Transgenic animals having main alzheimer's disease-related disorders
SUBSTANCE: invention relates to field of genetic engineering and medicine. Described is animal, non-human, having sequence of nucleic acid encoding presenilin 1, carrying mutations, corresponding to M233T and L235P mutations in PS1 protein of mouse. Animal also contains sequence of nucleic acid, encoding whole gene or part of gene, encoding APP. APP protein represents APP751, originates from human and carries mutations Swedish and London. Animal is intended for application in fight against Alzheimer's disease. Also described are PS1 protein and encoding it nucleic acid.
EFFECT: invention can be used in medicine for discovering compounds intended for Alzheimer's disease treatment.
20 cl, 50 dwg, 1 tbl, 8 ex
The present invention relates to transgenic animal models of Alzheimer's disease (AD). Its object is, in addition, the use of these animals.
Alzheimer's disease is a progressive neurodegenerative disease that affects a wide proportion of the population aged. This disease is characterized by clinical plan memory loss and decline in cognitive function, and neurotic diseases in the plan expressed by neuronal loss and the presence in the brain intracellular neurofibrillary deposits and extracellular deposits of β-amyloid peptide (Aβ), which forms the amyloid plaques.
Amyloid plaques are composed mostly of peptides Aβ 40 or 42 residues, which are formed during proteolysis of the precursor peptide Aβ (APP). Extracellular deposits of Aβ very characteristic of disorders associated with Alzheimer's disease. They represent an early and invariant feature of all forms of Alzheimer's disease, including family form (FAD). Family forms occur relatively early (between 30 and 60 years) and is caused by mutations in the APP gene in 5% of cases of FAD with eight identified single or double missense mutations in the gene presenilin 1 (PS1) in 50-70% of cases of FAD with more than 100 different mutations identified to date, and in the gene presenilin 2 more rare the cases FAD with two described missense mutations. It was shown that mutations in these three genes induced changes in proteolysis of APP, leading to increased production of Aβ, especially long form Aβ42, and the early appearance of the pathology and symptoms similar to the symptoms of sporadic forms of Alzheimer's disease.
Animal models designed to reproduce some characteristics of the pathology of Alzheimer's disease, were already described in the literature.
It is, on the one hand, transgenic mice carrying mutations in the APP gene. They have developed a pathology, such as Alzheimer's, since one year. So, the PDAPP mouse, superexpression human APP carrying the V717F mutation, develop with age deposits of Aβ in the brain, but not observed neuronal death outside the location of the plaques (Irizarry et al., 1997, J. Neurosc. 17(18): 7053-7059). This phenomenon can be called "plaques".
Also, the mouse Tg(HuAPP695. K670N-M671L)2576 expressing human isoforms of APP K670N-M671L (APPSw mutation type Swedish), shows deposits of amyloid type, but does not detect neuronal death (Irizarry et al., 1997, J. Neuropathol. Exp. Neurol 56:695-973).
In the study by Calhoun et al. (1998, Nature 395:755-756) loss of neurons has been shown in some areas of the brain near amyloid plaques in APP23 transgenic mice at the age of 14-18 months, expressing the mutated isoforms of human APP. This observation is contradictory, is sustainable, since the death of neurons are weak and occurs in animals in relatively large age and mainly in the vicinity of the plaques that could be detected previously in effect plaques". In addition, it is almost or quite is not mentioned in recent comments, which emphasizes that these animal models do not represent the full similarity to all known characteristics of the pathology of Alzheimer's disease, including death of neurons (Trojanowski, 2002, Am. J. Pathol. 160: 409-411).
On the other hand, aware of transgenic mice carrying mutations in the PS1 gene. They, apparently, do not develop diseases such as Alzheimer's, but there is a high number of peptide Aβ42 (increase of factor 2 with respect to PS1 wild-type), which is recognized as highly pathogenic.
In addition, in the above models, transgenic animals carrying mutations FAD P264L or M146L PS1 gene mouse ("knock-in"), mutant PS1 protein is not expressed in a sustainable manner (Siman et al., 2000, J. Neurosci., 20: 8717-8726; Flood et al., 2002, Neurobiol. Aging 23: 335-348; Rozmahel et al., 2002, 23: 187-194). These mice are also a high number of Aβ42 peptide.
In the application WO 02/0008407 described such transgenic mice in which the gene encoding presenilin 1 was mutated by the introduction of the P264L mutation.
Because of the role of PS1 protein in the formation of Aβ42 forms were also received double transgenic carrying mutations in the genes APP and PS1. As simple transgenic mouse, as described above, these mice demonstrate deposits of Aβ, but not show neuronal death (Takeuchi et al., 2000, Am. J.Pathol. 157: 331-339).
Thus, existing animal models of Alzheimer's disease are not satisfactory because they cannot be used to simulate the death of neurons, which is one of the main characteristics of neurodegenerative diseases, including Alzheimer's disease.
The applicant sought thus to obtain animals that represent the main characteristics of Alzheimer's disease, including death of neurons.
He showed that it was possible to get such animals, by introducing specific mutations in the gene encoding the protein PS1 mice, and crossing them with mice carrying out the super-expression of human APP gene.
The first aspect of the invention relates, therefore, non-human animal having, preferably, in its genome at least one nucleic acid sequence encoding presenilin 1, having at least one of the two mutations, the corresponding mutations M233T and L235P in the protein PS1 mouse.
Preferably, the animal carries both mutations.
Preferably, PS1 protein carrying mutations M233T and L235P, comes from the mouse.
Particularly preferably, the mutant protein presenilin 1 is endogenous.
Thus, the animal according to the present from which briteney preferably produces a protein including the sequence of SEQ ID NO:2. Preferably, it produces a protein that represents the sequence of SEQ ID NO:3. It preferably comprises in its genome the sequence of the nucleic acid SEQ ID NO:1 or the sequence SEQ ID NO:8.
Sequence SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:8 follow respectively from mutations introduced in the sequence of wild-type SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:9. Sequence of SEQ ID NO:5 - residue sequence 229-237 mouse protein presenilin 1 wild type. Sequence of SEQ ID NO:9 is the sequence of exon 7 wild-type mouse gene encoding the protein presenilin 1, i.e., not a mutant.
Preferably, the animal according to the present invention in conjunction expresses APP, preferably, a human APP. Such a gene may include one or more mutations FAD. Thus, mutations in the APP gene can be one of the various mutations described to date in the literature. Mutations in the APP gene can be selected among mutations "Swedish" (S), "London" (L) and "Dutch" (D), separately or in combination.
These mutations are well described in the literature and are generally the following changes:
|The nature and position||The Swedish mutation||Mutation Dutch||Mutation London|
|In relation to ARR||670N and 671L||E693Q and/or 692G||V717I|
|In relation to ARR||K651N and M652L||E674Q and/or 673G||V698I|
|In relation to ARR||K595N and M596L||E618Q and/or A617G||V642I|
|In relation to peptide|
|E22Q and/or A21G||V46I|
Under the mutation London understand all the substitutions, in addition to the replacement of the isoleucine, which are located at position 717 in relation to APP770, such as, for example, mutations V717G and V717F.
Of course, the APP, which can be used within the invention may be in various isoforms and, in particular, in the forms of 695, 751 and 770 or in a truncated form, such as, for example, isoform APP99, excluding the Swedish mutation for this isoform.
Preferably, the specified animal includes, in addition, preferably in its genome a sequence of nucleic acids encoding all or part of the gene encoding APP751. Preferably, the protein has a human APP751 PR is the procession. It is preferably mutation K670N and M671L (Swedich) and V717I (London).
In the framework of the present invention APP gene is preferably under the control sequences, ensuring its strong expression in neurons and in particular the sequences of the promoters of transcription, such as an exogenous promoter. As the promoting sequences can, in particular, to call the HMG promoter (Gautier et al. (1989), Nucleic Acids Res 17:20, 8389.), as well as the PDGF promoter (Sasahara et al. (1991)Cell 64, 217-27), the promoter of Thy-1 (Luthi et al. (1997, J. Neurosci 17, 4688-99) and the promoter of the gene Prion (Scott et al. (1992), Protein Sci 1, 986-97).
According to a particularly preferred variant of the invention, the animal model includes APP gene with the mutation S, D and/or L, under the control of the promoter Thyl.
Thus, the animal according to the present invention preferably produces a protein comprising the sequence of SEQ ID NO:7. It may contain a sequence of nucleic acids SEQ ID NO:6.
Preferably, it is about transgenic mice derived from crosses between transgenic mouse ThyAPP (TG53)bearing a nucleic acid sequence encoding a human protein APP751SL, and transgenic mouse bearing a nucleic acid sequence encoding a protein PS1 mice carrying mutations M233T and L235P.
Animals according to the present invention reproducing the first time one of the most important characteristics of neurodegenerative diseases, which is the early death of neurons.
They show besides other characteristics, traditionally described for these pathologies. Animals demonstrating accelerated deposition of amyloid plaques, is clearly visible from the age of 2 months and especially 6 months.
They also show the ratio of Aβ42 forms to total Aβ, β42/Aβ more than ~ 0.9, and it is observed with 2.5 months. This attitude is very high compared with those described in literature for other transgenic mice.
Already seen in mice at 6 months of age, the death of neurons becomes pronounced in 10 months.
PKR (Double strand RNA-dependent Protein Kinase is a kinase that is activated by stress and fosforiliruyusciye IF2 involved in apoptosis.
PKR is found in hippocampi (structure, where there is a loss of neurons) mouse APPxPS1KI according to the invention at the age of 10 months. It doesn't appear in hippocampe transgenic mice APPxPS1M146L at the age of 12 months, which, moreover, is not observed neuronal death.
New features of animals according to the present invention make them research model, a more complete and typical disorders observed in patients affected by Alzheimer's disease than previously described. These animals are particularly suitable, therefore, for the detection of neuroprotective properties of the link is, designed for the treatment of neurodegenerative diseases, preferably Alzheimer's disease.
Preferably, the animals according to the present invention have a ps1 mutant alleles in the homozygous state and APP - in the heterozygous state. Meanwhile, the same characteristics of this animal can be described by an animal with one mutant allele of the two ps1 alleles in the heterozygous state and APP - in the heterozygous state, however, expressed or manifested later phenotype.
Another advantage of the animals according to the present invention is that the number of mutant PS1 protein, expressed this transgenic mouse, equivalent to the amount of endogenous PS1 protein, normally expressed normal (non-transgenic) mouse expressing nematanthus PS1. This characteristic makes them best research model without superexpression PS1 protein was used to identify compounds for the treatment of neurodegenerative diseases.
These compounds may constitute, in particular, compounds that act on the regulation of PS1 gene at the transcriptional, posttranscriptional, translational, post-translational level or at the protein PS1, changing or adjusting one or more of its properties, or acting in a similar way to the partner who s interaction or on the target protein PS1, or compounds that act on the regulation of APP and, more generally, any molecule above signals initiated PS1 and APP during the neurodegenerative process.
In the framework of the present invention, the animals are preferably mammals, such as rodents. In particular, it may be a mouse, rat or rabbit.
Mouse and designs, ensuring their receipt obtained by the methods known to the expert.
They can be obtained according to the classical transgenic methods. As an example, illustrating one method of transgenesis can be called the method of electroporation gene construct containing modified genes in embryonic stem cells of the mouse, and, after selection, migration of cells carrying the desired genetic event in the blastocyst-recipient, such as described in the examples. In this respect, animals mutant for PS1, according to the invention obtained by electroporation of expression cassette comprising the nucleic acid. Preferably, this nucleic acid is a DNA, which can be genomic DNA (gdnc) or complementary DNA (cDNA).
Modification of the genome may follow from changes or modifications to one or some of a gene "knock-in". This modification can be caused by the action of the classical modify or mutagenic agents or can the t to be carried out by directed mutagenesis. In the present invention that relates to mutant ps1 gene, it is preferably about homologous recombination using a targeting vector carrying a pre-mutated using site-directed mutagenesis of the transgene, as described in the following examples.
Animals expressing mutant APP protein obtained by microinjection of genetic structure in the nucleus of the zygote.
Double transgenic animals produced by crossing animals mutant for ps1, and animals mutant for the APP.
Animals according to the present invention can be used preferably for the detection of neuroprotective properties of compounds intended for the treatment of neurodegenerative diseases, mainly Alzheimer's disease. These compounds can be chemical molecules, peptide or protein molecules, antibodies, chimeric molecules, as well as antimuslim RNA or ribozymes. Identified compounds can be used as medicines, tel Quel or in combination with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition. They can be, in particular, saline solution (phosphate monolatry, disodium, sodium chloride, potassium, calcium or magnesium, etc. or mixtures of such salts), sterile, isotonic or dry compositions, castnet is, liofilizovannye that you add, depending on the circumstances, sterilized water or physiological serum allow to obtain solutions for injection. Injections can be performed stereotactic, topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. by.
Another object of the invention relates to such method of identifying compounds for the treatment of neurodegenerative diseases, comprising at least the following stages:
- the introduction of the test compound or mixture of compounds to animals according to the present invention, and
- observation of the evolution of one or several characteristic markers, reproducing neuropathology observed in humans.
Another object of the invention relates to a method of identifying compounds for the treatment of neurodegenerative diseases, comprising at least the following stages:
the bringing into contact of cells derived from animals according to the present invention, with the compound or mixture of compounds, and
- measurement of the effect or effects of the compounds on the level of whole cells, cellular homogenizate or subcellular fractions.
Another object of the invention applies to any biological product, originating from about the tion, two animals according to the invention and their use for identifying compounds for the treatment of neurodegenerative diseases, preferably Alzheimer's disease. Under "biological product" is understood, in particular, cells, protein extracts, DNA, RNA, or antibodies.
Thus, an object of the present invention are cells or cell line, derived from an animal, such as described above, in particular embryonic stem cells.
The object of the present invention, furthermore, is a protein PS1 mice carrying mutations of amino acids M in T and L in P, respectively, at positions 233 and 235. Preferably such protein comprises the sequence of SEQ ID NO:2. Preferably, it has a sequence of SEQ ID NO:3.
Another object of the present invention is a nucleic acid encoding a protein PS1 mice carrying mutations of amino acids M in T and L in P, respectively, at positions 233 and 235.
Preferably, such nucleic acid according to the formula of the invention includes the sequence of SEQ ID NO:1 or the sequence SEQ ID NO:8.
The object of the present invention, moreover, are sequences complementary to these nucleic acids and vectors comprising these nucleic acid or the complementary sequence them.
Another aspect of the invention concerns the application of these proteins to identify neuroprotective properties of the compounds, designed for the treatment of neurodegenerative diseases.
The present invention is illustrated by the following examples, however it is not restricted to these examples.
In these examples describe the results show the advantage of PS1KI mice and confirm the preferred model PS1KIxAPP in therapeutic strategies, since its advantage is that it is capable of voproizvodit ' the main features of the currently known neurodegenerative diseases.
Description of figures
Figa: Schematic representation of the structure of the gene ps1 mouse and fundamental restriction enzymes cut sites around exon 7 wild-type (upper line) and used the targeting vector (middle line). Replacement of nucleotide bases to obtain mutations of codons MT and L235P in exon 7 (*) is represented in a dotted frame. Mutant allele PS1KI containing the cartridge resistance to neomycin (Neo), depicted on the bottom line. Also indicates the position of the probe 230 base pairs, used to identify newborns.
Figw: southern blotting using a probe 230 base pairs to distinguish alleles WT wild-type (lane 9,2 base pairs) from heterozygous PS1KI (He, dual band) and homozygous (But the band 7,4 base pairs) from different mice.
Figs: Immunoparesis C-terminal fragm the NTA PS1, showing that the levels of expression of PS1 protein is not degraded by the presence of mutations in allele PS1KI.
Figa, 2B and 2C: Determine the total number of Aβ42 and relationship total Aβ/Aβ42, at the age of 2.5, 4, 6 and 10 months.
Figure 3: Accelerating the deposition of Aβ peptide in mice APP751SLPS1KI Ho. Indicative picture of the regional distribution of extracellular deposits of Aβ peptide in the brain in 6 months. The images illustrate immunoreactive Aβ (Ac 4G8) 3 APP751SL mice (figa, 3B, 3C) and 3 mice APP751SLxPS1KI Ho (fig.3D, 3E, 3F). Monomachine reveals the emergence at the age of 6 months the first rare deposits in the cortex (Cx) and hippocampi (Hp) APP751SL mice. For comparison, mice APP751SLxPS1KI Ho of the same age, the number of deposits in these areas is significantly increased. It should be noted that these mice deposits present in significant quantities in the thalamus (T).
Figure 4: Progression with age of deposition of the Aβ peptide. Indicative picture of the regional distribution of Aβ deposits in the brain at the age of 10 months. Image correspond to the 2 mice APP751 SL (figa, 4B) and 2 mice APP751SLxPS1KI Ho (figs, 4D). APP751SL mice monomachine reveals a significant increase in the number and size of deposits in the cortex (Cx) and hippocampi (Hp) at the age of 10 months compared to 6 months of age and the appearance of the first deposits in the thalamus (see figure 3). The density and size of the sediments also Bo is significant at 10 months in the cortex, in hippocampi and thalamus of mice APP751SLxPS1KI Ho. It should be noted that in these mice, a small number of deposits can be detected in the striatum (St).
Figure 5: the Process of neuronal death in CA1 in mouse APP751SLxPS1KI Ho. Illustrative image of a lesion of the pyramidal neurons in hippocampe mice APP751SLxPS1KI Ho at the age of 10 months. Images are staining purple Crésyl, at low magnification in hippocampe, 2 PS1KI mice Ho (figa, 5B), 2 APP751SL mice (figs, 5D) and 2 mice APP751SLxPS1KI Ho (file, 5F). The density and thickness of the layer of pyramidal cells in hippocampe qualitatively comparable in mice and APP751SL mice PS1KI Ho at the age of 10 months. On the contrary, at the same age, they are clearly reduced in mice APP751SLxPS1KI Ho, especially in layer 1 Corne d Ammon (CA1). It should be noted that the number of small cells stained blue (glial cell type) increased in hippocampi mice APP751SLxPS1KI Ho.
6: the Process of neuronal death in CA1 in mice APP751SLxPS1KI Ho. Illustrative picture neural lesions in CA1 at the age of 10 months using other neural markers, methyl green and monomachine BIP. Images represent staining with methyl green, a strong increase in CA1, not mutant mouse (figa), mouse PKS1KI Ho (pigv), APP751SL mice (figs) and mouse APP751SLxPS1KI Ho (fig.6D). They are immunoreactive BIP in a strong increase in CA1 in PS1KI mouse Ho (fige) and mouse APP751SLxPS1KI Ho (fig.6F). Along with the avanyu with necroshine mice, PS1KI Ho and APP751SL, the number of neurons stained with methyl green, markedly reduced in area CA1 of the mouse APP751SLxPS1KI Ho. It should be noted the presence of stained cells glial types in large quantities in the parenchyma of the hippocampus in this double transgenic mouse. Monomachine BIP also confirms a significant loss of pyramidal neurons in CA1 in mouse APP751SLxPS1KI Ho at the age of 10 months.
Fig.7: the Death of neurons in CA1 and intracellular deposition of Aβ peptide. Revealing picture of both pathological processes, neural lesions and abnormal vnutricerepnae accumulation of Aβ peptide in the age of 10 months. Images are in a strong increase in CA1, immunoreactive Aβ 2 mice APP751 (figa, 7B) and 2 mice APP751SLPS1KI Ho (file, 7F). They represent, in a strong increase in CA1, staining purple Crésyl the mouse APP751 (figs, 7D), mouse APP751SLPS1KI Ho (fig.7G, 7H) and 2 PS1KI mice Ho (Fig, 7J). At the age of 10 months as simple APP751SL mice, and double APP751SLxPS1KI Ho, extracellular deposits of Aβ observed in the majority on one side or another layer of neurons in CA1. On the contrary, in CA1 (characterized neural lesions, expressed along the entire length of the layer of the mouse APP751SLxPS1KI Ho (figs, 7D), immunoreactive Aβ granular form (corresponding to the abnormal vnutriryadnoi accumulation of Aβ peptide, see arrows) more intensively in mice APP751SLxPS1KI Ho. This is true so the e at 6 months of age (see Fig).
Fig: Early start the process of neuronal death in CA1 at APP751SLxPS1KI Ho. Illustrative image area CA1 of the hippocampus at the age of 6 months. Images are in a strong increase in CA1, immunoreactive Aβ from 3 mice APP751 (figa, 8B, 8C) and 3 mice APP751SLPS1KI Ho (fig.8G, 8H, 8I). They represent staining purple Crésyl in mice APP751 (fig.8D, 8E, 8F) and mice APP751SLxPS1KI HO (fig.8J, 8K, 8L). In 6 months the CA1 region of the hippocampus in mice APP751SLxPS1KI Ho already characterized by a significant number of extracellular deposits of Aβ (Fig), intense intracellular granular tagging of Aβ (see arrows) and loss of neurons, painted purple Crésyl with a concomitant increase in the number of glial cells type (fig.8L). 2 other mice APP751SLxPS1KI Ho layer of CA1 neurons, painted purple Crésyl weak (fig.8J not disorganized (FIGC). It should be noted that for these two mice intracellular immunoreactive Aβ seems to be less intense and more diffuse (fig.8G, 8H)than the 3rd mouse (Fig).
Construction of the targeting vector
carrying mutations M233T and L235P
The aim was to introduce two mutations in exon 7 of the gene PS1 mouse replacement residues M233 on T and L235 to P. Both new codon correspond to the mutations identified in patients with Alzheimer's disease with early onset (FAD).
The mouse line PS1 knock-in (PS1KI) received IP is by using a two-stage strategy of mutagenesis, similar to that described in Kwok et al. (1997, Neuroreport 8; 157-42) and Champion et al. (1996, Neuroreport 7, 1582-4).
The strategy was to design a targeting vector carrying a replacement nucleic acid codons 233-235 geneps1 mouse (see figa).
Briefly, genomic fragment 17 base pairs gene PS1 mice was isolated by screening the Bank genomic DNA mouse 129SvJ, constructed in bacteriophage lambda (Stratagene, catalog number 946313). Analysis by cleavage with restriction enzymes, sequencing and comparison with the available partial sequences of the mouse PS1 gene (Mitsuda et al. 1997, JBC 272, 23489-97) showed that this fragment contains the region from intron 5 to exon 11 of the gene PS1 mouse.
SubfragmentBamHI-HindIII (9.8 thousand base pairs)containing part of intron 5, exon 6, intron 6, exon 7, and part of intron 7 was subcloned into the plasmid pGEM-llZf (+) (Promega, France) (figa). Mutagenesis 2 codons was performed using the kit Editor Gene (Promega) DNA fragment containing exon 7, and confirmed by nucleotide sequencing.
Long arm (5') of the homologous recombination vector was obtained by cloning the fragmentBamHI-XbaI (7 thousand base pairs)containing exon 6. Short one shoulder (3') was obtained by sublimemovies fragmentXbaI-EcoRI (1.8 thousand base pairs)containing mutagenically exon 7. The positive selection cassette (pMCI-Ne cassette) was introduced in the site XbaI, located in intron 6 in position - 470 base pairs at the 5'end of exon 7 (see figa).
Obtaining cells containing PS1KI
Targeting vector described in example 1, linearizability splitting withNotI and electroporative in a line of embryonic stem cells (ES) CK35 provided by Dr. Charles Babinet, Institute Pasteur, Paris, France.
Cells were cultured as described previously (W. Wurtz and A. Joyner, Gene Targeting: A Practical Approach by Alexandra L. Joyner (Editor). Oxford University Press; 2nd edition (February 15, 2000)).
430 cell clones capable of bearing homologous recombination, were selected in the presence of G418. Genomic DNA of these clones was analyzed by southern-blotting, as described previously (Sambrook, Fritsch and Maniatis, Molecular Cloning; A Laboratory Manual, Cold String Harbor Laboratory Press,2ndedition, 1989) using a probe PS1 located outside the region recombination (figa) long arm. Four cell clone carrying a targeted mutation in the PS1 gene, were thus identified. These cell clones were used to produce transgenic lines PS1KI mice.
Design line PS1KI mice
Clone 18C5 were injected with blastocyte mice C57B1/6.
Five of the obtained chimeric mice showed transmission of the mutant alleleps1 the germ line (and, thus, their offspring).
Based on these founders, received the iniu PS1KI mice with a pure 129SV genetic Fund and mixed Fund 129SV-C57B1/6.
The presence of the mutant allele in PS1KI heterozygous (He) or homozygous (Ho) state was determined by southern-blotting using a probeps1 230 base pairs (pigv). These mutant mice are viable and fertile.
The determination of the number of PS1 in line PS1KI
After euthanizing the mice brain were collected and weighed. One hemisphere was saved for immunohistochemistry (post-fixation), and the other was frozen and then homogenized individually on ice using a Potter in 2 ml of buffer solution of 0.32 M sucrose, Tris-HCl, 4 mm, pH 7,4 containing a cocktail of protease inhibitors (CompleteTMRoche Diagnostics). The concentration of protein was determined by BCA method (Pierce). The homogenate was stored at -80°C.
To detect PS1 25 µg of protein extract marrow were incubated at 56°C for 20 min in the Deposit Laemmli buffer containing 8M urea and dithiothreitol 50 mm. Proteins were fractionally electrophoresis on the gel NuPAGE 4-12% Bis-Tris polyacrylamide (SDS-PAGE) in MES buffer (2-(N-morpholino)econsultancy acid). After transfer of proteins to nitrocellulose filter (Amersham, France) the filter was heated in PBS for 5 min to increase the sensitivity and immediately became saturated with 5% (wt./about.) powdered skim milk in PBST buffer (PBS, 0.05% of (about./about.) Tween 20)for 1 h and incubated overnight at 4°C with primary antibody in a single puff is re PBST. Binding of antibodies was detected with the antibody anti-IgG (anti-mouse)conjugated to horseradish peroxidase (Amersham, France) at 1/10000 dilution in PBST, followed by use of a detection system by chemiluminescence (Amersham, France) according to manufacturer's instructions. To detect PS1 primary antibody MAB1563 (Chemicon, USA) was used at a dilution of 1/10000. For semiquantitative analysis, fluorescent signals were digitized using a CCD camera GeneGnome 16 bits (Syngene, Cambridge, UK) and analyzed with the software Genetools (Syngene). The linearity of the signal was checked with a standard curve established with samples from 2.5 to 10 µg of homogenate on the strip.
This analysis immunoparesis allowed to determine that the levels of expression of C-terminal fragment of mutant PS1 remained normal and not reduced in mouse PS1KI233/235 (figs).
The receiving line PS1KIxAPP crossing lines PS1KI and APP
PS1KI mouse (described in examples 1-4) were crossed with a transgenic line of mice, superexpression human form cDNA APP751bearing mutations FAD Swedish (mutation K670N; M671L) and London (V717I) under the control of the promoter of Thy-1. Mouse superexpression human form cDNA APP751bearing mutations were obtained as described in patent application WO 01/20977.
In all the following experiments were used mouse is the same genetic Fund to minimize the effects associated with the changes in the gene pool.
Determination of total amyloid peptide β and β42 method immunoelectrophoresis
To determine the number of General population β in the brain (soluble form and aggregated or insoluble forms) aliquots of brain homogenate was treated with 2 volumes of a solution of 9M hydrochlorate guanidine (GH) in 50 mm Tris pH7,4. The homogenates were stirred for 1 h with 3 periods of treatment with ultrasound for 15 min followed by centrifugation at 50000×g at 4°C for 2 hours guanidine Extracts were diluted to the extent 1/20 in 20 mm buffer Tris-HCl, pH to 7.6, 150 mm NaCl, 0,5% BSA (wt/about.) and 0.05% Tween 20 (weight/vol.). The concentration of Aβ peptide in the fractions was determined using immunoelectrophoresis (Yang et al., 1994, Biotechnology (NY) 12(2), 193-194)) using 2 monoclonal antibodies antipeptide Aβ (4G8 and 6E10) and the reader Origen M8 Analyzer (IGEN Europe Inc. Oxford) according to the recipe, modified by Khorkova et al. (J. Neurosci. Methods 82, 159-166 (1998)).
Monoclonal antibody 4G8 (Senetek PLC)recognizes an epitope of residues 17-24 of Aβ peptide, was retailiatory complex air TAG-NHS according to the recipe vendor (IGEN Europe Inc., Oxford). EN-4G8 and biotinylated antibody 6E10, epitope 1-10 of Aβ peptide (Senetek PLC) was administered in the presence of soluble brain fraction and determined the amount of three complexes of Ru-4G8β/6E10-biot using reader Origen. Range of synthetic Aβ peptide (Bachem) was used for calibration of each experience. The amount of Aβ peptide was calculated in nanograms per gram of initial weight of brain tissue.
To measure specific forms of Aβ peptide ending at position 42 (Aβ42), antibody 6E10 was replaced by a monoclonal antibody 22F9, which is recorded specifically for the C-end of Aβ42 (Wirths et al., 2002, Brain Pathol. 12, 275-286).
In conclusion, the presence of a gene ps1 knock-in (PS1-KI) leads to
- to accelerate the accumulation of Aβ (figa) and Aβ42 (pigv) in the brain with the effect more pronounced when the allele PS1KI is present in the homozygous state (the effect of gene dosage). The effect PS1KI (Ho) is stronger than in the case of transgenic mice, superexpression PS1M146L, previously described in the application WO 01/20977;
- massive increase in the proportion of Aβ peptide having the end β42, which represents the vast majority of Aβ, when the mutation PS1KI is present in the homozygous state, as shown figs (the ratio of Aβ42/total Aβ is 0.92, at the age of 2.5 months, against 0.25 in the absence of PS1KI and the intermediate value of 0.70 in the presence of a single allele PS1KI: the effect of gene dosage). In the literature it is believed that the species of Aβ peptide-terminated end β42, represent the most pathological forms of the peptide. Line PS1KIxAPP is thus a model, especially enriched pathological f is Mami.
Analysis of the deposits of Aβ peptide using immunohistochemistry
For testing immunohistochemistry/histology after selection and then postfixation in paraformaldehyde (4%) hemisphere of the brain has been cryoprotective for 1 night at 4°C in a buffer of sodium phosphate and 0.2 M (NaH2RHO4, 2H2O/Na2HPO412H2Oh, a pH of 7.4)containing sucrose 20% (wt./vol.). Then they were frozen for 1 min in isopentane having a temperature of -30°C in dry ice. Slices with a thickness of 25 μm, obtained in the cryostat, cooled at -30°C (LEICA CM3000), placed ultimately in PBS buffer 0,02M, then maintained at 4°C.
These sections carry out the immunoassay detection of Aβ peptide with the help of a detection system, with the formation of complexes of avidin-Biotin-peroxidase (ABC), in which horseradish peroxidase combined with Avidya, biotinylation. Briefly, after incubation for 30 min in buffer lock (normal goat serum (Chemicon) in 10% PBS containing 0.1% Triton (Sigma), the slices are placed in the presence of a solution of H2About20,3% in order to remove endoperoxides present in the tissue. Then these slices are incubated in a solution of primary antibody containing 0.3% Triton and 2% normal serum (overnight at 4°C). Used primary anti-Aβ antibody (4G8, Senetek) (monoclonal antibody, napravlennoe residues 17-24 of Aβ peptide) biotinylating. After washing, the slices are placed directly in the presence of complex ABC, within 1 hour according to the manufacturer's instructions (ABC Kit Vectastin, Vector Laboratories, Burlingame, CA). 3-3'-Diaminobenzidine was used as the Chromogen for peroxidase enzyme.
Thus, the acceleration of the abnormal accumulation of Aβ peptide in the brain of double transgenic mice APP751SLxPS1KI Ho found earlier biochemical tests in homogenates of cerebral hemispheres, was confirmed by immunohistochemistry. Indeed, microscopic analysis of immunodeciency Aβ obtained on sections of the cerebral hemispheres, proved the existence of the accelerated deposition of Aβ peptide in the parenchyma of the brain of these mice. Indeed, while the first deposits appear in the bark and in hippocampe to 6 months of age APP751SL mice (figure 3), they can be detected from the age of 2 months at double APP751SLxPS1KI in the homozygous state. Compared to the simple transgenic APP751SL, double transgenic (APP751SLxPS1KI Ho) at the age of 6 months, the density of Aβ deposits significantly more in hippocampi and bark. In addition, the distribution of sediments is broader, in particular, deposits are already found in the thalamus and in the bridge (figure 3).
With age, particularly in the age of 10 months, density, and size of deposits increased in the brain of transgenic simple APP751SL mice (figure 4. The distribution of these sediments also wider, as they are present in the thalamus. The double transgenic APP751SLxPS1KI Ho at the age of 10 months is similar to the progression of the deposition of Aβ peptide is observed in hippocampi, the cortex, the thalamus and the bridge. First deposits can be found in limited quantities in the striatum (figure 4). In contrast, the cerebellum is not affected by the deposition of Aβ. It should be noted that in the brain of mice PS1KI Ho at the age of 10 months (n=4) no deposits of Aβ peptide not detected.
Analysis of neuronal death using histology and immunohistochemistry
The presence of very significant proportions of pathological peptide Aβ42 resulted in the analysis develops with age in line APP751SLxPS1KI Ho, besides accelerating the deposition of the Aβ peptide, the death of neurons. This was carried out 3 types of stains, allowing to visualize the disappearance of neural cells in slices of brain tissue: a) histology in purple crésyl, which stains the body Nissl (organelles in the cytoplasm, associates with ribosomes rough endoplasmic reticulum) and allows you to discover on sections of brain neuronal and glial cells; (b) histology in methyl green, which stains the DNA of all cells; (c) immunohistochemistry in BIP, which finds expression in cells shapero the nogo protein to the endoplasmic reticulum.
For dyeing purple crésyl slices of brain tissue was placed on gilotinirovaniya plates, then incubated for 10 minutes in a solution of purple crésyl (C 1791, Sigma) 0.5% in distilled water. After washing in acidic environment the sections were obezvozhivani.
For staining with methyl green slices placed on gilotinirovaniya plates, then incubated for 10 minutes in a solution of methyl green (M5015 Sigma) 1% in distilled water, washed, then obezvozhivani.
For immunohistochemistry BIP (polyclonal antibody, SPA-826, Stressgen), a Protocol identical to those used for immunohistochemistry peptide Aβ (see above), excluding additional incubation (1 h at ambient temperature) slices in a solution of biotinylated secondary antibodies (antibody anti-rabbit IgG, obtained from goats, Vector) before incubation in ABC complex.
Microscopic analysis revealed through the use of different histological/immunohistochemical markers reducing the thickness of the pyramidal cells of the hippocampus, including CA1, in the brain of mice APP751SLxPS1KI Ho (n=3/3) (figure 5 and 6). This decrease indicates the loss of the neurons that are already taking place at the age of 10 months. In 6 months the death of neurons in the brain 1/3 mice, suggesting an early start neurotoxic process (Fig). Analysis of parallel hippocampe and, particularly in CA1, 2 pathological processes, abnormal accumulation of Aβ peptide in the brain and neural lesions, suggests a more likely role in the neurotoxic process, intracellular accumulation of Aβ (a phenomenon already described in mice thy1APP751SLxPS1 M146L) compared to its accumulation in the form of extracellular deposits (Fig.7). Indeed, the neurons still present in CA1, there is an abnormally high expression of the Aβ peptide. In addition, neural lesions in CA1 clearly observed in areas deprived of extracellular deposits. It should be noted the existence of probable effect of gene dosage in the process of neuronal death in CA1. Neural defeat was indeed detected in mice APP751SLxPS1KI in a very great age (>15 months), with only one allele PS1KI.
1. Animal, not a human, with a sequence of nucleic acid encoding presenilin 1 carrying mutations corresponding to the mutations MT and L235P in the protein PS1 mice, as well as the sequence of the nucleic acid encoding the entire gene or a portion of the gene encoding the RDA, characterized in that the protein RDA is ARR, comes from the person, and bears a mutation Swedish and London, and the specified animal intended to be used to combat Alzheimer's disease.
2. Animal according to claim 1, characterized in that the mutation or mutations are located in the GOM the zygote state.
3. Animal according to claim 1, characterized in that it expresses the number of mutant PS1, comparable to the number of endogenous PS1 animal expressing nematanthus PS1.
4. Animal according to claim 1, characterized in that the protein presenilin 1 carrying mutations MT and L235P, comes from the mouse.
5. Animal according to claim 1, characterized in that it is a rodent.
6. Animal according to claim 5, characterized in that it is a mouse, rat or rabbit.
7. Animal according to claim 1, characterized in that it produces a protein comprising the sequence of SEQ ID NO:2.
8. Animal according to claim 1, characterized in that it has in its genome the nucleic acid sequence SEQ ID NO:1.
9. Animal according to claim 1, characterized in that the expression of the gene encoding the RDA, is under the control of an exogenous promoter.
10. Animal according to claim 1, characterized in that it is observed the death of neurons.
11. Animal according to claim 1, characterized in that it has Abeta/Abete total more than ~ 0.9.
12. Animal according to claim 1, characterized in that the mutant protein presenilin 1 is endogenous.
13. The cell or cell line, obtained from the animal according to one of claims 1 to 12, designed to be used to combat Alzheimer's disease.
14. The cell or cell line, containing a nucleic acid sequence encoding a mouse is th gene presenilin 1 in mutant form with mutations MT and L235P, moreover, the specified cell or cell line is intended for use to combat Alzheimer's disease.
15. Embryonic stem cell obtained from an animal according to one of claims 1 to 12, designed to be used to combat Alzheimer's disease.
16. Protein PS1 mice carrying mutations in the form of replacement of amino acids M on T and L on R, respectively, at positions 233 and 235, characterized in that it comprises the sequence of SEQ ID NO:2.
17. Nucleic acid encoding a protein PS1 mice carrying mutations in the form of replacement of amino acids M on T and L on R, respectively, at positions 233 and 235, characterized in that it comprises the sequence of SEQ ID NO:1.
18. The vector comprising the nucleic acid according 17 to be used to combat Alzheimer's disease.
19. The use of animal in one of claims 1 to 12 for identifying compounds for the treatment of Alzheimer's disease.
20. The use of protein P16 to identify compounds for the treatment of Alzheimer's disease.
SUBSTANCE: method involves sampling with further sample degassing. Further, if luminescent sea bacterium Photobacterium phosphoreum B-17 677f is added to test and reference samples, then sample mineralisation level is defined and NaCl is added to the test sample to reach total mineralisation level of 30 g/l. Reference samples are prepared. Further, 0.01 solution of NaCl is added to test and reference samples to reach pH 7.5. Then luminescent sea bacteria Photobacterium phosphoreum B - 17 677f or recombinant luminescent bacteria Esherichia coli K12 TGI with luminescent system genes of Photobacterium leognathi are added to test and reference samples. Further the luminescence level is measure, and toxicity index is calculated by the formula T=((Ir-It)/Ir)*100%, where Ir is luminescence intensity in reference sample, It is luminescence intensity in test sample. With T<20% water is assessed as non-toxic, with T within 20 to 50% water is considered toxic, and with T>50% it is considered highly toxic.
EFFECT: improved accuracy of method.
2 tbl, 1 ex
SUBSTANCE: method involves application of diphtherial antitoxin to nutrient medium. Further the tested strain culture is seeded after the bacteria culture processing with low-intensity laser radiation (LILR) of λ=650-670 nm wavelength and 2.5-3.5 mW/cm2 radiation intensity at bacteria colony surface for 4.5-5.5 minutes.
EFFECT: enhanced method efficiency.
1 tbl, 1 ex
SUBSTANCE: invention relates to quality evaluation of products of animate and inanimate nature, namely to biological quality evaluation of food products for a man, animal feeding staff, food supplements and other substances contacting human organism, natural and waste water, fisheries water, soil and ground. Besides, invention can be used to evaluate degree of different illness severity of a man and animals. Device for evaluation of products of animate and inanimate nature consists of biodetector contained in dust- and drip-proof housing and computer with installed controlling software. Biodector contains board with two rows of holes for placement of materials under examination and test objects, table with possibility of step-by-step motion; video system interfaced with computer for transfer of images of holes for materials under examination and test objects to monitor and RAM and consecutive calculation of toxicity degree for material under examination; lighting installation with two highlight bars. Video system includes two small-size uncased video cameras with microscope objectives having variable focal distance; step-by-step motion node of the table includes microcontroller connected with step motor and computer serial port. Device is also equipped with three-colour status indicator of biodetector. Quality evaluation of different objects and severity degree of illnesses is carried out based on comparison of multiple calculations of animated test objects in samples within set period of time by means of special computer program. Device allows simultaneous evaluating of large number of different objects and identifying time history of test objects number in one experiment.
EFFECT: device is simple and reliable to operate; it does not require start-up operations and high qualification of an operator.
SUBSTANCE: mixed suspensions of microbes and erythrocytes are incubated at 37-39°C on a shake-flask propagator during 30 mines, centrifuged to erythrocyte sedimentation and with using photoelectrocolorimeter tested for optical density of the supernatant fluid. bacteria fixing activity of erythrocytes - "БФАЭ" (%) is derived from the difference between DC (optical density of the supernatant fluid in control system) and total DT (optical density of the supernatant fluid in test system) and DT* (optical density of the supernatant fluid in the system containing erythrocyte suspension and physiological solution of sodium chloride instead of microbial suspension and being in the same environment) divided by DC and multiplied by 100 %.
EFFECT: application of the method allows for erythrocyte analysis for their ability to fix on their surface various microorganisms, including agents of bacterial infections and makes it possible to study adhesive properties of microbes.
SUBSTANCE: present invention relates to biotechnology and genetic engineering. Proposed here is a set of lux-biosensors, consisting of Escherichia coli cells obtained using recombinant DNA technology, containing plasmids with bacterial luxCDABE genes controlled by stress inducible promoters PgpoE and PgrpE and non-inducible Plac promoter.
EFFECT: invention can be used in evaluating degree of contamination of the environment by hydrophobic detergents.
SUBSTANCE: claimed is system of rating, including 5 criterions. If three criterions are positive, said gland is suitable source of therapeutically applicable islets.
EFFECT: increase of qualitative and quantitative output of islets.
13 cl, 5 ex, 2 tbl, 1 dwg
FIELD: chemistry, biochemistry.
SUBSTANCE: invention refers to medical microbiology. Method provides as follows. Analysed microorganism culture is grown in liquid nutrient medium. Then supernatant fluid is separated from microbial cells by filtration through membranes of pore size 0.2-0.45 mcm. Then antimicrobial thrombocyte protein (β-lysine) is added to supernatant fluid. It is combined with preparing two check samples: antimicrobial thrombocyte protein (β-lysine) is added to check sample 1; while physical solution instead of antimicrobial protein is added to check sample 2. Test and check samples are incubated that is followed with adding test culture Bacillus subtilis SRDE No.010011 to all the samples then incubated once more. Thereafter test and check samples are centrifuged. Deposited cells of test culture are re-suspended in 0.75 M saccharose solution and incubated. Then test and check samples are measured for optical densities. Change in test culture growth in test sample compared to that in check sample indicate producing inhibitors of antimicrobial thrombocyte protein (β-lysine) by microorganisms.
EFFECT: higher accuracy of microorganism analysis for ability to produce inhibitors of antimicrobial thrombocyte protein (β-lysine).
2 cl, 2 tbl, 2 ex
SUBSTANCE: invention is related to the field of medicine, namely to medical toxicology, microbiology, and is related to method for assessment of clinical significance of clostridia in conditions of polymicrobial infection. Method consists in the fact that standard amount of daily clostridia culture is incubated in conditions of anaerostat in SKS medium together with monolayer of human embryo skin fibroblasts, and after triple washing of monolayer cells by Eagle's medium, method of microscopy is used to assess availability and effect of toxin according to extent of monolayer damage in compliance with USA Pharmacopoeia scale.
EFFECT: higher accuracy of detection of clostridial toxins and determination of their effect.
FIELD: physics, measurement.
SUBSTANCE: invention relates to quality control equipment for animate and inanimate nature products and can be used to evaluate safety of food and feed products, natural and effluent waters, grounds, soil as well as to develop maximum permissible concentration values for pollutants including products of oil production and refining etc. Device for evaluating quality of animate and inanimate nature products consists of a computer with bundled software and a biodetector comprising a motion controller of plate 1 with containers for tested products, light source 2, optical system 3 with telecamera 4 which is connected to the respective computer input by an image acquisition videocard. Containers for tested products are made as truncated cones and are set on the plate in two rows; the containers for tested products in the outer row are by 2.5 times bigger in volume than the containers 6 in the inner row. The light source - light emitting diode 2 - is set under the measured plate container and fitted by transparent cap 7 in the form a truncated cone with the smaller base at the container side. Electronic control circuit of movements in the device is mounted in a protective housing, fed from the computer and involves higher speed USB port operation.
EFFECT: increasing measurement accuracy.
FIELD: chemistry, biochemistry.
SUBSTANCE: offered invention refers to biotechnology and can be used for ecological toxic analysis and aquatic ecosystem monitoring. Strain Vibrio fischeri "ВКПМ" B-9579 highly sensitive to multichemical toxic substances can be used in toxicity testing of bottom sediments.
EFFECT: higher sensitivity of test-cultures to multichemical toxic substances.
4 tbl, 4 ex
SUBSTANCE: vitamin K dependent protein is made by separating a cultivated eukaryotic cell that contains an expressing vector that contains a nucleic acid molecule coding vitamin K dependent protein and associated sequences regulating expression. The associated sequences contain the first promoter and the nucleic acid molecule coding gamma-glutamylcarboxylase, and the second promoter. The first promoter represents a pre-early promoter of human cytomegalovirus (hCMV), and the second promoter is a pre-early promoter SV40. Herewith the expressing relation of vitamin K dependent protein and gamma-glutamylcarboxylase is 10:1 to 250:1.
EFFECT: invention allows for making gamma-carboxylated vitamin K dependent protein in production quantities.
29 cl, 5 dwg, 6 tbl, 7 ex
SUBSTANCE: invention claims synthetic DNS molecule encoding L1 HPV58 protein, where codons are optimised for high expression level in yeast cell. Also invention claims expression vector, yeast host cell, HPV58 virus-like particle and method of its obtainment, and pharmaceutical composition including such VLP.
EFFECT: invention can be applied in medicine for efficient immune prevention of papillomavirus infection by neutralising antibodies and cell-mediated immunity, and for treatment of developed HPV infections.
10 cl, 10 dwg, 8 ex
SUBSTANCE: dendritic cells presenting hepatitis B antigen are separated from peripheral blood of patients suffering from chronic hepatitis B virus and cultivated nonadherent fraction of mononuclear cells with proinflammatory cytokine "рчИЛ"-18 added.
EFFECT: invention allows generating cells with high cytotoxic potential with respect to the hepatitis B infected cells.
SUBSTANCE: invention relates to field of biotechnology, namely, to obtaining genetically modified cell lines and can be applied in medicine for immunotherapy and immuno-prophylaxis in patients with malignant neoplasms. By means of recombinant method line of cells of human melanoma KG is obtained, which secretes recombinant granulocytic-macrofagal colony-stimulating human factor. Obtained line is deposited with Specialised cell culture collection of vertebrates of Russian cell culture collection under number RCCC ("П") 699"Д".
EFFECT: line of human melanoma cells KG possesses stable cultural, morphological and immunological characteristics and possesses ability to secrete recombinant human GM-CSF, remaining after cell inactivation with ionising irradiation.
SUBSTANCE: invention concerns virology and medicine area. The synthetic DNA-molecule coding protein L1 HPV45 is presented. Thus DNA-molecule was Codonum-optimised for high-level protein expression in a yeast cell. The given synthetic molecules can be used for reception of virus-like particles (VLP) HPV45 and for reception of vaccines and the pharmaceutical compositions containing VLP-particles HPV45.
EFFECT: effective immunologic prophylaxis of infections with papilloma virus due to neutralised antibodies and cellular immunity.
9 cl, 6 dwg, 8 ex
FIELD: biology, genetic engineering.
SUBSTANCE: invention concerns molecular biology and genetic engineering. Invention claims synthetic DNA molecule encoding HPV31 L1 protein, virus-like HPV31 particles, vaccine and pharmaceutical composition including virus-like HPV31 particles.
EFFECT: papilloma virus therapy.
17 cl, 6 dwg, 8 ex
SUBSTANCE: present invention relates to biotechnology and immunology. Proposed here is a polynucleotide, encoding a cyclic single-stranded tri-specific antibody. The antibody is directed against human ovarian carcinoma in vitro, has mass of approximately 84 kD and consists of three components: an antibody against human ovarian carcinoma cells, anti-CD3 antibody and anti-CD28 antibody, which are joined together by peptide interlinks such that, they form a cyclic antibody. Invented is an expression vector, containing a coding polynucleotide and versions of E.coli host cell based on the polynucleotide and expression vector.
EFFECT: use of the invention provides for a stable antibody molecule, optimum for activation of T-cells, which can be used in curing human ovarian carcinoma.
8 cl, 12 dwg
FIELD: chemistry, biochemistry.
SUBSTANCE: invention refers to molecular biology, virology and genetic engineering. There are disclosed method of producing recombinant influenza virus with mutant gene NB of membrane protein, recombinant influenza virus produced by this method, and vector composition based on parts of recombinant influenza virus.
EFFECT: developed method of recombinant influenza virus with mutant gene NB of membrane protein.
24 cl, 8 dwg
FIELD: chemistry, biochemistry.
SUBSTANCE: invention refers to biotechnology, specifically, to cell differentiation and can be used in medicine. Differentiated stromal cell is made of adipose tissue. This cell expresses one or more proteins, characteristic for endocrine pancreas cell, such as insulin, glucagon, somatostatin and pancreatic polypeptide.
EFFECT: production of differentiated and functionally relevant cell for researches, implantation, transplantation and development of tissue products for treating pancreas diseases and repairing pancreas tissues.
24 cl, 8 ex
FIELD: pharmacology; biotechnology.
SUBSTANCE: invention concerns biotechnology area, in particular to the gene-engineering way providing mass production multimeasured recombinant human Mannan-Binding Lectin (MBL), also can be used in the biomedical industry. The offered way includes a) a cultivation stage of cells-owners lines CHO, transfectant with a recombinant vector pMSG-MBL, containing sequence of human MBL coding area, and b) a clearing stage of the recombinant protein. Thus at the first stage preferably use new cellular line CHO MBL D1-3, deposited with the Korean collection of sample cultures at number KCTC 10472BP which is cultivated in a protein-free medium with bioreactor use, and on the second selective clearing of high-molecular form MBL of medium cultivation is spent, providing separation of samples with the help anion exchange chromatography, their drawing on a column with an immobilised MBL-binding protein on it , in particular with glycosilated protein of a cover of a virus pre-S, in the presence of ions of calcium and the subsequent elution using a buffered solution with EDTA or EGTA.
EFFECT: high output of functionally active product opens possibilities of its application by working out of therapeutic agents for treatment of virus, bacteriemic or fungoid infections.
6 cl, 11 dwg, 9 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, specifically to production of versions of the Gla domain of human factor VII or human factor VIIa, and can be used in medicine. Amino acid sequence of the FVII or FVIIa version is obtained, which differs on 1 to 15 amino acid residues with amino acid sequence of the human factor VII (hFVII) or human factor VIIa (hFVIIa), in which a negatively charged amino acid residue is introduced by substitution in position 36. Obtained variants of FVII or FVIIa are used in a composition for treating mammals with diseases or disorders, where blood clotting is desirable.
EFFECT: invention allows for producing versions of FVII or FVIIa with high clotting activity and/or high activation of factor X, compared to natural form of hFVIIa.
42 cl, 3 dwg, 5 tbl, 11 ex