RU2701964C2 - Recombinant protein having protective action on meningococcus (embodiments), polynucleotide coding recombinant protein, recombinant plasmid dna containing said polynucleotide, host cell containing said recombinant plasmid dna, method of producing recombinant protein - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology. Disclosed is a recombinant protein having protective effect on meningococcus having amino acid sequence SEQ ID NO: 2, consisting of three fragments of primary structure of IgA protease Neisseria meningitidis serogroup B, connected in a single polypeptide chain. Also disclosed is a recombinant protein variant having the amino acid sequence SEQ ID NO: 4, consisting of two fragments of primary structure of IgA protease Neisseria meningitidis serogroup B, connected in a single polypeptide chain. Invention also discloses a polynucleotide encoding a corresponding recombinant protein, a recombinant plasmid DNA comprising a corresponding polynucleotide, a host cell transformed with recombinant plasmid DNA and producing a recombinant protein, and a method of producing a recombinant protein.

EFFECT: group of inventions provides producing highly purified immunogenic proteins with a high level of protective action on meningococci.

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Description

FIELD OF THE INVENTION

The invention relates to the field of biotechnology, in particular genetic engineering, and for the preparation of a new recombinant protein SEQ ID NO: 2 or its variant SEQ ID NO: 4, which has a protective effect on meningococci of various serogroups, consisting of polypeptide fragments of the primary structure of a full-sized IgA protease Neisseria meningitidis serogroup B linked into a single polypeptide chain; a new polynucleotide SEQ ID NO: 1 or a variant thereof SEQ ID NO: 3 encoding a recombinant protein; a new recombinant plasmid DNA pET28-IgA1-11-3 containing the polynucleotide SEQ ID NO: 1 or a variant of the recombinant plasmid DNA pET28-IgA1-13-1 containing the polynucleotide SEQ ID NO: 3; the host cell, especially the new bacterial strain Escherichia coli BL21 (DE3) / pET28-IgA1-11-3 producing the recombinant protein SEQ ID NO: 2 or its variant BL21 (DE3) / pET28-IgA1-13-1 producing the recombinant protein SEQ ID NO: 4; and a method for producing a recombinant protein of SEQ ID NO: 2 or SEQ ID NO: 4, intended for use in the preparation of a monocomponent vaccine to protect a person from meningococcal infection.

State of the art

Bacterial meningitis, an infectious disease leading to inflammation of the membranes of the human brain and higher primates, is characterized by a severe course, numerous complications and high mortality. The causative agents of bacterial meningitis are meningococci, pneumococci and hemophilic bacilli (93%) and other pathogens of infections. The main causative agents of meningococcal meningitis are bacteria N. meningitidis: of the 12 identified serogroups of N. meningitidis, six - A, B, C, W, X and Y, are epidemically dangerous. To prevent the disease, there are several polysaccharide and conjugate vaccines. Polysaccharide vaccines are available in divalent (A, C), trivalent (A, C, W135) and tetravalent (A, C, W135, Y) formulations, conjugated in monovalent (A or C) and tetravalent (A, C, W135, Y) formulations as well as combinations (N.

meningitidis serogroup C and Haemophilus influenzae type b) (Crum-Cianflone N., Sullivan E. Infect Dis Ther, 5: 89-112, 2016). There are no polysaccharide vaccines to protect against serogroup B meningococci due to the structure of their capsular polysaccharide, but there are two multicomponent vaccines based on the surface proteins of this pathogen (Bianchi A. et al. J prev med hyg, 56: E140-E143, 2015; Gandhi A. et al. Postgraduate medicine, 128 (6): 548-556, 2016; Donald RGK et al. Hum vaccine immunother, 13 (2): 255-265,2017). There are no vaccines against meningococci of serogroup X, but the search for antigens that can provide protection against meningococci of this serogroup continues (RU 2015100889, 06/14/2013 Pizza M. et al.). Most currently used vaccines have a narrow focus on a specific pathogen. To protect a person from the whole variety of circulating and continuously mutating strains of meningococcus, it is necessary to use multicomponent vaccines, which leads to a high immune load on the body. Thus, despite the existence of a significant number of meningococcal vaccines, the development of a monocomponent vaccine against a wide range of meningococcal pathogens, in particular, against serogroup B meningococcus, remains relevant. The use for this purpose of IgAl proteases (or fragments thereof) secreted by a number of gram-negative (N. meningitidis, N. influenzae, N. gonorrhoeae) and gram-positive (Str. Pneumoniae, Str. Sanguis, Str. Oralis) bacteria and which are one of the main factors the virulence of these bacteria seems to be very promising.

IgA1 proteases have unique specificity for immunoglobulins A1: they cleave the peptide bonds of the prolyl series or prolyl-threonine in the hinge regions of human and higher primate (IgA1) and secretory (sIgA1) immunoglobulins A1 (Plaut AG, Bachovchin WW Methods Enzymol, 244: -151, 1994; Kilian M. et al. Mol Immunol, 20: 1051-1058.1983). The surface of human mucous membranes contains a significant amount of sIgA1, which inhibit the adhesion of bacteria and viruses on the cells of the mucous membrane and are the first line of defense of the body from foreign agents. It is believed that IgA1 proteases secreted by pathogenic bacteria that inhabit the human mucosa break down sIgA1 and thereby contribute to the adhesion of pathogenic bacteria on the surface of the mucous membrane and the onset of the inflammatory process. The neutralization of IgA1 proteases at this stage of invasion may be an obstacle to the development of infections caused by

pathogens secreting this enzyme, hindering the adhesion of bacteria on the surface of the mucosa.

Serine-type IgA1 proteases are synthesized in the form of precursor proteins - full-sized IgA proteases, Gram-negative bacterial autotransporters, consisting of three functional domains: the N-terminal signal peptide (SP), the transported domain, and the C-terminal translocation domain (TD). The transported domain is localized between SP and TD and contains the protease subdomain, γ- and α-peptides. On the outer membrane of the bacterium, the transported domain of the full-sized IgA protease undergoes autocatalytic processing for prolyl-serine bonds localized in the case of the full-sized IgA protease N. meningitidis strain H44 / 76 with the general sequence Met ¹ - (Lys ² -Phe ¹⁵⁶⁸ ), in the amino acid sequence γ-peptide Val ⁹⁷⁵ -Pro ¹⁰⁰⁶ (Van Ulsen P. and Tommassen J. FEMS Microbiol Rev., 30: 292-319, 2006), forming a mature enzyme, IgA1 protease (about 1000 amino acid residues). The numbering of amino acid residues of IgA protease fragments hereinafter in the text corresponds to the sequence of the full-length IgA protease N. meningitidis H44 / 76, available at http://www.ncbi.nlm.nih.gov/protein/ADC80147).

Analysis of the primary structures of bacterial IgA proteases from N. meningitidis, N. gonorrhoeae and N. Influenza indicates their high homology (up to 70%) (Lomholt, H. et al. Mol. Microbiol. 15: 495-506. 1995). The most conserved regions of these proteins are located in the translocative domain and the protease subdomain. Homology of the amino acid sequences of IgA proteases from N. meningitidis and N. gonorrhoeae in the Ala ²⁸ -Pro ¹⁰⁰⁴ section of the primary structure of the full-length immunoglobulin A protease of N. meningitidis serogroup B, strain H44 / 76, including the protease domain and γ peptide that we selected for research in as the basic structure, it is more than 90%. Native and recombinant meningococcal IgA1 proteases from N. meningitidis serogroups A and B are known that protect mice from infection by the virulent culture of meningococcal serogroups A, B and C (Yagudaeva E.Yu. et al. Biooo rgan, chemistry, 36: 96-105, 2010; Serova O.V. et al. Biopharm. Journal, 3 (6): 42-47, 2011; Kotelnikova O.V. et al. Biomed. Chem. 60: 479 -486, 2014; Zinchenko A.A. et al. J. Meningitis, 2015, 1: 102). ELISA showed that protective antibodies produced by immunizing animals with IgA1 protease are able to bind to an enzyme not only in solution but also to an enzyme exposed on the surface of dead meningococcus cells (O. Kotelnikova et al. Bulletin of experimental biology and medicine , 161 (3): 369-372, 2016). The results, combined with the high homology of the amino acid sequence portion of the Ala28-Pro1004 primary structure of the full-length immunoglobulin A proteases from various sources, allowed us to consider the IgA1 protease as a promising basis for the anti-meningococcal mono-vaccine.

The prior art describes multiple compositions, conjugates and candidate vaccines based on lipopolysaccharides, capsular oligosaccharides and / or polypeptides of a number of meningococcal surface proteins to immunize people against meningitis caused by the pathogenic bacteria N. meningitidis of various serogroups (US Pat. US 9561269, GW Zlot 02/07/2017; US Pat. No. 9,724,402, AS Anderson et al. 08/08/2017; US Pat. No. 9,475,864, S. Ram et al. 10/25/2016). However, these compositions are multicomponent and do not provide effective protection against meningococcal infection of serogroup B.

It is known to obtain immunogenic compositions against N. meningitidis serogroup B (Pat. RU 2498815 Kontorni M. et al) containing adjuvant in various containers, including an oil-in-water emulsion and a cocktail of lyophilized antigens, serogroup B meningococcal membrane proteins, which may include conjugated polysaccharides from N. meningitidis serogroups A, C, W135 and / or Y, as well as natural membrane vesicles, recombinant proteins and lipopolysaccharides from N. meningitidis serogroup B; It is also known to obtain polypeptide vaccines (US Pat. RU 2333007, Pizza M, September 10, 2008), which are compositions of five antigens (NadA protein, protein 741, protein 936, protein 953 and protein 287) of the outer membrane of N. meningitidis, and are capable of inducing humoral immune response against some supervirulent series of generations A4, ET5 and a number of generations 3 of N. meningitidis serogroup B.

The disadvantages of the above compositions are the complexity of manufacture, multicomponentness and, as a result, a high immune load on the human body, a narrow focus on the meningococcus of a particular serogroup.

The listed compositions do not use meningococcal IgA1 protease or its fragments.

It is known to produce immunogenic polypeptides, nucleic acids encoding these polypeptides and antibodies immunogenic for meningococcal infections,

binding to these polypeptides based on protein antigens of strain M004-240196 N. meningitidis serogroup B having at least 95% homology with proteins of meningococcal serogroups A and C (US Pat. RU 2450019 R. Rappuoli et al., 10.05 2012), or polypeptides on based on Neisseria protein ORF2086 (US Pat. US 08563006, BJ Metcalf et al. November 22, 2013), which can be used to treat or prevent diseases caused by N. meningitidis. The authors consider polypeptides mainly as additional components for use in improved vaccines for the prevention and / or treatment of meningococcal meningitis. Among the sources of immunogenic peptides IgA1 protease or its components are absent.

It is known to obtain recombinant chimeric proteins created on the basis of fragments of antigenic domains of three different families of the surface protein NMB1870 N. meningitidis expressed in the structure of one molecule to form immunity against diseases and / or infections caused by meningococci, in particular, serogroup B. (US Pat. RU 2375374 Mazignani V. et al., 10.12.2009). The authors cite several variants of recombinant chimeric proteins and compositions based on them that can induce a humoral bactericidal response against meningococcal strains of serogroup B and at least one of serogroups A, C, W or Y.

The authors consider chimeric proteins as candidate vaccines, however, they are quite narrowly targeted and several components must be used to obtain an effective universal anti-meningococcal vaccine. It is known to obtain IgA1 protease fragments (US Pat. US 7,235,242, M. Achtman et al., June 26, 2007), which are used as peptides carriers of carbohydrate components of the cell walls of various microorganisms: Neisseria, Streptococcus, Klebsiella, Salmonella, Shigella and Haemophilus. The synthesis of N-terminal fragments of an IgA1 protease from N. meningitidis serogroup A containing from 40 to 104 amino acid residues is described.

However, the conjugation of these peptides with polysaccharides of meningococci of other serogroups does not expand the spectrum of action of the obtained compositions, they provide protection from meningococcus only one serogroup.

A known method of obtaining a protein of the precursor of IgA1 protease. The article (S. Vitovski and JR Sayers, Infection and Immunity, 75 (6): 2875-2885, 2007) describes the cloning of a full-sized gene encoding a signal peptide, a secreted protease form and a transport domain, and the mechanism of autoprocessing of IgA1 protease from its predecessor is studied , a series of mutated forms of IgA protease N. meningitides

serogroup B. It was found that these mutants do not have autoproteolytic activity and do not secrete mature protease. In this work, precursors of IgA1 protease proteins were obtained only for the purpose of studying the mechanism of autocatalytic activation of the enzyme, they were not isolated in a purified state, immunogenic and protective properties of proteins were not studied, the possibility of their use in order to create a vaccine was not considered.

It is known to obtain bacterial IgA1 proteases and their use for the treatment of diseases associated with the accumulation of IgA1 in the tissues and organs of the body (US Pat. US 7407653, Plaut et al., 05.08.2008). Soluble IgA1 proteases containing various labels were prepared using recombinant DNA methods. The resulting preparations were used as an enzyme for the treatment of autoimmune diseases associated with the accumulation of IgA1. However, the resulting recombinant protein had enzymatic activity and its use as a vaccine in the patent was not discussed.

The article (Gupta SK et al, Vaccine. 28: 7092-7097, 2010) describes the results of a computer analysis of potential T-cell epitopes of three serogroup B meningococcal proteins: T-cell stimulating protein (TspA) A, autotransport protein A (AutA) and IgA1 proteases. The study revealed six nine-membered T-cell epitopes. The authors believe that these peptides can be used as promising agents for protection against serogroup B meningococci, but experimental data confirming this hypothesis are not presented and the possibility of obtaining a vaccine based on them is not discussed.

Known methods for producing enzymatically active DNA using recombinant DNA technology (Serova O.V. et al. Biopharm. Journal, 3 (6): 42-47, 2011; pat. RU 2453599, Rumsh L.D. et al. 20.06.2012 ), or inactive (mutant at the serine residue of the active center, Ser267Ala) meningococcal IgA1 protease (Kotelnikova O.V. et al. Biomed. chem. 60: 479-486, 2014; pat. RU 2486243, Rumsh L.D. et al. June 27, 2013). The work described the preparation of polynucleotides encoding these proteins, recombinant plasmid DNAs including these polynucleotides, and cells transformed with the corresponding recombinant plasmid DNAs to obtain purified target proteins with high immunogenicity and effective protective effect against meningococcal infection

the main epidemic serogroups A, B and C, in order to use them as promising agents for protection against meningococci of various serogroups. However, a high molecular weight (more than 100 kDa), a significant amount of B- and T-cell antigenic determinants in the structure of these enzymes and, accordingly, a high antigenic load on the body during immunization, low solubility of recombinant IgA1 protease variants, both in active and in mutant forms, as well as the ability of the IgA1 protease to bind the C1 inhibitor of the human complement system (Pat. RU 2475756, Kozlov L.V. et al. 02/20/2013), which can lead to uncontrolled activation of human complement along the classical pathway in the process of immunization, limit the possibility of using IgA1 proteases as the basis of the anti-meningococcal mono-vaccine.

A known method of producing, using recombinant DNA technology, two variants of recombinant IgA1 protease Met1- (Lys2-Asn963) -Leu-Glu-His6 and Met1- (Ala28-Asn963) -Leu-Glu-His6 from N. meningitidis serogroup B, strain H44 / 76 and low molecular weight recombinant protein Met1- (Glu135-His328) -Leu-Glu-His6 with a mol. mass of 23,367 Yes, a fragment of the N-terminal portion of this enzyme with a high density of B-cell epitopes (Zinchenko A.A. et al. J. Meningitis, 2015, 1: 102). All three proteins were immunogenic and ensured high survival of mice immunized by them after infection with the virulent strain H44 / 76 N. meningitidis of serogroup B. It was suggested that the low molecular weight recombinant protein Met1- (Glu135-His328) -Leu-Glu-His6 be used as the basis of the meningococcal vaccine, however, data on the protection of animals from meningococcal infection of other serogroups are not given. In addition, when studying the behavior of immunocompetent cells CD4 +, CD8 +, CD19 in the blood of animals immunized with this compound, it was found that the immunoregulatory index, CD4 + / CD8 +, significantly increased (1.28 ± 0.10) 6 months after immunization compared with control (1.00 ± 0.07). This indicates undesirable changes in the state of general homeostasis of immunized animals, which is a serious drawback for the candidate vaccine. A method of obtaining a shortened recombinant protein (Zinchenko A.A. et al. J. Meningitis, 2015, 1: 102) based on the primary structure of IgA1 protease of N. meningitidis serogroup B, strain H44 / 76 was chosen as the closest prior art as a prototype.

Disclosure of invention

The invention solves the problem of producing immunogenic recombinant proteins based on the amino acid sequence of the Ala28-Pro1004 fragment of the primary structure of the full-length IgA protease of N. meningitidis serogroup B strain H44 / 76, which have an effective immunogenic and protective effect against meningococci of various serogroups, including meningococci of serogroup B comparable with the action of recombinant IgA1 protease containing the sequence Ala28-Pro1004, not leading to undesirable changes in the overall homeostasis of the body immunized with these proteins of animals, for use as the main component of a monovaccine to protect against infections caused by meningococci of various serogroups and other pathogens, the virulence of which is due to the IgA1 protease.

To solve this problem using the service http://www.iedb.org

in the sequence Ala28-Pro1004, which we selected for studies as the base structure, we studied the distribution of B and T cell epitopes and showed that the highest density of antigenic determinants is concentrated in three parts of the base structure: N-terminal (Ser120-Thr360), central (Lys415-Val710) and C-terminal (Gly820-Ser990). Considering the results of animal studies of the immunogenic and protective properties of low molecular weight recombinant proteins obtained on the basis of the structure of these sites, which showed that only the recombinant protein Met1- (Glu135-His328) -Leu-Glu-His6 c had the effective protective effect on meningococcus serogroup pier mass of 23,367 Yes, obtained on the basis of the amino acid sequence from the N-terminal portion of the base molecule Ser120-Thr360 (Zinchenko A.A. et al. J. Meningitis, 2015, 1: 102), while recombinant proteins based on the Lys415 sequences -Val710 or Gly820-Ser990: Met1- (Trp329-His622) -Leu-Glu-His6, mol. mass 33 460 Yes and Met1- (His835-Pro1004) -Leu-Glu-His6, mol. weight 19 808 Yes, poorly protected animals from infection by serogroup B meningococci (the bacteremia level in the group of mice immunized with these proteins was 80 and 60%, respectively), we selected polypeptide fragments from the N-terminal portion of the base structure with high protective activity against meningococci (Trp140-His328 or Trp140-Gln299) and from sites with a low protective effect: Trp412-Asp604 and Tyr866-Pro1004, and then combined them into different polypeptide chains in various combinations. Fragments were

chosen so that each of the selected polypeptide fragments contained in its structure one of the three T-cell epitopes of the basic structure, capable of binding to the largest number of HLA-DR alleles http://www.iedb.org. As a result, a recombinant protein with the sequence SEQ ID NO: 2, consisting of three fragments of the base sequence of Ala28-Pro1004 IgA protease, Trp140-His328, Trp412-Asp604 and Tyr866-Pro1004 (Trp412-Asp604 and Tyr866-Pro1004) molasses 59 407 Da) and a variant of a recombinant protein with the sequence SEQ ID NO: 4, consisting of two fragments of the basic sequence connected into a single polypeptide chain: Trp140-Gln299 and Tyr866-Pro1004 (molmass 34 690 Da). In addition, a recombinant IgA1 protease with the sequence SEQ ID NO: 11 (reference protein) containing the base sequence Ala28-Pro1004 was designed and prepared.

The resulting combined recombinant proteins SEQ ID NO: 2 and SEQ ID NO: 4 had an effective protective effect against meningococci of various serogroups, including bacteria N. meningitidis serogroup B, not inferior to the recombinant IgA1 protease SEQ ID NO: 11. Recombinant proteins SEQ ID NO: 2 and SEQ ID NO: 4 did not change the immunoregulatory index, CD4 + / CD8 +, of immunized animals, while maintaining it at the control level. Thus, the obtained recombinant proteins SEQ ID NO: 2 and SEQ ID NO: 4 can be considered as the basis of a monocomponent vaccine for protection against meningococcal infection and other pathogens whose virulence is due to the IgA1 protease.

The obtained recombinant proteins of SEQ ID NO: 2 and SEQ ID NO: 4 with different molecular weights are objects of the same species, as they contain parts of the same base sequence of Ala28-Pro1004 IgA protease N meningitidis serogroup B, and protein SEQ ID NO: 4 is a component of not only the recombinant protease SEQ ID NO: 11, but also the protein SEQ ID NO: 2. Proteins SEQ ID NO: 2 and SEQ ID NO: 4 are objects for the same purpose: both are intended for use as the main component of a monocomponent vaccine for the prevention of diseases caused by meningococci ra personal serogroups, including serogroup B meningococcus and other pathogens, virulence of which is due IgA1 protease.

Proteins SEQ ID NO: 2 and SEQ ID NO: 4 provide one result: both are immunogenic, have a protective effect against meningococci of various serogroups, including bacteria N. meningitidis serogroup B, not inferior to the recombinant IgA1 protease SEQ ID NO : 11, and do not change the immunoregulatory index, CD4 + / CD8 +, of immunized animals.

The problem is solved by:

A: recombinant protein of SEQ ID NO: 2 with a molecular weight of 59,407 Da, calculated by an isoelectric point of 8.75, with an amino acid sequence of 530 amino acid residues, obtained from three sections of the primary structure of the Ala28-Pro1004 fragment of the primary structure of the full-length IgA protease N. meningitidis, connected into a single polypeptide chain serogroup B strain H44 / 76: Trp140-His328, Trp412-Asp604 and Tyr866-Pro1004 encoded by the nucleotide sequence of SEQ ID NO: 1, which has a high level of protective effect against meningococci of various serogroups that does not change the immune regulatory index, CD4 + / CD8 +, immunized animals, destined to produce monocomponent vaccine for preventing meningococcal infections caused by pathogens of various serogroups, including bacteria N. meningitidis serogroup B, and other pathogens, virulence of which conditioned IgA1 protease;

polynucleotide (SEQ ID NO: 1) encoding the recombinant protein of SEQ ID NO: 2; pET28-IgA1-11-3 recombinant plasmid DNA, comprising the nucleotide sequence of SEQ ID NO: 1 and providing expression of the recombinant protein SEQ ID NO: 2 in a host cell, characterized by the following features:

chain length is 6799 bp .;

encodes a recombinant fusion protein of SEQ ID NO: 2 with a hexahistidine tag at the C-terminus of the protein;

the nucleotide sequence of SEQ ID NO: 1 encoding the recombinant protein SEQ ID NO: 2 in plasmid pET28-IgA1-11-3 flanks the unique restriction site with XhoI endonuclease at the 3'-end;

the nucleotide sequence of SEQ ID NO: 1 encoding the recombinant protein SEQ ID NO: 2 in the plasmid pET28-IgA1-11-3 contains a unique restriction site by endonuclease AgeI;

contains a T7lac transcription promoter;

the genetic marker is the nucleotide sequence (kan), which determines the resistance of bacterial cells transformed with plasmid pET28-IgA1-11-3 to kanamycin;

a host cell, especially a producer strain of E. coli BL21 (DE3) / pET28-IgA1-11-3, containing a recombinant plasmid DNA producing the recombinant protein SEQ ID NO: 2;

and also due to the method of producing the recombinant protein SEQ ID NO: 2, which consists in the fact that the host cell is transformed with recombinant plasmid DNA pET28-IgA1-11-3 containing the polynucleotide SEQ ID NO: 1 encoding the recombinant protein SEQ ID NO: 2 , the producer strain is cultured, inclusion bodies are obtained, from which the target product is isolated and purified using a combination of chromatographic methods.

The task is also solved by:

B) a variant of the recombinant protein SEQ ID NO: 4 with a molecular weight of 34 690 Da, calculated by an isoelectric point of 8.7, with an amino acid sequence of 308 amino acid residues of SEQ ID NO: 4, obtained from two sections of the primary structure fragment Ala28-Pro1004 connected into a single polypeptide chain IgA proteases of N. meningitidis serogroup B strain H44 / 76: Trp140-Gln299 and Tyr866-Pro1004 encoded by the nucleotide sequence of SEQ ID NO: 3, which has a high level of protective effect against meningococci of various serogroups that does not change immunoregul the index, CD4 + / CD8 +, of immunized animals, intended for the production of a monocomponent vaccine for the prevention of meningococcal infections caused by pathogens of various serogroups, including bacteria N. meningitidis serogroup B, and other pathogens whose virulence is due to the IgA1 protease;

polynucleotide SEQ ID NO: 3 encoding the recombinant protein SEQ ID NO: 4, recombinant plasmid DNA pET28-IgA1-13-1, comprising the nucleotide sequence of SEQ ID NO: 3 and allowing expression of the recombinant protein SEQ ID NO: 4 in the host cell, characterized by the following features:

chain length is 6133 bp .;

encodes a recombinant protein of SEQ ID NO: 4 with a hexahistidine tag at the C-terminus of the protein;

the nucleotide sequence of SEQ ID NO: 3 encoding the recombinant protein SEQ ID NO: 4 in the plasmid pET28-IgA1-13-1 at the 3'-end flanks a unique restriction site with XhoI endonuclease;

the nucleotide sequence of SEQ ID NO: 3 encoding SEQ ID NO: 4 in the plasmid pET28-IgA1-13-1 contains a unique restriction site by endonuclease AgeI;

contains a T7lac transcription promoter;

the genetic marker is the nucleotide sequence (kan), which determines the resistance of bacterial cells transformed with plasmid pET28-IgA1-13-1 to kanamycin;

a host cell, especially a producer strain of E. coli BL21 (DE3) / pET28-IgA1-13-1 containing a recombinant plasmid DNA that produces the recombinant protein SEQ ID NO: 4;

and also due to the method for producing the recombinant protein SEQ ID NO: 4, which consists in the fact that the host cell is transformed with the recombinant plasmid DNA pET28-IgA1-13-1 containing the polynucleotide SEQ ID NO: 3 encoding the recombinant protein SEQ ID NO: 4 , the producer strain is cultured, inclusion bodies are obtained, from which the target product is isolated and purified using a combination of chromatographic methods.

Recombinant IgA1 protease SEQ ID NO: 11 containing the amino acid sequence of the primary structure Ala28-Pro1004 fragment of the primary structure of the full-length IgA protease of N. meningitidis serogroup B strain H44 / 76 is used as the comparison protein. One object of the present invention includes a recombinant protein of SEQ ID NO: 2 with a molecular weight of 59,407 Da, calculated by an isoelectric point of 8.75, with an amino acid sequence of 530 amino acid residues, consisting of three sections of the Ala28-Pro1004 fragment of the primary IgA primary structure connected into a single polypeptide chain proteases of N. meningitidis serogroup B strain H44 / 76: Trp140-His328, Trp412-Asp604 and Tyr866-Pro1004 encoded by the polynucleotide SEQ ID NO: 1, obtained by the above method and intended for use as the main component of monovaccines for the prevention of meningococcal infections of various serogroups (A, B, C, etc.), as well as any other infections, the pathogenicity of which is caused by the IgA1 protease secreted by them, such as: Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Haemophilus aegyptius, as well as Streptococonia pneum Streptococcus sanguis; Streptococcus oralis; Streptococcus mitis; Streptococcus viridans; Streptococcus canis; Streptococcus suis; Staphylococcus aureus; Staphylococcus epidermidis; Clostridium ramosum;

Another object of the present invention includes the polynucleotide SEQ ID NO: 1 encoding the amino acid sequence of the recombinant protein SEQ ID NO: 2. To obtain the nucleotide sequence of SEQ ID NO: 1 encoding the recombinant protein SEQ ID NO: 2, the megaprimer method was used. For this, specific primers f1 (SEQ ID NO: 5), f2 (SEQ ID NO: 6), f3 (SEQ ID NO: 7) and f4 (SEQ ID NO: 8) encoding fragments of the nucleotide sequence (https: / /www.ncbi.nlm.nih.gov/nuccore/GU190729.1) IgA protease of N. meningitidis H44 / 76 with a deletion of nucleotides encoding the amino acid sequence Trp ^329- Val ⁴¹¹ (SEQ ID NO: 6), and nucleotides encoding the amino acid the sequence of residues Trp ^605- Gln ⁸⁶⁵ (SEQ ID NO: 7). For primer ligation with the vector pET-28 (a +), the sequence of the XhoI restriction enzyme recognition site in the vector polylinker was introduced into the primer (SEQ ID NO: 8). The structure of the primer SEQ ID NO: 8 allows to obtain a recombinant protein modified in the C-terminal region with a hexahistidine tag.

The recombinant protein gene fragment was amplified in three stages by PCR using pET31-IgA1-1004 plasmid DNA as described in SEQ ID NO: 10 described in Supplementary Example 1, and the resulting DNA fragment was obtained at about 1570 bp.

Another object of the present invention includes a recombinant plasmid DNA containing the nucleotide sequence of SEQ ID NO: 1 and allowing expression of the protein SEQ ID NO: 2 in the host cell. A DNA fragment of about 1570 bp in length was cloned into pET-28a (+) vector (Novagen, cat. No. 69864-3) using unique NcoI (blunted) and XhoI restriction sites so that the protein produced contained a hexahistidine tag in the C-terminal region for subsequent protein purification with using metal chelate chromatography.

Sequencing of the obtained DNA confirmed compliance with the target structure. The resulting recombinant plasmid DNA was named pET28-IgA1-11-3. Another object of the present invention includes a host cell, especially an E. coli strain producing the recombinant protein SEQ ID NO: 2. As a host cell, E. coli strain BL21 (DE3) was used, on the basis of which the producer strain BL21 was obtained (DE3) / pET28-IgA1-11-3, described below.

Another object of the present invention includes a method for producing a recombinant protein SEQ ID NO: 2, which is that the host cell of E. coli BL21 (DE3) is transformed with the above recombinant plasmid DNA pET28-IgA1-11-3 with the polynucleotide SEQ ID NO: 1 encoding the recombinant protein of SEQ ID NO: 2, the producer strain is cultured as described below, and then the target product is isolated and purified.

Another object of the present invention includes a variant of the recombinant protein SEQ ID NO: 4 with a molecular weight of 34,690 Da, calculated by an isoelectric point of 8.7, with an amino acid sequence of 308 amino acid residues, consisting of two sections of a primary structure fragment Ala28-Pro1004 connected into a single polypeptide chain IgA protease N. meningitidis H44 / 76: Trp140-Gln299 and Tyr866-Pro1004 encoded by the polynucleotide SEQ ID NO: 3, obtained by the above method and intended for use as the main component of a monovaccine for the prevention of and meningococcal infections of various serogroups (A, B, C, etc.), as well as any other infections, the pathogenicity of which is caused by the IgA1 protease secreted by them, such as: Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Haemophilus aegyptius, as well as Streptococcus pneumonia Streptococcus sanguis, Streptococcus oralis, Streptococcus mitis, Streptococcus viridans, Streptococcus canis, Streptococcus suis, Staphylococcus aureus, Staphylococcus epidermidis, Clostridium ramosum, Bacteroides melaninogenicus, Capnocyercherhobhorah orachocerhobhorah orachlochrachocerhobria campis

Another object of the present invention includes a polynucleotide SEQ ID NO: 3 encoding the amino acid sequence of the recombinant protein SEQ ID NO: 4.

To obtain the nucleotide sequence of SEQ ID NO: 3 encoding the recombinant protein SEQ ID NO: 4, the megaprimer method was used. For this, specific primers f1 SEQ ID NO: 5, f4 SEQ ID NO: 8, and f5 SEQ ID NO: 9 encoding fragments of the nucleotide sequence (https://www.ncbi.nlm.nih.gov/nuccore/GU190729. 1) N. meningitidis H44 / 76 IgA protease with a deletion of nucleotides encoding the amino acid sequence Glu300-Gln865 SEQ ID NO: 9. A recognition site sequence was introduced into primer SEQ ID NO: 8 for subsequent ligation with pET-28 (a +) vector XhoI restriction enzymes available in the polylinker of the vector. The structure of the primer SEQ ID NO: 8 allows to obtain a recombinant protein modified in the C-terminal region with a hexahistidine tag.

The recombinant protein gene fragment was amplified in two stages by PCR using the pET31-IgA1-1004 plasmid DNA as described in SEQ ID NO: 10 described in Supplementary Example 1, and the resulting DNA fragment of about 900 bp was obtained.

Another object of the present invention includes a recombinant plasmid DNA containing the nucleotide sequence of SEQ ID NO: 3 and for the expression of the protein SEQ ID NO: 4 in the host cell. A DNA fragment of about 900 bp in length was cloned into pET-28 (a +) vector (Novagen, cat. No. 69864-3) using unique NcoI (blunted) and XhoI restriction sites so that the protein produced contained a hexahistidine tag in the C-terminal region for subsequent protein purification using metal chelate chromatography. Sequencing of the obtained DNA confirmed compliance with the target structure. The resulting recombinant plasmid DNA was named pET28-IgA1-13-1. Another object of the present invention includes a host cell, especially an E. coli strain producing the recombinant protein SEQ ID NO: 4. As a host cell, E. coli strain BL21 (DE3) was used, on the basis of which strain BL21 (DE3) was obtained ) / pET28-IgA1-13-1, described below.

Another object of the present invention includes a method for producing a recombinant protein of SEQ ID NO: 4, which is that the host cell of E. coli BL21 (DE3) is transformed with the above recombinant plasmid DNA pET28-IgA1-13-1 with the polynucleotide SEQ ID NO: 3 encoding the recombinant protein of SEQ ID NO: 4, the producer strain is cultured as described below, and then the desired product is isolated and purified.

To obtain a producer strain of the recombinant protein SEQ ID NO: 2 or a variant of the recombinant protein SEQ ID NO: 4 competent cells of Escherichia coli BL21 (DE3) (E. coli B F ^- dcm ompT hsdS (r _B ^- m _B ^- ) gal λ ( DE3)) (Stratagene Cat. No. 200131) is transformed with the corresponding plasmid DNA pET28-IgA1-11-3 or pET28-IgA1-13-1.

The resulting strain, named E. coli BL21 (DE3) / pET28-IgA1-11-3 or a variant strain E. coli BL21 (DE3) / pET28-IgA1-13-1, 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, mineral ammonium 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 pET28-IgA1-11-3 and variant pET28-IgA1-13-1.

Storage conditions.

The bacterial strain E. coli BL21 (DE3) / pET28-IgA1-11-3 or the E. coli BL21 (DE3) / pET28-IgA1-13-1 variant 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.

Cells of producer strains exhibit resistance to kanamycin (up to 100 μg / ml) due to the presence of the resistance gene in recombinant plasmid DNA pET28-IgA1-11-3 or pET28-IgA1-13-1.

Eukaryotic cells (e.g., yeast cells, plant cells, animal cells and human cells), or prokaryotic cells (e.g. bacterial cells) can be used as the host cell to produce the recombinant protein SEQ ID NO: 2 or a variant of the recombinant protein SEQ ID NO: 4 cells, etc.), or cells that induce an immune response. Specialists in this field know many of these cells and cell lines (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 developed for laboratory research, such as, for example, hTERT-MSC, HEK293, Sf9 or COS cells. Suitable cells can be obtained in a similar way from eukaryotic microorganisms such as yeast, for example, Saccharomyces cerevisiae (Stinchcomb DT et al. Nature, 282 (5734): 39-43.1979), Schizosaccharomyces pombe, Candida, Pichia and hyphomycetes of the genera Aspergillus Pen 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.

The technical result of the invention is to obtain highly purified recombinant protein SEQ ID NO: 2 with a molecular weight of 59,407 Da and a purity of at least 93% according to SDS-PAGE and a variant of highly purified recombinant protein SEQ ID NO: 4 with a molecular weight of 34,690 Da and a purity not less than 95%. The recombinant protein SEQ ID NO: 2 and its variant SEQ ID NO: 4 are immunogenic and are characterized by high protective activity against meningococci of various serogroups, comparable to the activity of the recombinant IgA1 protease SEQ ID NO: 11. The obtained proteins have antimicrobial activity, protecting animals from meningococcal infections of various serogroups A, B and C.

A significant decrease in the molecular weight of the obtained recombinant proteins (59,407 Da and 34,690 Da compared to 109,019 Da for the recombinant IgA1 protease SEQ ID NO: 11) and, as a result, a decrease in the number of B- and T-cell epitopes while maintaining immunogenic properties and protective action against meningococci of various epidemic serogroups will allow the use of each of these proteins as the main component of a monovaccine and to lower the immune load on the human body during immunization. In addition, the proteins SEQ ID NO: 2 and SEQ ID NO: 4 do not change the immunoregulatory index (CD4 + / CD8 +) in the blood of immunized animals (1.00 ± 0.08) and (1.02 ± 0.05), respectively, which is an important indicator for the candidate vaccine.

The invention is illustrated by graphic materials.

FIG. 1. Physical map of recombinant plasmid DNA pET28-IgA1-11-3.

Recombinant plasmid DNA pET28-IgA1-11-3, containing 6799 bp encoding the recombinant protein SEQ ID NO: 2, consisting of: IgA1-11-3 - the region encoding the recombinant protein SEQ ID NO: 2 and hexahistidine label, flanked by the unique XhoI endonuclease restriction site (158) (numbering is given according to the numbering in the pET-28a vector), IgA1-11-3 also contains the unique restriction site by the EndI nucleonase I (252), ori is the recombinant plasmid DNA replication initiation site, T7lac promoter - hybrid transcription promoter, Stop - ribosomal transcription terminator Peron E. coli, kan - gene that determines a microorganism resistance to kanamycin, ƒ1 origin - lot, allowing to obtain single-stranded DNA, lacI - the region encoding the lac repressor.

FIG. 2. Physical map of the recombinant plasmid DNA pET28-IgA1-13-1.

Recombinant plasmid DNA pET28-IgA1-13-1 containing 6133 bp encoding the recombinant protein SEQ ID NO: 4, consisting of:

IgA1-13-1 is the region encoding the recombinant protein SEQ ID NO: 4 and a histidine tag flanked by a unique restriction site of the endonuclease XhoI (158) (the numbering is given in accordance with the numbering in the vector pET-28a), IgA1-13-1 also contains a unique restriction site by endonuclease AgeI (252), ori is the recombinant plasmid DNA replication initiation site, T7lac promoter is the hybrid transcription promoter, Stop is the transcription terminator of the ribosomal operon E. coli, kan is the gene that determines the resistance of the microorganism to kanamycin, ƒ1 origin is the site allowing you to get od otsepochechnuyu DNA, lacI - the region encoding the lac repressor.

FIG. 3. Electrophoretic analysis of the purity of the obtained recombinant proteins by the method of DDS-PAG (10% polyacrylamide gel, non-reducing conditions). 1 - SEQ ID NO: 2 (93%), 2 - SEQ ID NO: 4 (95%), 3 - SEQ ID NO: 11 (90%), 4 - MWM - molecular weight marker proteins (Thermo Scientific), kDa.

Table 1. The level of antibodies (AT) to recombinant IgA1 protease SEQ ID NO: 11 in the serum of mice immunized with recombinant proteins SEQ ID NO: 2, SEQ ID NO: 4 or recombinant IgA1 protease SEQ ID NO: 11.

Table 2. The number of CFU (%) in the blood of mice immunized with recombinant proteins SEQ ID NO: 2, SEQ ID NO: 4 or recombinant IgA1 protease SEQ ID NO: 11, when infected with meningococcus serogroups A, B and C.

The implementation of the invention

The invention is illustrated by the following examples:

Example 1

Obtaining polynucleotide SEQ ID NO: 1 encoding the amino acid sequence of the recombinant protein of SEQ ID NO: 2

Based on the known sequence of the iga gene of strain H44 / 76 encoding a full-sized IgA protease (https://www.ncbi.nlm.nih.gov/nuccore/GU190729.1), primers are designed to obtain the nucleotide sequence of SEQ ID NO: 1 encoding the amino acid sequence of the recombinant protein of SEQ ID NO: 2, and its subsequent ligation with the 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 recombinant proteins in E. coli strains lysogenic by T7 bacteriophage.

To obtain the nucleotide sequence of SEQ ID NO: 1, the megaprimer method is used. For this, specific primers f1, f2, f3, f4 are designed (f1 - direct - 5'-TGGCATCACGGAAATCAAGGTC-3 'SEQ ID NO: 5, f2 - reverse - 5'-CCATTCGGATTTTTTGACTTGCCAATGATGTTCTCCATTACCTTTGAT 3 - NO 3: - 5'-TGATTGATTTTGAGCGTGTGGTAGTCGTTATTTTCTGTTACACCGT-3 'SEQ ID NO: 7, f4 - inverse - 5'-GCGCTCGAGAGGCGGTGCGACGACGATAT-3' SEQ ID NO: 8. The primer fn gov / nuccore / GU190729.1) a full-length IgA protease of N. meningitidis serogroup B H44 / 76 with a deletion of nucleotides encoding the amino acid sequence Trp ³²⁹ -Val ⁴¹¹ , primer f3 encodes a fragment of the nucleotide sequence of the IgA protease with a deletion residue Trp ⁶⁰⁵ -Gln ^865. For fusion primer f4 with vector pET-28 (a +), the sequence of the XhoI restriction enzyme recognition site located in the polylinker of the vector is introduced.The structure of primer f4, which does not contain termination codons, allows one to obtain a recombinant protein modified in C-terminal region with a hexahistidine tag.

Amplification of the gene fragment of the recombinant protein is carried out in three stages by PCR using the plasmid DNA pET31-IgA1-1004 as described in Supplementary Example 1 (SEQ ID NO: 10) as a template. At the first stage, a fragment of this gene is amplified using primers f1 and f2, resulting in a DNA fragment of about 590 bp in length. The obtained DNA fragment was isolated from an agarose gel and used as the first primer in the second PCR step, and f3 primer was used as the second primer in the second PCR step. A DNA fragment of about 1170 bp is obtained. In the third step, PCR uses the fragment of PCR obtained in the second step and primer f4 as primers. As a result, a target DNA fragment of about 1570 bp is obtained.

The target DNA fragment is digested with XhoI restriction enzyme and cloned into pET28 (a +) vector (Novagen, Cat. No. 69864-3) using the restriction site NcoI (dull) and XhoI so that the protein produced contains a hexahistidine tag in the C-terminal region . Amplification of nucleic acid fragments is carried out according to the standard protocol for 25 cycles of PCR. The annealing temperature of the primers is selected experimentally. Processing of the PCR fragment and vector with restriction enzymes, ligation and transformation of TOR10 competent cells is carried out according to standard procedures. By sequencing the obtained DNA, the correspondence to the target structure of SEQ ID NO: 2 was confirmed. The obtained plasmid DNA was named pET28-IgA1-11-3.

Example 2

Obtaining recombinant plasmid DNA pET28-IgA1-11-3 and producer strain E. coli BL21 (DE3) / pET28-IgA1-11-3

Recombinant plasmid DNA was obtained from an overnight culture of E. coli TOP 10 cells using a standard alkaline method (Maniatis, T., Fritsch, EF and Sambrook, J. 1982. Molecular Cloning: a Laboratory Manual, Cold Sping Harbor Laboratory Press). A physical map of the recombinant plasmid DNA pET28-IgA1-11-3 is shown in FIG. 1. The strain E. coli BL21 (DE3) / pET28-IgA1-11-3 is obtained by introducing plasmid DNA pET28-IgA1-11-3 into competent cells of E. coli BL21 (DE3) 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 in a ratio of 1: 100. The culture is grown to the early logarithmic phase on a rocking chair with intensive aeration (to an optical density at 600 nm of 0.4 ÷ 0.5 OE), it is rapidly cooled in ice, and cells are precipitated by centrifugation in for 10 min at 5000 rev / min at + 4 ° C. The precipitate was washed twice with 50 ml of 100 mM CaCl _2. The precipitated cells were resuspended in 2 l 100 mM CaCl _2, supplemented with glycerol to 10%. Divide into aliquots of 50 .mu.l, quickly frozen and stored at -70 ° C.

To introduce pET28-IgA1-11-3 plasmid DNA into competent cells, 100 ng - 1 μg of plasmid DNA was added to 50 μl of a competent cell suspension and incubated for 40 minutes on ice. The resulting mixture was incubated at 42 ° C for 2 min, then in ice for 2 min, 1 ml of LB medium was added and incubated for 1 h at 37 ° C. Discrimination of cells (strain) containing recombinant plasmid DNA pET28-IgA1-11-3 is carried out on LB agar medium with kanamycin. For this, 100 μl of a suspension of bacterial cells are plated on Petri dishes with agar in LB medium containing 50 μg / ml kanamycin. The plates are incubated overnight at 37 ° C, the bacterial cells of several grown colonies are 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 ( night culture). 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.

Example 3

Determination of the productivity of the producer strain of the recombinant protein of SEQ ID NO: 2 E. coli BL21 (DE3) / pET28-IgA1-11-3

In 15 ml of LB liquid medium containing 50 μg / ml kanamycin, 1% inoculum of overnight cell culture is introduced and grown at 37 ° C on a shaker at 180 rpm for 2 hours until the optical absorption (OD) of 0.8. The isopropylthiogalactopyranoside inductor (IPTG) is then added to a final concentration of 0.5 mM and incubation is continued for 2.0 hours under the same conditions. A 1 ml sample is taken and centrifuged for 6 minutes at 12,000 rpm, after which the cells are suspended in 100 μl of deionized water, add 33 μl of a buffer solution containing 125 mm Tris-HCl, pH 6.8, 20% glycerol, 3% sodium dodecyl sulfate (SDS), 3% mercaptoethanol and 0.01% bromphenol blue, heated for 10 min up to 95 ° C. An aliquot was taken and analyzed by electrophoresis on a 10% polyacrylamide gel containing 0.1% SDS. The gel is stained with Coomassie R250 and scanned on a laser scanner. The productivity of strain E. coli BL21 (DE3) / pET28-IgA1-11-3 is 40% of the recombinant protein SEQ ID NO: 2 of the total cellular protein.

Example 4

Preparation, Isolation, and Purification of Recombinant Protein SEQ ID NO: 2

A) Cultivation of E. coli strain BL21 (DE3) / pET28-IgA1-11-3

10 ml of the corresponding inoculum of the overnight cell culture are introduced into 1 L of LB liquid medium containing 50 μg / ml kanamycin, and cells are grown at + 37 ° C on a shaker at 200 rpm to an optical density of 0.8 at 600 nm. Then the isopropylthiogalactopianoside inducer (IPTG) is added to a final concentration of 0.5 mM and the cells are continued to incubate at + 37 ° C on a shaker for two hours.

B) Cell destruction and lysate fractionation

To collect cells, 1 l of culture of the producer strain of E. coli BL21 (DE3) / pET28-IgA1-113 (10 min, 3.5 thousand rpm) is centrifuged and the precipitated cells are suspended in a seven-fold volume of 20 mM Tris-HCl buffer solution, 0.15 M NaCl, pH 8.5, containing lysozyme (0.002%), and then incubated for 30 min at room temperature. A cell suspension (2 g of cells with 1 l of culture) is sonicated five times, centrifuged (1 hour, 20,000 g) to obtain soluble protein fractions (cell-free extract) and an insoluble portion of the cell lysate (debris and insoluble proteins). 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 precipitate containing TB after centrifugation is washed with a buffer solution (60 ml of 20 mM Tris-HCl, pH 8.5, 1% Triton X-100, 0.1% SDS) and centrifuged for 40 min 10,000 g. The newly obtained precipitate was washed with buffer (60 ml of 20 mM Tris-HCl, 1 M NaCl) and centrifuged for 40 minutes, 10,000 g. The yield of washed TB recombinant protein SEQ ID NO: 2 is 0.96 g. The washed precipitates of TV recombinant protein SEQ ID NO: 2 are used for chromatography on Ni-agarose under denaturing conditions.

B) Purification of the dissolved bodies of the inclusion of the recombinant protein SEQ ID NO: 2 using metal-chelate chromatography on a Ni-agarose sorbent TB, isolated from 2 g of E. coli BL21 (DE3) / pET28-IgA1-11-3 cells, dissolved in 100 ml of 50 mm Tris-HCl buffer solution containing 8 M urea, 0.15 M sodium chloride and 0.02 M imidazole, pH 7.0. After centrifugation and filtration, the resulting solution was applied to a packed Ni-agarose column (HiTrap Chelating HP, GE Healthcare, 2 × 5 ml), equilibrated with the initial buffer solution, and the sorbent was washed with 50 ml of the initial buffer solution. Bound proteins are eluted with imidazole in a concentration gradient from 20 to 400 mM in the initial buffer solution. Fractions containing purified protein of SEQ ID NO: 2 are combined and used in the renaturation and purification step on Q-Sepharose.

D) Renaturation and purification of the recombinant protein of SEQ ID NO: 2 on Q-Sepharose

The combined eluate of the recombinant protein SEQ ID NO: 2 obtained after chromatography on Ni-agarose (protein concentration 0.35 mg / ml) is subjected to stepwise dialysis, gradually reducing the urea concentration to 1.5 M in 20 mm Tris-HCl buffer solution containing 0.15 M sodium chloride, pH 7.0. The resulting solution was centrifuged (30 min, 5000 g), filtered through a 0.2 μm pore membrane and applied to a packed Q-Sepharose column (GE 5 mlX2), equilibrated in 20 mM Tris-HCl buffer solution containing 0.15 M chloride sodium and 1.5 M urea, pH 7.0. The target protein that did not bind to the sorbent was collected in a single fraction, sorbitol (5 wt%) was added, filtered through a sterile membrane with a pore size of 0.2 μm and packed in sterile containers of 2 mg. The purity of the target protein is about 93% according to electrophoretic analysis (Fig. 3), the protein concentration is 0.2 mg / ml, the yield is 67 mg from 2 g of cell biomass.

Example 5

Obtaining polynucleotide SEQ ID NO: 3 encoding the amino acid sequence of the recombinant protein of SEQ ID NO: 4

Based on the known sequence of the iga gene of strain H44 / 76 encoding a full-sized IgA protease (https://www.ncbi.nlm.nih.gov/nuccore/GU190729.1), primers are designed to obtain the nucleotide sequence of SEQ ID NO: 3 encoding the recombinant protein SEQ ID NO: 4, and its subsequent ligation with the vector. The selected structure of the primers allows amplification of the product of the polymerase chain reaction (PCR), suitable for subsequent integration into expression vectors of the pET family (Novagen). Further, these vectors are used to obtain recombinant proteins in E. coli strains lysogenic at the T7 bacteriophage. To obtain the nucleotide sequence of SEQ ID NO: 3, the recombinant protein of SEQ ID NO: 4, the megaprimer method is used. For this, specific primers f1, f4, f5 are designed (f1 - direct - 5'-TGGCATCACGGAAATCAAGGTC-3 'SEQ ID NO: 5, f4 - reverse - 5'-GCGCTCGAGAGGCGGTGCGACGACGATAT-3' SEQ ID NO: 8, 5 - reverse '-ATGATTGATTTTGAGCGTGTGGTATTGCCAAGAGTTTTTTTGATAGCC-3' SEQ ID NO: 9). Primer f5 encodes a fragment of the nucleotide sequence (https://www.ncbi.nlm.nih.gov/nuccore/GU190729.1) of the N. meningitidis H44 / 76 IgA protease with a deletion of the nucleotides encoding the amino acid sequence Glu ³⁰⁰ -Gln ⁸⁶⁵ . The primer f4 for subsequent ligation with the vector pET-28 (a +) is introduced the sequence of the recognition site of the restriction enzyme XhoI, available in the polylinker of the vector. The structure of primer f4, which does not contain termination codons, allows one to obtain a protein modified in the C-terminal region with a hexahistidine tag. Amplification of a gene fragment is carried out in two stages, using pET31-IgA1-1004 plasmid DNA as a template described in Supplementary Example 1 (SEQ ID NO: 10). At the first stage, a gene fragment amplification reaction is carried out using primers f1 and f5, as a result, a DNA fragment of about 480 bp is obtained. The obtained DNA fragment was isolated from an agarose gel and used as the first primer in the second PCR step, and f4 primer was used as the second primer in the second PCR step. A target DNA fragment of about 900 bp was obtained. The target fragment is digested with XhoI restriction enzyme and cloned into pET28 (a +) vector (Novagen, Cat. No. 69864-3) using the restriction site NcoI (blunted) and XhoI so that the protein produced contains a hexahistidine tag in the C-terminal region. Amplification of nucleic acid fragments is carried out according to the standard protocol for 25 cycles of PCR. The annealing temperature of the primers is selected experimentally. Processing of the PCR fragment and vector with restriction enzymes, ligation and transformation of TOR10 competent cells is carried out according to standard procedures. Sequencing of the obtained DNA confirmed compliance with the target structure (SEQ ID NO: 4). The resulting plasmid DNA was named pET28-IgA1-13-1.

Example 6

Obtaining recombinant plasmid DNA pET28-IgA1-13-1 and producer strain E. coli BL21 (DE3) / pET28-IgA1-13-1

Recombinant plasmid DNA was obtained from an overnight culture of E. coli TOP 10 cells using a standard alkaline method (Maniatis, T., Fritsch, EF and Sambrook, J. 1982. Molecular Cloning: a Laboratory Manual, Cold Sping Harbor Laboratory Press). A physical map of the recombinant plasmid DNA pET28-IgA1-13-1 is shown in FIG. 2. The E. coli BL21 (DE3) / pET28-IgA1-13-1 strain is obtained by introducing pET28-IgA1-13-1 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 in a ratio of 1: 100. The culture is grown to the early logarithmic phase on a rocking chair with intensive aeration (to an optical density at 600 nm of 0.4 ÷ 0.5 OE), it is rapidly cooled in ice, and cells are precipitated by centrifugation in for 10 min at 5000 rev / min at + 4 ° C. The precipitate was washed twice with 50 ml of 100 mM CaCl _2. The precipitated cells were resuspended in 2 l 100 mM CaCl ₂ with the addition of glycerol to 10%. Divide into aliquots of 50 .mu.l, quickly frozen and stored at -70 ° C.

To introduce pET28-IgA1-13-1 plasmid DNA into competent cells, 100 ng - 1 μg of plasmid DNA was added to 50 μl of a competent cell suspension and incubated for 40 minutes on ice. The resulting mixture was incubated at 42 ° C for 2 min, then in ice for 2 min, 1 ml of LB medium was added and incubated for 1 h at 37 ° C. Discrimination of cells (strain) containing recombinant plasmid DNA pET28-IgA1-13-1 is carried out on LB agar medium with kanamycin. For this, 100 μl of a suspension of bacterial cells are plated on Petri dishes with agar in LB medium containing 50 μg / ml kanamycin. The plates are incubated overnight at 37 ° C, the bacterial cells of several grown colonies are 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 ( night culture). 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.

Example 7

Determination of the productivity of the producer strain of the recombinant protein of SEQ ID NO: 4 E. coli BL21 (DE3) / pET28-IgA1-13-1

In 15 ml of LB liquid medium containing 50 μg / ml kanamycin, 1% inoculum of overnight cell culture is introduced and grown at 37 ° C on a shaker at 180 rpm for 2 hours until an optical absorption of 0.8 OE. The isopropylthiogalactopyranoside inductor (IPTG) is then added to a final concentration of 0.5 mM and incubation is continued for 2.0 hours under the same conditions. A 1 ml sample is taken and centrifuged for 6 minutes at 12,000 rpm, after which the cells are suspended in 100 μl of deionized water, add 33 μl of a buffer solution containing 125 mm Tris-HCl, pH 6.8, 20% glycerol, 3% sodium dodecyl sulfate (SDS), 3% mercaptoethanol and 0.01% bromphenol blue, heated for 10 min up to 95 ° C. An aliquot was taken and analyzed by electrophoresis on a 10% polyacrylamide gel containing 0.1% SDS. The gel is stained with Coomassie R250 and scanned on a laser scanner. The productivity of strain E. coli BL21 (DE3) / pET28-IgA1-13-1 is 35% of the recombinant protein SEQ ID NO: 4 of the total cellular protein.

Example 8

Preparation, Isolation, and Purification of Recombinant Protein SEQ ID NO: 4

A) Cultivation of E. coli strain BL21 (DE3) / pET28-IgA1-13-1

10 ml of the corresponding inoculum of overnight cell culture are added to 1 L of LB liquid medium containing 50 μg / ml kanamycin, and cells are grown at + 37 ° C on a shaker at 200 rpm to an optical density of 0.8 OE at 600 nm. The isopropylthiogalactopianoside inducer (IPTG) is then added to a final concentration of 0.5 mM and the cells are continued to incubate at + 37 ° C on a shaker for two hours.

B) Cell destruction and lysate fractionation

To collect cells, 1 liter of culture of the producer strain E. coli BL21 (DE3) / pET28-IgA1-13-1 (10 min, 3.5 thousand rpm) is centrifuged and the precipitated cells are suspended in a seven-fold volume of 20 mM Tris- buffer solution HCl, 0.15 M NaCl, pH 8.5, containing lysozyme (0.002%), incubated for 30 min at room temperature. A cell suspension (2 g of cells with 1 l of culture) is sonicated five times, centrifuged (1 hour, 20,000 g) to obtain fractions of soluble proteins (cell-free extract) and insoluble part of the cell lysate (debris and insoluble proteins). 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 precipitate containing TB after centrifugation is washed with a buffer solution of 60 ml of 20 mM Tris-HCl, 1% Triton X-100, 0.1% SDS, pH 8.5 and centrifuged for 40 min 10,000 g. The newly obtained precipitate was washed with a buffer solution of 60 ml of 20 mM Tris-HCl, 1 M NaCl and centrifuged for 40 minutes, 10,000 g. The yield of washed TB recombinant protein SEQ ID NO: 4 is 0.64 g. The washed precipitates of TV recombinant protein SEQ ID NO: 4 are used for chromatography on Ni-agarose under denaturing conditions.

C) Purification of the dissolved bodies of the inclusion of the recombinant protein SEQ ID NO: 4 using metal-chelate chromatography on a Ni-agarose sorbent TB, isolated from 2 g of E. coli BL21 (DE3) / pET28-IgA1-13-1 cells, dissolved in 100 ml of 50 mm Tris-HCl buffer solution containing 8 M urea, 0.15 M sodium chloride and 0.02 M imidazole, pH 9.0. After centrifugation and filtration, the solution was applied to a packed Ni-agarose column (HiTrap Chelating HP, GE Healthcare, 2 × 5 ml), equilibrated with the initial buffer solution, and the sorbent was washed with 50 ml of the initial buffer solution. The bound proteins are eluted with imidazole in a concentration gradient from 20 to 400 mM in the initial buffer solution, the gradient volume is 70 ml (35 × 35). Fractions containing purified protein of SEQ ID NO: 4 are combined and used at the stage of refolding and purification on Q-Sepharose.

D) Renaturation and purification of the recombinant protein of SEQ ID NO: 4 on Q-Sepharose

The combined eluate of the recombinant protein SEQ ID NO: 4 obtained after chromatography on Ni-agarose (protein concentration 0.4 mg / ml) is subjected to stepwise dialysis, gradually reducing the urea concentration to 1.5 M in 20 mm Tris-HCl buffer solution containing 0.15 M sodium chloride, pH 7.8. The resulting solution was centrifuged (30 min, 5000 g), filtered through a 0.2 μm pore membrane and applied to a packed Q-Sepharose column (GE 5 ml × 2), equilibrated in 20 mM Tris-HCl buffer solution containing 0.15 M sodium chloride and 1.5 M urea, pH 7.8.

The target protein that did not bind to the sorbent was collected in a single fraction, sorbitol (5% wt.) Was added, filtered through a sterile membrane with a pore size of 0.2 μm and Packed in sterile containers of 1 mg. The purity of the target protein is about 95% according to electrophoretic analysis (Fig. 3), the protein concentration is 0.2 mg / ml, the yield is 33 mg from 2 g of cell biomass.

Example 9

Determination of immunogenic and protective activity of recombinant proteins

The immunogenic and protective activity of recombinant proteins SEQ ID NO: 2, SEQ ID NO: 4 and recombinant IgA1 protease SEQ ID NO: 11 was evaluated on female Balb / C mice (16-18 g) from Pushchino nursery, Moscow region. Mice are kept in accordance with the IACUC protocol (IBCH 58a, 05/15/15). Experimental animals are divided into 4 groups of 21 mice in each - three groups are immunized with a recombinant protein SEQ ID NO: 2, obtained as described in example 4, or a recombinant protein SEQ ID NO: 4, obtained as described in example 8, or recombinant IgA1 protease SEQ ID NO: 11, prepared as described in Supplementary Example 1, one group was left to control (non-immunized mice). Mice are immunized intravenously by the administration of proteins at a dose of 40 μg per mouse into the retro-orbital sinus. Antigens are administered twice at 45-day intervals between injections. An indicator of the immunogenicity of the obtained recombinant proteins is an increase in the level of specific antibodies. The levels of specific antibodies in mouse sera are determined on the 30th day after the first immunization and on the 10th day after the second immunization with the method of solid-phase ELISA adsorbed onto microplate recombinant IgA1 protease SEQ ID NO: 11 using horseradish peroxidase conjugate with goat antibodies against total mouse immunoglobulins (O. Kotelnikova et al. Bulletin of Experimental Biology and Medicine, 161 (3): 369-372, 2016). The optical density is determined using a microplate reader for enzyme immunoassay at a wavelength of 492 nm. Antibody titers are calculated based on the optical densities obtained for the sera of immunized mice in comparison with the corresponding values obtained by titration of the sera of non-immunized animals.

The values of antibody levels (1 / antibody titer) in the serum of mice immunized with the studied recombinant proteins are shown in Table 1. Table 1 shows that after the first immunization, the level of specific antibodies to recombinant IgA1 protease SEQ ID NO: 11 is low in all three drugs, however, after repeated immunization, antibody titers increase by two orders of magnitude, which indicates the development of immunological memory in animals, the highest level of antibodies being for the shortest recombinant protein SEQ ID NO: 4.

The protective activity of recombinant proteins is evaluated on the 12th day after re-immunization of animals. Mice immunized with each of the preparations (21 animals) are divided into subgroups of 7 animals for intraperitoneal infection with live virulent cultures of meningococcus serogroup A (strain A208), B (strain H44 / 76) or C (strain C0638), the most common among circulating in Russia, at a dose of 0.25 × 10 ⁶ microbial cells. 4 hours after infection with a meningococcal culture, blood is sown individually for each animal in 12-well plates with a solid nutrient medium.

The protective activity of the drugs is evaluated by the level of bacteremia (the number of colony forming cells - CFU) in immunized mice in comparison with control (non-immunized) animals.

Statistical processing of the results is carried out using MS Office Excel Software using Probit Analysis and Student t-test. The results are presented as mean values ± standard deviation for p≤0.05 (%).

Table 2 shows the bacteremia level in mice immunized with the above recombinant proteins after infection of serogroups A, B, and C with meningococcus as compared to control non-immunized animals (% of the number of CFU in the control group of mice). Table 2 shows that the bacteremia level in the group of mice immunized with the recombinant proteins SEQ ID NO: 2 and SEQ ID NO: 4 is comparable or lower than the control recombinant IgA1 protease SEQ ID NO: 11 and does not exceed 40% of the control .

Example 10

Determination of the immunoregulatory index (CD4 + / CD8 +) in mice immunized with recombinant proteins of SEQ ID NO: 2 and SEQ ID NO: 4

The population composition of lymphocytes (CD4 + T-helpers and CD8 + cytotoxic T-lymphocytes) and the immunoregulatory index of immunity (CD4 + / CD8 +) in the blood of mice immunized with recombinant proteins SEQ ID NO: 2 and SEQ ID NO: 4 are evaluated by flow cytometry with corresponding monoclonal antibodies (Biolegend, USA) on a FACScan flow cytometer (BD Biosciences, USA) 6 months after re-immunization of animals. Analysis of the data obtained is carried out using the program Flowing Software 2.5.1 (Turku Center for Biotechnology, Finland). The immunoregulatory index is presented as a deviation from the control. The immunoregulatory index (CD4 + / CD8 +) in the blood of mice immunized with the recombinant proteins SEQ ID NO: 2 and SEQ ID NO: 4 is (1.00 ± 0.08) and (1.02 ± 0.05), respectively, and in the blood of control mice - (1.00 ± 0.05), which indicates the absence of violations of general homeostasis in the body of immunized animals after immunization of animals with the obtained recombinant proteins.

Additional example 1

A) Obtaining the nucleotide sequence of SEQ ID NO: 10 encoding the recombinant IgA1 protease of SEQ ID NO: 11

Based on the known sequence of the iga gene of strain H44 / 76 encoding the full-length IgA protease of N. meningitidis serogroup B strain H44 / 76 (https://www.ncbi.nlm.nih.gov/nuccore/GU190729.1), primers are designed to obtain the nucleotide the sequence of SEQ ID NO: 10 encoding the recombinant IgA1 protease of SEQ ID NO: 11, and its subsequent ligation with the 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) and used to produce proteins in E. coli strains lysogenic by T7 bacteriophage. For ligation with the vector pET-31 (b +) (Novagen Cat. No. 69952-3) to the primers (f6 - direct - 5'-CGAGACAGCCATATGGCATTGGTCAGAGACGATGTC-3 ', SEQ ID NO: 12, and f4 - reverse - 5'-GCGCTCGAGAGGCGGGGGGGGGGGGGGGGGGGGGGGG -3 ', SEQ ID NO: 8), flanking the target region of the iga gene of strain H44 / 76, introduces the sequence of restriction enzyme recognition sites NdeI and XhoI (respectively, in f6 and f4) present in the polylinker of the vector. The structure of primer f4, which does not contain termination codons, allows one to obtain a recombinant IgA1 protease modified in the C-terminal region with a hexahistidine tag. 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, their ligation and transformation of competent E. coli TOP 10 cells with a vector containing the nucleotide sequence of SEQ ID NO: 10 is carried out according to standard procedures. By sequencing the obtained plasmid DNA, the correspondence to the target structure of the recombinant protein SEQ ID NO: 11 was confirmed. The obtained plasmid DNA was named pET31-IgA1-1004.

B) Obtaining recombinant plasmid DNA pET28-IgA1-11-3 and producer strain E. coli BL21 (DE3) / pET31-IgA1-1004

Recombinant plasmid DNA was obtained from an overnight culture of E. coli TOP 10 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 for introduction into competent BL21 cells (DE3) by heat shock.

To obtain competent cells, the bacterial cells of the original E. coli strain BL21 (DE3) (E. coli B F-dcm ompT hsdS (rB-mB-) gal λ (DE3)) are grown in LB liquid medium overnight at 37 ° C and used for seeding 100 ml of the same medium in a ratio of 1: 100. The culture is grown to the early logarithmic phase on a rocking chair with intensive aeration (to an optical density at 600 nm equal to 0.4-0.5 OE), it is rapidly cooled in ice, the cells are precipitated by centrifugation for 10 min at 5000 rpm at +4 ° C. 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%, divided into aliquots of 50 μl, quickly frozen and stored at -70 ° C.

The strain E. coli BL21 (DE3) / pET31-IgA1-1004 is obtained by introducing plasmid DNA pET31-IgA1-1004 into competent cells of E. coli BL21 (DE3) using the heat shock method. 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 in ice for 2 min, 1 ml of LB medium was added and incubated for 1 h at 37 ° C. The genetic marker in the pET31 vector is the nucleotide sequence (amp), which determines the resistance of ampicillin to bacterial cells transformed with the pET31-IgA1-1004 plasmid. After that, 100 μl of a suspension of bacterial cells are plated on Petri dishes with agar in LB medium containing 100 μg / ml ampicillin. The plates are incubated overnight at 37 ° C, the bacterial cells of several grown colonies are used for independent inoculation of microbiological tubes with 5 ml of LB liquid medium containing 100 μg / ml ampicillin, followed by growth of bacterial cells on a rocking chair at 37 ° C overnight ( night culture). 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.

C) Determination of the productivity of the producer strain of the recombinant IgA1 protease SEQ ID NO: 11 E. coli BL21 (DE3) / pET31-IgA1-1004

In 15 ml of liquid LB medium containing 50 μg / ml ampicillin, 1% inoculum of overnight cell culture was added and grown at 37 ° C on a shaker at 180 rpm for 2 hours to an OD of 0.8. The isopropylthiogalactopyranoside inductor (IPTG) is then added to a final concentration of 0.5 mM and incubation is continued for 2.0 hours under the same conditions. A 1 ml sample is taken and centrifuged for 6 minutes at 12,000 rpm, after which the cells are suspended in 100 μl of deionized water, add 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, heated for 10 min to 95 ° FROM. An aliquot was taken and analyzed by electrophoresis in 10% PAG containing 0.1% sodium dodecyl sulfate. The gel is stained with Coomassie R250 and scanned on a laser scanner. The productivity of E. coli strain BL21 (DE3) / pET31-IgA1-1004 is 40% of the recombinant protein of the total cellular protein.

D) Preparation, Isolation and Purification of Recombinant IgA1 Protease SEQ ID NO: 11

1) Cultivation of E. coli strain BL21 (DE3) / pET31-IgA1-1004

10 ml of the corresponding inoculum of the overnight cell culture are introduced into 1 L of LB liquid medium containing 100 μg / ml ampicillin, and cells are grown at + 37 ° C on a shaker at 200 rpm to an optical density of 0.8 OE at 600 nm. Then the isopropylthiogalactopianoside inducer (IPTG) is added to a final concentration of 0.5 mM and the cells are continued to incubate at + 37 ° C on a shaker for two hours.

2) Cell destruction and lysate fractionation.

To collect cells, centrifuged 1 l of the culture of the producer strain E. coli

BL21 (DE3) / pET31-IgA1-1004 (10 min, 3.5 k rpm) and the precipitated cells are suspended in a seven-fold volume of 20 mM Tris-HCl buffer solution, 0.15 M NaCl, pH 8.5, containing lysozyme (0.002%), incubated for 30 minutes at room temperature. A cell suspension (2 g of 1 L of the induced culture) is sonicated five times and centrifuged (1 hour, 20,000 g), resulting in a supernatant and a pellet. The supernatant contains soluble proteins (cell-free extract) and sediment - the insoluble part of the cell lysate (debris and insoluble proteins). According to the results of electrophoretic analysis, the target protein is an insoluble protein and forms TB, regardless of the temperature conditions of culture induction and the type of buffer used to destroy cells. The precipitate containing TB after centrifugation is washed with a buffer solution of 60 ml of 20 mM Tris-HCl, 1% Triton X-100, 0.1% SDS, pH 8.5, and again centrifuged for 40 min 10,000 g. The resulting precipitate was washed with a buffer solution of 60 ml of 20 mM Tris-HCl buffer, IM NaCl, pH 8.5, centrifuged for 40 minutes, 10,000 g. The precipitate obtained after centrifugation is used for chromatography on Ni-agarose under denaturing conditions.

3) Purification of the dissolved bodies of the inclusion of the recombinant IgA1 protease SEQ ID NO: 11 using metal-chelate chromatography on a Ni-agarose sorbent TB, isolated from 2 g of E. coli BL21 (DE3) / pET31-IgA1-1004 cells, dissolved in 100 ml 20 mM Tris-HCl buffer solution containing 0.15 M NaCl, 8 M urea and 0.02 M imidazole, pH 8.1. After centrifugation and filtration, the solution was applied to a packed Ni-agarose column (HiTrap Chelating HP, GE Healthcare, 2 × 5 ml), equilibrated with the initial buffer solution, and the sorbent was washed with 50 ml of the initial buffer solution. The bound proteins are eluted with imidazole in a concentration gradient from 20 to 400 mM in the initial buffer solution, the gradient volume is 100 ml (50 × 50).

Fractions containing the purified protein of SEQ ID NO: 11 are combined and used in the renaturation and purification step on Q-Sepharose.

4) Renaturation and purification of the recombinant IgA1 protease SEQ ID NO: 11 on Q-Sepharose

The combined recombinant protease eluate of SEQ ID NO: 11, obtained after chromatography on Ni-agarose (protein concentration 0.3 mg / ml), is subjected to stepwise dialysis, gradually reducing the urea concentration to 1.5 M in 20 mm Tris-HCl buffer solution containing 0.15 M sodium chloride, pH 7.0. The resulting solution was centrifuged (30 min, 5000 g), filtered through a 0.2 μm pore membrane and applied to a packed Q-Sepharose column (GE Healthcare 2 × 5 ml), equilibrated in 20 mM Tris-HCl buffer solution containing 0.15 M sodium chloride and 1.5 M urea, pH 7.0. The target protein that did not bind to the sorbent was collected in a single fraction, sorbitol (5 wt%) was added, filtered through a sterile membrane with a pore size of 0.2 μm and packed in sterile containers of 2 mg. The purity of the target protein is about 90% according to electrophoretic analysis (Fig. 3), the protein concentration is 0.25 mg / ml, the yield is 34 mg from 2 g of cell biomass.

Thus, recombinant proteins consisting of three SEQ ID NO: 2 or two SEQ ID NO: 4 (variant) polypeptides from different parts of the Ala28-Pro1004 fragment of the primary structure of the IgA protease of N. meningitidis serogroup B, strain H44 / 76, combined into a single polypeptide chain, capable of protecting animals from infection by a live virulent culture of meningococcus of the main epidemic groups A, B and C and forming immunological memory as effectively as the recombinant IgA1 protease SEQ ID NO: 11. These data, together with the absence of disturbances in general homeostasis in animals, immunized with recombinant proteins SEQ ID NO: 2 and SEQ ID NO: 4, indicate the possibility of using any of these proteins as the basis for potential

 monocomponent vaccine to protect against meningococcal infection and other pathogens, the virulence of which is due to the IgA1 protease.

Claims (6)

1. Recombinant protein with a protective effect against meningococci, having the amino acid sequence of SEQ ID NO: 2, consisting of three fragments of the primary structure of IgA protease Neisseria meningitidis serogroup B, connected in a single polypeptide chain. 2. Recombinant protein having a protective effect against meningococci, having the amino acid sequence of SEQ ID NO: 4, consisting of two fragments of the primary structure of IgA protease Neisseria meningitidis serogroup B, connected in a single polypeptide chain. 3. The polynucleotide encoding the recombinant protein according to claim 1 or 2, with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. 4. Recombinant plasmid DNA, comprising the polynucleotide according to claim 3, which provides expression of the recombinant protein according to claim 1 or 2 in the host cell. 5. A host cell of Escherichia coli BL21 (DE3), transformed with the recombinant plasmid DNA according to claim 4, producing a recombinant protein according to claim 1 or 2. 6. The method of producing the recombinant protein according to claim 1 or 2, which consists in the fact that the host cell is transformed with the recombinant plasmid DNA according to claim 4, the transformed cells according to claim 5 are cultured to obtain the recombinant protein according to claim 1 or 2, and the target the product is isolated from inclusion bodies and purified.

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