RU2486243C1 - 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 - Google Patents

Abstract

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

Description

The invention relates to the field of immunology, enzymology, microbiology and genetic engineering and relates to a new polynucleotide encoding a functionally active recombinant IgA1 protease Neisseria meningitidis, a new recombinant plasmid DNA pBIGAPS1-M and a host cell, especially a new bacterial strain Escherichia coli BL21 DE (DE pBIGAPS1-M, which can be used to obtain highly purified genetically engineered IgA1 protease and as a component of a multivalent vaccine designed to protect humans, especially children, from meningococcal infection uu and other microorganisms which are pathogenic due IgA1 protease.

The problem of effective vaccine prophylaxis of meningococcal infection caused by meningococcus serogroup B has not yet been resolved. The WHO registry of registered meningococcal vaccines has not recorded a single drug against meningococcus of this serogroup. The lack of an effective meningococcal group B vaccine prevents the eradication of this infection in our country and beyond. It is known that serine IgA protease is secreted by some dangerous bacteria, primarily Neisseria meningitidis, and breaks down class A immunoglobulins on the surface of mucous membranes, destroying the first protective barrier of the host organism's immunity. Known meningococcal IgA1 protease, which in experiments on laboratory animals proved to be a highly effective meningococcal polivaccine, and therefore the vaccine against serogroup B meningococcus (US Pat. RU 2407792, Alliluyev A.P. et al., 16.08.2010 g). It was found that IgA1 protease isolated from the culture fluid or from the purification products of the vaccine (meningococcal group A) is highly effective enzymatically and immunologically, but is contained in the indicated amounts in micro quantities, therefore, it may be difficult to obtain the protease in preparative amounts.

The prior art describes many attempts to create vaccines designed to protect against infection of group B meningitis. In the 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 offer compositions and candidates for vaccines based on lipopolysaccharide, capsular oligosaccharides and a number of surface protein meninges for immunization of a patient against a disease caused by Neisseria meningitidis of various serogroups.

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

WO 2004/102199 A2, LEENHOUTS et al., November 25, 2004, describes a method for identifying, selecting and isolating a vaccine component for passive and active immunization against a Streptococcus pneumonia microorganism that is destroyed by opsonophagocytic cells. The method of computer simulation (in silico) identified antigens inducing opsonophagocytosis, which are proposed to be used as components of a vaccine intended for immunization. Three pneumococcal proteins have been identified: streptococclipoproteinaromatase (SlrA), metalloprotease class IgA1 protease and pneumococcal surface adhesion protein A (PsaA) from Streptococcus pneumonia, and their recombinant variants have been obtained and studied as vaccine components. It was found that the composition containing all three recombinant variants protects the mice from the specified infection, however, each component individually does not have protective properties.

The article by Shishir K. Gupta et al., Vaccine, 2010, v. 28, pp. 7092-7097, describes the results of computer simulation of serogroup B meningococcal protein sequences and identifies 6 peptides of T-helper cell epitopes of three proteins: stimulating T- protein A cells, motor protein A and IgA1 protease. The authors suggest that these peptides can be used as promising agents against group B meningococcus. However, experimental data confirming this hypothesis are not presented.

It is known to obtain a fragment of an IgA1 protease that is used as a carrier peptide (US Pat. US 7,235,242, Achtman et al., June 26, 2007). The synthesis of an IgA1 protease fragment containing from 40 to 200 amino acid residues and including 40 amino acid residues of the sequence SEQ ID NO: 1 is described using an automatic synthesizer. However, when conjugated with an IgA1 polysaccharide, the protease provides protection only against serogroup C meningococcus and is not effective against serogroup B meningococcus.

The use of bacterial IgA1 proteases for the treatment of diseases associated with IgA1 deposits in the tissues and organs of the body is known (US Pat. US7407653, Plaut et al., 05.08.2008). Soluble IgA1 proteases containing various labels, for example a polyhistidine fragment, are produced by a recombinant method. The enzyme is isolated by a known method 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 the accumulation of IgA, but not as a vaccine.

There is also known a method of obtaining a precursor of IgA1 protease. In an article by S. Vitovski and J.R. Sayers, Infection and Immunity, 2007, v. 75, No. 6, pp. 2875-2885, describes the cloning of a full-sized gene encoding a signal peptide, a secreted form of a proteinase and an autotransporter domain, and an investigation of the autoprocessing mechanism of an IgA1 protease from its predecessor. To this end, a series of mutated forms of the serum group B Neisseria meningitidis serum B (NMB) precursor protein IgA1 were created, including a mutant with a deletion of a 32-membered peptide at the site of the autoprocessing site, as well as a mutant involving the replacement of the Ser residue in the active center with the Val residue with the aim of obtaining a precursor that does not have autoproteolytic activity. To obtain the mutants, NMB strains isolated from the cerebrospinal fluid of patients with meningococcus were used. It was found that these mutants did not possess autoproteolytic activity and the formation of secreted mature protease was not observed. But in this work, only the precursor of IgA1 protease was obtained. The protective and immunogenic properties of IgA1 protease itself have not been investigated, and the possibility of creating a vaccine based on it has not been considered.

In the application LONG and others, WO 20101183337 (A2), 10/14/2010, describes a method for producing a soluble recombinant IgA1 protease using direct and / or indirect production through inclusion bodies with a significantly higher yield compared to previously known methods, the yield Between 20 and 500 mg of soluble IgA1 protease / L culture medium. However, the preparations and compositions of IgA1 proteases obtained by this method are enzymatically active and are 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. In addition, the work received IgA1 protease of H. influenzae expressed in E. coli.

A known method for producing recombinant IgA1 protease Neisseria meningitidis serogroup B (NMB) (Serova OV and others, Biopharmaceutical journal, 2011, t.3, No. 6), according to which received a full-sized enzymatically active IgA1 protease with immunogenic and protective properties in experiments on mice with respect to meningococci of serogroup B. However, this drug has enzymatic activity against human IgA1, which can lead to undesirable consequences when splitting immunoglobulin A1 on the mucous membranes of the body, for example p development of inflammatory response. This method as the closest prior art was selected as a prototype.

The invention solves the problem of obtaining a recombinant full-sized IgA1 protease with enhanced immunogenic and protective properties against Neisseria meningitidis of the main epidemic serogroups A, B and C, but lacking enzymatic activity against human IgA1, to create a multivalent vaccine to protect a person from meningococcal infection , and from other pathogens caused by IgA1 protease.

The problem is solved by polynucleotide encoding IgA1 protease Neisseria meningitidis serogroup B strain H44 / 76 with a mutation of the catalytically active residue Ser267 on Ala (SEQ ID NO: 1);

recombinant plasmid DNA comprising the above nucleotide sequence and providing expression of the IgA1 protease in the host cell (pBIGAPS1-M), characterized by the following features: chain length is 8123 bp; encodes a secreted form of IgA1 protease with a mutation of the catalytically active Ser267 residue on Ala, with a signal sequence at the N-terminus and a histidine tag at the C-terminus of the protein;

the nucleotide sequence encoding the IgA1 protease in the plasmid pBIGAPS1-M is flanked by unique restriction sites by endonuclease NdeI and XhoI;

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

contains a T7 lac transcription promoter;

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

a host cell, especially a producer strain of E. coli BL21 (DE3) / pBIGAPS1-M, containing recombinant plasmid DNA that produces a mature form of a mutant IgA1 protease;

mutant IgA1 protease with a molecular weight of 107.6 kDa and an calculated isoelectric point of 7.4, with the amino acid sequence (SEQ ID NO: 2) encoded by the above nucleotide sequence having immunogenic and protective properties, designed to obtain a multivaccine for the prevention of meningococcal infections, in including those caused by bacteria N. meningitidis serogroup B;

and also due to the method of obtaining a mature form of IgA1 protease of Neisseria meningitidis serogroup B, which consists in the fact that the host cell is transformed with a recombinant plasmid DNA encoding the mutant IgA1 protease Neisseria meningitidis serogroup B, with a polynucleotide (SEQ ID NO: 1), cultured producer, isolate and purify the target product from inclusion bodies.

The technical result of the invention is to obtain a highly purified full-sized enzymatically inactive mutant IgA1 protease of N. meningitidis serogroup B (40% of the enzyme from the total cellular protein, 20 mg from 2 g of cell biomass, the purity of the drug is not less than 90%), characterized by increased immunogenic and protective activity in Comparison with enzymatically active native and recombinant proteases. The resulting preparation of mutant IgA1 protease has a multivalent effect with respect to protection against meningococcal infection of the animal organism, regardless of its source (serogroup A or B). The lack of enzymatic activity against human immunoglobulin A1 mutant IgA1 protease will avoid additional complications during human immunization.

One aspect of the present invention includes a recombinant plasmid comprising the aforementioned nucleotide sequence and allowing expression of a mutant IgA1 protease in a host cell.

To obtain the mutant form of IgA1 proteinase, in which the catalytically active Ser267 residue is replaced by Ala, the SOE method (splicing by overlap extension, splicing during the expansion of partially overlapping sequences) was used. For this, specific primers were designed (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6), overlapping in the region of the mutation site, in addition, the BamHI endonuclease restriction site was introduced into these primers . The amplification of the gene fragment was carried out in two stages, using the plasmid DNA pBIGAPSI as described earlier in the application RU 2453599, L. Rumsh. et al., dated February 18, 2011. A DNA fragment of about 2900 bp was obtained. The target PNR product was cloned into pET-24a (+) vector (Novagen, Cat. No. 69749-3) using unique NdeI and XhoI restriction sites so that the protein produced contained His tag in the C-terminal region for subsequent purification protein using metal chelate chromatography. Sequencing of the obtained DNA confirmed the presence of a mutation. The resulting plasmid DNA was named pBIGAPS1-M.

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

Another object includes a method for producing a mature mutant form of IgA1 protease of Neisseria meningitidis serogroup B, which is that the host cell of E. coli BL21 (DE3) is transformed with the above recombinant plasmid DNA encoding IgA1 protease of Neisseria meningitidis serogroup B with a polynucleotide (SE NO: 1), host cells are cultured as described below, the target product is isolated and purified from inclusion bodies.

Another object of the present invention includes an IgA1 protease with a molecular weight of 107.6 kDa and an calculated isoelectric point of 7.4 and the amino acid sequence (SEQ ID NO: 2) encoded by the polynucleotide described above and obtained by the above method, designed to obtain a multivaccine for the prevention of infections , including those caused by bacteria N. meningitidis serogroup B.

IgA1 protease as an active component can be used to obtain a multivaccine for the prevention of meninococcal infection of serogroups A, B, C, as well as for infections whose pathogenicity is due to IgA1 protease, such as Clostridium ramosum, Bacteroides melaninogenicus, Capnocytophaga ochracea, Streptocococcus pneumonia, Streptocococcus pneumonia, Streptocococcus pneumonia, Streptocococcus pneumonia , 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 a producer strain of IgA1, proteases transform competent Escherichia coli cells BL21 (DE3) (E. coli B F ^- dcm ompT hsdS (r _B ^- m _B ^- ) gal λ (DE3)) (Stratagene Cat. No 200131) plasmid DNA pBIGAPS1 -M.

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

Cultural and morphological characters.

The cells are small, rod-shaped, gram-negative, 1 × 3.5 μm, motile. The strain grows well on ordinary nutrient media (LB-broth, LB-agar). When growing on LB agar medium, the colonies are rounded, smooth, translucent, shiny, yellowish. The edge is even, the diameter of the colonies is 2-3 mm, the consistency is pasty. Growth in a liquid LB medium is characterized by even turbidity, the sediment easily sedimentes.

Physiological and biochemical characteristics.

Cells grow at 4-42 ° C; optimum pH is 6.8-7.6. As a source of nitrogen, both ammonium mineral salts and organic compounds are used: amino acids, peptone, tryptone, yeast extract.

Genetic signs.

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

Storage conditions.

The bacterial strain E. coli BL21 (DE3) / pBIGAPS1-M is stored on plates and jambs at 4 ° C. Reseeding on fresh media is carried out once a month. It can be stored for at least one year in LB medium containing 15% glycerol at -70 ° C.

Antibiotic resistance.

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

Eukaryotic cells (e.g., yeast cells, plant cells, animal cells and human cells), or prokaryotic cells (e.g., bacterial cells, etc.), or cells that induce an immune response can be used as the host cell. Specialists in this field know many of these cells and cell lines of (higher) eukaryotes, for example 293T (line of embryonic kidney cells), HeLa (human cervical carcinoma cells), CHO (Chinese hamster ovary cells) and other cell lines, including these cells and cell lines designed for laboratory research, such as, for example, hTERT-MSC, HEK293, Sf9 or COS cells. Suitable eukaryotic cells also include cells or cell lines affected by diseases or infections, for example cancer cells, especially cancer cells of any type of cancer mentioned in this context, cells infected with HIV and / or cells of the immune system or central nervous system (CNS). Suitable cells can be similarly obtained from eukaryotic microorganisms such as yeast, for example Saccharomyces cerevisiae (Stinchcomb et al., Nature, v. 282, p. 39, (1997)), Schizosaccharomyces pombe, Candida, Pichia and hyphomycetes of the genera Aspergillus, Penicillium etc. Suitable cells likewise include prokaryotic cells, such as, for example, bacterial cells, for example Escherichia coli, or bacteria of the genus Bacillus, Lactococcus, Lactobacillus, Pseudomonas, Streptomyces, Streptococcus, Staphylococcus, preferably E. coli, and the like. Human cells or animal cells, for example animals referred to in this context, are primarily preferably eukaryotic cells.

The invention is illustrated by graphic materials.

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

Recombinant plasmid DNA pBIGAPS1-M containing 8123 bp encoding a secreted mutant form of IgA1 proteinase, consisting of:

bigaps1-M is a region encoding a signal peptide secreted by a mutant form of IgA1 protease and a histidine tag and flanked by unique restriction sites by the NdeI (3051) and XhoI (158) endonucleases (the numbering is according to the numbering in the pET-24a vector), bigaps1-M also contains a unique restriction site with BamHI endonuclease, ori - a site for the initiation of replication of recombinant plasmid DNA, T7lac promoter - a hybrid transcription promoter, Stop - a terminator for transcription of the ribosomal operon E. coli, kan - a gene that determines the resistance of a microorganism to kanamits Well, f1 origin - land, which yields a single-stranded DNA, lacI - the region encoding the lac repressor.

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

Figure 3. The protection of mice immunized with an IgAl protease after infection with serogroup B meningococcus strain H44 / 76.

Figure 4. IgA1 immunoglobulin level detected in a commercial preparation.

Figure 5. Electrophoresis in SDS-PAGE.

Example 1

Determination and preparation of a polynucleotide encoding IgA1 protease of Neisseria meningitidis serogroup B strain H44 / 76 with a mutation of the catalytically active residue Ser267 (SEQ ID NO: 1)

Based on the known sequence of the iga gene fragment of strain H44 / 76 encoding a protease form that is close in size to the secreted one (approximately 900-1000 amino acid residues, amino acid residues), which is described in Supplementary Example 1, primers were designed to replace the catalytically active residue Ser267 on Ala and subsequent cloning into an expression vector. The selected primer structure allows amplification of the product of polymerase chain reaction (PCR), suitable for subsequent incorporation into expression vectors of the pET family (Novagen), used to obtain proteins in E. coli strains lysogenic by T7 bacteriophage.

To obtain the mutant form of IgA1 proteinase, in which the catalytically active Ser267 residue is replaced by Ala, the SOE method (splicing by overlap extension, splicing during the expansion of partially overlapping sequences) was used. For this, specific primers f3 and f4 were designed (f3 - direct - 5'-GCTGTGTTAGGCGATGCCGGATCCCCATTATTTGCTTATG-3 'SEQ ID No. 5, f4 - reverse - 5'-CATAAGCAAATAATGGGGGATCCGGCATCG6QACAC site 3'-sites) of this, a BamHI endonuclease restriction site was introduced into these primers.

For subsequent cloning in the pET-24 vector (a +) into primers (f1 — direct — 5-CGAGACAGCCATATGAAAACCAAACGTTTTAAAATTAAC-3 SEQ ID No. 3, f2 — reverse — 5-GGGCTCGAGATTGTACAATCGGGTAATACCGma-3 SEQ ID NO. 3) H44 / 76, the sequences of restriction enzyme recognition sites NdeI and XhoI (respectively for f1 and f2), introduced in the vector polylinker, were introduced. The structure of primer f2, which does not contain termination codons, allows one to obtain a protein modified in the C-terminal region with a hexahistidine tag for subsequent metal chelate chromatography during isolation.

Amplification of the gene fragment was carried out in two stages, using the plasmid DNA pBIGAPS1 as described previously in the application RU 2011106149 as a matrix.

At the first stage, two amplification reactions of the gene fragment were carried out in parallel, in one using primers f1 and f4, in the other, f2 and f3. As a result, two DNA fragments with a length of about 800 and 2100 bp were obtained, respectively. At the second stage, a DNA fragment of about 2900 bp was obtained. when used in the amplification reaction as a matrix of DNA fragments obtained in the first stage, as well as primers f1 and f2.

Amplification of nucleic acid fragments was carried out according to the standard protocol for 25 cycles of PCR. The primer annealing temperature was selected experimentally. Processing of the PCR fragment and vector with restriction enzymes, ligation and transformation of competent cells was carried out according to standard procedures. The desired PCR product was cloned into pET-24a (+) vector (Novagen, Cat. No. 69749-3) using unique NdeI and XhoI restriction sites so that the protein produced contained His tag in the C-terminal region for subsequent purification protein using metal chelate chromatography. Sequencing of the obtained DNA confirmed the presence of a mutation in the protein (SEQ ID NO: 2). The resulting plasmid DNA was named pBIGAPS1-M.

Example 2

A) Obtaining recombinant plasmid DNA pBIGAPS1-M

Recombinant plasmid DNA was obtained from overnight cells by the standard alkaline method (Maniatis, T., Fritsch, EF and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual, Cold Sping Harbor Laboratory Press) and used to introduce into competent cells using heat shock. For this, 100 ng - 1 μg of plasmid DNA was added to 50 μl of a suspension of competent cells 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, 1 ml of cold LB broth was added and incubated for 1 h at 37 ° C. After that, 100 μl of a suspension of bacterial cells were plated on Petri dishes with agar containing 50 μg / ml kanamycin. The cups were incubated overnight at 37 ° C, the bacterial cells of several grown colonies were used for independent inoculation of microbiological tubes with 5 ml of LB liquid medium containing 50 μg / ml kanamycin, followed by growth of bacterial cells on a shaker at 37 ° C overnight. After that, glycerol was added to the tubes to a final concentration of 15%, the cell suspension was poured into plastic tubes and stored at -70 ° C.

A physical map of recombinant plasmid DNA is shown in figure 1.

B) Obtaining strain

The E. coli BL21 (DE3) / pBIGAPS1-M strain was obtained by introducing pBIGAPS1-M plasmid DNA into competent E. coli BL21 (DE3) cells using the heat shock method. To obtain competent cells, the bacterial cells of the original strain Escherichia coli BL21 (DE3) (E. coli B F ^- dcm ompT hsdS (r _B ^- m _B ^- ) gal λ (DE3)) were grown in LB liquid medium overnight at 37 ° C and used for seeding 100 ml of the same medium. The culture was grown to the early logarithmic phase on a rocking chair under intensive aeration (to an optical density at 600 nm = 0.4 ÷ 0.5), it was quickly cooled in ice, the cells were precipitated by centrifugation for 10 min at 5000 rpm at + 4 ° С. The precipitate was washed twice in 50 ml of 100 mm CaCl ₂ . The precipitated cells were resuspended in 2 ml of 100 mm CaCl ₂ , with the addition of glycerol up to 10%. Aliquots of 50 μl were divided, quickly frozen and stored at -70 ° C.

Example 3

Determination of the productivity of the producer strain of IgA1 protease

In 15 ml of LB liquid medium containing 50 μg / ml kanamycin, a 1% inoculum of overnight cell culture was added and grown at 37 ° C on a shaker at 180 rpm for 2 h until a turbidity of 0.8. Then the isopropylthiogalactopyranoside inducer (IPTG) was added to a final concentration of 0.5 mM and the incubation was continued for 2.0 hours under the same conditions. A 1 ml sample was taken and centrifuged for 6 min at 12,000 rpm, after which the cells were suspended in 100 μl of deionized water, 33 μl of a buffer solution containing 125 mM Tris-HCl, pH 6.8, 20% glycerol, 3% sodium dodecyl sulfate, 3% mercaptoethanol and 0.01% bromophenol blue was added, heated for 10 min to 95 ° FROM. An aliquot was taken and analyzed by electrophoresis on a 10% polyacrylamide gel containing 0.1% sodium dodecyl sulfate. The gel was stained with Coomassie R250 and scanned on a laser scanner. The productivity of E. coli strain BL21 (DE3) / pBIGAPS1 is 40% of the enzyme from the total cellular protein.

Example 4

Isolation of Recombinant Mutant IgA1 Protease

Cell disruption and lysate fractionation

Thawed cells after storage (2 g from 1 L of the induced culture) were resuspended in a seven-fold volume of 20 mM Tris-HCl buffer, pH 8.5, containing lysozyme. The cell suspension was five times sonicated and centrifuged (1 hour, 20,000 g). Separated fractions of soluble proteins (cell-free extract, BE) and the insoluble part of the cell lysate (debris and insoluble proteins) were isolated. According to the results of electrophoretic analysis, the target protein forms insoluble inclusion bodies (TB), regardless of the temperature conditions of the culture induction and the type of buffer used to destroy cells. The insoluble portion of the cell lysate containing TB after centrifugation was washed: a) 60 ml of 20 mM Tris-HCl buffer, 1% Triton X-100, 0.1% SDS, centrifuged for 40 min 10,000 g; b) 60 ml of 20 mM Tris-HCl buffer, IM NaCl, centrifuged for 40 min, 10,000 g. The precipitate was used for chromatography on Ni-agarose under denaturing conditions.

Metal Chelate Chromatography on Ni-Agarose

TB isolated 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 as determined by the Bradford method. After centrifugation and filtration, the solution was incubated overnight with 10 ml of Ni-agarose (Qiagen, Cat No. 30410), equilibrated with the same buffer. Then the sorbent was washed with a 10V column with buffer containing 20 mM Tris-HCl pH 8.5, 8 M urea, 250 mM NaCl. Bound proteins were eluted with 5 column volumes with buffer containing 20 mM Tris-HCl pH 8.5, 8 M urea, 250 mM NaCl, 400 mM imidazole. Fractions containing purified protein were combined and used to convert the IgA1 protease to a soluble form. The yield of a partially purified preparation of the target protein at this stage is 30 mg from 2 g of cell biomass.

IgA1 protease refolding

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

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

Example 5

Determination of immunogenic and protective activity

The immunogenic and protective activity of the obtained IgA1 protease was evaluated in Balb / C mice. To evaluate these parameters, animals were immunized with various IgA1 protease preparations (native IgA1 protease isolated from serogroup A Neisseria meningitidis culture, obtained as described in patent RU 2407792, recombinant serogroup B IgA1 protease obtained as described in Supplementary Example 1, mutant serogroup IgAl protease In, obtained as described in the present description, example 4) once at a dose of 20 μg / mouse. According to the enzyme immunoassay carried out as described in RF patent No. 2310853, the mutant recombinant IgA1 protease, in contrast to the native and recombinant non-mutant IgA1 protease, does not have specific enzymatic activity, i.e. does not hydrolyze human IgA1.

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

Figure 2 shows a diagram of the immunogenic activity of the above preparations of IgA1 protease. The ordinate shows the optical density of the sera at λ = 492 nm. After a single immunization, the level of specific antibodies in the sera of mice immunized with a mutant IgA1 protease increases to 1: 5000, which exceeds this indicator in mice immunized with a native and recombinant IgA1 protease (1: 2560).

1 month after immunization, the mice were divided into three groups and infected with a live virulent culture of meningococcus serogroup A strain A208, B strain H44 / 47 and C strain 0638.

The protective activity of IgA1 proteases was evaluated by the level of bacteremia in immunized animals 4 hours after infection with meningococcus compared with the same indicator in control (non-immunized) mice.

The diagram of Fig. 3 shows the bacteremia level (in%) in mice immunized once with the above IgA1 protease preparations (shaded bars) after infection of serogroups A, B and C with meningococcus as compared to control non-immunized animals (black bars). The bacteremia level in the group of mice immunized with a mutant protease was respectively for serogroups A, B and C 13, 19 and 17 CFU, in terms of 100 CFU in the control (in non-immunized mice).

Additional example 1

A) Determination and preparation of a nucleic acid encoding an IgA1 protease of Neisseria meningitides serogroup B strain H44 / 76 (SEQ ID NO: 7)

An analysis of the data of the genomic bank (http://www.ncbi.nlm.nih.gov/Genbank) revealed that so far the only genome of strain MC58 is known for Neisseria meningitidis serogroup B. Based on the assumption that the structure of IgA1 genes of proteases of related strains is similar, according to the known sequence of the iga MC58 gene, primers were designed for cloning a fragment of a nucleic acid (gene) of strain H44 / 76 encoding a protease form that is close in size to the secreted (approximately 900-1000 amino acid residues , a.o.). The selected structure of the primers allows amplification of the product of polymerase chain reaction (PCR), suitable for subsequent incorporation into expression vectors of the pET family (Novagen) used to obtain proteins in E. coli strains lysogenic by T7 bacteriophage.

For subsequent cloning in the pET-24 vector (a +) into primers (f1 — direct — 5-CGAGACAGCCATATGAAAACCAAACGTTTTAAAATTAAC-3 SEQ ID No. 3, f2 — reverse — 5-GGGCTCGAGATTGTACAATCGGGTAATACCG-4 SEQ ID NO: 3) strain H44 / 76, introduced the sequence of restriction enzyme recognition sites NdeI and XhoI (respectively for f1 and f2), available in the polylinker of the vector. The structure of primer f2, which does not contain termination codons, allows one to obtain a protein modified in the C-terminal region with a hexahistidine tag for subsequent metal chelate chromatography during isolation.

Amplification of a nucleic acid fragment is carried out according to the standard protocol for 25 cycles of PCR using the genomic DNA of strain H44 / 76 as a template. The annealing temperature of the primers is selected experimentally. Processing of the PCR fragment and vector with restriction enzymes, ligation and transformation of competent cells is carried out according to standard procedures. Next, the cloned DNA is sequenced from several primary clones, as a result, the nucleotide sequence of a nucleic acid fragment encoding the secreted form of the IgA1 protease from Neisseria meningitides H44 / 76 was first established, based on which the amino acid sequence of the protein was determined (SEQ ID NO: 8). The resulting plasmid DNA was named pBIGAPS1.

B) Obtaining recombinant plasmid DNA pBIGAPS1

Recombinant plasmid DNA is obtained from overnight culture cells by the standard alkaline method (Maniatis, T., Fritsch, EF and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual, Cold Sping Harbor Laboratory Press) and used to introduce into competent cells using heat shock. For this, 100 ng - 1 μg of plasmid DNA is added to 50 μl of a suspension of competent cells and incubated for 40 minutes on ice. The resulting mixture was incubated at 42 ° C for 2 min, then incubated in ice for 2 min, 1 ml of cold LB broth was added and incubated for 1 h at 37 ° C. After this, 100 μl of a suspension of bacterial cells are plated on Petri dishes with agar containing 50 μg / ml kanamycin. The plates are incubated overnight at 37 ° C, the bacterial cells of several grown colonies are used for independent plating of microbiological tubes with 5 ml of LB liquid medium containing 50 μg / ml kanamycin, followed by growth of bacterial cells on a shaker at 37 ° C overnight. After that, glycerin is added to the tubes to a final concentration of 15%, the cell suspension is poured into plastic tubes and stored at -70 ° C.

A physical map of recombinant plasmid DNA is shown in figure 1.

C) Obtaining strain

The E. coli BL21 (DE3) / pBIGAPS1 strain is obtained by introducing pBIGAPSI plasmid DNA into competent E. coli BL21 (DE3) cells using the heat shock method. To obtain competent cells, the bacterial cells of the original strain Escherichia coli BL21 (DE3) (E. coli B F ^- dcm ompT hsdS (r _B ^- m _B ^- ) gal λ (DE3)) are grown in LB liquid medium overnight at 37 ° C and used for seeding 100 ml of the same medium. The culture is grown to the early logarithmic phase on a rocking chair with intensive aeration (to an optical density at 600 nm = 0.4-0.5), it is quickly cooled in ice, the cells are precipitated by centrifugation for 10 min at 5000 rpm at + 4 ° С. The precipitate is washed twice in 50 ml of 100 mm CaCl ₂ . The precipitated cells are resuspended in 2 ml of 100 mm CaCl ₂ , with the addition of glycerol up to 10%. Separate into aliquots of 50 μl, quickly freeze and store at -70 ° C.

D) Isolation of Recombinant IgA1 Protease

Cell disruption and lysate fractionation

Thawed cells after storage (2 g from 1 L of the induced culture) are resuspended in a seven-fold volume of 20 mM Tris-HCl buffer, pH 8.5, containing 1% Triton-X100, or in detergent-containing 20 mM potassium phosphate buffer, pH 7.5. The cell suspension was sonicated five times and centrifuged (1 hour, 20,000 g). Separated fractions of soluble proteins (cell-free extract, BE) and the insoluble part of the cell lysate (debris and insoluble proteins) are isolated. According to the results of electrophoretic analysis, the target protein forms insoluble inclusion bodies (TBs) regardless of the temperature conditions of the culture induction and the type of buffer used to destroy cells. The insoluble portion of the cell lysate containing TB, after resuspension in 1% Triton-X100 with repeated sonication and subsequent centrifugation, is used for chromatography on Q-Sepharose under denaturing conditions.

Anion exchange chromatography on Q-Sepharose

TB, isolated from 2 g of cells induced at 25 ° C, are 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 as determined by the Bradford method. After centrifugation and filtration, the solution was applied at a rate of 1 ml / min per 10 ml of anion exchange sorbent (two pre-packed HiTrap Q Sepharose HP cartridge columns, GE Healthcare, Sweden), equilibrated with the same buffer. The fractions containing purified protein and obtained as a result of gradient elution with sodium chloride (100 ml, 0-1 M NaCl) in the starting buffer are combined (20 ml in total) and used to convert the IgA1 protease into a soluble form. The yield of a partially purified preparation of the target protein at this stage is 40 mg from 2 g of cell biomass.

IgA1 protease refolding

The combined eluate (20 ml, 2 mg / ml) obtained after chromatography on Q-Sepharose was diluted 10 times with 20 mM Tris-HCl buffer, pH 8.5, by rapid dilution (digging out the protein solution into the buffer). After slowly mixing the solution (at a protein concentration of 0.2 mg / ml) for 20 hours and subsequent concentration by ultrafiltration to the initial volume, the protein solution is dialyzed three times against 1 L of 20 mM Tris-HCl buffer, pH 8.5. The solution is then centrifuged (30 min, 5000 g) and filtered through a sterile membrane with a pore size of 0.2 μm.

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

Additional example 2

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

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

An analysis of the enzymatic activity of the obtained recombinant IgA1 protease samples showed (Fig. 4) the ability of active IgA1 protease to catalyze the hydrolysis of IgA1 immunoglobulin and the absence of proteolytic activity in the mutant sample.

The level of proteolytic activity of IgA1 protease was also determined by electrophoretic method in SDS-PAGE. Figure 5 shows the hydrolysis of IgA1 in the presence of various concentrations of the active enzyme and its mutant form (sample 1 and sample 2, respectively).

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

Assessment of species specificity of IgA1 protease samples Sample Number IgA1 protease sample IgA1 antibody titer in mice * in humans ** one active 1/1280 1/20 2 mutant 1/1280 1/640

Thus, based on the obtained plasmid DNA pBIGAPS1-M, an E. coli expression strain super-producer of serogroup B IgA1 protease was created, a protein was isolated and refolded, which made it possible to obtain IgA1 protease in an amount of 10-40 mg from 2 g of cell biomass.

For the first time, results have been obtained indicating a higher immunogenic and protective activity of the mutant IgA1 protease in comparison with the recombinant meningococcal protease of serogroup B, as described in the article by Serova O.V. et al., Biopharmaceutical Journal, 2012, vol. 2, No. 1. The resulting preparation of mutant IgA1 protease has a multivalent effect with respect to protection against meningococcal infection of the animal organism, regardless of its source (serogroup A or B). The lack of enzymatic activity against human immunoglobulin A1 mutant IgA1 protease will avoid additional complications during human immunization. The presented data on immunogenicity and protectiveness give reason to believe that it can be used as a multivaccine for the treatment of bacteriocarriers and the prevention of meningococcal infection of the main epidemic serogroups A, B and C, primarily for the prevention of meningococcal infections caused by bacteria of N. meningitidis serogroup B, as well as other pathogens whose activity is due to the presence of IgA1 protease.

Claims (6)

1. Polynucleotide encoding a mutant IgA1 protease of Neisseria meningitidis with a nucleotide sequence (SEQ ID NO: 1). 2. Recombinant plasmid DNA comprising the polynucleotide according to claim 1 and providing expression of the mutant IgA1 protease in the host cell. 3. A host cell transformed with the recombinant plasmid DNA according to claim 2, producing a mature mutant form of IgA1 protease. 4. The host cell according to claim 3, where the prokaryotic host cell is selected from the series including bacteria of the genus Escherichia, Bacillus, Lactococcus, Lactobacillus, Pseudomonas, Streptomyces, Streptococcus, Staphylococcus. 5. A method of obtaining a mature mutant form of IgA1 protease Neisseria meningitidis serogroup B, which consists in the fact that the host cell is transformed with the recombinant plasmid DNA according to claim 2, the transformed cells according to claim 3 are cultured to obtain a mature mutant form of IgA1 protease, and the target product is isolated from inclusion bodies and cleanse. 6. Mutant IgA1 protease N.meningitidis serogroup B with the amino acid sequence of SEQ ID NO: 2, which has an increased level of immunogenicity compared to the wild-type enzyme.

Images