20). The source did not explain the creation of anticancer drug.

Two independent strains of the virus SeV: Z/H/Fushimi and Ohita M/Hamamatsu (doc.rero.ch/record/9055/files/these.pdf). The nucleotide sequence of the genome of strains within each line are identical to 99%, while the relationship between the different lines is only 89%.

Line strains Z/H/Fushimi comes from the viruses isolated in Japan in 1956. These strains continuously-passaged in eggs for several decades. They are moderately virulent for mice (50% lethal dose [LD50]=103up to 104The COMBAT).

Strains of lines Ohita M (SeVM) and Hamamatsu source highly virulent for mice (LD50<102), but after 30 passages in chicken embryos occurs their attenuate and loss of virulence (Kiyotani, K., et al., Attenuation of a field Sendai virus isolate through egg-passages is associated with an impediment of viral genome replication in mouse respiratory cells, Arch. Virol., 2001, v.146, pp.893-908).

A number of works performed in Japan showed that a weakened genetic engineering techniques are not pathogenic for rodents, the virus can intensively spread in tumor cells without affecting surrounding normal cells. The�th distribution leads to inhibition of tumor growth in mice. Among the tested models xenografts were fibrosarcoma cells, pancreatic epithelioid carcinoma cells and colon cancer (Kinoh H., et al., Generation of a recombinant Sendai virus that is selectively activated and lyses human tumor cells expressing matrix metalloproteinases. Gene Ther., 2004, v.11, pp.1137-1145).

The object of the invention is to obtain a strain of Sendai virus, the most suitable for use as a cancer drug. This problem is solved by obtaining the strain of Sendai virus adapted to growth in culture of human cells. Adaptation to growth in culture is accompanied by an increase replicative capacity of the virus during infection of human cells and loss of virulence for laboratory animals. In the process of adaptation observed increase in ecosections of the virus, which consists in the acquisition of greater selectivity against a broad spectrum of human tumor cells compared to cultures of normal human cells. Selection of a new variant strain of Sendai virus was carried out by 20 the adaptation of passages on cells NECK, during which there was an increase in replicative activity of the virus, manifested in 30-50-fold increase in the titers of virus in the final passages, compared with the titers of virus produced in cells NECK when infected with equal doses of virus Senda� (Moscow strain M-SeV), received on chicken embryos. Received a new attenuated strain of Sendai virus got the name Sen293nsk1. This strain is deposited in the State collection of the Federal service for pathogens of viral infections and rickettsial located in the Federal STATE institution of science "State research center of Virology and biotechnology "Vector" (FBSI SRC VB "Vector", 630559, R. p. Koltsovo, Novosibirsk region) under number V-648. The strain is designed to create anti-cancer oncolytic drug.

SeV may have oncolytic effect by several independent mechanisms. It can directly kill malignant cells, replicases in them. In the process of reproduction SeV causes the formation of a syncytium of 50-100 cells. The cells fused in the syncytium, can't share and are doomed to collective simultaneous death. In addition, the virus can cause mediated by the immune system destroying malignant cells. This occurs by activating anti-tumor activity of natural killer cells, due to increased ofwholesale cytotoxic activity of T cells, stimulation of antigen presenting dendritic cells and suppress the immunosuppressive activity of T-cells. The virus contains in its outer membrane enzyme neuraminidase, which is able to cleave sialic acid from the surface� malignant cells, making relevant cancer antigens more visible to the immune system.

The Protocol capacity of Sendai virus.

Capacity drugs Sendai virus produced in cells NECK cultivated on the dextran microbeads Cytodex. Cells NECK are immortalisant kidney cells of green monkeys Cercopitecus aethiops (F. L. Graham et al., Characteristics of a human cell line transformed by DNA from human adenovirus type 5, J. Gen. Virol., 1977, v.36, pp.59-72). They are certified for the production of viral preparations used as vaccines. Cells are well supported monolayer after reaching confluently that allows you to repeatedly harvest the virus. It monoclona culture, which is also able to grow on the surface of dextran microbeads Cytodex. The cultivation of the granules Cytodex characterized by maximum economy environment and allows the build-up of cells and obtaining viral drug in suspension in flasks with stirring using a magnetic stirrer.

For transplanting cells into pellets grown in monolayer culture, cells treated with 0.2% trypsin solution prepared in phosphate buffer solution containing 0.1% EDTA. After counting the cells in hemocytometer 106cells were placed in 10 ml of MEM culture medium in the modification of Dulbecco with the addition of 10% fetal serum of large horns�CSOs cattle, containing 1% suspension of granules Cytodex (GE-Healthcare, USA). The suspension was placed on a magnetic stirrer in a closed flask and incubated at 37°C. After 2 days there is an overgrowth of the surface of the granules by a monolayer of cells NECK that is controlled by microscopy.

Before infection, the granules are washed with medium without serum. The pellets were incubated in 1/10 the original volume in the medium without serum added drug Sendai virus (100 physical particles per cell). Adsorption of the virus was carried out for 30 minutes, after which the cells are again washed with medium without serum and placed in flasks with medium containing 1% fetal serum and 10 μg/ml trypsin. The addition of trypsin is required for efficient maturation of infectious Sendai virus, which requires exogenous protease activity for cleavage of F-protein (Heminaway V. R., et al., Role of basic residues in the proteolytic activation of Sendai virus fusion glycoprotein, Virus Res., 1995, V. 36, pp.15-35). Control of infection is produced daily by visual inspection under a microscope. Collecting the virus produced on third-fourth day after infection, when formed sincitii begin to undergo apoptosis. The titer of virus produced is determined in the reaction of hemagglutination.

Morphological properties of the strain

When electron-microscopic study in virusdatabases find material wie�asnie particles, the size of 90-100 nm, morphologically identical to the representatives of SeV. He pathogenic for humans, for potentially susceptible laboratory animals (mice of the line Balb/c) are not virulent (LD50>105The COMBAT).

Determination of the nucleotide sequence

It is known (Lamb R. A., Parks G. D., Paramyxoviridae: The viruses and their replication. In: Knipe, D. M., Howley, P. M. (Eds.), Fields Virology. 2007. 5th ed. Lippincott Williams & Wilkins, Philadelphia, pp.1449-1496.), that the genome of Sendai virus includes 7 genes encoding viral proteins (table 1).

The nucleotide sequence of the strain Sen293nsk1 given in the Appendix.

A comparative analysis of the genome of strains of Sendai virus, the original M-SeV and adapted Sen293nsk1, allowed to conclude that the new proposed strain is characterized by a 10 nucleotide substitutions, which are located in the coding region, and 6 of nucleotide substitutions are accompanied by changes in the amino acid sequence of amino acid viral proteins. Learning the differences between the viral proteins of different strains of Sendai virus showed that all the substitutions result in changes of grade amino acids. It should be noted that a single amino acid substitution detected in the protein F (replaced by valine to methionine), and five substitutions accounted for the HN protein. Thus, all amino acid substitutions are concentrated in virion proteins located on topically� the shell of the virus, what could alter the efficiency of penetration of the virus into cells.

The resulting strain is characterized by a high reproductive capacity, harmlessness, absence of contamination by bacteria, fungi, Mycoplasma and extraneous virus. The strain has a high reproductive and oncolytic activity. Strain can be used to produce medication that will improve the effectiveness of cancer treatment by direct destruction of malignant cells under the action of the virus, and stimulate antitumor immunity

App

The nucleotide sequence of the strain Sen293nsk1 Sendai virus

TAA ATT TCC TTG TCT CTT TGT AAG TTT TTC TTA TTG CTA TCA TAT 45

GGA TAA GTC CAA GAC TTC TAG GCA CCG TGC ACC CTA CAC ACT CCC 90

GCA TGA TAA TGA TTA CTT ATT CCG AGC CGT CAT ATG GCT CGA TGT 135

CAT ACG CCA ATC CAT CAT CAA CGG CCA TCA TGT ACT CAT TAT GCC 180

TGG CTC CGA ACA TCT CAG TGA CTC CTA AAA TCT TCA TCA AAA TCT 225

TTA TCT CTT TAG TGA GGA ATC ATG TAT TTA TAC TAT GTA TAT TAA 270

TCT CAA ACC TAT CTA ATA CAG TTT TTG TGA TAA CCC TCA TCC AGT 315

TGA TTT CAC GAG ATG GTG ACA AGA CTA TCC ATT CCA GAA GTC TCG 360

CCT CAA ACT TCT GGT CAG GGA ACC ACG CGG TTA CCA GTC TTG TAG 405

ACA TAG ATA ATG ATA TCC AAG ACA ACA CAA GTC TCC TGG AGA TTG 450

TCT TTA ACT TGC CTG CTC AGT TAA CAG GGT ACA AGT CAT ACT TAC 495

CGG TCA GTT TGA GCC TCT TGT CTG ACT CGA TTA TTC TGG CCC ATT 540

CAA AGA TTG TAT CCT TTA AAA CTC TAT TGA AAG CTG TCA TGC AGT 585

TGT GTG TGT CAG CTA TGT TCA TGG TGG ACA AGA AAT CCC TGC TCA 630

ATT TAT ATA AGT TGG GTT CAA AAC CAA ACG TTT GCG GTA CCT GGT 675

GGT AAG GTC TAA GCA TCC CTG ACG ACG AGC TTC CTT CTC TTA ACT 720

CTC GGG TAACCC ATT CGT GAG CCT TTG CTT TCT CTA TTA GGA TCC 765

ACT TTT CTA TCT TGA TGC TAT CTC CCT TCG ATA GAG GGT GAC TGA 810

TGG CCA GCA CTG TCT TGC TAT CTT CTA TAA TAT CAG ACT TAG GGT 855

GCC GTG ATA GTA GAT ACA TCT CTG CAG AAG CAG GAT TAG ATG TTT 900

TAA GTA CAA CCA AGT TGA CCT CAT CCC AGT ACC TCA GGT GGC ATA 945

TAA GCT GCC TGG AAT TCC CTG TGC CCA GCT TAG GAG CAA TCT TGC 990

TTA TCA CAA GCA CGT CTC GAT CCC CCA CCA ACG GAT CGA TCC TGA 1035

TCA CAC TGT AAT GCT CAT GCA GTA CAA CTT GAT CAT CCT TAT GGT 1080

CCC CTC CCT CCA TGT CGC AAT GGA CTA CTA GAC TCG AGC TAT TCT 1125

GCA GTT CAT TCC AAA TCA AAG CCT CAC CAT ACT CAT TCC TCC CAA 1170

ATG TCG AGC CAG GAT TTC CGT TGA ACA ACA CCT TAA CTC TCT GAC 1215

CCA GAC TAA TAG CAT TGT TTA GTT TCT TCC CCA ATG CTA CCA CCT 1260

CAG CAG GAT ATA TAT TTA CTC ACT TCT GCC CAT TGA CAT CAC AAG 1305

AGT ATA CTC CCG AGT TAT AAT AGT TGA TGC ATG GGC CAA GAG TAG 1350

CGT CAT AAC AGG AAA GCA CCC TAG CGG CTC CTT CCC CTA AAA ACA 1395

GCC TGT CCT TAT CCT TGT CAA CCA AAG GGC TTA ATA AGT AGG TAA 1440

GTT CAA GCG CCT TCA AGC AGC TGC TAG TGT TAA TAC CAA AGA GCC 1485

TCA GCT GGT GAG ATA GGT ACC TTC CAT ATG CGA GTA ACG TCA GAC 1530

CAA CAC CTG ATC TGC CTA CCT CTC CTT TTG TGA TCT CCT CGT ACC 1575

CCC GTA GAC GCA GTA CCT GTG ACT TGG ATA CTC TCA CCC AAA GCC 1620

AGG GGG CCC TAG TCT GAT GTC CTA AGG GAA TAT TCT GTT GTA TTT 1665

CTG CCG CGA TGC CGT CTA GCA GTG TAT CAT CCG CGG CAT GAT CCC 1710

AAT CCT CTA AGA CTT CGG GGA CTC CTA TAC CTC TTC TCG TTA ATA 1755

CTT TTA TAG ATG ACT TCC GGA TGT AGG TCA GAG ACG TTG GGA ATA 1800

CTT TGA TGA CTA AGC CAG TCA ATT GAC ACC TGT TCA GTA TAG GGT 1845

CAT CAA GAA ATG TGA GTT CCA GAT AGC TCT TTA AAG AGT CTA GCC 1890

TCT CAA GGG ATG TCA TTG ATT CTA GTC TTG CAT GCC CCC TAG ATA 1935

TCT CTG CCA AGC TGC ACA GGT ATG CAA GAT GTC TAG CCA AGA AAG 1980

AGG TCC ACC TCA TGT CAG CCA CAT CTG GGT CAT CAC TAT AGA TAA 2025

AGA CCT CAA GCG GTA CAC CTT CCC GCC AGT CAT GCA TAA ATA GAT 2070

CTA CAG AAT ATT CAC ATA CAG AGA GGG CCA GGA GTG TCT TAT CCT 2115

GAT TTG AGA GGT TAG GCC CAT ACA CAG GTT CTA CAA CAC CTG CAT 2160

TCC AAA ATC GCT TGA AGA TCT TTG GAT GGG ATA AAG CAT TAG ATA 2205

AGA CTT TCA GAA CTG CGT GGG AGG TAT CTT TCA GAA GGA TCC CTA 2250/p>

CAT GTC CCC ATA TTT CTT CTC TCC CTC TGA TGT TCA ATC CGT AGA 2295

GTG ATG AGT CAA ACT GAT CTA TGA GGA TAC CCC ATG CGA AGC TTG 2340

AAA GAG ATA GAA CAT CAA CCA CCA TGA ACT CAG TAA TCA AGC TGT 2385

TAA CGT CAT CAT CAT TTA CTA GTG CTA TCA TCT CTT TTA GAT TGT 2430

CTC TAT ATT CTA GAG ACA TTG TGT CGG TTG CTA TCA TTG CAG TAC 2475

ATA TGC TGG TCG CCC TGA TGA CTT CGT CAT CCG ACC AAT AAG TCA 2520

TGT CAA CTG TAT GTA CGA CAT CTC TGA CCT TGC TAA ATA GCT CAA 2565

GGT CCA CAT CCT TGA GTG GAT CAG GGT CAT AGA TCA ATT TAT TGT 2610

TTT CCT GTG TGA TCT CCA AAT CCA GTG TAG ATC TTG GAG GGA TAT 2655

TTG CCT CCT GTG GGG ACT TTA CCA TAC AGC ACC CGT TAT TAA GAT 2700

GTA AGT GTA ATA TCA GTG GCT TCT CTA AGG AAC CTT TCT TAT ACC 2745

TCA TAT TGA ATT CGA ACA AGC TTA GCC CAG TTA GCA TAA GCT TCT 2790

GAT ACA CGA GAT TAG TGT CCT TCG ACT CTC CTG CTT CCT GTG TGA 2835

CCA TGT TGT CAT TTG ATA TTG TTA TGA GAC ACC TTG CAC GGA CCA 2880

GTG TTG CAC TGG AGA ACT TCA TCT GGG TTG CTG TGT CCT TTA ACC 2925

TAT GAG AGA GAT TCG TGG ATG TTG AAA CAG GAG TCA GCA GTT TTA 2970

GAT TTT CCA GGC TCA GAT CTC TAG TTG TTT GGG CTA TGA GAG CAG 3015

CTT CCA TCC ACG ATA CCT CAT CGG TCC CGT AGG CCC ACG TGT ACA 3060

CCA CTA TAG GGA TCC TAG CTG CCT TTG CTG GTT TGC TTA GAT TCC 3105

TTA CGT ATC CAA GTT GAG CTT CCG ATC TTT CAT CAG TGG ATC CTG 3150

CGA AGG AGT GAA TCC TTA TAG CCG GGC TCC CGT TTG TAA GTG TAT 3195

CCA AGT CTA TAT TGT CCG GGT GGA AAA ACC ATG TAT AGA TAG GGT 3240

CCG TTC CTT CAG ACC TGC AAA GCT TGC AGA CCT CCG AGC CCT CAA 3285

TAA TCC ATG CCC TCA AGA GCT CTA AAG GAT CTG GTG TTT CTA GCC 3330

CAT GTA GTC TGG TCC ACG CAT TCA GGT GGA TCC ACA TTT TCT GCC 3375

TTA AAC CGA CAG CCA CAA GCT CGG AAC ACA TAT CAT ACT ACT CAA 3420

TGT TAT CTT CCG TCA GCT TCC TGA GAG TTC TAG TCA GTG TCT CGT 3465

ACT GCA ACA GAT CAT AGT TGA CAA GTC TCC TCA ATA TCC CAT ATG 3510

ACA ACC CTC CCT TCT TAA CGC TTG CCC TCA CCA GAG ACT TGG TTG 3555

TAT CAA GCA TCC CTG CAA TTG CTT CTC TAA CCC CTG TTA AGG AAT 3600

TGC CCA GGA CAT TCT GAG CTA CTC TCG GTA GGA TGA CTT TCC GGT 3645

CCA TAA GGA ACG CGG CCA GGT TGA GAT CCT CTT CGC CAC TGG ACT 3690

CTG TGA AAA GAC CAG ACA GCA AGG GGT TCG GGG ATT CCT GCA GCA 735

CAG ACC TAG CAG TGA TAT TCT TTA TAA TTG TAG TTA TAC TCT GGG 3780

AAT GCG GAA GGT TAC ATG AAT AGG GAT CTG CCC AAG AAT CTA GGA 3825

AGC TAG AAT CAC CGG GTT CTT GAT TCA TGA CCC TGT ACA ACA CTT 3870

GCT TAT CTA GCA GAT CCG CTC TGA TGA ACC TCT TGA CGG GAT CCA 3915

GGG CCG CTA CTG CAG GAT CCC CGA TAT TCC TGA CGA AGC ACC TAG 3960

ATG TAG ACA TGT AGT TGA ATC CCC CAA CAT TGG CCG GAA TTA ATA 4005

CTG CAC ATC TCA GCC AGT TTT TAC CCT TAA AGT ACT GAT CTC TTA 4050

CGG TCG GGT TTG TGA TTG GAT TTA TAG TCA TCC CCA GTG AAA TAC 4095

ACA CCT GCT GAC AGG TCT TGT ACA ACG CAA TGC AGC AGT CCA GTA 4140

TAG GAG AAT ACC CAT TTT CTA TGG CCT TGG CTA TAG ATG TTG AGA 4185

TAT TTG AGC AGG CAG ACC TGT TCT CAT CTA CCA ACG CAG TCT ACC 4230

AGA ATA CAC ATC TCA TGG AGG CTT TTA GAC ATT GTG GTA TTT GGA 4275

TCC AAT CAT CAT ATA TCC TTT TGC TGT CAA AGA ACA TTT TGC TAC 4320

TAA TGA TAG CAT TCT TCA ACT TTA GCT CAT GCC CTA TAT CAA ACA of 4,365

TGA CAT GTC TCA GAG CAC TGA AAT AGC GAG TGA TCT CTT CAT AGA 4410

CAT GAT TTT TCT TCT GCT TAT AAG TCT GGG CTA CAG GTA CCC TTG 4455

ATG CCG TCA CTA TAG CTT GAT TGT CGC CCT GGA CCA TTG CAG AGA 4500

CCC TGA CAC CTC CCA TCA CAG CTG GGT CTA TAG GGA CAC TGA TTG 4545

AGA TTA AGG TCC ATA GTT TCT GGC AAT AAC CTT CTA TTC CCC CCC 4590

TAG GAT TGT GTA TGA AGA TGC CAG AGT CTG CAT GAT CTT GCA GTT 4635

GTT GAT GCA TCC TGT CAG CGA CCG GAC AGT AAG GGT CTC CAA CAT 4680

ATA TTG TAC ACT TTT CGA GGA CTG GAT GCA TCC AGT TAA AAA AGG 4725

TCT TGA AGC CAA ATA TCT CGT TGC ACC TCT GCC CGA GTG AAA CAG 4770

TGC TCT CAA ATC AAT TCC TTA AGC AGT ACT TCT TAA GGT CTG TTG 4815

TTA GGA AGC AAC TTA ACG TCT CAT ATC CGT CTG TTG ATG CTG AAT 4860

TTG CCT TAA ATT CAT GTC TCG ATC TCT TCT CGT TTT CCC AGT ACC 4905

CCT TTG AGT TCC TCT TCC TGC TTA CTT CAT TTC TCT TCT CTG ATG 4950

ATC TAG GGT TAT TGT ACA CTG AAT TCC CAG TAG GGA CTC CTG AGA 4995

CAG AGA GGG TAG TTA GTC TTT GTA TAA AGT CTA TCT CTC CTT TTA 5040

CCA TTC CAT TCT CGC TGA ACA ATT CTC CTA TTC CTT TCG CCA GCA 5085

GTG TCT CCG CTA GTA CCT GTA CGG CCC GCA TTT TAT AAG TCA TCT 5130

TTG CGA ACA GAC CCT GGC CTT GTT TGA TCT CTT TCT CTT TCA AAC 5175

TAT ACG AGA TGT TGA ACT TCT CAT CTT TCA ACC AGT CTC CTG ATT5220

CCA CGT AAT CTA TTA TGT CCT CTG GGT TGA AGT TCT CAT CAT TTA 5265

TAA ATA CGA CTT TGA GCC GCC TGG TCT CCT CAG ACT CTG GAA CTT 5310

TGT AGT ACA GGT TAC TAT CTG GAT ACA CAG AGT CCC ATG CCT CTC 5355

TCC TAG GCG ATA ATG CTT TGT CCT TCA TAT ATA TCG TGA GAT CTT 5400

CAT CTA GTT GTG GTT TAA CTA ACT TCC GAA ACT TGA AGC CTA TGA 5445

AAC TTG TAT AAT TGT CTA CGG CAC ACT CGT AGG AGA TAG CCG TAT 5490

TGG ACC CTT CAT GAG TTC TAA GTT CCA GGC ACA CAT GGT CAG GGA 5535

AAT CAC AGG GGG GCC ACT GTC CGC CAT GCC TTT CCC TGT ATC CAT 5580

TTA TGA TGA TTG TAC AGA ATA CTG CGT GAC ACT CGT GTA AGG TCT 5625

TAA GCT TTA TTG CCT TCT GTG CAT ACA TAT GAG CCC GTA CCT TGT 5670

CAG CTG CAG CGG TGA CCT CTA AGC TAG GAT CGA GAC TCC ATG GAA 5715

AGA ATG AAA AGA TCT CTG CTT CAT TCT CAA TAG AAG TTC CAT GGA 5760

AGA TAG GTA CCA ACG ACT CCA CAA TGG CAT CTG CCT CAG GAT CCG 5805

TAT ACA CGT CCT TGT TTG TTA AAA CAG TCT GAA GCT CTG TCA ACA 5850

CAT GCC TCA TGA ATG CAC CAC GTA GAG GTA TAA CAG GAT CAC TTA 5895

ACT GTA TAA GAG CAA GTG ATA AAG GCT CGA GTA ATG CGA TGA CAT 5940

TGT ATA TCT CTT CCC CGA GAC TCG AAA AGA GGG AAT CCA CTA GCC 5985

TCC ATA GCT CCT CTT CCC TGC TTG TTA TCC CAG AGG ACC TCT TAT 6030

CTA ACT GCC CTG CAG CAG ACA TGT TCC ACC TCC CTT CCA CAA CAT 6075

CAC AAT ACA TTA ACA CCA GCT CAG GTG TCA GAA TAT ACC CTG TCA 6120

GTG TCA ACT TAT TCA GTA TCA TGA CAA GAT CTC CAT ATG TTA CTA 6165

GAG ATG TGT ATT TGC ACT CTA GGA GAT TAT GTG AGT TGG AGG TAT 6210

CAA GAG GTC CCC CTG GCC TAG TTT TTT GCA TCC ACC GCA TGT CAT 6255

ATT TGA TAC TGA ACC AAG TTA GGA ATG GCC TAT ACC ATT TCC TGT 6300

TGC TAT TAT ACT CAG TTA TCT CAG GGA TGG TGC TAA TGT CCT GCA 6345

ATG GAT CGT ACC CCT CTC TTC CTT CTA TAT TAC CTA GTT GCT TAA 6390

AGA TAT TCA ACC AGA GAT CCT GAA ACC CAT TAG ACT ATA CCT TTG 6435

TTA GCC GAT CCG CTG AGA AAA CGA CAG ATC GGA TCT TGT CAC ATA 6480

TCT CTG TAT GTA CGA GTC TAA ATA ACT CCT GAG AAT AGG TTG GGT 6525

ACG GTT CAA ACG ACC TGT GGT GAT CCA CCT TTA TTG TGC GTT GGA 6570

GAG CCT TGC CTA GAG ATC TGA TCT TAA TTT GGC GAG GGG GTC ATA 6615

CAC CAT TCC TCT TGA TGT GTT TTG TGA TAT TGA TTA TGC TGT CGT 6660

CTT TCA TAT GAC AAG GCT GGT CAT TAA CTA ACA AAA CGT GCA GCT6705

GTG CAA TCT TCC CCC TCA CTA TAG GGG AGT TCA GGT GAC ATT CTG 6750

GAT AGA GTA TAT CAG AAG GGT TTT GGG AGG ACT CTT GCC TGT CCA 6795

TGG CCT ATG ACA GGC TTC TTC CCA ACC CTG GGT TTT TCT TAA TAC 6840

TAT GGG AGA TGT GAG ATA CCA GGA GAT ATT GTG GAT GTA CAT TGA 6885

TGA CCC CCG TGC CAT CAA GGC CGA CCA GCC CGC TGA TTA GTG AAG 6930

TTT ATG GCT CGA CCC TGC ATA GTT TCG GGA TGC TGG TCT TAA AGA 6975

GCA TCG GCA GTT GGG TGT TCA AGC TCT TTT GGT TCT TGA CGA TGA 7020

TGT GGA AAC AGT AGC CTT TGC CAA AGT GCG TGC TGA ACG ATG TTG 7065

TGG ATG TGT CAG CCT CCA GTC CGT GGA CTT TCC TTA TCA ACA TAT 7110

TTA TAA TGT TGG TAG TGG TGT AAT ACA TGA TCG TTG GAT TAA CAC 7155

GTG AAG TGT TGG ATA CAT ACG TGA CGG TGG CTA CAT TAG CTG CAT 7200

CAG GGG ATA GGG GAT AAG CGT CGG TAT ACA CAC CTG ATA TGC ATT 7245

CCC TCG GAC AAG TAT TGT ACC AAT TGC CTC ACT CTT TTC CTG GCC 7290

TAG ACA AGG CCT CAT GAG GCG TCC AGT TGA TAG TCA AGG GGT GGC 7335

TGA TAT CAA GTA CCC CTA TCT GCA GTT GAG AGT GCC AAC CTG ACG 7380

ATC TCG TGT AGA TGT ACA CTT GAT CAC ATT CCA TTA ATA ATC TGC 7425

CCT CCG CTC GGT CGA AGT TCT GAG TGA CTG GAA TAG TTG TGA CTC 7470

TTA TCT TTG GTC TCT CTG AGA GAT AGT CGT TGA CTT GGA TGA GCA 7515

CAC TGA CCA CCT GCT TCC CCC CCA GCC ATG TAA TCT TCA GGG CTT 7560

CAT TGC ATG TGT CTT GCG ACA CCT GTT GGC ATC CTT GGG TCC TAC 7605

ACT TCG TGT CAC CTT GCA ATG GGG TGG TTA GCC CAC ACC CAT CGA 7650

GGA ACA TCA AAC GTG CTT CTG TTG CAA TGC CGC TGC CCA CAC TGG 7695

GGT ATA GTG CAG AAT ACG GGT GGT CAA AAT CTA CCT CGC TGC TGT 7740

TAT GGT ACC GGG ACT TTC TGG TTC CTT TGA GAT CTA GGA TGT CAA 7785

GGA CCA CAT CTT CTA TGC CAT CAC TTG AGT AGT TTC CAG TTT CGT 7830

CTA CGG GCA TCG AGC TGG AAA GCT GAT AAC CCC TCC TAG CGG TTG 7875

CCA CTA CCG AGC ACG ATT TCC GGT TAT CAT TGA TGT CAT AAG TGT 7920

GAG ACA CTA CTG GGT TGA GGT CAG GAA ACA TAT CTG AAT GTG TGA 7965

ATA TGT ACC CTA GCT GCA GGA GAT CCT ATG ACT TCC CAA TGT CAG 8010

TAC AGC CTT GTG TGA TGA GGT TTG ACG AAT AGG CAT AGA TCG CCT 8055

CAC CGA TTG AAA GTG GGA AGG GTC TAA CAC ATC CAG TCG AGA TTG 8100

TCG AAC CAG GCA GGC ATA TTG GAC CAG GCA ACA ATG AGA TTT CAG 8145

GGT CCG GGC TGA GAT ACG GTT CCC CTG CAG GGC ATC TCC AGA AGC8190

TAT GTG GTT CAA GTG GGG CGA TTC CCT CAG CAT GAT GAA CTG CAA 8235

TTG TAC TAT CGC AGA GCT GCG TGA GAT CCT GTC TGT TGC ACG ACT 8280

TTT CAA TCA GCT GGG TAA CAT CTC TGC TGT TCT TGT TCA ACA AGA 8325

CAG GGA TCC CAG TTT GCA CAG AAC TCT GAA TGT TAA CGG CTC TAG 8370

TGA TAA CCT CTT GCC TTA TTA GAC TGG GTG TGA ACT CCT TTA CTT 8415

CTT TAC TGC TTA TGT TCA GTG CCT CTA TAG TTG TTG AAT ATT TTC 8460

TCA TAC TAT GCC CTT GCC TAG CAA CGA TCA CGA TGC AAA TGA TCA 8505

CTG TAG CAA TCG ACA AGG CCC ATT GAG TGA ATG CGA TCA GAA ACA 8550

ACC AGG TGT CAA TTT TAC CTG ACC TCC CCC AGT CTG ATA ATT CTR 8595

TTG TAG TGC TAC CAC TAG GGG AGG TAG ACC AAT AAT ACG GCT CCC 8640

TGC GTT TAT CAC CAT CCA TGA TCT GTA CCT GTT TGA GGT GAA AGC 8685

TAT AGT GTT AAG CGG CCC AAC TTT CAC AAG CCT TTT TTC TTA TTA 8730

TGT ACA GAC CTC AAC TAC AAA ACC CAT TAC TTT ATC AAG CAT CCA 8775

ACA AGA ATT GTG ATC ATC TTT TCC CAG TCA TAG CAT TAA ATC CAC 8820

CGT TGG TAT ACA TAT GCC TTA TCT TTG GCT CTA ATG ATG TGT TGT 8865

CTC TTG ATA CCC GAC CAG CCG GAT TAC CCG TCA GCA TTG TCC ATC 8910

TGA GCC TGT GCA AGA CGA TGA CAA TCA TAA CTA TGA CTA CCA ATA 8955

TCA CAA TCA CGA CTA CTA TGA TCG TAA TCA AAG TCG CCC CTG AAT 9000

TGT ACC ATC TAC CTA CTT CAG ATA GGA TTT TTC GTG CCC TCT CAA 9045

GCT CAA CTC TAG AGT CTT GCA GGA AAT CCG TAG CAG CAG CAA GGT 9090

TGA GAG AAA TAT CCA CGG GCC TGA TAG CGA TTG CAG CAA GAC CCG 9135

TCA AAT TCT GGA CCC CCC AAG TGG CAT CAC CCC CTT TCC GGT TAG 9180

CAT ACA ATT CTA TCC CGT TGA CTC TCA CTA GAC CAC AGT TAT CAT 9225

GGG TTA AGA ACA CCA CAC CTT TAG AGC GAT CTT GAC TTG TGA GTC 9270

TTC GGC TTA TTC CGC AGG TAC ACG AGG ATG CTA TGC AAT TAG CGA 9315

CAA CAC CTC CGT TCA CAA CAA AGG ATT TGG GGA TGA GGC TAT CCA 9360

CAA CCC TTG CAG TGA GAC ACC TTG TTG TAT CCC CCA AGA TAC ATT 9405

TTT GCT GAC CAG TGT GTA TCA ATT GTG CAG GAT CCC TGG GGC ATA 9450

TGT AGG TCA ACC TGG ACT CTA CAC AAT CGA CTA TGT TTG CAC CCC 9495

CAA GGA AGG AGG CAC GAC TGA GTA TAT GAC TGG GGA CAG TTA CAT 9540

ACC ACT CCT CCC CGT TAT CTA TAT AAG AAA TAG ACG ATG CCT TGT 9585

GTA TAA GCA CGC CTG GGA CCT CAG AAA GAA GGA TGG TCT TCA CAG 9630

ACA GGG TAA CCA TGT ATC TCT GGT CTA CTA CAT CAA TCA CAG TTC9675

CCT TCT TGA GTT CTG TAT AAA TGA AGA CAT CAT TGT TAG ACT GCC 9720

CTG TCC CAG TGA TCA TGG TAA CAG TCT TGA TGT TAG CAG AGT AGA 9765

GTG AAG ACA CCT GCG GCA GTG GGC TGA TCT TTT CTC CGA TTC TAG 9810

CAA AAT TGG ATC CGA ATG CAG TCA GCA ACT CAG AGT AAT GTT GTG 9855

TCA GTT TTA TCC CCA GTC TTA GAG CGG CAG TTT CAC AAC CTA ATT 9900

CTC TTA TTG CGG GTT TGA TCT TCA CAT CAT CAA AAT CCT GGA GTG 9945

TCT TTA GAG CAA GAA TTT GTT CCC CCA CAG CGT TTT GCA GCA GTT 9990

CTA TGG ACT TGT GTG TTT TTG TCA TTG ATT CTT TTA TGA GTG CTA 10035

TGC CTC TCT TGG CTT CCC TCG CTT CGG CTA GCG CGA TCC CTG CGG 10080

TTA TCT GTG CTG ATG TTG CTA CTC CAA GTG CAA TGG TAC CAA TCA 10125

CGG CAC CGA AGA ATC TTG CCT GTG GAA CAC CGG CAT TTG TCA TCG 10170

TGT CAT TAG CAG TGA TTA TCA GTG CTT CCT GAA GGT CTA AGG CAT 10215

CTC TTA GTG GGA TTA ACA GGT ACC TCA GTA GGC TCT TAT ACT GGA 10260

TGA CCT CAG GAA TTC CGC ACC CAT TCT GGT CGA CAA CCC CTG GGA 10305

CTA AAC GTA TTA CGA TGT ACC TCG CGT ATT GGG ATC CAG CAA TCT 10350

TCA GTG ATT TCC CTT CAT CTA CTA TGA CCC TAT CTA TAG AGA GCT 10395

TAT CCC TAG GAA TTT GAC ACG AGG CTA GTT TTA GCA TGA CAA TTG 10440

TCA ATA ATG CAG ATA TGC ACT GTA CTC TCA GAG TGT AGG TCG CCA 10485

TGT TTC TCA GGG GAG AAG TTT TAC AAT GCA AAG CGA ACA AGG GAC 10530

TTT ATC AAG CCT TTT TTC TTA TTT GAG ACA AGG AGC AAT GCT GTG 10575

GCT CCA ACT GCA AAG TGG TGG CTG CTT GTA AAA GTG CCA CAG GTC 10620

CCT GAT TGC AGA ACA TTT ATA GCT TCC TGA TCC TTC CTA TGT TCC 10665

TTG CCA CGA CAT TAG GGT AGC AGT GGA AAT CAC GAG GGA TGG CCG 10710

GTT GGA ACA CCG CAT CAA CAT CTG TGA TCT CAA CAG ATG CAG CCC 10755

AAA TCA CCA AGT TCA TAT GAG GAT TCA CGT TTA CCA ATG GGA AGC 10800

AGA CCG CCC TCT TCC ATG CGA GCT GAC TCA ATG TAA TAG TCT ATA 10845

GGG TCC CAG TAA GAA CCT CAT GGA AAC TGC TTA CGC CGA TCA ACC 10890

CAA GGG AGA AAA TAA GCA GCC TCT TTT CAA TGC TCT TGC TCT AGT 10935

ACT CAA CGG AGT ATA TTT TCC CGA CTT TTC TTC TGA TCA ACC CAA 10980

GGT GCA CCA TGA AAT TGA GCT TCT TCT CTC CTT GAT CAT CAA GTA 11025

CCG GGA GTA CCC CCT TTT GTT CTG TGG GAA TTC CGG TCT TAA GTG 11070

TCA CTA GCA GGT CAG TAA AGA TAG AAT TAG GCA ATG CAA GGT CTG 11115

CAA GAG TTT TCG GGA TCT TGG CTA TT TGA TTG CCC CCA ATG GAG 11160

TCC CAT TGA CAA ACA CTC CCA TGA TTA ATC TGT CCT TAT CCA CCG 11205

GGA GGC ACT GAG GGG CGA GTG CGA CCT TGT TAG CAT TAA ATA TCA 11250

TTC CCT GTC TCA GCC TGC CTG ACC ATG GTA GGA GTG GAG CAC CGA 11295

TCG AAT CCA CCA TGT ATA CTA TCA TTT CTC CTG GAA CTC CAG TCC 11340

TCC TCA CTG TAA TTT TGA GAT CGG TGC AGG CCT TTA AGA GTT CCT 11385

GAT CAG ACC CGT AAT ATT TGG CAC CTA CTA TGG GTA ACG ACC CGG 11430

ATC CGC AGA TTG AGC AGT CAG TGT GCT CTG TTA AAT CGG ACA CAC 11475

TCC CTA GGC TAG CTG TTT GTT TCG GAG TCT CAA AGA AGC CCA AGA 11520

GCA ATA AAT CTA AGT ATC TCA CTC CAT GCT TCG GAG GGT CTC CTA 11565

CCT TGA TCC CAA TGA TGT GAG GGA TAG CTT TAT TCT CTG GGC CAG 11610

TTC TTA GCA GAG GAG GTT CCA CAG TAC CGT TGT CCT CAT ATG AGA 11655

ATT TGG GGA ATT TGT AGA TGT CGG CCA TTG TGT CTT GTT GGG TGA 11700

AAT TTC TTT CAC AAG CCT TTT TTC TTA ATC ACT TCT GGC TGC TGA 11745

TAA CAA AGG GGG ATT TCG CTC CAT GAC TGA GGA TGA TTA GAT GCC 11790

TCA TCT TCC AGT TAA CCA GTC AGT GAT ATG TCT TCT TCT TCT ACG 11835

AGC TCC ATG ACT TCC TTA ACC TCT CGA TCT GTC TTG CAC TTG GAT 11880

AAC AAT TTT ACA TAT GCT GCT TTC TCA GCT CTG CTT GGA AGG CTG 11925

CTC TCT ATA ACG AGC CTG AGG GAG TGC ATT GTA GGC TTC TCC TTT 11970

GAG GGG AGC AGA CGC GCG GAT TTG GAG GCT CTG GAT GTC TCG TCT 12015

CTT TCC TGG TAC ACC GGG TTG CGG ATC TCC TCA CTA AAT TCA TCC 12060

TCT CGG AGT AGG TCC GGC TTA TAC TTC ATA TCC CCC AAG GTC TCC 12105

ATA GGG GAC TCA AAC CTG ATC ACT TTC TTG TTG CTC TCT TTT GTT 12150

TTT GCA AAG ACA GAG GGA GAT CTT GTA GGG GAA TCT GTG TTG TCG 12195

GTC TTG CCA CCT CTA TCT GTG ATG ATA TGA AGT GTA GAT TTG AGG 12240

GAC ATC AGT AAT GAG TTT TGT TCC TTC TGA TAC TCG GAG AAC CTC 12285

TTR TAT ATA TCC CGA AAT GAT TCC ACA CTT TCT TGG ATC TGT TTA 12330

AGC AGC TGT TTA TTC TCA TCT ACC CTA TTA GCG AAA GAC TTC TCG 12375

GCA GAA AGG AGA ATT CCG CAT ACA TTA AAT GTC ATC TCT GCA TAG 12420

TTT GCA GAT TTT AGG GCA CGC CTT GCA AAC ACA CTC TAA GCG TCT 12465

CGG GAT GAC AAT TCG TCT TGA GCA GAC TGG ATT ACA CCA CTC AGA 12510

TTT AAC AAT GTA GCC ATC TTC TCT ATA GAT GAT GTG CTC CCT CCT 12555

GTG TTT CCC TTT GTT GCG TCG GTC GGA TCC CCT GGA GTT TGA GGA 12600

TGA TCG CCG GCC GGA CGG TCTGGG GCA GAG CGG GTC CCT GCT GGT 12645

AGT CTC CGG TCC TTG GTC CTA ACA GCG GGG GCA TTT CTG GAG TTG 12690

GTG TAC TCA CCC TGT GTG GTT GGG GGT TTC CCT GGT GGT GAC CTT 12735

GTG CTG TTG TCA CCC TGA GGT GAT GAG GAC TGA ATG CCG GGC GCG 12780

GCC GCT GGT GTG AGG GGT CTG GAC CCA CTG ACG TTG GGT CGT CTC 12825

TTG TTC CTT TGT AGT ACA GCC TCT TCG AGC TCA GGA CTA GGG ATC 12870

ACC AAG CCG ACT GTC ACT CTT GTA CTA TGT GGG CTG CCA GAC TCC 12915

ATG CCT CTT CCA TTA TTA TCT GCT CTA CCT TCT CCT TCA TCA GGT 12960

AAG GGT GCA CTT CTT CGT TCC ACC TCT GGA AAT CCT TCA GCT TGG 13005

TCT TCT CCC CTC TTA TCA GGG TTC GCA GCC ATC TCT CGG TTT TCA 13050

TTT TCA GCA GAT CCT CTC GGA TGT CCT CCC TCA TTT GGA GGA TCT 13095

TCA AGG ATT CCG GAG TTT CCT CCA TTG CCC TCC AGA TGA GAT ACA 13140

GCG TTT GTA CCA GCT CCT CTC CCA GCG CGG GTC TGT ATA TTT TGT 13185

TTA TCA ACA TCT CTA GCA TGT GCT TCT GCC TCT GAC TTG CTT GTT 13230

CTC CCA GGG ACT CTA CTC TCC TCA CCT GGT TTA CGA TCT TGG GTC 13275

GGA GAT GTC GAG ACT TCT CCC TCC CCA TCA CTT TTG ATT CTG TAG 13320

GTA GAG CCT GGC CCT TGG AGG GTG TCG ATA GTG TTG TAG AGC CAG 13365

CTT CTG TCT CCT CCG ATT TCA GTT GGT TCA CTT AGG GAT ACT GCA 13410

TCG AGG AAT CCG ATA ACA TCC GAG GAC AGC CGT TCT CCT CCT GAC 13455

GCC TCC CTC TCA ACT TCA GAA TCT TCT TTA ATG GGA AGG GCA TCT 13500

TGA TCC ATG CGG GAA ATG TAG CCG AAG CCG TGA TTG TCT GGA CTG 13545

CTG AGT GTG GTC GAT GTT GGG GGA TGT GGC ATC CTG TGG GCG GAT 13590

CGG TGG ATG AGC TTT CAC AAG CCT TTT TTC TTA CTA GTC TAG ATG 13635

TAT CCC GGA ACC TAG TGT GAT CTT TCC AGA CTC CTA TTC CAG CTG 13680

CTG CTG CGG CAT CAT CCT CTT CAT CCT GAT CAA CAT CGT TGT TAC 13725

GGC CTC CGT CTT CGT GGG TTG TAG TGC CAT CTT GCC TTC TCT CTG 13770

CAA GTC TCC TGG CTA TTC TCC TCT CTA TGT CTG AGA CAT CCT CAA 13815

CTT TAG TTT CCT CCT ACC CCA GTT CTG CCC CGT GTA ATG TGA TAA 13860

AAT GAC CAC TGC TTG CCG GGC GAG CCC ATC TGT CAC CAC TGT CCT 13905

CGC CCC AAC CCC TGA CGT CCT GGT CTG TGT GAG TTT CAG GTT CTA 13950

GGT CTA TGT CAG CAT TAT CTA CTA TAG CTT CGA TTG CAC CGC CAC 13995

CCG GCT TCG TGT GGT ATG CGC CAT CTC CAC CAG ACA GAT TAG CCA 14040

AAT GAT GTC TGA GTC TCT CCT TAG CCG TAT CTG TCA CTC CTA GTT 14085

CAT CCT CCA GAG CAC TGC TGATCT TAG ATT CAG CGT CCT TTG CTA 14130

CGG CTT GTC CTA GTA GGA ACA TCT CCA TAT CAA GGT TTC ATG TTC 14175

CTG TGA CAT ACT GCT GCA TCG CCT TGT TCT GTA CAA CGA CGG CCC message 14220

CCA TTG AGC CAT TCC AGA GTG CTG AGT GAT TGC CTG GTG CAA ACT 14265

CTC CAT GGA CAG GGT CCT TAA GGA TAC AAA TAA AAG GGG CTC TGG 14310

GAC CTT TTG ACA AGT AGG TGT CTA GGC TGA TTC TCA GCT TAT TTA 14355

TAT CAG GCC TCA AGT TTG ATA GTG TTA GAG CCG CCA TCT TGG TCT 14400

CTA CCC CAT ATT TAG TGA TGT TCA TGA AGG AAG CTA GCC CTG CAT 14445

CTC GGA TGT TCC AGT CGA CGA TCT GGA TGT TCT TCT CTA GCG TAG 14490

TGA GGT CGG ATC CAG TCG TGT TCA TAG TCA CAA TCT GGG CAA CCA 14535

TGA GAG ATA CCA AGC TTT GCT GAG ATC TCA TAA CCG AAC CTA TCC 14580

CCT CAA CTG TCT CTC CAG TGA AAA CTA ATG CAC CTT TCA CGG TCC 14625

CAT CTT GTC GGA ACG CCT CTA ATC TGT TGA AAA ATC CTT TCC TTA 14670

GAC CGG CGC TAC TTG TTG TGA CCT TCA CCA ACA TCC CAA AGA CCT 14715

GGA CAA TTA TTG CTC CTA GGC ATG GAT CCG ACC CAT AGA TTT GAA 14760

GGA GTG CAT CAG GAT CTG CAG AAT CTC GTT GTC CCT GGA AAA GCG 14805

GGC TCT TGT TGA CCA TGG GTC CAA ACA GCC ATT CTG TCC TGG TCT 14850

CAT ACT CCA TAT CTC TAG TCT TCA CAA TGA ATC CAT CTG TCT TTG 14895

TCC TAG TCT GGT CCT TTT CTA TGT TGT AGA CGT TCA ATT TAA CAT 14940

CGG CAT TTA CCC CGT TCG TTG TCA GGT ATA ATT CTG GAC TAC TGT 14985

AAG CCA TGG CAA GGG GTA AGA CTA GGA AAC CTC CTC TCT GAG AGT 15030

GCT GCT TAT CTG TGT CCA ATG AGT GAG CTA AGA AGG TGG TTG CAA 15075

TGA ATA ACT TGT CTG CAT CGT CAG CCA CAC TTG GGC CTA GTA CGA 15120

ACA CTG AGA CTG TGC TCC TCT GGC CAG GGA TAA CAG CAC CTC CCC 15165

CTG ACT TAT TAA TAC TTT CAC TCC TCC TAG AGC TAA ATG TAT CAA 15210

AAG TGC TCA ACA GCC CAG CCA TCG ATT TAA CCG GCA GCA AAG CTA 15255

AAG GTC TGA AAC CTG CTC TTC AGG GTG GAT ACT ACC TTG CTA AAA 15300

TCC TGT CCA 15309

Attenuated culture strain of Sendai virus Sen293nskl to develop anticancer drug deposited in the Public collections of the CPS pathogens of viral infections, rickettsial Federal STATE institution of science "State research cent� of Virology and biotechnology "Vector" under number V-648.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to such compositions and pharmaceutical compositions, which include poxviruses, and namely to those, which include extracellular enveloped viruses. Claimed invention also relates to such method, which is intended for production of poxviruses, as well as poxviruses, obtained in accordance with claimed invention. In addition, claimed invention also relates to application of claimed poxviruses and said composition for medication preparation.

EFFECT: obtaining pharmaceutical compositions, which include poxviruses.

11 cl, 3 dwg

FIELD: biotechnology.

SUBSTANCE: method of detection and differentiation of a genome of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates comprises: extraction of the DNA from the biological material, posing a multiplex polymerase chain reaction using synthesised primers complementary to regions of genes M130R and M151R of the myxoma virus of rabbit, and having the following nucleotide composition: MF 5'TGG-AGC-TTT-TCA-AGC-ATT 3', MR 5'ATA-TCT-CGG-CTC-TAG-GGC-GAG 3', MZ 5' [FAM]-AG-CGT-CGG-ACG-TCT-TCG-TT-[RTQ1] 3', VF 5'AGC-CCT-ATA-AAC-CCG-TAG-ACG-AAC 3', VR 5'CAA-GCT-TTT-TTT-TAT-CCT-CGT-CCG 3', VZ 5' [R6G]-TCG-ACG-GTT-TCG-TCC-GCC-TTC-TTG-[BHQ2] 3', DNA amplification of the virus and evaluation of the reaction. The present inventions may be used in veterinary virology for the detection and differentiation of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates.

EFFECT: increase in accuracy.

2 cl, 6 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, virology and immunology. Particularly, the present invention refers to a new avian astrovirus; to antibodies and their fragments targeted to the above new virus; to antigen preparations, proteins and DNA molecules of new avian astrovirus; to vaccines based on the above new virus or to its antigen preparations, protein or DNA; to methods for producing such vaccines and to diagnostic kits. The present invention can be used in veterinary science.

EFFECT: preparing the new avian astrovirus.

11 cl, 5 dwg, 4 tbl, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for preparing influenza A or B viruses in a cell culture, and a composition of the cell culture for preparing influenza A or B viruses.

EFFECT: invention provides the serum-free culture medium, avoids the need for the stage of cell culture medium replacement, prepares the influenza viruses with the high live virus recovery and can be used for the active immunisation of individuals and for producing the antibodies for various applications, including passive immunisation and diagnostic immunoassays.

22 cl, 24 dwg, 47 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for producing viral-like influenza virus particles (IVP) in a plant or a part thereof. The method involves the expression of a new influenza virus protein HA in plants and purification thereof. The invention also aims at IVPs containing the influenza virus protein HA and herbal lipids. The invention also refers to nucleic acids coding an improved influenza virus HA, as well as to vectors. The IVPs can be used in developing the influenza vaccines or for the treatment of existing vaccines.

EFFECT: presented group of inventions can be used in medicine.

18 cl, 44 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention relates to medical virology and deals with an influenza virus strain. The vaccine strain B/60/Massachusetts/2012/10 is a reassortant, obtained by crossing the "wild" virus B/Massachusetts/2/2012 with the cold-adapted temperature-sensitive virus B/USSR/60/69 -attenuation donor. The strain B/60/Massachusetts/2012/10 is deposited in the State Collection of Viruses FSBI D.I.Ivanovsky Scientific Research Institute of Virology Russian Ministry of Health under No 2740, actively reproduces in developing chicken embryos at the optimal temperature of 32°C, is characterised by temperature sensitivity and cold-adaptedness and safety for laboratory animals. Reassortant inherited genes, which code surface antigens of virus hemagglutinin (HA) and neuraminidase (NA), from the epidemic parent virus and remaining six genes, which code internal non-glycosylated proteins, from the attenuation donor.

EFFECT: claimed invention can be applied in practical healthcare for the prevention of influenza morbidity among adults and children.

4 tbl

FIELD: veterinary medicine.

SUBSTANCE: presented subline of cells A4C2/9k is highly sensitive to the ASF virus. The growth medium is used as medium Needle-MEM with 10% blood serum of swine. The inoculating concentration is 80-100 thousand cells/ml. The mitotic index to 2-3 days of cultivation is 25-35. The subline of cells is deposited at the Specialized collection of cell cultures of agricultural and game animals at the All-Russian research institute of experimental veterinary medicine under the number of 87.

EFFECT: invention enables to isolate the African swine fever virus without prior adaptation in reaction of hemadsorption and provides its accumulation in titre.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention relates to medical virology and deals with a strain of influenza virus. The vaccine strain A/17/Indiana/2011/72 (H3N2v) - reassortant, obtained by crossing of the "wild" virus A/Indiana/10/2011 (H3N2v) with the cold-adapted temperature-sensitive virus A/Leningrad/13 4/17/57 (H2N2) - attenuation donor. The strain A/17/Indiana/2011/72 (H3N2v) is deposited in the State Collection of Viruses FSBI D. I. Ivanovsky Scientific Research Institute of Virology Russian Ministry of Health, under No 2739, actively reproduces in developing chicken embryos at the optimal temperature of 32°C, is characterised by temperature-sensitivity and cold-adaptedness and safety for laboratory animals. Reassortant inherited genes, which code surface antigens of virus hemagglutinin (NA) and neuramindase (NA), from the epidemic parent virus and remaining six genes, which code internal non-glycosylated proteins, from the attenuation donor.

EFFECT: claimed invention can be applied in practical healthcare for the prevention of influenza disease morbidity among adults and children.

4 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medical virology and concerns a vaccinating influenza virus strain. The characterised strain B/60/Wisconsin/2010/125 is a reaccortant produced by crossing the epidemic virus B/Wisconsin/1/2010 with the cold-adapted heat-sensitive virus B/USSR/60/69 that is an attenuation donor. The strain B/60/Wisconsin/2010/125 is characterised by heat-sensitivity and cold-adaptation. The reassortant has inherited genes coding the surface antigens, haemagglutinine (HA) and neuraminidase (NA), from the epidemic virus and rest six genes coding internal non-glycosylated proteins, from the attenuation donor. The strain is deposited in the State Collection of Viruses of Federal State Budgetary Institution D. I. Ivanovskiy Research Institute of Virology, under No. 2723.

EFFECT: invention can be used in practical health care for preventing the influenza rate in adults and children.

1 dwg, 4 tbl

FIELD: medicine.

SUBSTANCE: strain is deposited from pulmonary tissue homogenate of guinea pigs infected by the 7th passage of smallpox virus. The strain is deposited in the State Collection of Agents of Viral Infections and Rickettsial Diseases of State-Financed Research Institution State Scientific Centre of Virology and Biotechnology Vector of Federal Service for Supervision for Consumer Rights Protection and Human Welfare, registration No. V-624. The strain causes an acute lethal infection with the more manifested clinical pattern in guinea pigs 200-300g heavy infected intranasally, thereby modelling smallpox in a human.

EFFECT: strain can be used for studying in vivo the efficacy of the antiviral preparations and assessing the reduction routines of undesired vaccine reactions and complications accompanying primary smallpox vaccination.

3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: CEA, CA 19-9, CA 72-4, M2-PK cancer-specific markers and Ki-67 proliferation index are pre-determined; combined positron emission and computer tomography (PET/CT) are performed. Up to two procedures of neoadjuvant intra-arterial chemoembolisation are conducted with the first one performed by administering a half single systemic dose of irinotecan in lipiodol 4-6 ml; no sooner than 2-3 weeks later, the cancer-specific markers and Ki-67 proliferation index and PET/CT SUVmax are checked; if at least one of this factors tends to increase as compared to references, the intra-arterial chemoembolisation is conducted again by administering at least 1/3 single systemic dose of irinotecan in no more than lipiodol 5 ml. Not later than 10-14 days following the second chemoembolisation or no sooner than 2-3 weeks after the first chemoembolisation, a surgical intervention is required if the tumour keeps its metabolic and proliferative activity on these days.

EFFECT: method enables decreasing the tumour process activity and performing the surgical intervention at the moment of extreme devitalisation of the tumour and micro-disseminates, and thereby improving the remote therapeutic effects.

2 cl, 4 dwg, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to biochemistry, particularly to monoclonal antibodies binding to c-Met and able to inhibit both ligand-dependent, and ligand-independent c-Met activation. The above antibodies, as well as a composition containing them can be used for producing a therapeutic agent for treating cancer.

EFFECT: invention enables specifically inhibiting both ligand-dependent, and ligand-independent c-Met activation, particularly inhibiting c-Met dimerisation.

34 cl, 111 dwg, 4 tbl, 27 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of organic chemistry, namely to novel heterocyclic compounds of formula (1) and/or to their pharmaceutically acceptable salts, where A1 represents CH; A4and A5 independently represent CR2 or N; A2 and A3 together with ring B represent 5-membered heteroaryl or heterocycle, with said 5-membered heteroaryl or heterocycle being selected from where t represents 1 or 2; and R3 is independently selected from H, C1-C6 alkyl, C6-aryl, C3-C6-membered cycloalkyl, C(O)NRcRd, -ORb, heteroaryl, representing pyridine, and heterocycle, representing piperidine and tetrahydropyran; and each of said alkyl, aryl, cycloalkyl, heteroaryl and heterocycle can be substituted with one group, independently selected from C1-C6 alkyl, possibly substituted with one substituent, selected from -CONMe2, C3-membered cycloalkyl, -CN, -OMe, -pyridine, tetrahydropyran, -CO-morpholine, -CO-pyrrolidine, (3-methyl)oxetane; -OH; -C(O)Ra; -CN; -C(O)NRcRd; -NRcRd; -ORb; -S(O)nRe; halogen, and substituted with one group -COMe heterocycle, representing piperidine, on condition that when A4 represents CR2, A2 and A3 together with ring B are selected from structure (3), (5) or (6); represents single bond or double bond; R1 represents heteroaryl, representing 6-membered or 9-10-membered aromatic mono- or bicyclic ring, containing 1-3 heteroatoms, selected from nitrogen, oxygen and sulphur; possibly substituted with one or two groups, independently selected from C1alkyl, C2alkinyl, -NRcRd, -NRcS(O)nRe, -ORb, halogen, halogenalkyl; R2 is independently selected from H; each Ra, Rb, Rc, Rd, and Re is independently selected from H; C1-C4alkyl, possibly substituted with one substituent, selected from -OH, -OMe, -CN, -NH2, -NMe2, C3-cycloalkyl; C2-C3alkenyl; C3alkinyl; C6aryl, possibly substituted with one or more substituents, selected from fluorine or methyl group; C3-membered cycloalkyl, possibly substituted with one substituent, selected from -OH and -CN; halogenalkyl; heteroaryl, representing pyridine; and substituted with one methyl group heterocycle, representing piperidine, or Rc and Rd together with atom (atoms) which they are bound to form 5-6-membered heterocyclic ring, representing pyrrolidine or morpholine; and in each case n is independently equal 2. Invention also relates to particular compounds, pharmaceutical composition, based on claimed compounds; method of inhibiting PI3K and/or mTOR activity and to application of claimed compounds.

EFFECT: novel compounds, useful for inhibiting PI3K and/or mTOR activity have been obtained.

15 cl, 16 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to such compositions and pharmaceutical compositions, which include poxviruses, and namely to those, which include extracellular enveloped viruses. Claimed invention also relates to such method, which is intended for production of poxviruses, as well as poxviruses, obtained in accordance with claimed invention. In addition, claimed invention also relates to application of claimed poxviruses and said composition for medication preparation.

EFFECT: obtaining pharmaceutical compositions, which include poxviruses.

11 cl, 3 dwg

FIELD: medicine.

SUBSTANCE: presented invention refers to immunology. There are described versions of antibodies or antigen-binding fragments binding to human 4-1BB. One of the versions is characterised by the presence of respective 3 CDR light chain sites and 3 CDR of heavy chain sites. The other version is characterised by the presence of the heavy and light chain with respective amino acid sequences. There are described versions of a pharmaceutical composition for reducing tumour growth or for treating cancer in an individual, as well as methods for reducing the tumour growth or treating cancer in the individual using the versions of antibodies or antigen-binding fragments in a therapeutically effective amount. What is described is a method of treating cancer with using a combination of the antibody and an immunotherapeutic agent. There are disclosed: versions of coding nucleic acids, an expression vector and a host cell containing the antibody expression vector. What is disclosed is a method for producing the antibody with using the cell.

EFFECT: invention provides the new agonist anti-human 4-1BB (also called CD137 or TNFRSF9) antibodies, which recognise an epitope within the amino acid residues K115, C121, R134, R154, V156 of the antigen, have Kd affinity measured by the BIACORE method and approximated to nM, eg 0,4 nM or 8 nM (for the anti-IgG1 antibody format) that can find application in the therapy of cancer and cancerous diseases.

19 cl, 8 dwg, 11 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to betulinic acid derivatives, producing them and using for cancer. For producing derivatives of formulas

,

1.R1=OAc; R2=H; R3=Me; X=Br 2. R1=H; R2= OAc; R3=Me; X=Br 3. R1=OH; R2= OAc; R3=Me; X=Br 4. R1=H; R2= OH; R3=Me; X=I 5. R1=OAc; R2=H; R3=H; X=Br 6. R1=OAc; R2=H; R3=Bn; X=I betulin is transformed into betulonic acid, then into dihydrobetulonic acid and respective dihydrobetulonates to be acetylated; the produced compounds are hydroborated and oxidised, then transformed into respective halogen derivatives; that is followed by hydrogenolysis and triphenylphosphine reaction to produce the derivatives of the above formulas. Cytotoxic activity of all the derivatives exceeds anticancer activity of betulinic acid substantially.

EFFECT: there are presented new effective anticancer agents.

6 cl, 1 dwg, 3 tbl, 11 ex

FIELD: chemistry.

SUBSTANCE: invention relates to organic chemistry, specifically to N-(4-(4-((2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohex-1-ene-1-yl)methyl)piperazine-1-yl)benzoyl)-4-(((1R)-3-(morpholine-4-yl)-1-((phenylsulphanyl)methyl)propyl)-amino)-3-((trifluoromethyl)sulphonyl)benzenesulphonamide (ABT-263) in a solid crystalline form. The invention also relates to a pharmaceutical composition on the basis of the above crystalline forms, suitable for the treatment of diseases, characterised by the apoptosis disorder and/or overexpression of one or more anti-apoptotic Bcl-2 family proteins.

EFFECT: obtained new crystalline forms of ABT-263 with useful biological properties.

35 cl, 2 dwg, 22 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to the field of organic chemistry, namely to the field of obtaining 5,9-eicosadienoic acid of formula (1), demonstrating an inhibiting effect on human topoisomerase (5Z,9Z)-5,9- The essence of the method consists in the following: tetrahydropyran ether of 5,6-heptadien-1-ol (4) and 1,2-tridecadiene (5) interact with the Grignard reagent RMgX (R=Me, Et, Pr, Bu, Oct; X=Cl, Br, I) in diethyl ether in the presence of metal Mg (powder) and the catalyst titanocenedichloride Cp2TiCl2, with the molar ratio of (4):5): RMgX:Mg:Cp2TiCl2=10:12:(30-50):32:(0.4-0.6), in an argon atmosphere at a temperature of 0-35°C and atmospheric pressure for 6-10 h, after which the reaction mass is processed with a 5% water HCl solution with obtaining 2-[(5Z,9Z)-5,9-eicosadien-1-yloxy]tetrahydro-2H-pyrane (6), which is oxidised with the Jones reagent.

EFFECT: eicosadienic acid is promising as the medication, possessing anti-tumour, antiviral and antibacterial action.

2 cl, 1 tbl, 1 dwg, 15 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to quinolines substituted by phosphorus-containing group of formula and applicable in medicine, wherein Z represents V1 and V2 are independently specified in hydrogen or halogen; one of R and R` represent phosphorus-containing substitute Q; the other one is specified in hydrogen or methoxyl; wherein the phosphorus-containing substitute Q represents A represents O; L represents C1-6alkyl; J represents NH or C3-6heterocycloalkyl and J is optionally substituted by G3; X is absent or represents -C(=O)-; X is absent or represents C1-6alkyl; each of R1 and R2 are independently specified in C1-6alkyl or C1-6alkoxy; G3 represents C1-6alkyl, R3S(=O)m-, R5C(=O)- or R3R4NC(=O)-; R3, R4 and R5 are independently specified in 3 or C1-6alkyl; m is equal to 0-2.

EFFECT: there are presented new protein kinase inhibitors effective for treating the diseases associated with abnormal protein kinase activity.

20 cl, 42 ex, 8 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to neurosurgery, neurooncology, and can be used for the treatment of glial brain tumours of a supratentorial localisation. For this purpose photodithazine in a dose of 1 mg/kg of body weight is introduced to a patient 2 hours before the tumour ablation. After that, surgical access to the tumour is performed. The operation wound is illuminated by blue colour with a wavelength of 400 nm, and the tumour boundaries are determined by means of fluorescence of photodithazine, selectively accumulated in the tumour tissue. The tumour is ablated under control of the tumour luminescence in blue colour with the application of an operation microscope. After that, a flexible light guide from a radiation source with a wavelength of 662 nm and power of 2.0 W with a light dispersing nozzle is placed into the tumour bed and the perifocal zone of the tumour is irradiated. The dose of irradiation is determined by the disappearance of fluorescence.

EFFECT: method provides an increase of the treatment efficiency due to the reliable clear determination of the tumour tissue boundaries with the normal brain substance independent on the malignancy degree and character of the tumour growth, with an increase of its ablation radicality, as well as due to the destruction of cells, located in the perifocal zone.

2 cl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves intravenously administering 0.1-1% aqueous solution of khlorin, selected from group containing photolon, radachlorine or photoditazine at a dose of 0.2-0.5 mg/kg or 0.2-1% aqueous solution of porphyrin like photogem at a dose of 0.2-1 mg/kg. Laser irradiation of blood is carried out 5-15 min later after beginning photosensitizer injection into cubital vein of one arm via laser light guide set in advance in the cubital vein of the other arm during 10-40 min at wavelength of 661-666 nm and power of 20-50 mW one session per day during 3-10 days with the aqueous solution of khlorin used as the photosensitizer, or laser irradiation of blood with wavelength equal to 630-633 nm during 10-45 min with power of 20-50 mW one session per day with the aqueous solution of porphyrin used as the photosensitizer. Repeated intravenous administration of photosensitizer is carried out 1-3 months later combined with repeated laser irradiation of blood.

EFFECT: reduced risk of tumor cells dissemination and metastasis development.

2 cl

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