Polynucleotide encoding mutant recombinant iga1 protease of neisseria meningitidis of serogroup b, recombinant plasmid dna comprising said polynucleotide, host cell containing said plasmid dna, recombinant iga1 protease of neisseria memingitidis of serogroup b, method of producing mature form of iga1 protease

FIELD: biotechnology.

SUBSTANCE: proposed enzymatically inactive IgA1 protease with replacement Ser267Ala for use as a component of a polyvalent vaccine designed to protect people against meningococcal infection and other microorganisms, which pathogenicity is caused by IgA1 protease. The invention includes a polynucleotide encoding the said mutant form of IgA1 protease of Neisseria meningitidis of serogroup B, comprising the said polynucleotide, a recombinant plasmid DNA, the strain Escherichia coli - producer of a mutant form of IgA1 protease according to the invention, the method of preparing a recombinant form of the enzyme using a technology of recombinant DNA, and recombinant inactivated IgA1 protease.

EFFECT: increased level of immunogenicity.

6 cl, 1 tbl, 5 dwg, 7 ex

 

The invention relates to the field of immunology, Enzymology, Microbiology and genetic engineering and the new polynucleotide encoding functionally active recombinant IgA1 protease of Neisseria meningitidis, a new recombinant plasmid DNA pBIGAPS1-M and the host cell, especially a new strain of the bacteria Escherichia coli BL21(DE3)/pBIGAPS1-M, which can be used to obtain high purity genetically engineered IgA1 protease and as a component of a multivalent vaccine intended to protect individuals, especially children, from meningococcal infection and other microorganisms, the pathogenicity of which is due to the IgA1 protease.

The problem of effective vaccine prevention of meningococcal disease caused by Neisseria meningitides serogroup B, still not solved. In the register of meningococcal vaccines who have not recorded a single drug against meningococci this serogroup. The lack of effective meningococcal group b vaccine prevents the elimination of this infection in our country and abroad. It is known that IgA serine protease secreted by some dangerous bacteria, especially Neisseria meningitidis, and cleaves immunoglobulin on the surface of the mucous membranes, destroying the first protective barrier of the immune system of the host body. Known meningococcal IgA1 proteases is, in experiments on laboratory animals showed himself as a highly effective meningococcal polio, and hence the vaccine against meningococcus serogroup B (U.S. Pat. EN 2407792, Alliluev A.P. and others, 16.08.2010 g). It was found that isolated from culture of fluid or cleaning products vaccine (meningococcal group a) IgA1 protease highly efficient enzymatic and immunological but is contained in these sources trace, so there can be complications when receiving protease in preparative amounts.

In prior art describes numerous attempts to create a vaccine designed to protect against infection meningitis group C. In applications WO 2005/105141 20051110, WO 95/03413, WO 2005/102384 20051103, WO 03/094960 20031120, WO 2006/024954 20060309, WO 2004/014417 20040219, WO 2005/033148 20050414, WO 2004/032958, WO 03/028661 20030410, WO 2004/014419 A1, WO 02/083711 available compositions and vaccine candidates on the basis of lipopolysaccharide, capsular oligosaccharides and several surface proteins of Neisseria meningitides to immunize the patient against a disease caused by Neisseria meningitidis different serogroups.

However, these compositions do not include IgA1 protease and do not provide adequate effective protection against meningococcal infection and, in particular, from meningitis group C.

In the application WO 2004/102199 A2, LEENHOUTS and others, 25.11.2004, describes how to identify, select, and you the population vaccine component for passive and active immunization against microorganism Streptococcus pneumoniae, which is subject to destruction opsonophagocytosis cells. In computer simulations (in silico) identified antigens that induce opsonophagocytic proposed to be used as components of a vaccine designed to immunize. Identified three pneumococcal protein: streptococcuspneumoniae (SlrA), IgA1 protease class metalloprotease and pneumococcal surface protein adhesion A (PsaA) of Streptococcus pneumoniae obtained with recombinant variants that have been studied as components of vaccines. It is established that the composition containing all three recombinant variant, protects mice from this infection, however, each individual component has no protective properties.

In article Shishir K. Gupta and others, Vaccine, 2010, V.28, str-7097, describes the results of computer simulation of sequences of proteins of Neisseria meningitides serogroup b and identified 6 peptide epitopes T-helper cells of three proteins: stimulating T-cells protein a, a motor protein a and IgA1 protease. The authors suggest that these peptides can be used as promising agents against meningococcus group C. However, the experimental data confirming this hypothesis, are not given.

Known to produce fragment of IgA1-protease, which is used as peptide-wear is El (U.S. Pat. US 7235242, Achtman and others, 26.06.2007). Described the synthesis of a fragment of IgA1-protease, containing from 40 to 200 amino acid residues and comprising 40 amino acid residues of the sequence SEQ ID NO:1, using an automatic synthesizer. However, conjugation with the polysaccharide IgA1 protease provides protection only from the meningococcal serogroup C and not effective against meningococcal disease serogroup C.

Known application of bacterial IgA1 proteases for the treatment of diseases associated with IgA1 deposits in the tissues and organs of the body (U.S. Pat. US7407653, Plaut and others, 05.08.2008). Soluble IgA1 protease containing different labels, for example polyhistidine fragment, receive recombinant method. The enzyme produce by well-known methods using ultrafiltration, ion exchange and metal-chelate chromatography. The resulting preparations can be used as an enzyme for the treatment of autoimmune diseases associated with accumulation of IgA, but not as vaccines.

Also known is a method of obtaining predecessor IgA1 protease. Article S. Vitovski and J. R. Sayers, Infection and Immunity, 2007, CH, No. 6, str-2885, described the cloning of a full-sized gene encoding the signal peptide, secreterial form proteases and autotransporters domain, and study of the mechanism of autoprocessing IgA1 protease from its predecessor. This purpose was created a number of mu the new forms of the protein, precursor of the IgA1 protease of Neisseria meningitidis serogroup B (NMB), including a mutant with a deletion of 32-membered peptide at the site scope of autoprocessing and mutant, which includes the replacement of the Ser residue in the active center for the remainder of the Val with the aim of obtaining predecessor, does not have autoproteolytic activity. To obtain mutants used NMB strains isolated from cerebrospinal fluid of patients with meningococcal disease. It was found that these mutants had no autoproteolytic activity and was not observed the formation of Sekretareva Mature protease. But in this work received only predecessor IgA1 protease. Protective and immunogenic properties actually IgA1 protease was not investigated and does not consider the possibility of creating a vaccine based on it.

In the application and other LONG, WO 20101183337 (A2), 14.10.2010, describes a method for soluble recombinant IgA1 protease using direct and/or indirect production through Taurus enable significantly higher yield compared to known previously developed methods, and the output is from 20 to 500 mg of soluble IgA1 protease/l of culture medium. However, the specified preparations and compositions IgA1 protease is enzymatically active and is intended only for the destruction of IgA1 in the treatment of diseases associated with the deposition of this protein in the tissues of the body. The AOC is e, the work was received IgA1 protease H. influenzae, expressed in E. coli.

A method of obtaining recombinant IgA1 protease of Neisseria meningitidis serogroup B (NMB) (Serova, O., and others, Biopharmaceutical journal, 2011, Vol.3, No. 6), which received full enzymatic active IgA1 protease having immunogenic and protective properties in mice against meningococci of serogroups Century, However, this drug has enzymatic activity against human IgA1, which can lead to undesirable consequences in the cleavage of immunoglobulin A1 on the mucous membranes of the body, such as the development of inflammatory reactions. This way as the closest prior art was chosen as a prototype.

The invention solves the problem of obtaining recombinant full-IgA1 protease with enhanced immunogenic and protective properties against Neisseria meningitidis major epidemic serogroups - a, b and C, but do not possess enzymatic activity against human IgA1, to create a multivalent vaccine to protect people from meningococcal infection and other pathogens, due to the IgA1 protease.

The problem is solved by polynucleotide encoding IgA1 protease of Neisseria meningitidis serogroup In strain N/76 mutation catalytically active is the STATCOM Ser267 on Ala (SEQ ID NO:1);

recombinant plasmid DNA comprising the above nucleotide sequence, and providing the expression of IgA1 proteases in the cell-master (pBIGAPS1-M), characterized by the following features: chain length is 8123 BP; encodes secreterial form IgA1 protease with mutation of the catalytically active residue Ser267 on Ala, with the signal sequence at the N end of the his-tag and tag-end of the protein;

the nucleotide sequence encoding IgA1 protease plasmid pBIGAPS1-M, flank the unique sites for the restriction endonucleases NdeI and XhoI;

the nucleotide sequence encoding IgA1 protease plasmid pBIGAPS1-M contains a unique restriction site of the endonuclease BamHI;

contains the T7 lac promoter transcription;

a genetic marker is a nucleotide sequence (kan), which determines the stability of the transformed plasmid pBIGAPS1-M of bacterial cells to kanamycin;

host cell, first of all producer strain E. coli BL21 (DE3)/pBIGAPS1-M containing recombinant plasmid DNA, which produces the Mature form of mutant IgA1 protease;

mutant IgA1 protease with a molecular mass of 107,6 kDa and a calculated isoelectric point of 7.4, with the amino acid sequence (SEQ ID NO:2)encoded by the above nucleotide sequence having immunogenic and about the objective properties designed to take poliomyelitis for prevention of meningococcal infections, including those caused by bacteria N. meningitidis serogroup B;

and by way of getting a Mature form of IgA1 protease of Neisseria meningitidis serogroup B, which consists in the fact that the cell host transformed with recombinant plasmid DNA that encodes a mutant IgA1 protease of Neisseria meningitidis serogroup B, with polynucleotides (SEQ ID NO:1), cultivated producing strains, isolate and purify the desired product from Taurus enabled.

The technical result of the invention to provide high purity full-enzymatically inactive mutant IgA1 protease of N. meningitidis serogroup B (40% of the enzyme from the total cellular protein, 20 mg of 2 g cell biomass purity of the drug - not less than 90%), characterized by an increased immunogenic and protective activity compared to the enzyme active native and recombinant proteases. The resulting preparation mutant IgA1 protease has a polyvalent action in protection against meningococcal infection of an animal's body regardless of the source of its receipt (serogroup a or b). The lack of enzymatic activity in respect of immunoglobulin A1 human mutant IgA1 protease will allow to avoid additional complications of immunization of man.

One object of us who Otsego of the invention includes a recombinant plasmid, comprising the above nucleotide sequence, and providing for the expression of mutant IgA1 protease in the cell host.

To obtain a mutant form of IgA1 proteases in which the catalytically active residue Ser267 replaced by Ala, method was used SOE (splicing by overlap extension, splicing when extending partially overlapping sequences). For this we have designed specific primers (SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6), overlapping in the field site mutations, in addition, these primers introduced a restriction site for the endonuclease BamHI. Amplification of a fragment of the gene was performed in two stages, using as template the plasmid DNA pBIGAPSI described earlier in the application EN 2453599, Rooms L.D., and others, from 18.02.2011 was obtained DNA fragment with a length of about 2900 BP Target NDP-product was cloned into the vector pet-24A(+) (Novagen, cat. No. 69749-3) when using unique restriction sites NdeI and XhoI so that the produced protein contained a His-tag at the C-terminal region for subsequent protein purification using metallogenetic chromatography. Sequencing the DNA confirmed the presence of mutations. The obtained plasmid DNA was named pBIGAPS1-M.

Another object of the present invention includes a cell-master, first of all, the strain of E. coli containing a recombinant nucleic acid sequence (see above), which is producing the Mature form of mutant IgA1 protease. As the host cell used, first of all, the strain E. coli BL21(DE3), on the basis of which was obtained strain BL21(DE3)/pBIGAPS1-M, described below.

Another object includes a method of obtaining a Mature mutant form of IgA1 protease of Neisseria meningitidis serogroup B, which consists in the fact that the cell host E. coli BL21(DE3) transformed described above recombinant plasmid DNA, encoding IgA1 protease of Neisseria meningitidis serogroup B, with polynucleotides (SEQ ID NO:1), cultured host cell, as described below, isolate and purify the desired product from Taurus enabled.

Another object of the present invention includes IgA1 protease with a molecular mass of 107,6 kDa and a calculated isoelectric point of 7.4 and amino acid sequence (SEQ ID NO:2)encoded described above polynucleotides and received the above method, designed to produce poliomyelitis prevention of infections, including those caused by bacteria N. meningitidis serogroup C.

IgA1 protease as an active ingredient can be used for poliomyelitis prevention meningoccocal infection of serogroups a, b, C, and infections, the pathogenicity of which is due to the IgA1 protease, such as Clostridium ramosum, Bacteroides melaninogenicus, Capnocytophaga ochracea, Streptococcus pneumoniae, Streptococcus sanguis, Streptococcus oralis, Streptococcus mitis, Haemophilus influenzae, Haemophilus influenzae, Haemophilus aegyptius, Neisseria gonorrhoeae,Neisseria meningitidis, Haemophilus influenzae, Streptococcus viridans, Streptococcus canis, Streptococcus suis, Staphylococcus aureus, Staphylococcus epidermidis, Moraxella catarrhalis, Helicobacter pylori, Campylobacter jejuni.

To obtain strain-producer IgA1 protease transform competent cells of Escherichia coli BL21(DE3) (E. coli F-dcm ompT hsdS(rB-mB-) gal λ(DE3)) (Stratagene Cat. No 200131) plasmid DNA pBIGAPS1)

The resulting strain called E. coli BL21(DE3)/pBIGAPS1-M, is characterized by the following properties.

Cultural and morphological characteristics.

Cells are small, rod-shaped, gram-negative, 1×3.5 µm, motile. The strain grows well on ordinary nutrient media (LB-broth, LB agar). During growth on agar LB medium, the colonies are round, smooth, translucent, shiny, yellowish color. The edge is smooth, the diameter of the colonies 2-3 mm thick, pasty consistency. Growth in LB liquid medium is characterized by smooth turbidity, sediment easily sedimentary.

Physiological and biochemical characteristics.

Cells grow when 4-42°C, the optimum pH of 6.8 to 7.6. As the source of nitrogen used as mineral ammonium salts, and organic compounds: amino acids, peptone, tripton, yeast extract.

Genetic traits.

Cells are resistant to kanamycin (100 μg/ml), due to the presence of the gene of resistance to the antibiotic in the recombinant plasmid DNA pBIGAPS1-M.

Storage conditions.

Strain is acteri E. coli BL21(DE3)/pBIGAPS1-M store on cups and jambs at 4°C. Subcultures to fresh medium is conducted once a month. Can be stored for at least one year in LB medium containing 15% glycerol at -70°C.

Resistance to antibiotics.

The cells of the producer strain are resistant to kanamycin (100 μg/ml), due to the presence of resistance gene in the recombinant plasmid DNA pBIGAPS1-M.

As a host cell, you can use the cells of eukaryotes (such as yeast cells, plant cells, animal cells and human cells)or prokaryotic cells such as bacterial cells and the like), or cells, which induce the immune response. Specialists in this field there are many of these cells and cell lines (higher eukaryotes, for example T (line human embryonic kidney cell), HeLa (cell carcinoma of the cervix person), SNO (cells Chinese hamster ovary) and other cell lines, including cells and cell lines developed for laboratory studies, such as, for example, cells hTERT-MSC, HEK293, Sf9 or COS. Suitable eukaryotic cells also include cells or cell lines affected by diseases or infections, such as cancer cells, especially cancer cells of any type of cancer, referred to in this context, cells affected by HIV, and/or cells of the immune system or the Central nervous system (CNS). Fit the notches can similarly be obtained from eukaryotic microorganisms, such as yeast, for example Saccharomyces cerevisiae (Stinchcomb and others, Nature, t, p.39, (1997)), Schizosaccharomyces pombe, Candida, Pichia and hyphomycetes genera Aspergillus, Penicillium and other Suitable cells in a similar way include prokaryotic cells, such as cells, bacteria, such as Escherichia coli or bacteria of the genus Bacillus, Lactococcus, Lactobacillus, Pseudomonas, Streptomyces, Streptococcus, Staphylococcus, preferably E. coli, and other human Cells or cells of animals, such as animals mentioned in this context are preferably eukaryotic cells.

The invention is illustrated graphics.

Figure 1. Physical map of recombinant plasmid DNA pBIGAPS1-M.

Recombinant plasmid DNA pBIGAPS1-M containing 8123 BP encoding secreterial mutant form of IgA1 proteases, consisting of:

bigaps1-M - region encoding a signal peptide, secreterial mutant form of IgA1 protease and his-tag tag and flokiruemaya unique sites for the restriction endonucleases NdeI (3051) and XhoI (158) (the numbering given in accordance with the numbering in the vector pet-24A), bigaps1-M also contains a unique restriction site of the endonuclease BamHI, ori - the site of initiation of replication of the recombinant plasmid DNA, T7lac promoter is a hybrid promoter transcription Stop the terminator of transcription of ribosomal operon of E. coli, kan gene that determines resistance of the microorganism to kalamitsi is, f1 origin - plot, which allows to obtain single-stranded DNA lacI - region encoding the lac repressor.

Figure 2. The level of specific antibodies in the blood of immunized mice.

Figure 3. Protection of mice immunized with IgAl-protease after infection with meningococcus serogroup In strain N/76.

Figure 4. The level of immunoglobulin IgA1 found in the commercial product.

Figure 5. Electrophoresis in LTO-PAG.

Example 1

Determining and obtaining polynucleotide encoding IgA1 protease of Neisseria meningitidis serogroup In strain N/76 mutation catalytically active residue Ser267 (SEQ ID NO:1)

Based on the known sequence of the gene fragment of iga strain N/76 encoding form of the protease, similar in size to Sekretareva (about 900-1000 amino acid residues, S.A.), which is described in additional example 1, primers were designed to replace the catalytically active residue Ser267 on Ala and subsequent cloning in the expression vector. The structure chosen primers allows you to amplify the product of the polymerase chain reaction (PCR), suitable for subsequent embedding in the expression vectors of the family pet (Novagen), used for protein in E. coli strains, lysogenic on bacteriophage T7.

To obtain a mutant form of IgA1 proteases in which the catalytically active residue Ser267 replaced Ala, method was used SOE (splicing by overlap extension, splicing when extending partially overlapping sequences). For this we have designed specific primers f3 and f4 (f3 - direct - 5'-GCTGTGTTAGGCGATGCCGGATCCCCATTATTTGCTTATG-3' SEQ ID No. 5, f4 - reverse - 5'-CATAAGCAAATAATGGGGATCCGGCATCGCCTAACACAGC-3' SEQ ID No. 6), overlapping in the field site mutations, in addition, these primers introduced a restriction site for the endonuclease BamHI.

For subsequent cloning into the vector pet-24 (+) primers (f1 - direct - 5-CGAGACAGCCATATGAAAACCAAACGTTTTAAAATTAAC-3 SEQ ID No. 3, f2 - back - 5-GGGCTCGAGATTGTACAATCGGGTAATACCG-3 SEQ ID No. 4)flanking the target gene iga strain N/76, the input sequence of recognition sites of restricted NdeI and XhoI respectively for f1 and f2), available polylinker vector. The structure of the primer f2 do not contain termination codons, allows you to get the protein that has been modified in the C-terminal region getservletinfo label for subsequent metallogenetic chromatography selecting it.

Amplification of a fragment of the gene was performed in two stages, using as template the plasmid DNA pBIGAPS1 described earlier in the application EN 2011106149.

In the first stage, conducted two parallel reaction amplification of a fragment of the gene, one using primers f1 and f4, the other with f2 and f3. This has resulted in two DNA fragments with a length of about 800 and 2100 BP, respectively. The second stage was p the obtained DNA fragment of about 2900 BP when used in the amplification reaction as template DNA fragments obtained in the first stage, as well as primers f1 and f2.

Amplification of fragments of nucleic acids was performed according to standard Protocol for 25 cycles of PCR. The annealing temperature of the primers were selected experimentally. The processing of the PCR fragment and the vector restrictase, ligation and transformation of competent cells was performed according to standard procedures. The target PCR product was cloned into the vector pet-24A(+) (Novagen, cat. No. 69749-3) when using unique restriction sites NdeI and XhoI so that the produced protein contained a His-tag at the C-terminal region for subsequent protein purification using metallogenetic chromatography. Sequencing the DNA confirmed the presence of mutations in the protein (SEQ ID NO:2). The obtained plasmid DNA was named pBIGAPS1-M.

Example 2

A) preparation of recombinant plasmid DNA pBIGAPS1-M

Recombinant plasmid DNA was obtained from cells of the night culture standard alkaline method (Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual, Cold Sping Harbor Laboratory Press) and used for introduction into competent cells by heat shock. To do this, 50 μl of a suspension of competent cells were added to 100 ng - 1 µg of plasmid DNA and incubated for 40 min on ice. The resulting mixture was incubated at 42°C in the course is 2 min, then incubated in ice for 2 min, was added 1 ml of cold LB broth and incubated for 1 h at 37°C. then 100 μl of the suspension of bacterial cells were sown on Petri dishes with agar containing 50 μg/ml kanamycin. The cups were incubated over night at 37°C, bacterial cells several grown colonies were used for independent seeding microbiological test tubes with 5 ml of liquid LB medium containing 50 μg/ml kanamycin, followed by growing bacterial cells on the rocking chair at 37°C over night. Then the test tube was added glycerol to a final concentration of 15%, the cell suspension was poured into plastic tubes and stored at -70°C.

Physical map of recombinant plasmid DNA is shown in figure 1.

B) Obtaining strain

The strain E. coli BL21(DE3)/pBIGAPS1-M was obtained by introducing plasmid DNA pBIGAPS1-M in competent cells of E. coli BL21(DE3) using the method of thermal shock. To obtain competent cells, bacterial cells, the source of the Escherichia coli strain BL21(DE3) (E. coli F-dcm ompT hsdS(rB-mB-) gal λ(DE3)) were grown in liquid LB medium overnight at 37°C and used for inoculation of 100 ml of the same medium. The culture was grown to early logarithmic phase in a rocking chair under intensive aeration (up to an optical density at 600 nm=0.4÷0.5), quickly cooled in ice, tile and besieged by centrifugation for 10 min at 5000 rpm at 4°C. The precipitate was twice washed with 50 ml of 100 mm CaCl2. Precipitated cells resuspendable in 2 ml of 100 mm CaCl2with the addition of glycerol to 10%. Was divided into aliquots of 50 μl, were rapidly frozen and stored at -70°C.

Example 3

Determination of productivity of the producer strain IgA1 protease

15 ml liquid LB medium containing 50 μg/ml kanamycin, contributed 1% of the inoculate overnight culture of cells was grown at 37°C on a shaker at 180 rpm for 2 h until the turbidity of 0.8. Then added the field isopropylcyclopentadienyl (IPTG) to a final concentration of 0.5 mm and continued incubation in the same conditions during 2,0 hours, a sample was Taken 1 ml and centrifuged for 6 min at 12000 rpm, after which the cells suspended in 100 μl of deionized water was added 33 μl of buffer solution containing 125 mm Tris-HCl, pH 6.8, 20% glycerol, 3% sodium dodecyl sulphate, 3% mercaptoethanol and 0.01% bromophenol blue, heated for 10 min to 95°C. was Selected aliquot was analyzed by electrophoresis in 10% polyacrylamide gel containing 0.1% sodium dodecyl sulphate. The gel was stained with Kumasi R250 and scanned by a laser scanner. The productivity of the strain E. coli BL21(DE3)/pBIGAPS1 is 40% of the enzyme from the total cellular protein.

Example 4

Selection of recombinant mutant IgA1 protease

Cell disruption and fractionation of the lysate

Razor is defined after storage cells (2 g of 1 l of induced culture) resuspendable in nine-fold volume of 20 mm Tris-HCl-buffer, pH 8.5, containing lysozyme. Suspension of five cells were treated with ultrasound and was subjected to centrifugation (1 hour, 20000 g). Separated fractions of soluble proteins (cell-free extract, NFE) and the insoluble portion of the cell lysate (debris and insoluble proteins) were isolated. According to the results of electrophoretic analysis of the target protein forms insoluble bullock inclusion (TV), regardless of the temperature conditions of the induction culture and the type of buffer used for the destruction of cells. The insoluble portion of the cell lysate containing the TV after centrifugation, washed: a) 60 ml of a buffer of 20 mm Tris-HCl, 1% Triton X-100, 0.1% SDS, and centrifuged for 40 min, 10000 g; b) 60 ml of a buffer of 20 mm Tris-HCl, I M NaCl, centrifuged for 40 min, 10000 g. The sediment used for carrying out chromatography on Ni-agarose under denaturing conditions.

Metallokhelaty chromatography on Ni-agarose

TV, extracted from 2 g of cells was dissolved in 20 ml of 20 mm Tris-HCl, pH 8.5, containing 8 M urea and 5 mm dithiothreitol (DTT). The total amount of protein contained in the TV, was about 40 mg when determining by the method of Bradford. After centrifugation and filtration, the solution was incubated overnight with 10 ml Ni-agarose (Qiagen, Cat No 30410), equilibrated with the same buffer. Then the sorbent was washed 10V column buffer containing 20 mm Tris-HCl pH 8.5, 8 M urea, 250 mm NaCl. Bound peroxidase is white and has suirable 5 volumes of column buffer, containing 20 mm Tris-HCl pH 8.5, 8 M urea, 250 mm NaCl, 400 mm imidazole. Fractions containing purified protein were pooled and used for transferring the IgA1 protease in soluble form. The outlet of a partially purified preparation of the target protein at this stage is 30 mg of 2 g of biomass cells.

Refolding IgA1 protease

The combined eluate (30 ml, 1 mg/ml)obtained after chromatography on Ni-agarose, were subjected to a stepwise dialysis, gradually reducing the concentration of urea to the value of 1.5 M (which allows to obtain a preparation with a high concentration of protein) in 20 mm Tris-HCl buffer, pH 8.5, 1 mm DTT. Then the protein solution was subjected to concentration using ultrafiltration method. Then the solution was subjected to centrifugation (30 min, 5000 g) and filtered through a sterile membrane with pore size 0.2 μm. The concentration of protein in the final preparation is 1.8 mg/ml, the volume of 10ml

The protein yield at the final stage is 18 mg of 2 g cell biomass. The purity of the drug - not less than 90%.

Example 5

The definition of immunogenic and protective activity

Immunogenic and protective activity obtained IgA1 protease was evaluated in mice of Balb/C. For the evaluation of these indicators, animals were immunized with various preparations IgA1 protease (native IgA1 protease isolated from the culture of Neisseria meningitidis serogroup a, obtained as described in patent RU 2407792, recombinant IgA1 protease serogroup B, obtained as described in supplementary example 1 mutant IgAl protease serogroup B, obtained as described in the present description, example 4) a single dose of 20 μg/mouse. According to ELISA analysis as described in the patent of Russian Federation №2310853, mutant recombinant IgA1 protease in contrast to native and recombinant nematanthus IgA1 protease does not have a specific enzymatic activity, i.e. not hydrolyzes human IgA1.

Indicator of immunogenicity is obtained IgA1 protease is the increase of specific antibodies in the blood of mice on the 30th day after immunization, as determined by ELISA.

Figure 2 shows a diagram of immunogenic activity of the above drugs IgA1 protease. The ordinate axis is the optical density of the serum at λ=492 nm. Already after a single immunization, the level of specific antibodies in sera of mice immunized with mutant IgA1 protease, increases to 1:5000, which is higher than in mice immunized with native and recombinant IgA1 protease (1:2560).

1 month after immunization, the mice were divided into three groups and infected live virulent culture of Neisseria meningitides serogroups And strain A208, strain N/47 and strain 0638.

Protective activity of IgA1 protease was assessed by the level of bacteremia at immunizer the bathrooms animals 4 hours after infection with meningococcal disease compared with the same indicator control (not immunized) mice.

In figure 3 presents the level of bacteremia (%) in mice immunized once the above mentioned drugs, IgA1 protease (shaded bars)after infection with meningococcus serogroups a, b and C compared to the control, not immunized animals (black bars). The level of bacteremia in the group of mice immunized with mutant protease, respectively for serogroups a, b and C 13, 19 and 17 SOME, in terms of 100 CFU in the control (non-immunized mice).

Additional example 1

A) Determining and obtaining nucleic acid encoding IgA1 protease of Neisseria meningitides serogroup In strain N/76 (SEQ ID NO:7)

The analysis of genomic data Bank (http://www.ncbi.nlm.nih.gov/Genbank) revealed that at the present time for Neisseria meningitidis serogroup known In only the genome of strain MS. On the basis of assumptions about the proximity of the structure of genes IgA1 proteases related strains on the known sequence of the gene iga MZ designed primers for cloning of the fragment of the nucleic acid (gene) strain N/76 encoding form of the protease, similar in size to Sekretareva (about 900-1000 amino acid residues, S.A.). The structure chosen primers allows you to amplify the product of the polymerase chain reaction (PCR), suitable for subsequent embedding in the expression vectors of the CE is ASTA pet (Novagen), used for protein in strains of E. coli, lysogenic on bacteriophage T7.

For subsequent cloning into the vector pet-24 (+) primers (f1 - direct - 5-CGAGACAGCCATATGAAAACCAAACGTTTTAAAATTAAC-3 SEQ ID No. 3, f2 - back - 5-GGGCTCGAGATTGTACAATCGGGTAATACCG-3 SEQ ID No. 4), potentially flanking the target gene iga strain N/76, the input sequence of recognition sites of restricted NdeI and XhoI respectively for f1 and f2), available polylinker vector. The structure of the primer f2 do not contain termination codons, allows you to get the protein that has been modified in the C-terminal region getservletinfo label for subsequent metallogenetic chromatography selecting it.

The amplification of the fragment of the nucleic acid is performed by standard Protocol for 25 cycles of PCR using as template genomic DNA of strain N/76. The annealing temperature of the primers are selected experimentally. The processing of the PCR fragment and the vector restrictase, ligation and transformation of competent cells performed by standard procedures. Next, perform sequencing of the cloned DNA with multiple primary clones in the first set of the nucleotide sequence of the fragment of the nucleic acid encoding secreterial form IgA1 protease from Neisseria meningitides N/76, on the basis of which is determined amino acid sequence of the tree (SEQ ID NO:8). The obtained plasmid DNA was named pBIGAPS1.

B) preparation of recombinant plasmid DNA pBIGAPS1

Recombinant plasmid DNA is obtained from cells overnight culture standard alkaline method (Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual, Cold Sping Harbor Laboratory Press) and used for the introduction of competent cells by heat shock. To do this, 50 μl of a suspension of competent cells was added 100 ng - 1 µg of plasmid DNA and incubated for 40 min on ice. The resulting mixture was incubated at 42°C for 2 min, then incubated in ice for 2 min, add 1 ml of cold LB broth and incubated for 1 h at 37°C. then 100 μl of the suspension of bacterial cells plated on Petri dishes with agar containing 50 μg/ml kanamycin. Cup incubated over night at 37°C, bacterial cells several grown colonies used for independent seeding microbiological test tubes with 5 ml of liquid LB medium containing 50 μg/ml kanamycin, followed by growing bacterial cells on the rocking chair at 37°C over night. Then in a test tube add glycerol to a final concentration of 15%, the cell suspension is poured into plastic tubes and stored at -70°C.

Physical map of recombinant plasmid DNA is shown in figure 1.

C) Obtaining strain

The strain E. coli BL21(DE3)/pBIGAPS1 get PU is eating the introduction of plasmid DNA pBIGAPSI in competent cells of E. coli BL21(DE3) using the method of thermal shock. To obtain competent cells, bacterial cells, the source of the Escherichia coli strain BL21(DE3) E. coli In F-dcm ompT hsdS(rB-mB-) gal λ(DE3)) grown in liquid LB medium overnight at 37°C and used for inoculation of 100 ml of the same medium. The culture is grown to early logarithmic phase in a rocking chair under intensive aeration (up to an optical density at 600 nm=0.4-0.5), quickly cooled in ice, the cells are precipitated by centrifugation for 10 min at 5000 rpm at 4°C. the Precipitate washed twice in 50 ml of 100 mm CaCl2. Precipitated cells resuspended in 2 ml of 100 mm SAS2with the addition of glycerol to 10%. Divided into aliquots of 50 µl, quickly frozen and stored at -70°C.

G) Isolation of recombinant IgA1 protease

Cell disruption and fractionation of the lysate

Thawed after storage cells (2 g of 1 l of induced culture) resuspending in nine-fold volume of 20 mm Tris-HCl buffer, pH 8.5, containing 1% Triton-X100, or in a detergent-containing 20 mm potassium-phosphate buffer, pH 7.5. Suspension of five cells treated with ultrasound and subjected to centrifugation (1 hour, 20000 g). Separated fractions of soluble proteins (cell-free extract, NFE) and the insoluble portion of the cell lysate (debris and insoluble proteins) are isolated. According to the results of elec is reparationswho analysis of the target protein forms insoluble bullock inclusion (TV) regardless of the temperature conditions of the induction culture and type of buffer, used in the destruction of the cells. The insoluble portion of the cell lysate containing TV, after resuspendable in 1% Triton-X100 in the repeated sonication and subsequent centrifugation is used for chromatography on Q-sepharose under denaturing conditions.

Anion-exchange chromatography on Q-sepharose

TV, extracted from 2 g of cells, induced at 25°C, dissolved in 20 ml of Tris-HCl buffer, pH 8.5, containing 6 M urea and 20 mm dithiothreitol (DTT). The total amount of protein contained in the TV, is about 50 mg when determining by the method of Bradford. After centrifugation and filtration, the solution is applied at a rate of 1 ml/min to 10 ml of anion-exchange sorbent (two pre-Packed cartridge-column HiTrap Q Sepharose HP, GE Healthcare, Sweden)equilibrated with the same buffer. Fractions containing purified protein, and the resulting gradient elution with sodium chloride (100 ml, 0-1 M NaCl in the starting buffer, combine (total 20 ml) and used for transferring the IgA1-protease into a soluble form. The outlet of a partially purified preparation of the target protein at this stage is 40 mg of 2 g of biomass cells.

Refolding IgA1 protease

The combined eluate (20 ml, 2 mg/ml)obtained after chromatography on Q-sepharose, diluted 10 times in 20 mm Tris-HCl-buffer, pH 8.5, using a rapid dilution (in capitania of protein solution in buffer). After slow stirring of the solution (at a concentration of 0.2 mg protein/ml) for 20 hours and subsequent concentration by ultrafiltration until the original volume of the protein solution three times cialiswhat against 1 l of 20 mm Tris-HCl buffer, pH 8.5. Then, the solution is subjected to centrifugation (30 min, 5000 g) and filtered through a sterile membrane with pore size of 0.2 microns.

The protein yield at the final stage is 20 mg of 2 g cell biomass. The purity of the drug - not less than 90%.

Additional example 2

Evaluation of the enzymatic activity of samples recombinantly active and mutant IgA1 protease

In two samples of active recombinant and mutant IgA1 protease (sample 1 and sample 2) were determined by enzymatic activity by ELISA using as substrate myeloma IgA1 person and conjugate anti-human IgA with horseradish peroxidase, as described in the patent of Russian Federation №2310853.

Analysis of the enzymatic activity of the samples recombinant IgA1 protease showed (figure 4) the ability of the active IgA1 proteases to catalyze the hydrolysis of immunoglobulin IgA1 and lack of proteolytic activity in the mutant sample.

The level of proteolytic activity IgA1 protease was determined by electrophoretic method in LTO-PAG. Figure 5 shows the hydrolysis of IgA1 in the presence of different concentrations of active enzyme is its mutant forms (sample 1 and sample 2, respectively.

Species-specific activity of the obtained preparations IgA1 protease was evaluated by ELISA for the level of IgA1 in the sera of mice and humans. The level of antibodies to the IgA1 protease in the blood of animals immunized with mutant drug was 1:1280 and not depended on the presence of enzymatic activity in the sample of active protease (see table).

Assessment of species specificity samples IgA1 protease
Sample numberSample IgA1 proteaseTitle IgA1 antibodies
in mice*man**
1active1/12801/20
2mutant1/12801/640

Thus, on the basis of the obtained plasmid DNA pBIGAPS1-M created the expression strain E. coli superproducer IgA1 protease serogroup B, isolated protein and implemented its refolding, allowing you to get IgA1 protease in the number of level 10-40 mg of 2 g of biomass cells.

First the results obtained, widelystudied higher immunogenic and protective activity of mutant IgA1 protease in comparison with recombinant meningococcal protease serogroup B, as described in the article Serov O.V. and other, life science journal, 2012, volume 2, No. 1. The resulting preparation mutant IgA1 protease has a polyvalent action in protection against meningococcal infection of an animal's body regardless of the source of its receipt (serogroup a or b). The lack of enzymatic activity in respect of immunoglobulin A1 human mutant IgA1 protease will allow to avoid additional complications of immunization of man. Presents data on the immunogenicity and protectively suggest possible to use it as poliomyelitis for the treatment of bacteria carrier and prevention of meningococcal infection major epidemic serogroups a, b and C, primarily for prevention of meningococcal infections caused by bacteria N. meningitidis serogroup B, as well as other pathogens, the activity of which is due to the presence of IgA1 protease.

1. Polynucleotide encoding mutant IgA1 protease of Neisseria meningitidis with the nucleotide sequence (SEQ ID NO:1).

2. Recombinant plasmid DNA, including polynucleotide according to claim 1 and providing for the expression of mutant IgA1 protease in the cell host.

3. A host cell transformed with recombinant plasmid DNA according to claim 2, producing the Mature mutant form of IgA1 protease.

4. A host cell according to claim 3, where procario is practical a host cell selected from the series including bacteria of the genus Escherichia, Bacillus, Lactococcus, Lactobacillus, Pseudomonas, Streptomyces, Streptococcus, Staphylococcus.

5. The method of obtaining Mature mutant form of IgA1 protease of Neisseria meningitidis serogroup B, which consists in the fact that the cell host transformed with recombinant plasmid DNA according to claim 2, culturing the transformed cell according to claim 3 with obtaining Mature mutant form of IgA1 protease, and the target product is separated from Taurus enable and clear.

6. Mutant IgA1 protease N.meningitidis serogroup In the amino acid sequence SEQ ID NO:2, having a high level of immunogenicity compared with the wild-type enzyme.



 

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