Immunogenic polypeptide inducing protective immune response against bacillus anthracis (variants), method for its preparing, nucleic acid encoding thereof, expression vector (variants), method and vaccine for prophylaxis of infection caused by bacillus anthracis
FIELD: biotechnology, molecular biology, microbiology, genetic engineering.
SUBSTANCE: invention relates to a method for preparing an immunogenic polypeptide inducing immune response that represents the protective response against infection with Bacillus anthracis. Proposed immunogenic polypeptide comprises from one to three domains of the full-scale Protective Antigen (PA) from B. anthracis or their variants and at least one of indicated domains represents domain 1 or domain 4 from PA or its variant. These variants of immunogenic polypeptide and full-scale PA are produced as result of expression in E. coli. Also, invention proposes a vector for expression in bacterial cells that comprises nucleic acid encoding abovementioned immunogenic polypeptide. Also, invention the developed method for prophylaxis of infection caused by B. anthracis based on administration of sufficient amount of immunogenic polypeptide. Also, invention proposes a vaccine for prophylaxis of infection caused by B. anthracis that comprises the effective amount of immunogenic polypeptide and a suitable carrier. Invention provides preparing the effective agent used for prophylaxis of infection caused by B. anthracis.
EFFECT: improved preparing method and valuable properties of polypeptide and vaccine.
22 cl, 5 dwg, 3 tbl, 6 ex
The invention relates to polypeptides that cause an immune response which is protective against infection with Bacillus anthracis, to methods for their preparation, to the recombinant Escherichia coli cells used in these ways, and used to nucleic acids and to transforming vectors.
Existing systems for the expression of Protective Antigen (PA) for vaccine systems as expression host use scarce on the protease of Bacillus subtilis. Despite the fact that such systems are suitable from the point of view of quantitative characteristics and purity of the product, they have a significant drawback. First, the regulatory system, as a rule, is alien to this host, and this may inhibit the expression. The more important point is that currently used strains of Bacillus subtilis form thermostability disputes for which you want to use a specially designed plant.
In particular, in WO 00/02522 describes the VEE replicons virus that Express the RA or some immunogenic fragments.
The bacterium E. coli is well known as an expression system for a number of human vaccines. Despite the ability of E.coli easy to ferment at very high cell densities makes this bacterium a perfect host for ekspressirovali many of the x protein, previous attempts to Express and isolate recombinant RA from cytosol E.coli was difficult because of the low yield of protein and proteolytic degradation (Singh et al., J. Biol. Chem. (1989) 264; 11099-11102, Vodkin, et al., Cell (1993) 34; 693-697 and Sharma et al., Protein Expr. purif. (1996), 7, 33-38).
Recently described approach to sverkhekspressiya RA in the cytosol of E. coli in the form of a stable soluble protein (Willhite et al., Protein and Peptide Letters, (1998), 5; 273-278). The approach applied is one of increasing the affinity tag sequence to the N-end of RA, which allows to obtain a simple allocation system cleaning.
The difficulty inherent in this system is that it requires the use of additional auxiliary process stage in order to remove the tag-label before using RA.
The codon optimization is a technique which is now well known and are used for constructing artificial genes. There is some redundancy of the genetic code, because most amino acids are encoded by more than one radonaway sequence. Different organisms preferentially use one or the other of these different codons. We should expect that as a result of optimization of codons levels of expression of individual proteins will increase.
Generally, it is desirable, except in the case of RA, when increasing the n levels of expression will lead to proteolytic degradation and/or cell toxicity. In such cases, increasing levels of expression can be counterproductive and lead to significant cellular toxicity.
However, to the surprise of applicants they found that this does not happen in the case of E. coli, and that in this system the optimization of codon leads to unexpectedly high levels of expression of recombinant RA, regardless of the presence or absence of proteolytic enzymes in this strain.
Moreover, it is clear that the expression of the protective domain of the RA does not suppress the expression in E.coli.
Was elucidated crystal structure of native RA (C. Petosa et al. Nature 385: 833-838, 1997) and it was shown that RA consists of four distinct and functionally independent domains: domain 1, is divided into 1A, amino acids 1-167, and 1b, 168-258 amino acids; domain 2, 259-487 amino acids; domain 3, 488-595 amino acids and domain 4, 596-735 amino acids.
Applicants have found that some domains, while their individual use, when used in fused proteins or in combination with each other, in all probability, have a surprisingly pronounced protective effects.
In accordance with the present invention, has created an immunogenic reagent that causes an immune response that protects against Bacillus anthracis, and the specified reagent includes one or more polypeptides, which together represent up to Tr the x domain full Protective Antigen (PA) of the Century anthracis or their variants, and at least one of these domains includes domain 1 or domain 4 of the RA or their variants.
In this case, this reagent should include a mixture of polypeptides or fused peptides, in which a separate polypeptides include one or more individual domains of RA.
In particular, this reagent contains the polypeptide(s)comprising a domain 1 or domain 4 of the RA or its variant in the form different from the full RA. In this case, the domains are presented, respectively, in full, in particular, domain 1 is presented in its entirety.
Used herein, the term "polypeptide" includes proteins and peptides.
Used here, the expression "variant" refers to sequences of amino acids that differ from the base sequence having one or several amino acids in the sequence delegated or replaced by other amino acids, but which still produce an immune response that protects against Bad 11 us anthracis. Amino acid substitutions can be considered as "conservative", if one or the other amino acid is replaced by a different amino acid, in General, with similar properties. Non-conservative substitutions are those substitutions, where amino acids are replaced by amino acids of another type. In General, the few who konservativnye replacement will be possible without altering the biological activity of the given polypeptide. The relevant options must be identical, at least 60%, preferably identical, at least 75%, and more preferably identical, at least 90% sequence RA.
The identity of the individual variant sequence RA can be defined, in particular, using the method of multiple alignment described by Lipman and Pearson (Lipman, D.J. & Pearson, W.R. (1985) Rapid and Sensitive Protein Similarity Searches, Science, vol.227, pp.1435-1441). "Optimized" percentage score must be calculated according to the algorithm of Lipman-Pearson using the following parameters:
ktup = 1, the penalty for gap = 4 and the penalty for the length of the gap = 12. Sequences for which assesses the similarity should be used as a "test sequence", that is, the base sequence for comparison, (SEQ ID NO 1), you must first be included in this algorithm.
Preferably, if the reagent according to the invention includes a polypeptide that has the sequence of domain 1 and/or domain 4 of the RA wild-type.
The most preferable implementation of the present invention includes domain 4 of PA B.ankhracis.
These domains contain the following sequences shown in Table 1.
|Domain/td>||Amino acids full RA*|
These rooms amino acids belong to the sequence that is shown Welkos et al. Gene 69 (1988) 287-300, and below, respectively, in SEQ ID No 15 (Figure 4) and 3 (Fig 3).
Domain 1 contains two regions, denoted by 1A and 1b. Region 1A includes amino acids 1-167, and the region 1b consists of amino acids 168-258. Apparently, the area 1A essential for obtaining good protective response, and the full domain name may be preferred.
In the most preferred embodiment of the present invention the combination of domains 1 and 4 or their protective areas used as immunogenic reagent, which causes the increase of the protective immune response to B.. This combination, for example in the form of chimeric (hybrid) of a peptide can be expressed using the expression system according to the invention, which are summarized below.
When using domain 1 he accordingly merges with the domain 2 sequence RA, but, apparently, preferably merges with the domain 2 and domain 3.
Such combinations and their use for the prophylaxis and therapy creates another aspect of the present invention.
ACC is respectively above domains are part of the slit, (chimeric or hybrid) protein, preferably with N-terminal glutathione-s-transferase protein (GST). Protein GST not only contributes to the purification of fused protein, but may also create adjuvant (immunostimulatory) action, probably by increasing its size.
The polypeptides according to the invention receive the traditional ways. For example, it is possible to synthesize or they can be obtained using the techniques of recombinant DNA. In particular, nucleic acids that encode these domains include expression vector used to transform the host cell. Then the traditional ways you can culturing this host cell, followed by separation of the desired polypeptide. Nucleic acids, vectors and transformed cells used in these ways, create additional aspect of the present invention.
In General, the host cell used shall be those cells that are traditionally used to obtain RA, for example Bacillus subtilis.
Applicants unexpectedly discovered that the domains separately or in combination, under certain conditions, can be successfully expressed in E. coli.
So, in the present invention, a method of obtaining immunogenic polypeptide is, which causes an immune response which is protective against B. moreover, this method involves transforming a host E. coli cell with a nucleic acid that encodes either (a) protective antigen (PA) of Bacillus anthracis or a variant of it that can induce a protective immune response, or (b) a polypeptide comprising at least one protective domain of this protective antigen (PA) of Bacillus anthracis or a variant of it that can induce a protective immune response, as described above, culturing the transformed host and the extraction of this polypeptide, provided that in cases where the polypeptide is a protective antigen (PA) of Bacillus anthracis or a variant of it that can induce a protective immune response, the percentage guanidino and casinovip residues in a specified nucleic acid exceeds 35%.
Using these options, and with the use of proper expressing of the owner it is possible to achieve high product yield.
The table representing the codons and the frequency with which they appear in the genomes of Escherichia coli and Bacillus anthracis, is shown in figure 1. It is clear that the guanidine and cytosine occur more frequently in E. coli than in .anthracis. The content analysis used in this codon finds the following:
|Types||1st letter of the codon GC||2nd letter of the codon GC||The 3rd letter of the codon GC||Overall GC-content|
Thus, apparently, the codons, preferably occurring in E. coli, such codons, which meet in yourself, if possible, guanidine or cytosine.
With the increase in the percentage of nucleotides of guanidine and cytosine in the sequence used to encode immunogenic protein, in excess of what is usually found in the gene .anthracis wild type, the use of this codon will improve expression in E.coli.
Accordingly, the percentage guanidino and casinovip residues in the coding nucleic acid used in the present invention, at least in cases where the polypeptide is a protective antigen (PA) of Bacillus anthracis or a variant of it that can induce a protective immune response, more than 40%, preferably greater than 45%, and most preferably greater than 50-52%.
High levels of expression of protective domains can be achieved by using a sequence of .anthracis of decorative, the coding of these structural units. However, the performance of expression can be enhanced further by increasing the GC content in a given nucleic acid, as described above.
In a specific embodiment of the present invention, this method includes the expression of PA B..
Further, in accordance with the present invention, has created a recombinant cell of Escherichia coil, which is transformed with nucleic acid which encodes a protective antigen (PA) of Bacillus anthracis or a variant of it that can induce a protective immune response, wherein the percentage of residue of guanidine and cytosine in a given nucleic acid exceeds 35%.
As before, corresponding to the percentage of residue of guanidine and cytosine in this coding nucleic acid is greater than 40%, preferably greater than 45% and most preferably greater than 50-52%,
Accordingly, the nucleic acid according to the invention used for transformation of E.coli cells, is a synthetic gene. In particular, this nucleic acid is a SEQ ID NO 1, which is shown in Figure 2, or its modified form.
Expressed by the "modified form" refers to other nukleinovokisly sequences that encode RA or its fragments or variants, which is what's called a protective immune response, but which use different codons, providing a requirement for the percentage of GC in accordance with the present invention. The relevant modified forms should be similar to at least 80%, preferably between 90%and most preferably similar in at least 95% with SEQ ID NO 1. In particular, this nucleic acid comprises SEQ ID NO I.
In an alternative embodiment of the present invention, the present invention has created a recombinant Escherichia coli cell that has been transformed with nucleic acid which encodes a protective domain protective Anthea (PA) of Bacillus anthracis or a variant of it that can induce a protective immune response.
Preferably, this nucleic acid encodes domain 1 or domain 4 .anthracis.
Further, in accordance with the present invention, has developed a method of obtaining immunogenic polypeptide that induces an immune response that is protective against .anthracis, and the method includes culturing cells that described above, and extract the desired polypeptide from the culture. Such methods are well known in the art.
In accordance with another aspect, the present invention has created a vector, transforming E. coli containing the nucleic acid to which I encodes the protective antigen (PA) of Bacillus anthracis or a variant which can induce a protective immune response, wherein the percentage guanidino and casinovip residues in a given nucleic acid exceeds 35%.
Another aspect of the present invention relates to a vector, transforming E. coli and containing nucleic acid which encodes a protective domain of the protective antigen (PA) of Bacillus anthracis or a variant of it that can induce a protective immune response.
The corresponding vectors used for transformation of E. coli are well known in the art. For example, the T7-expression system provides high levels of expression. But, especially preferred vector contains pAG163 obtained from Avecia (UK).
Nucleic acid SEQ ID NO 1, or its variant, which encodes RA and which has at least 35%, preferably at least 40%, more preferably at least 45%, and most preferably 50-52%GC content, creates an additional aspect of the present invention.
By having these options RA or domains can be expressed in the form of combination with another protein, such as protein, which provides some immunity, with protein, which facilitates the cleaning of this product, or vysokoagressivnyh protein (e.g., thioredoxin, GST), which guarantees the right of initiats the Yu or broadcast.
If necessary, this expression system, add additional systems, such as T7-lysozyme, which improves the repression of this system, although in the case of the present invention the difficulties associated with cellular toxicity, is not marked.
In the method according to the invention can use any suitable strain of E. coli. Suitable strains, which are characterized by a failure on a number of proteases (e.g., Ion-, ompT-), and in respect of which they are assumed to minimize proteolysis. However, applicants have found that it is not necessary to use such strains to achieve a good yield and that other strains, such as K12, ironically, produce large quantities of product.
Fermentation of strain is usually performed in standard conditions, which are known in the art. For example, the fermentation can be done in the form of periodic cultures, preferably in large flasks for shaking, using a complex medium containing antibiotics to save the plasmid and with the addition of IPTG for induction.
After cultivation, the cells are harvested and stored at -20°until then, until you need surgery to allocate cleanup.
Appropriate allocation scheme clearing expressed E.coli PA (option either on the exchange) can be adapted for diagrams used for .subtilis. The individual used the stage of allocation of cleaning depend on the physical characteristics of the recombinant RA. Usually carry out the separation using ion-exchange chromatography under conditions that allow for the greatest differential binding to the column and followed by collecting fractions with a small gradient. In some cases, to obtain a product with desired technical characteristics may be sufficient to only one chromatographic step.
If necessary, fractions can be analyzed for availability of this product using SDS-PAGE or Western blotting.
Below is illustrated the successful cloning and expression of a panel of chimeric proteins, representing the intact or partial domains of rPA. Immunogenicity and protective efficacy of these chimeric proteins from infection STI-spores estimate on the model of A/J mice.
All Dominova rPA proteins are immunogenic to A/J mice and provide at least partial protection against infection compared with the control GST-immunized mice. Protein-carrier, GST is attached to the N-end domain proteins without compromising immunogenicity fused proteins or in vivo, as shown or antibody-based test response, induced in immunized animals, or in vitro in fidelity proteins, which can be detected using antisera to the rPA after Western blotting, suggesting that GST-tag does not prevent the recognition of rPA-epitope. Immunization large chimeric proteins gives the highest titers. In particular, mice immunized with full-sized chimeric protein GST 1-4, on average, produce anticigarette rPA with eight-fold concentration compared to the group immunized with rPA (Figure 5). Immunization of mice domains 1-4 rPA with GST split gives approximately half the titer produced by immunization indicated chimeric protein. Why this protein should be more immunogenic, remains outstanding. Possibly, the increased size of this protein may have increased immunogenic effect on immune effector cells. But it does not stimulate such a response in the same degree in other chimeric proteins, in addition, any enhancing the immunogenicity of action GST-tag does not increase protection from infection, because otsepleniya proteins similarly protected them chimeric analogues.
Despite the presence of high titers of anti-rPA, in groups of animals immunized with GST1, split 1, GST1b-2, GST1b-3 and GST1-3, with a reduced level of infection of 102MLD has some crash protection, and immunization with these proteins do not prolong the time of survival of the ex mice, which did not survive the infection, in contrast to the control mice immunized with GST. This suggests that the immune response was not properly raised these proteins for complete resistance to this infection. As has been shown in other studies in mice and Guinea pigs (Little S.F. et al. 1986. Infect. Irnmun. 52: 356-363), there is no exact correlation between the antibody titer against RA and protection from infection. However, to protect requires some threshold antibodies (S. Cohen et al., 2000 Infect. Immun. 68: 4549-4558), and this suggests that cell-mediated components of the immune response also need to encourage protection (Williamson 1989).
As the results of the SDS-Paqe and Western blotting, obtained chimeric protein GST1, GST1b-2 and GST1-2 was the least stable, which is probably related to the fact that these proteins, in the absence of domain 3, is more susceptible to degradation, and this instability may have led to the loss of protective epitopes.
Certain structural conformation of these proteins also appear to be substantial to stimulate a protective immune response. Remove from the data chimeric protein Domain 1A leads to lower titers of antibodies to the weakening of protection during infection compared to their intact counterparts GST1-2 and GST1-3. Similarly, mouse, IMM is nozirovania using only the GST 1, were only partially protected from infection, but when combined with domain 2, in the form of a chimeric protein GST1-2, showed complete protection when the level of infection of 102MLD. However, the immune response induced as a result of immunization with chimeric protein GST1-2, was insufficient to produce complete protection at a higher level of infection in 10 MLD, which again could be due to loss of protective epitopes in the degradation of this protein.
All groups of animals immunized with truncated proteins containing domain 4, including separate GST 4, taken separately derived domain 4, and a mixture of two individually expressed domains 1 and GST GST 4, were completely protected from infection STI-spores in 103MLD (table 1). Brossier et al. showed reduction in protection in mice immunized with the mutant strain of C. anthracis, which expresses RA without domain 4 (Brossier f, et al. 2000. Infect. Immun. 68: 1781-1786), and this was confirmed in this study, where immunization with GST1-3 resulted in crash protection, despite high antibody titers. These data suggest that domain 4 is an immunodominant subunit of RA. Domain 4 presents 139 amino acids at the carboxy-end of the polypeptide RA. It contains the receptor binding site of the host cell (Little S.F. et al., 1996 Microbiology 12: 707-715), identified as being within and near a small loop located between amino acid residues 679-693 (Varughese M., et al. 1999 Infect. Immun. 67: 1860-1865).
For this reason, toxicity to the host cell is significant, although it has been shown that expressing in the form of RA that contains the mutation (Varughese 1999, above) or deletions (Brossier 1999, above) on the plot of domain 4 are not toxic. Crystal structure of RA representing domain 4, and in particular, the loop this domain, consisting of 19 amino acids (703-722), largely not protected than the other three domains, which are tightly associated with each other (Petrosa 1997, above). This structural arrangement can transform domain 4 in very bold epitope for recognition by immune effector cells, and therefore, chimeric proteins containing domain 4, should be the most immune response.
In addition, this study shed light on the role of RA in stimulating a protective immune response, demonstrating that protection from infection anthrax can be attributed to the individual domains of RA.
Further, the present invention is described in detail in the examples, with reference to the accompanying drawings.
Figure 1 shows the frequency of codons found in E. coli and B..
In figure 2, in accordance with the present invention, the above pic is egovernance nucleic acid, which encodes RA .anthracis, as published in Welkos et al. above.
Figure 3 shows SEQ ID No 3-14, which are sequences of amino acids and DNA used for encoding different domains or combinations of domains of RA, which is detailed below.
Figure 4 shows SEQ ID No 15-16, which are, respectively, amino acid sequences and DNA domain 4 RA.
Figure 5 is a table showing the concentration of IgG against rPA in A/J mice immunized intramuscularly in the 1st and 28th days of 10 μg chimeric protein containing RA-fragment, after 37 days after the first immunization; the results presented correspond to the average ± standard error (sem) samples from 5 mice in the experimental group.
The study of expression in E. coli
Plasmid pAG163::rPA expressing rPA, modify, replacing KmRtoken source TCR-gene. This plasmid transform in expressing the cell-host E. coli BLR (DE3), and then evaluate the expression level and solubility. This strain is deficient in the intracellular protease La (gene product Ion) and the outer membrane protease Ompt.
However, studies on the expression did not show any improvement in the accumulation of soluble protein in this strain compared to the host strain K12 Ion+ (i.e. NAC the attack is overcome by excessive proteolysis). The conclusion is that any intracellular proteolysis rPA is not due to the action of La-protease.
Analysis of fermentation
Performed additional analysis of fermentation, which were performed using strain K12 UT5600 (DE3) pAG163::rPA.
Found that rPA in this culture is distributed between the soluble and insoluble fractions (presumably 350 mg/l for insoluble rPA, 650 mg/l for full-length soluble rPA). In used conditions (37°C, 1 mm IPTG for induction) soluble rPA in shake cultures in flasks detection is not difficult, but, given the results described above in Example 1, are surprised by the presence of large amounts of soluble rPA. Be that as it may, it appears that regulation of the fermentation run, and the moment of harvesting cells grown in culture, allows stably accumulate rPA in expressing strains of E.coli K12.
Sample rPA derived from material originally allocated in the form of insoluble intracellular inclusions (intracellular cells) from fermentive strain UT5600 (DE3) pAG163::rPA. Intracellular inclusions washed twice with 25 mm Tris-HCl pH 8.0, and once with the same buffer + 2M urea. Then solubilizer in the buffer +8 M urea, and debris precipitated. Urea is removed by dilution in 25 mm Tris-HCl rn,0 is statically incubated over night at 4° C. the Diluted sample is applied on a column of Q-separate, and protein elute NaCl gradient. The fractions containing the most pure rPA, unite, take an aliquot and freeze at -70°C. Testing of this sample using 4-12% MES-SDS NuPAGE gel against a known standard is evidence of its high purity and low endotoxin contamination.
Additional characteristics of the obtained product
N-terminal sequencing of the obtained product shows that the N-terminal sequence consists of
MEVKQENRLL (SEQ ID NO 2)
This confirms that this product, as expected, is to the left relative to the initiating methionine.
Found that the resulting material reacts in Western band; according to MALDI-MS, the sample has a mass of approximately 82700 (compared with the estimated mass 82915). The difference between the large molecular weight of the obtained material and weight of the used standard (66 kDa) is considered as an indication that the material is not significantly shortened, but does not exclude microheterogeneity the analyzed sample.
Testing of individual domains of RA
Individual domains of RA obtained as recombinant proteins in E. coli in the form of a chimeric protein with protein carrier glutathione-s-transferase (GST) and using EC is pressional system Pharmacia pGEX-6R-3. Sequence of different domains and the DNA sequence used to encode them, are attached as Figure 3. The corresponding amino acid and DNA sequences are given below in Table 2.
Data chimeric proteins used for immunization of A/J mice (Harlan Olac) by intramuscular injection of 10 μg of the corresponding chimeric protein adsorbed on 20% vol/about alhydrogel in a total volume of 100 μl.
Animals twice subjected to immunization randomly, and the development of protective immunity determines by infecting spores Century anthracis (STI-strain) level indicator doses. In the following table 2 shows data on animals surviving to 14 days after exposure.
|The degree of contamination of spores/mouse|
|Domains||Amino acid SEQ ID NO||DNA SEQ ID NO||5×104||9×104||9×105||1×106||5×106|
|Domains||Amino acid SEQ ID NO||DNA SEQ ID NO||5×104||9×104||9×105||1×106||5×106|
|GST-1+2||5||6||4/4; 5/5||4/5; 5/5|
The data presented indicate that the combination of all 4 domains of RA, represented in the form of a chimeric protein with GST or without, has a protective effect, and with the highest level of infection. Remove domain 4 when you are 1+2+3, causes a crash protection at the highest tested level of infection, 9×105. Domains 1+2 eye of yaytsa protective combination of domains 1+2+3 when the level of infection 9× 104a dispute. However, deletion of domain 1A, when there is a fusion of GST with domains 1b+2, leads to violation of protection at the highest tested levels of infection (9×104), which is somewhat improved when adding the domain 3.
The data obtained indicate that protective immunity stimulated RA may be associated with individual domains (intact domain 1 and domain 4), or combinations of all 4 domains, taken in the modified order.
Amino acid sequence and DNA sequence of domain 4 is presented on Figure 4, respectively, in SEQ ID No 15 and 16.
Additional testing domains as vaccines
DNA from B. Sterne encoding the domains of RA, amino acids 1-259, 168-488, 1-488, 168-596, 1-596, 260-735, 489-735, 597-735 and 1-735 (respectively, shortened GST1, GST1b-2, GST1-2, GST1b-3, GST1-3, GST2-4, GST3-4, GST4 and GST1-4), amplified using PCR and clone into the XhoI/BamHI sites expressing vector pGEX-6-P3 (Amersham-Pharmacia) to the right from the starting point of transcription and under lac promoter. Proteins produced using this system, expressed as chimeric (hybrid) protein with protein glutathione-s-transferase (GST) at the N-end. Then the recombinant plasmid DNA carrying the above-mentioned DNA encoding the domains of RA, transformed into E. coli BL21 for the analysis of the expressed protein.
E.coll BL21, anchored plasmid is mi pGEX-6-P3, cultured in L-medium containing 50 μg/ml ampicillin, 30 μg/ml chloramphenicol and 1% weight/volume of glucose. Before induced with 0.5 mm IPTG to the culture incubated in the mode of shaking (170 rpm-1) at 30°and a600Nm0.4. Next, the culture incubated for 4 hours, followed by harvesting cells by centrifugation at 10,000 rpm for 15 minutes.
In the initial extraction of the truncated chimeric proteins RA discovered that they were obtained in the form of intracellular Taurus. Precipitated cells resuspended in phosphate-buffered saline (PBS) and treated with ultrasound for 4×20 seconds in a water bath with ice cubes. Treated with ultrasound, the suspension is centrifuged at 15,000 rpm for 15 minutes and then the precipitated cells are extracted by urea by suspension in 8M urea and stirring at room temperature for 1 hour. This suspension is centrifuged for 15 minutes at 15,000 rpm and the resulting supernatant cialiswhat against 100 ml of Tris, pH 8.0, containing 400 mm L-arginine and 0.1 mm EDTA before dialysis in PBS.
Successful re-packaging the truncated chimeric proteins RA allows their separation by purification on a column by glutathione affinity with Separate CL-4B. All the extracts (except shortened GSTlb-2, amino acid residues 168-487) NAS is placed in a 15 ml-new column with glutathione CL-4B Sepharose (Amersham-Parmacia), pre-equilibrated PBS and incubated with rocking overnight at 4°C. the column is washed with PBS, and chimeric protein elute 50 mm Tris, pH 7.0, containing 150 mm NaCl, 1 mm EDTA and 20 mm reduced glutathione. The fractions containing the shortened RA, identify by analysis in SDS-PAGE, pooled and cialiswhat against PBS. The protein concentration determined using ICA (Perbio).
However, shortened GST1b-2 cannot be eluted from the column affinity with glutathioneperoxidase CL-4B using reduced glutathione, and therefore emit by purification using ion exchange chromatography. In this case shortened GST1b-2 before loading into the column HiTrap Q (Amersham-Parmacia), balanced 20 mm Tris pH 8.0, dialist against the same buffer. Chimeric protein elute increasing gradient of 0-1 M NaCl in 20 mm Tris pH 8.0. The fractions containing the indicated GST-protein are pooled, concentrated and loaded onto a gel-filtration column HiLoad 26/60 Superdex 200 (Amersham-Parmacia), pre-equilibrated PBS. Fractions containing the chimeric protein are pooled and the concentration of a given protein is determined using ICA (Perbio). The output varies between 1 and 43 mg per liter of culture.
The molecular weight of the fragments and their recognition by antibodies against RA confirmed using SDS-PAGE and Western blotting. Analysis reproach the Chennai rPA using SDS-PAGE and Western blotting detects protein bands of expected size. Some degradation of all investigated shortened rPA finds their obvious similarity with recombinant RA, expressed .subtilis. Shortened rPA GST1, GST1b-2 and GST1-2, in the absence of domain 3 are particularly sensitive to degradation. This is similar to what was reported in relation to structures rPA containing mutations in domain 3, which cannot be allocated by purification from the culture of supernatants B. (Brossier 1999), which suggests that the domain 3, probably stabilizes the domains 1 and 2.
In this study, use of females totally healthy A/J mice (Harlan UK), as they are a suitable model for infection anthrax (Welkos 1986). By the beginning of this study, mice were sexually Mature at the age of seven weeks.
A/J mice subjected to immunization in the 1st and 28th days of this study, 10 μg of chimeric protein adsorbed to 20% 1,3%V/V Alhydrogel (HCI Biosector, Denmark) in a total volume of 100 μl PBS. Were also included in the group of animals immunized with rPA from B.subtilis (Miller 1998) together with recombinant control protein GST, or chimeric protein-coding domains 1, 4 and 1-4, which contain deleted GST-tag. Immunizing doses intramuscularly in areas on their hind legs. Mice take samples of blood through 37 days after the first immunization for analysis of serum antibodies using enzyme-dependent immunosor entogo assay (ELISA).
Tablets for micrometrology (Immulon 2, Dynex Technologies) cover over night at 4°With 5 μg/ml rPA expressed B.subtilis (Miller 1998), in PBS, with the exception of two rows on the tablet, which cover 5 µg/ml mouse antibodies against Fab (Sigma, Poole, Dorset). Tablets washed with PBS containing 1% V/V Tween 20 (PBS-T) and blocked with 5% weight/about skim milk powder in PBS (reagent for analysis Western blotting) for 2 hours at 37°C. In the wells coated with rPA, poured serum, diluted two times 1%reagent for the analysis of Western blot testing, and analyze repeat it with a standard mouse IgG (Sigma)added to the anti-Fab antibody, covering the wells, and incubated over night if 4°C. After washing to all wells add horseradish peroxidase, conjugate with antibodies goat against mouse IgG (Southern Biotechnology Associates Inc.), diluted 1:2000 in PBS, and incubated for 1 hour at 37°C. the Tablets washed again before the addition of substrate 2,2'-Azinobis (3-ethylbenzthiazolinesulfonic acid) (1,09 mm ABTS, Sigma). After 20 minutes of incubation at room temperature, measure the absorption in the wells at 414 nm (Titertek Multiscan, ICN Flow). Standard curves calculated using the software Titersoft version s. The titles were presented as μg IgG per ml of serum with the calculation of the group average ± the standard error for the average sem). The results are presented in figure 5.
All received shortened rPA was immunogenic and raised in A/J mice average concentrations of IgG antisera to the rPA in the range of 6 µg per ml in the group immunized with shortened GST1b-2, up to 1488 µg per ml in the group immunized with truncated GST 1-4 (Figure 5). The control mice immunized with GST antibodies against rPA were not found.
The mice were infected with spores Century anthracis STI on the 70th day of the mode of immunization. From strain were allocated a sufficient number of STI-dispute, washed with distilled water and resuspendable in PBS to a concentration of 1×1071×106dispute on ml, the Mice were infected intraperitoneally with 0.1 ml of business volumes, respectively, containing 1×1061×105the dispute on the mouse, and monitor them within 14 days after infection to determine their protective status. Humane treatment of animals was strictly adhered to in the sense that wypracowanie those animals in which it was observed clinical symptoms, combination of which indicates the presence of a lethal infection. The number of immunized mice that survived to the 14th day after infection, are shown in Table 3.
|Domain||Levels of contamination MLD survived the/a number of infected (%)|
|GST 1||3/5 (60)||1/5 (20)|
|GST 1b-2||1/5 (20)||Nd|
|GST 1-2||5/5 (100)||3/5 (60)|
|GST 1b-3||3/5 (60)||Nd|
|GST 1-3||4/5 (80)||Nd|
|GST 1-4||Nd||5/5 (100)|
|GST 2-4||Nd||5/5 (100)|
|GST 3-4||Nd||5/5 (100)|
|GST 4||5/5 (100)||5/5 (100)|
|GST 1 + GST 4||Nd||5/5 (100)|
|Ottsepleny 1||1/5 (20)||2/5|
|Ottsepleny 4||5/5 (100)||5/5|
|control||0/5 (0)||0/5 (0)|
|MLD = approximately 1×103STI-dispute|
Nd = not determined
Group infected with Id3MLD STI-dispute, were all fully protected, except in the groups immunized with GST1, GST1-2 and split domain 1 indicates some failure in the protection and control the group, immunized only GST, in which all were susceptible to infection with a mean time of death (MTTD) 2,4±0.2 days. At the lowest level of infection of 102MLD GST1-2, GST4 and split the domain 4 - all immunized groups were fully protected, but in other groups there was some crash protection. The mouse that died in these groups, was characterized by MTTD 4,5±0.2 day that is not significantly different from the control group immunized with GST, in which all were killed with the index MTTD 4±0.4 days.
1. Immunogenic polypeptide that causes an immune response which is protective response against Bacillus anthracis, where the specified polypeptide contains from one to three full-sized domains of Protective Antigen (PA) Century anthracis or their variants, which are the result of deletions and/or substitutions in the amino acid sequence that do not result in loss of immunogenicity, and where at least one of these domains is a domain 1 or domain 4 of the RA, or option.
2. Immunogenic polypeptide according to claim 1, which includes domain 4 RA of the Century anthracis.
3. Immunogenic polypeptide according to claim 1, which includes domain 1, merged with the domain 2 RA-sequence.
4. Immunogenic polypeptide according to claim 1, which includes domain 1, merged with the domain 2 and domain 3 of the RA sequence.
5. Immunogenic polypep is d, which includes a combination of domain 1, or its protective of section 1A and domain 4 full Protective Antigen (PA) from Century anthracis or their variants, which are the result of deletions and/or substitutions in the amino acid sequence that do not result in loss of immunogenicity.
6. Immunogenic polypeptide according to claim 5, which comprises a sequence of domain 1 and domain 4 of the RA wild-type.
7. Immunogenic polypeptide that causes an immune response which is protective response against Bacillus anthracis, where the specified polypeptide consists of a polypeptide under item 1, fused with GST.
8. Immunogenic polypeptide according to claims 1-6, used as active ingredient to obtain drugs for prevention or treatment of infections Century anthracis.
9. Immunogenic polypeptide according to claim 7, used as active ingredient to obtain drugs for prevention or treatment of infections Century anthracis.
10. Nucleic acid encoding the immunogenic polypeptide that causes an immune response which is protective response against Bacillus anthracis, and is characterized by a sequence of nucleic acid, which essentially corresponds to the amino acid sequence of the immunogenic polypeptide under item 1 or 3.
11. Vector for expression in bacterial cells containing nucleic what islote of claim 10, operatively associated with the regulatory region.
12. The method of obtaining immunogenic polypeptide according to any one of paragraphs. 1-6, includes a transform E. coli host nucleic acid on p. 10, culturing the transformed host and get the specific immunogenic polypeptide.
13. The method according to item 12, where the polypeptide is a domain 1 and/or domain 4 of the protective antigen RA from Bacillus anthracis.
14. The method according to item 12 or 13, where the nucleic acid is optimized to the conditions of expression in these cells so that the percentage of residue of guanidine and cytosine in a specified nucleic acid exceeds 35%.
15. The method according to 14, where the percentage of residue of guanidine and cytosine in a specified nucleic acid exceeds 45%.
16. The method according to clause 15, where the percentage of residue of guanidine and cytosine in a specified nucleic acid is from 50-52%.
17. Vector, transforming E. coli comprising the nucleic acid under item 10.
18. Vector, transforming E.coli on 17, where the nucleic acid is optimized to the conditions of expression in these cells so that the percentage of residue of guanidine and cytosine in a specified nucleic acid exceeds 35%.
19. Vector, transforming E.coli, p, where the percentage of residue of guanidine and cytosine in a specified nucleic acid exceeds 45%.
20. The vector t is informiruyuschii E. coli according to claim 19, where the percentage of residue of guanidine and cytosine in a specified nucleic acid is from 50-52%.
21. The way to prevent infection caused by C. anthracis, comprising the administration to a mammal sufficient immunogenic polypeptide according to any one of claims 1 to 6.
22. A vaccine to prevent infection caused by C. anthracis containing an effective amount of the polypeptide under item 1 and a suitable carrier.
FIELD: biotechnology, in particular gene engineering, pharmaceutical and food processing industry.
SUBSTANCE: DNA sequence (1341 n.p.) encoding fatty acid -desaturase (447 amino acid residue, 57 kD) of nematode Caenorhabditis elegants is isolated and characterized. Obtained DNA-sequence is expressed in bacterium and yeast cells to produce Biologically active enzyme recombinant form. Said recombinant form is capable to catalyze conversion of dihomo-γ-linolenic acid to arachidonic acid and eicosatetraenoate to eicosapentaenoate.
EFFECT: method for large-scale production of polyunsaturated fatty acid.
15 cl, 4 dwg, 1 ex
FIELD: biotechnology, microbiology, genetic engineering.
SUBSTANCE: invention proposes a new recombinant plasmid pR752 (5269 pair bases) comprising genetic construction under control of bacteriophage T5 promoter and encoding a module polypeptide consisting of 6 histidine residues, hemoglobin-like protein of E. coli, modified fragment of large T-antigen SV-40, translocation domain of diphtheria toxin, spacer sequence (Gly-Ser)5 and human epidermal growth factor (6 His-HMP-NLS-Dtox-(Gly-Ser)5-EGF) and designated for the directed transfer of photosensitizers into target-cell nuclei. By transformation of the strain E. coli M15 (rep 4) with plasmid pR752 the recombinant strain E. coli VKPM B-8356 as a producer of new polypeptide vector is prepared. The usage of this new strain is able to enhance the effectiveness of effect of photosensitizers by some orders. Invention can be used in medicinal-biological industry in preparing agents providing the directed transport of photosensitizing agents into tumor cell nuclei.
EFFECT: valuable biological and medicinal properties of polypeptide.
3 dwg, 4 ex
FIELD: biotechnology, genetic and protein engineering.
SUBSTANCE: invention reports construction of plasmid DNA pES6-1 based on plasmid pET22b(+) and DNA fragment comprising a sequence of artificial gene encoding human interferon β-1b providing expression of human recombinant interferon β-1b. Also, the strain Escherichia coli BDEES6 (BL21(DE3)/pES6-1) as producer of human recombinant interferonβ-1b is prepared. Invention provides enhancing yield of human recombinant interferon β-1b. Invention can be used in medicine and pharmaceutical industry.
EFFECT: valuable biological and medicinal properties of strain.
2 cl, 2 dwg, 2 ex
FIELD: genetic engineering, molecular biology, biochemistry.
SUBSTANCE: recombinant plasmid DNA pTES-His-OPH is constructed for expression of polypeptide eliciting properties of organophosphate hydrolase comprising Cla I/Hind III fragment of plasmid pTrcTEGF, fragment of plasmid pTES-OPH with nucleotide sequence that encodes amino acid sequence of the matured form of organophosphate hydrolase, and nucleotide sequence encoding 6 histidine residues that is located by 5'-end of nucleotide sequence encoding organophosphate hydrolase. Based on indicated plasmid the strain Escherichia coli TSKMIBKH-29 - a producer of polypeptide eliciting properties of organophosphate hydrolase is obtained. Applying the invention provides preparing polypeptide with properties of organophosphate hydrolase by simplified technology and this polypeptide elicits the improved catalytic effectiveness of action with respect to thio-containing phosphoric acid triesters. Invention can be used for carrying out hydrolysis of organophosphate compounds.
EFFECT: valuable biochemical properties of producer.
2 cl, 4 dwg, 2 tbl, 4 ex
FIELD: biotechnology, genetic engineering, pharmaceutical industry.
SUBSTANCE: plasmid DNA pET23-a(+)/PrxVIhumΔ178 with molecular weight of 19691.61 Da is constructed. DNA contains RNA-polymerase T7 promoter; replication initiation site; genetic marker which determinates resistance of cells transformed by said plasmid to ampicillin; and nucleotide sequence encoding N-terminal fragment of human peroxiredoxine VI containing 177 of amino acid residues. E.coli strain BL21/DE3/pET23-a(+)/PrxVIhumΔ178 being producer of N-terminal fragment of human peroxiredoxine VI is obtained by transformation of E.coli cells with plasmid DNA pET23-a(+)/PrxVIhumΔ178. Method of present invention makes it possible to obtain human peroxiredoxine VI fragment having reduced molecular weight, improved tissue permeability, and antioxidant activity of full-scale peroxiredoxine.
EFFECT: human peroxiredoxine VI fragment with improved tissue permeability.
2 cl, 3 dwg, 4 ex
FIELD: genetic and tissue engineering, biotechnology, medicine, agriculture.
SUBSTANCE: invention relates to the development of simple with constructive relation peptide vector (PGE-κ) consisting of polypeptide sequence of epidermal growth factor (EGF) and modified sequence of signal peptide T-antigen SV-40. New vector PGE-κ is able to provide the selective delivery of genetic material in target-cell cytoplasm carrying external receptors to EGF and the following its transport across nuclear membrane. Also, invention proposes a method for preparing peptide vector PGE-κ involving its expression as a fused protein "mutant thioredoxine-linker-vector" and cleavage of product expressed in E. coli in the linker region with specific protease. Invention provides preparing the recombinant strain E. coli B-8389 VKPM as a producer of the fused protein comprising PGE-κ. Proposed vector shows simple structure, absence of toxicity and immunogenicity and these properties provide its usefulness for the directed genetic modification of epithelial, embryonic and tumor cells in vivo.
EFFECT: improved preparing method, valuable medicinal properties of vector, improved genetic modification.
7 cl, 12 dwg, 4 tbl, 16 ex
FIELD: medicine, immunology, phthisiology.
SUBSTANCE: invention proposes a vaccine composition used in prophylaxis and treatment of tuberculosis infection. The composition comprises a mixture of fused recombinant protein ESAT-HSP70 from Mycobacterium tuberculosis, protein HSP65 from M. tuberculosis and an inert vehicle. Also, the proposed composition can comprise timogen additionally. Also, invention proposes recombinant plasmid DNA pE HSP70 and pQE HSP65 encoding corresponding proteins. The proposed vaccine composition enhances effectiveness in treatment and prophylaxis of tuberculosis infection. Genetic constructions provide carrying out the stable expression of indicated proteins and to enhance purity the end preparing proteins. Invention can be used in treatment and prophylaxis of tuberculosis.
EFFECT: valuable medicinal properties of composition.
5 cl, 6 dwg, 6 ex
FIELD: gene engineering, biotechnology, medicine.
SUBSTANCE: invention relates to production of polypeptides bearing specific antigen determinants of OspC protein and FlaB flagellin fragment appropriate to genus Borrelia garinii being prevalent in Russia. Recombinant plasmid pREB9-H6-F7 DNA providing synthesis of B.garnii FlaB protein fragment contains: fragment with size of 100 b.p. encoding signal amino acid sequence of Staphylococcus aureus α-toxin (CAT) and eight N-terminated amino acid residues of mature CAT; B.garnii flaB gene fragment with size of 500 b.p; nucleotide sequence in flaB gene reading frame encoding amino acid residues of glycin and 6 histidins; pIL-2/21 plasmid BamHI - EcoR1 DNA fragment with internal unique Xmal restriction site, containing β-lactamase bla gene and regulatory region of Proteus mirabilis recA gene; unique Xmal, EcoR I, BstEII and BamHI restriction sites. Recombinant plasmid pREB9-H6-C25 DNA providing synthesis of B.garnii OspC protein contains fragment with size of 660 b.p. encoding B.garnii OspC protein; nucleotide sequence in ospC gene reading frame encoding amino acid residues of glycin and 6 histidins; pIL-2/21 plasmid BamHI - EcoR1 DNA fragment with internal unique Xmal restriction site, containing β-lactamase bla gene and regulatory region of Proteus mirabilis recA gene; unique Xmal, EcoR I, BstEII and BamHI restriction sites.
EFFECT: high specific diagnosis method with improved accuracy.
2 cl, 2 dwg, 5 ex
FIELD: biotechnology, veterinary science, microbiology.
SUBSTANCE: strain is prepared by fusion of E. coli genes isolated from mink intestine that induce biosynthesis of microcine C51 and B-subunit of escherichia thermolabile enterotoxin into the genome. Microcine inhibits growth and development of enterobacteria producing thermolabile, thermostable and shiga-like (verotoxin) enterotoxins, and B-subunit screens intestine receptors for enterotoxin and initiates specific immunity in intestine as far as intestine comprises determinants of protective antigen of toxin molecule. The strain is used for preparing curative-prophylactic preparations with prolonged action. Microcines synthesized by the strain inhibit enterotoxic enterobacteria and synthesized B-subunits of escherichia thermolabile enterotoxin screen receptors for enterotoxin and stimulate specific immunity in intestine. Resident properties of a carrier don't cause rejection of the strain by the mink intestine microecosystem that allows applying these preparations based on thereof more rarely and in smaller doses.
EFFECT: valuable properties of preparation.
2 cl, 4 ex
FIELD: biotechnology, medicine, in particular treatment, prevention and diagnosis of diseases, associated with Neisseria meningitides.
SUBSTANCE: Claimed protein includes one or more N. meningitides protein fragments with known amino acid sequences, wherein said fragment contains one or more antigen determinants and has not more than 1977 amino acid from SEQ ID NO:1 and/or not more than 1531 amino acid from SEQ ID NO:2 which are described in specification with the proviso, that general protein sequence is not characterized by amino acid sequences represented in NO:1 and NO:2. Each claimed protein is encoded by nuclear acid (NA) with nucleotide sequence that defines protein amino acid sequence according to gene code. Peptides and nuclear acid of present invention are useful in drug production for treatment and prophylaxis of infections induced neisseria, as well as in production of diagnostic reagent for detection of neisseria or specific antibodies. Aldo disclosed is peptide- or NA-based pharmaceutical composition in effective amount with acceptable carrier. Said composition is useful for treatment of N. meningitides mediated infection. For prophylaxis composition is applied in vaccine form. Invention makes it possible to overcome diversity of neisseria antigen properties.
EFFECT: improved method for neisseria infection prophylaxis and treatment.
24 cl, 2 tbl
FIELD: biotechnology, in particular provision storage.
SUBSTANCE: bacteriocin represents polypeptide isolated from lactobacillus sakei 2512 and is capable to suppress lysteria growth and reproducing. Bactericin has specific amino acid sequence represented in claims. Nuclear acid sequence encoding said polypeptide is disclosed. Also disclosed is a vector including nuclear acid sequence for cloning and/or expression of polypeptide, for example in transformed cells, selected from Lactococcus, Lactobacillus, etc. Method for production of recombinant polypeptide is developed. Claimed bactericin or strain 2512 are used as component of bactericide composition, being capable to suppress growth of gram positive pathogenic bacteria, in particular Listeria monocytogenes.
EFFECT: large scale application of bacteriocin against pathogenic or undesired flora in food industry.
13 cl, 2 dwg, 1 tbl, 3 ex
FIELD: biotechnology, microbiology, medicine.
SUBSTANCE: invention represents group of Neisseria proteins eliciting antigen properties. Protein eliciting antigen properties comprises fragment of protein ORF 40 (amino acid sequence of ORF 40 is given in the invention claim) that involves 7 or more conservative amino acids arranging in succession. Proteins as components of proteins in the claimed group are used as a medicinal agent or for it manufacturing for treatment and prophylaxis of infection caused by Neisseria, and for manufacturing the diagnostic preparation. Invention relates also nucleic acid encoding the Neisseria protein. Nucleic acid is used as the protein. Applying the invention provides the maximal recognition and reactivity between strains of Neisseria. Invention can be used in manufacturing curative-prophylactic preparations with respect to Neisseria meningitides.
EFFECT: valuable biological and medicinal properties of antigen.
27 cl, 51 dwg, 10 ex
FIELD: biotechnology, biochemistry, enzymology, genetic engineering.
SUBSTANCE: invention relates to preparing new strain used for isolation of the new restriction endonuclease Bis I that can be used in detection of the modified DNA. The strain Bacillus subtilis 230 isolated from soil as result of search of producers of restriction endonucleases provides preparing the restriction endonuclease Bis I recognizing and cleaving both chain of DNA nucleotide sequence comprising at least one C5-methylcytosine base in the recognition site 5'-GCNGC-3'.
EFFECT: valuable properties of strain, new enzyme.
1 tbl, 3 dwg, 2 ex