RU2387715C2 - RECOMBINANT DNA PROVIDING PRODUCTION OF POLYPEPTIDES P6, P7, P8 EXHIBITING PROTECTIVE PROPERTIES AGAINST STREPTOCOCCUS AGALACTIAE AND SELECTIVELY CONNECTING IgA - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used in producing vaccines against Streptococcus agalactiae - a representative of streptococci group B (SGB) in diagnostics of the diseases - for creation of a detection system of immunoglobulin A level in biological fluids, in immunochemistry as accessible immunochemical reagents (affine recovered IgA fragments). Offered unique recombinant DNA are produced by polymerase chain reaction (PCR) with using chromosomal DNA of strain 219/4849 Ibc of serotype SGB and unique primers. One recombinant DNA contains three nucleotide substitutes in comparison with an initial site of chromosomal DNA. The following cloning of amplified fragments is carried out in a linear vector pGEM-T Easy, and at the final stage by the system of express ionic vectors pQE30/31/32 in E coli JM 109. The produced recombinant DNA code amino acid sequences of recombinant polypeptides exhibiting ability to connect selectively various molecular forms of IgA and designated as P6, P7, P8. Polypeptide P6 causes synthesis of long circulating high-affine anti-Rb antibodies possessing protective properties against SGB.

EFFECT: application of the invention provides production of recombinant polypeptides based N-terminal conservative part of surface Bac SGB of Ibc serotype and containing a first IgA-connecting site A with changed or native sequence MLKKIE, polypeptide exhibits immunologically relevant and protective properties, and they also high selectively connect IgA.

12 cl, 20 dwg, 4 tbl, 19 ex

Description

The invention relates to the field of microbiology and molecular genetics and can be used in the medical industry in the production of vaccines against Streptococcus agalactiae. The invention can also be used to create a diagnosticum for detecting the level of immunoglobulin A (IgA) in biological fluids and can be used in immunochemistry as available immunochemical reagents.

Streptococcus agalactiae, a representative of group B streptococci (HBV), refers to opportunistic microorganisms that cause severe human diseases: arthritis, endocarditis, pyelonephritis, mastitis, septicemia, necrotic fasciitis, ophthalmitis and others. SGV are one of the leading causative agents in the etiology of pregnancy pathology, severe infections of newborns and young children, causing the occurrence of sepsis and meningitis. Newborns acquire bacteria through the birth canal of their mother, who is a carrier of HBV. Streptococcal infection in older children is often expressed as arthritis, osteomyelitis, and skin lesions. Group B streptococci are also capable of causing miscarriages, intrauterine damage to the fetus, postpartum sepsis and other pathologies in adults. A common carriage of hypertension in the urogenital region and in the rectum is described. The most severe diseases occur in people with weakened immune systems. A wide range of serious diseases caused by GBV, mainly among children of perinatal and early childhood, as well as women of childbearing age, is considered a significant medical and epidemiological problem in most countries, including developed ones, despite preventive measures. Prevention of infectious diseases of streptococcal etiology is carried out using benzathine benzylpenicillin, erythromycin, ampicillin and sulbactam (Burbello A.T. et al. Sovrem. Lekar. Means, St. Petersburg: Neva, 323-324, 332, 352 (2003)). Antibiotics are used to treat patients with diseases caused by HBV (Strachunsky L.S., Kozlov S.N., Sovrem. Antimicrob. Chemioter., M .: Borges, 1-432 (2002), Polyak M.S. Basics of antibiotic therapy , St. Petersburg: Research Center, 1-53 (2003)). The most widespread representatives of the penicillin group are benzylpenicillin and ampicillin, as well as macrolides: erythromycin, vancomycin, azithromycin, clarithromycin, roxithromycin ((Semina N.A., Sidorenko S.V. et al., Klin. Microbiol. Antimicrob. Chemioter 6. (4): 306-359 (2004)), Zueva L.P., Polyak M.S. et al., Microbiological Monitoring, St. Petersburg: Medical Information and Analytical Center, 1-72 (2004)).

Difficulty in the treatment and prevention through antibiotic therapy arises due to the variety of side effects that appear in patients: a negative effect on the central nervous and cardiovascular systems, the body’s immune system, impaired microbiocenosis, and allergic reactions. The ineffectiveness of antibiotic therapy in some cases is due to the appearance of antibiotic-resistant strains of HBV.

The lack of safe and effective measures for the prevention and treatment of diseases caused by HBV makes the development of antibacterial vaccines designed, including those based on proteins of microbial origin, relevant.

The development of vaccines is not the only topical area for the use of bacterial proteins. For example, proteins G and L (surface proteins of streptococci C and G), protein A of staphylococci have found active use as immunochemical reagents, aimed mainly at the determination of IgG. Currently, interest has grown in the IgA molecule, the variety of functions of which, as well as the possibility of using it for diagnostic purposes, require the creation of highly selective reagents with IgA receptor activity.

A unique surface protein, Vas protein, or β antigen (130 kDa), which has no homologs among other bacterial proteins with IgA receptor activity, is considered as an essential factor in the virulence of HBV. One of the features of you protein is the presence in its structure of a region of homology with proteins of the eukaryotic immunoglobulin superfamily. Expressed by many strains of serotypes Ia, Ib, II, and V, you protein is practically not represented in strains of HBV of the third serotype.

For the implementation of biological functions, β antigen has a number of properties. It binds non-immune to the constant (Fc) region of IgA, the main immunoglobulin that protects the mucous membranes from the penetration of microorganisms. Also, you protein interacts with human factor H (FH) - a blood plasma protein, the binding of which leads to inactivation of the alternative complement pathway and promotes the adhesion of the microorganism to human cells. The ability to interact with these two components of the human immune system allows HBV to evade the immune response (Eur. J. Immunol. - 1985. - No. 15. - P.893-899; Adv. In host defense mechanisms. - 1985. - V .4. - P.31-61). The prevalence among SGV strains, an important role in ensuring the virulence of bacteria, and the conservation of protein structure have determined the choice of β-antigen as the basis for creating potential components of vaccine preparations.

For the first time, the IgA receptor properties of you protein extracted with detergents from the surface of bacteria and subjected to multi-stage purification were described by Russell-Jones et al. In 1984 (J. exp.med. - 1984. - V.160. - P.1467-1475) .

In 1987, a gene encoding a β antigen was first cloned by Swiss scientists. The source of DNA was the clinical isolate of HBV Ic serotype, which was the cause of meningitis in newborns. Cloning was carried out using the plasmid vector pUC18 in E. coli K-12 (Infection and immunity. - 1987. - V.55, No. 5. - P.1151-1155).

The prototype of the invention is the nucleotide sequence encoding the IgA-binding site A in the β-antigen of HBV. The DNA of the LA239 Ibc strain of the HBV serotype served as the source of such a nucleotide sequence that was cloned in E. coli K-12 537 using the expression plasmid vector pEx31 in 1991 (Mol Microbiol. - 1991. - 5 (4). - P.843- 9). As a result of this work, recombinant IABF protein (8.5 kDa) was expressed and purified.

In 1996, truncated fragments of the IABF protein were obtained, and the MLKKIE hexapeptide was identified. The MLKKIE region localized at the IgA-binding site A (73 amino acids) was believed to be responsible for binding to you with the IgA Fc region protein (Infection and immunity. - 1996. - V.64, No. 7. - P.2787-2793) . IABF derivatives obtained by the authors of this study did not possess IgA receptor activity, which does not allow their use for diagnostic purposes and as IgA binding agents.

The objective of this invention was to obtain, on the basis of the N-terminal conservative part of the surface of you HBV Ibc protein, a serotype of recombinant polypeptides comprising the first IgA-binding site A with an altered or native MLKKIE sequence. Moreover, the polypeptides should have immunogenic and protective properties in order to use them as components of a vaccine against group B streptococci. In addition, polypeptides had to selectively bind IgA to create IgA receptor immunochemical reagents. To determine the possibility of using polypeptides as receptors for a partially denatured IgA molecule, it was necessary to isolate the binding sites on the IgA molecule of the previously described P6 polypeptide, as well as the polypeptides designated as P7, P8, and to test their resistance to denaturing effects. Another objective of the invention was the creation of producer strains of recombinant derivatives of β antigen.

The result of the invention was to obtain the designated objects of biotechnology.

The problem was solved by constructing pairs of unique primers directed to the regions of the gene for you that encodes the HBV protein. Their use in the polymerase chain reaction made it possible to obtain the desired DNA fragments subsequently ligated with the plasmid vector pGEM-T Easy. The polylinker of the indicated commercial vector is localized in the region encoding β-galactosidase, which made it possible to select E. coli JM 109 transformants with the necessary construction. Subsequently, the cloning of β antigen fragments was carried out using the pQE30 / 31/32 expression vector system, which facilitates the isolation and purification of recombinant derivatives. The creation of producer strains designated as E. coli JM 109-P6, E. coli JM 109-P7 and E. coli JM 109-P8 was carried out by genetic transformation of the strain based on strains of E. coli JM 109. The resulting producer strains E .coli JM 109-P6, E. coli JM 109-P7 and E. coli JM 109-P8 include plasmids containing a portion of the bac gene sequence published by Pierre G. Jerlstrőm (Infection and immunity. - 1996. - V.64, No. 7 - P. 2787-2793) associated with the DNA region of the pQE32 vector. The plasmid in E. coli strain JM 109-P7 contains a genetic replacement for this sequence in the region encoding the region described by Jerlstrőm. Strain E. coli JM 109-P6 was identical to the strain created earlier (Journal of Microbiology, Epidemiology and Immunobiology. - 2005. - No. 2. - P.35-40), but includes a previously undetermined nucleotide sequence encoding the P6 polypeptide from previously unspecified amino acid sequence. The transformation of bacteria has led to the emergence of the ability of inducible expression of recombinant polypeptides P6, P7, P8.

The essence of the invention is the creation of unique fragments of the gene of you - from 406 bp 1287 bp (designated as p6), with 301 bp 1653 bp each (designated as p8) and recombinant DNA with 301 bp 1653 bp each (designated as p7) obtained by polymerase chain reaction. Moreover, the creation of recombinant p7 DNA was performed by ligation of amplified recombinant DNA with 301 bp 682 bp each (designated as p71) and from 662 bp 1653 bp each (designated as p72) gene of you . p71 and p72 include, in turn, three nucleotide substitutions in comparison with the gene sequence of you . Amplification of these fragments was carried out using the chromosomal DNA of strain 219/4849 Ibc of the SGV serotype (strains from the collection of the National Streptococcal Center of Russia) as a matrix, unique primers designated as Pr6 and Pr3 to create p6, Pr5 and Pr8 for p8, as well as Pr5 and Pr2 for p71, Pr7 and Pr8 for p72 (primer sequences are shown in table 1). Moreover, when creating p6, newly designed primers were used that are different from those given in previously published works (Journal of Microbiology, Epidemiology and Immunobiology. - 2005. - No. 2. - P.35-40). Subsequent cloning of the obtained fragments of chromosomal DNA and recombinant DNA occurred first with the pGEM-T Easy plasmid vector (Promega, USA) and then with the pQE-30/31/32 expression vector system (The QIAexpress System, Qiagen, USA) in E. coli JM 109. The obtained fragments of chromosomal DNA and recombinant DNAs encode unique amino acid sequences of recombinant polypeptides P6, P7, P8, and, depending on the expression vector used, the polypeptides have or do not have a covalent bond with six histidines, coding vector pQE32.

Also the essence of the invention is the creation of producer strains of E. coli JM 109-P6 ′, E. coli JM 109-P7 ′, E.coli JM 109-P8 ′, allowing under certain conditions to induce the synthesis of recombinant polypeptides P6, P7, P8.

The authors obtained gene fragments have a size 882 bp (p6), 1353 n.p. (p8), 382 n.p. p71 and 992 n.p. p72 (after ligation: 1353 n.p., p7) during the polymerase chain reaction based on the chromosomal DNA of strain 219/4849 Ibc serotype SGV using the corresponding pairs of primers. The authors also ligated p6, p7, p8 nucleotide sequences with pGEM-T Easy plasmid vectors, primary selection of bacterial clones containing the necessary inserts, and cloning of p6, p7, p8 fragments into pQE-30/31/32 expression vector systems with further transformation recombinant plasmid DNA in the heterological system of E. coli JM 109. As a result, the authors obtained producer strains of E. coli JM 109-P6 ′, E. coli JM 109-P7 ′, E. coli JM 109-P8 ′, respectively, recombinant proteins P6, P7, P8. The authors also performed the isolation and single-stage purification of recombinant derivatives of β antigen by inducible expression of P6, P7, P8 polypeptides and affinity chromatography on Ni-NTA agarose (Qiagen, USA).

In addition, the authors obtained an immunochemical characterization of the interaction of various molecular forms of IgA with P6, P7, P8 polypeptides, determined the relative position of the polypeptide binding site on the IgA molecule, its non-conservative nature and resistance to denaturing effects.

It is known from literature that the P6 polypeptide, with a molecular weight of 35 kDa, induces the production of specific class G antibodies in mice by intraperitoneal administration, with a maximum antibody titer of 1: 100,000. It was found that the immune response is heterogeneous for antibody affinity. The values of the binding constants ranged from 10 ⁸ M ^-1 to 10 ¹³ M ^-1 for P6 (Medical Immunology, 2004, T. 6, p. 493-498). The P6 polypeptide has the ability to synthesize long-circulating high-affinity anti-P6 antibodies that have protective properties against HBV (Journal of Microbiology, Epidemiology and Immunobiology. - 2005. - No. 2. - P.35-40). The protective properties of anti-P6 and anti-ScaAB (surface HBV lipoprotein) antibodies were previously studied when combined and separately administered in vitro model experiments (opsonophagocytic test using peritoneal mouse macrophages and group B streptococci) (Medical Immunology, 2006, v. 8, p. 109-478). Polypeptides P6 and ScaAB, due to the ability to provide protection at extremely low titers of antibodies with opsonizing activity, including against SGA, as well as strains of III serotype of HBV, are very promising as vaccine components.

The invention shows that recombinant polypeptides P6, P7, P8 have the ability to selectively bind various molecular forms of IgA. The obtained and characterized recombinant P7, P8 polypeptides are promising as components of a vaccine preparation specific for a wide range of HBV strains. Proteins P6 and P8 with selective IgA receptor activity may be useful for rapid diagnostics of the level of immunoglobulin A in various biological fluids to assess the clinical condition of patients or the general immune status, which may play a role in the diagnosis of diseases. Also, recombinant derivatives P6, P8 have a wide range of practical applications in immunology: as accessible and convenient IgA-binding immunochemical reagents based on proteins of bacterial origin in ELISA (both in the adsorbed state and in the role of detecting molecules), for manipulating a denatured molecule IgA - immunoblot, with the aim of affinity isolation of IgA fragments.

Obtaining recombinant polypeptides P6, P7, P8

The source of chromosomal DNA was strain 219/4849 Ibc serotype SGV. DNA isolated by phenol-chloroform extraction was used as a template in the polymerase chain reaction (PCR) to obtain fragments of the bac: p6 gene (from 406 bp to 1287 bp, a total of 882 bp), p7 (from 301 bp to 682 bp, a total of 382 bp and from 662 bp to 1653 bp, a total of 992 bp, after ligation of two fragments: 1353 bp n.), p8 (from 301 bp to 1653 bp, a total of 1353 bp), encoding polypeptides P6, P7 and P8, respectively. Moreover, it was decided to introduce two amino acid substitutions into the potential antigenic determinant of the P7 polypeptide (lysine at position 225 is replaced by arginine, and glutamate at position 227 is replaced by glutamine). As a result, a distinctive feature of the P7 polypeptide is the presence of an altered (compared to the MLKKIE region of the Bac protein) sequence of 6 amino acids MLKRIQ located on the IgA-binding site. With a view to modifying hexapeptide MLKKIE and, accordingly, creation area MLKRIQ, it was decided to carry out the amplification of two overlapping fragments of your gene 301 bp - 682 bp, 662 bp - 1653 bp, spanning the region encoding MLKKIE. To this end, reverse Pr2 and direct Pr7 primers were constructed by nucleotide substitutions, respectively participating in the amplification of the first (382 bp, p71) and second (992 bp, p71) DNA fragments and containing the EcoRI site. Oligonucleotide primers are shown in Table 1.

Moreover, three nucleotide substitutions were introduced in the initial primer sequences Pr2 ′ (CTT CG ATT T TTTTCAGCATAG) and Pr7 ′ (CTATGCTGAAAA A AAT CG AAG) in such a way that this led to the formation of Pr2 and Pr7 primers containing the G AA T TC region encoding EcoRI website.

The analysis of PCR results was carried out by separating DNA fragments in a 1% agarose gel using horizontal electrophoresis. The selection of the desired amplified DNA sections was carried out using the QIAquick Gel Extraction Kit (Qiagen, USA).

382 bp fragments isolated from agarose and 992 n.p. were digested with EcoRI restriction enzyme and then ligated to each other, resulting in the formation of the p7 nucleotide sequence (1353 bp) containing the EcoRI site.

The nucleotide sequence of p6 is presented in figure 1.

The nucleotide sequence of p7 is presented in figure 2.

The nucleotide sequence of p8 is presented in figure 3.

The p6 nucleotide sequence was ligated with the pGEM-T Easy vector (Promega, USA, 3018 bp), which did not require restriction sites, according to the manufacturer's instructions. The result was a recombinant plasmid DNA, designated as pGEM-p6 and containing a fragment of you gene 882 bp - p6, as well as the plasmid pGEM-T Easy.

Similarly, recombinant plasmid DNA, designated as pGEM-p7 and containing a 1353 bp fragment of you gene, was obtained. with three nucleotide substitutions - p7, as well as the plasmid pGEM-T Easy.

Similarly, recombinant plasmid DNA, designated as pGEM-p8 and containing a 1353 bp fragment of you gene, was obtained. - p8, as well as the plasmid pGEM-T Easy.

The ligation products carried out the calcium transformation of E. coli strain JM109, after which the transformants were sown on Petri dishes with the addition of ampicillin (Ap), 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) and isopropyl- β-D-thiogalactopyranoside (IPTG). In the vector plasmid pGEM-T, the polylinker is located in the region encoding β-galactosidase. When growing on a medium containing Ap, X-gal, and IPTG, the colonies containing the plasmid without insertion are blue, and the colonies containing the recombinant plasmid are white, which allowed the selection of transformants with the insert according to the color of the bacterial colonies. Bacterial clones were checked for presence of correct size insert your gene by PCR using primers Pr6 and Pr3, Pr5 and Pr8 and subsequent electrophoretic analysis of the resulting DNA fragments on a 1.5% agarose gel.

From the selected transformants designated as E. coli JM109-P6, E. coli JM109-P7, E. coli JM109-P8, using the Mini-prep plasmid DNA purification kit (Qiagen, USA), plasmids containing β antigen fragments were isolated .

To isolate DNA fragments encoding the P6, P7, P8 polypeptides, the obtained plasmid DNAs (designated as pGEM-p6, pGEM-p7, pGEM-p8) were restricted. Restriction was carried out at SphI-PstI sites, since restriction sites by SphI and PstI enzymes were originally laid down in the sequence of the pGEM-T Easy vector. The pQE30 / 31/32 expression vector system (The QIAexpress System, Qiagen, USA), into which each of the three cut out full-length inserts will be cloned, was also pretreated with SphI and PstI enzymes. Restriction products were separated by horizontal electrophoresis on a 1% agarose gel. The restricted plasmid pGEM-T and p6, p7, p8 flanked by restriction sites SphI and PstI were isolated from agarose using the QIAquick Gel Extraction Kit (Qiagen, USA). As a result of cloning into the pQE30 / 31/32 vector system, recombinant plasmid DNAs (designated pQE-p6, pQE-p7, pQE-p8) containing recombinant DNA corresponding to p6 ′ were obtained (consisting of p6 - 882 bp of the gene region you and 44 bp of the plasmid pQE-32 fragment), p7 ′ (consists of p7 - 1353 bp of the modified portion of you gene and 44 bp of the plasmid pQE-32 fragment), p8 ′ (consists of p8- 1353 bp of the you gene region and 44 bp of the plasmid pQE-32 fragment).

Recombinant plasmid DNAs pQE-p6, pQE-p7, pQE-p8 were transformed into E. coli JM109 using the calcium method and The QIAexpress System reagents from Qiagen, USA. Transformation of E coli JM 109 cells was performed with recombinant plasmid DNA pQE-p6, pQE-p7, pQE-p8 using positive (E coli JM 109, transformed with the original plasmid DNA pQE30 / 31/32) and negative (cell culture E coli JM 109, not transformed) controls. The transformation process was carried out by the method of transformation of Escherichia coli, involving the incubation of a culture of E coli JM 109 suspended in a solution of CaCl ₂ with plasmid DNA at different temperatures (Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory manual. - Cold Spring Harbor : Cold Spring Harbor Laboratory Press, 1982.- 867 p.). Selective transformant selection media contained 50 μg / ml ampicillin, 25 μg / ml kanamycin.

In each of the three variants of bacterial clones containing modified pQE plasmids carrying genes for resistance to these antibiotics, ampicillin- and kanamycin-resistant transformants were selected. The presence of recombinant plasmids pQE-p6, pQE-p7, pQE-p8 in transformed E. coli JM109 strains was determined by PCR using the original primers Pr6 and Pr3, Pr5 and Pr8 and subsequent electrophoretic analysis of the obtained DNA fragments in 1.5% agarose gel , on the basis of which the selection of the corresponding clones was carried out.

In order to identify transformants containing one of three variants of recombinant plasmids pQE-30, pQE-31, pQE-32, in which the translation frame of the E. coli gene coincided with the translation frame of the inserted gene, it was necessary to test bacterial clones for the ability to induce expression of recombinant polypeptides P6, P7, P8. Cultures of E. coli strain JM109 were cultured in Terrific Broth liquid medium supplemented with ampicillin and kanamycin (to avoid cantomination). PQE expression vectors contain resistance genes for ampicillin and kanamycin resistance, and a promoter for transcriptional induction of the recombinant protein by adding IPTG to the medium, and as a host pQE plasmids used E. coli strains with a mutation lacI ^Q, constitutively producing the lac-repressor, which prevents protein expression . The cultures continued to grow in order to increase the number of polypeptides, then E. coli cells were precipitated by centrifugation, washed and suspended in binding buffer A, pH 7.9, and the cell wall was destroyed by ultrasound on an ultrasonic disintegrator. The lysate was freed from cell debris by centrifugation. The polypeptides were purified by affinity chromatography on Ni-NTA agarose (Qiagen, USA). A high degree of protein purification is achieved due to the strong affinity interaction between Ni and the chain of six histidines associated with the recombinant polypeptide and the encoded region preceding the polylinker in pQE plasmids. Non-specifically bound proteins were removed with buffer A, elution of the recombinant derivatives of you protein was carried out using elution buffer B pH 7.9 containing 400 mM EDTA. The resulting polypeptide preparations were purified from the elution buffer by dialysis against phosphate buffered saline, pH 7.4 (PBS).

The isolated recombinant proteins were analyzed by SDS-polyacrylamide gel electrophoresis, which made it possible to conclude that the purification of polypeptides was satisfactory, as well as their molecular weight: in P6 - (35 ± 0.5) kDa, in P7 and P8 - (52 ± 0 , 5) kDa compared to the mileage of proteins of known molecular weight (Precision Plus Protein standards (161-0373), Bio-Rad, USA).

The expression of polypeptides P6, P7, P8 is realized only in a transformed E. coli culture, including the recombinant plasmid pQE32. This indicates that the translation frame of the E. coli gene coincides with the translation frame of the inserted gene.

The drawings illustrate the experiment.

Figure 1 (The nucleotide sequence of the p6 chromosomal DNA fragment) represents the nucleotide sequence of the p6 chromosomal DNA fragment - 882 bp encoding the amino acid sequence of the recombinant P6 polypeptide.

Figure 2 (The nucleotide sequence of recombinant DNA p7) represents the nucleotide sequence of recombinant DNA p7 - 1353 bp encoding the amino acid sequence of the recombinant P7 polypeptide.

Figure 3 (The nucleotide sequence of the p8 chromosomal DNA fragment) represents the nucleotide sequence of the p8 chromosomal DNA fragment - 1353 bp encoding the amino acid sequence of the recombinant P8 polypeptide.

Figure 4 (Amino acid sequence of the recombinant P6 polypeptide) represents the amino acid sequence of the recombinant P6 polypeptide, P6 consists of 294 amino acid residues.

5 (Amino acid sequence of the recombinant P7 polypeptide) represents the amino acid sequence of the recombinant P7 polypeptide, P7 consists of 451 amino acid residues.

6 (Amino acid sequence of a recombinant P8 polypeptide) represents the amino acid sequence of a recombinant P8 polypeptide, P8 consists of 451 amino acid residues.

Thus, the recombinant P6 polypeptide is a fragment of the amino acid sequence of the surface of you protein of the HBV strain of strain 219/4849 Ibc serotype 136 to 429 amino acid residues. Recombinant P7 polypeptide is a fragment of the amino acid sequence of the surface of you protein of the HBV protein of strain 219/4849 Ibc serotype 101 to 551 amino acid residues with two amino acid changes. Recombinant P8 polypeptide is a fragment of the amino acid sequence of the surface of you protein of the HBV protein of strain 219/4849 Ibc serotype 101 to 551 amino acid residues.

The amino acid sequences of the three indicated β antigen derivatives are covalently linked to six histidines encoded by the pQE32 vector (The QIAexpress System, Qiagen, USA). Polypeptides P6 and P8 are designed in such a way that, along with you, the protein contains an IgA-binding site, including the MLKKIE hexapeptide. The P7 polypeptide also contains a site responsible for the implementation of IgA receptor activity by protein, however, in the six-amino acid region, two of them were replaced - lysine 225 with arginine and glutamate 227 with glutamine, which significantly impairs the binding of the β antigen derivative to IgA according to the authors .

The location of the amino acid sequences encoded by p6, p7, p8, relative to the amino acid sequence of surface you protein HBV shown in Fig.7.

In Fig.7 (Scheme of the amino acid sequences of you of the HBV protein, recombinant polypeptides P6, P7, P8):

1 - signal sequence;

2 - plot responsible for IgA binding;

3 is a region similar to sequences of proteins of the eukaryotic immunoglobulin superfamily (IgSF) family;

4 - the site responsible for the binding of human factor H (FH);

5 - XPZ plot, has the structure of polyproline-II, consists of repeating triplets, is rich in proline;

6 - C-terminal anchor region (contains LPYTG motif).

The study of the interaction of recombinant polypeptides P6, P7, P8 with various molecular forms of IgA

The authors obtained the immunochemical characteristics of the recombinant molecules P6, P7, P8, which are fragments of the bacterial IgA receptor for you. The results of the work allow us to expand the scope of application of P6, P7, P8 polypeptides, supplementing it with the use of diagnosticums and for the affinity isolation of IgA fragments.

A comparison of the receptor activity of the recombinant derivatives P6, P7, P8 in relation to the molecular forms of IgA represented by the following samples: serum IgA (mIgA) - human polyclonal serum IgA labeled with horseradish peroxidase; polyclonal serum unlabeled IgA; IgA1 and IgA2 - blood serum of patients with myeloma, as well as secretory IgA (SIgA) - secretory polyclonal IgA isolated from colostrum; chigain.

It was found that P6, P7, P8 have the ability to selectively (under conditions of indirect ELISA, the absence of interaction with polyclonal IgG, as well as with monoclonal IgG samples from 1 to 4 subclasses) is associated with all of the studied forms of IgA in any of the used ELISA variants (direct ELISA, indirect ELISA, inhibitory ELISA, competitive inhibition). Such a result is logical if the properties of the β antigen are extended to its recombinant derivatives. Despite this, structural differences between the three polypeptides, implemented, as indicated above, by molecular genetics methods, contribute to a significant differentiation of the recombinant proteins P6, P7, P8 by IgA-binding properties. Protein P7, containing the altered sequence of MLKRIQ, but otherwise completely identical to the recombinant protein P8, has a less pronounced ability to bind all the considered molecular forms of IgA. The latter indicates the importance of the MLKKIE hexapeptide in the realization of IgA receptor activity by proteins. The minimal efficiency of binding to class A immunoglobulin among the studied proteins makes it possible to consider P7 as one of the most acceptable options for creating a vaccine against HBV. It was shown that the structure of the recombinant derivative of P6 is optimal for the implementation of IgA receptor activity among polypeptides of P6, P7, P8. The advantage of protein P6 in binding of class A immunoglobulin over P8 can be explained by the lack of optimal conformation in the latter or the generation of steric hindrances caused by the interaction of a large molecule with the ligand (P6 is shortened compared to P8 at both ends). Data on the IgA receptor activity of the three proteins are shown in Table 2.

The effect of the molecular form of IgA on ligand recognition by receptor molecules P6, P7, P8 was evaluated using indirect ELISA. The data obtained in the course of the work indicate a very weak manifestation of IgA receptor activity by proteins P6, P7, P8 against secretory IgA (SIgA), compared with the binding of serum IgA. Serum IgA and SIgA are equalized by concentration (equalized by weight).

table 2 Comparison of the binding efficiency of proteins P6, P7, P8 of various molecular forms of IgA Research method Molecular Forms of IgA The ratio of optical densities reflecting the efficiency of IgA binding to one polypeptide compared to another P6 compared to P8 P6 compared to P7 P8 compared to P7 direct IFA mIgA P horseradish peroxidase labeled lgA 1.33 3.36 2.62 indirect IFA P Unlabeled human IgA 1.49 3.34 2.74 indirect IFA P IgA1 (k + λ) 1,02 2.03 2.01 P IgA2 (k + λ) 1.09 1.82 1.87 M IgA1, 10 individual myeloma samples 1.13 3.74 3.31 M IgA2, 2 individual myeloma samples 1.17 4.07 3.18 SIgA 1.89 3.45 1.87 Note. mIgA - serum IgA; P - polyclonal preparation; M is a monoclonal drug.

On Fig shows a histogram showing a comparison of the IgA receptor ability of the polypeptides P6, P7, P8, as well as a comparison of protein binding of the secretory and serum forms of IgA.

On Fig (Comparison of the binding efficiency of polyproteins P6, P7, P8 with serum IgA and secretory IgA):

A is mIgA;

B - SIgA;

On the abscissa axis, polypeptides P6, P7, P8;

The ordinate is the optical density (OD ₄₉₂ ).

When studying the features of the interaction of myeloma serum IgA1 and IgA2 samples, in each case a mixture of sera containing κ- and λ-type paraproteins with recombinant proteins was used by indirect ELISA on adsorbed P6, P7, P8. It was shown that the myeloma paraproteins of the immunoglobulin A of the first subclass interact with the derivatives of you protein significantly more efficiently than the immunoglobulin A of the second subclass.

Features in the behavior of proteins under ELISA are noted. Polypeptides P6 and P8 are able to interact with IgA both in solution (inhibitory ELISA) and in an adsorbed state (direct, indirect ELISA). The latter property allows their use as immunochemical reagents in ELISA, both in immobilized form and in the role of detecting molecules in the presence of an enzyme label.

Some structural aspects of the binding of immunoglobulin A with β antigen fragments were also considered: the relative position of the binding site on the IgA molecule was determined (by means of indirect and inhibitory ELISA) - the site is topographically close to the epitopes of three McAt (from the collection of the laboratory of hybridoma technology Federal State Research Center for Pharmaceutical Technologies and Medical Technologies) 3B4, 1H , 1B12, the variability of the binding site of the polypeptides on the IgA molecule was determined in the analysis of 10 individual IgA1 myeloma paraproteins (indirect ELISA), the stability of this site was determined IgA partial change of the tertiary structure. The latter was carried out by studying the effect of partial reduction of disulfide bonds in an IgA molecule on the interaction of P6, P8 polypeptides with immunoglobulins of this class. The check was carried out under the control of MKAT. Denaturing treatment of serum IgA and chigain with dithiothreitol led to the disruption of disulfide bonds between the α chains of the IgA molecule, as a result of which there was a decrease in antibody binding of the McAt 1H9 and 1B12 epitopes, and IgA denaturation did not affect the detection of the CB4 epitope (competitive inhibition). At the same time, P6 and P8 polypeptides exhibit a more pronounced IgA receptor activity when interacting with a ligand preparation treated with dithiothreitol, compared with untreated IgA (Fig. 9). This indicates the safety of the binding site after partial denaturation, however, it undergoes some changes. The ability of proteins P6, P8 to bind a partially reduced ligand can be useful for manipulations with denatured IgA, including for the affinity isolation of the immunoglobulin molecule itself and its fragments.

Figure 9 (Effect of the restoration of disulfide bonds in an IgA molecule on its interaction with recombinant polypeptides P6 and P8):

B — binding of P6, P8 to treated IgA;

G - binding of P6, P8 to untreated IgA;

On the abscissa axis - polypeptides P6, P8;

The ordinate axis shows inhibition,%.

Thus, the ability of recombinant derivatives of you protein of HBV P6, P7, P8 to bind IgA was investigated. Polypeptides P6, P7, P8 selectively bind all considered molecular forms of IgA. The IgA receptor properties of the tested derivatives of the HBV protein correlate with the properties characteristic of a full-sized protein. At the same time, structural features of each of the three polypeptides determine the differences between them in IgA-binding activity.

The result of the invention are recombinant DNA p6, p7, p8, the nucleotide sequences of which are presented in figures 1, 2, 3, respectively; recombinant plasmid DNA pQE-p6, pQE-p7, pQE-p8, which are plasmids pQE-32, carrying recombinant DNA p6, p7, p8, respectively; E. coli JM109 strains transformed with recombinant plasmid DNA pQE-p6, pQE-p7, pQE-p8, and expressing recombinant polypeptides P6, P7, P8, respectively; recombinant polypeptides P6, P7, P8 having the amino acid sequences shown in figures 4, 5, 6, respectively.

Recombinant polypeptides P6, P7, P8 have the following properties:

a) P6, P7, P8 have the ability to selectively bind various molecular forms of IgA;

b) P6, P7, P8 interact with partially denatured IgA, and the restoration of disulfide bonds between the α-chains of the molecule does not lead to a change in the interaction with recombinant polypeptides;

c) P6 when introduced into the body of mammals (mice and rabbits) causes the synthesis of long-circulating anti-P6 antibodies;

d) the synthesized anti-P6 antibodies have protective properties against Streptococcus agalactiae.

The following are specific examples illustrating some embodiments of the invention, but not limiting it.

Example 1. Obtaining a p6 DNA fragment by the method of polymerase chain reaction (PCR) based on the gene you , which encodes a surface you protein HBV

The p6 DNA fragment was amplified using a pair of Pr6-Pr3 primers flanking this nucleotide sequence. In the course of work, 10 picomoles of each specific primer (Pr3, Pr6), a polymerase buffer with magnesium content, 2 mM each, were added to 0.25 μg of genomic DNA isolated by phenol-chloroform extraction from the IHC serotype 219/4849 Ibc strain. of 4 deoxyribonucleotide triphosphates, the volume was adjusted with water to 50 μl. 1 μl of thermostable DNA polymerase was added. 40 μl of mineral oil was layered on the surface of the liquid. The mixture was incubated for 3 minutes at + 94 ° C. The tubes were placed in a Tercik amplifier (Russia). The device was programmed for a denaturation cycle of + 94 ° C for 1 minute, an annealing cycle from 42 ° C to 44 ° C (depending on the G-C composition of the primers), and a DNA synthesis cycle of + 72 ° C for 1 minute. The sequence of such cycles was repeated 30 times. Oligonucleotide primers are shown in Table 1.

Analysis of the PCR results was carried out using agarose gel electrophoresis.

Example 2. Obtaining a p7 DNA fragment by the method of polymerase chain reaction based on the gene you , coding for surface you protein HBV

The DNA fragments p71, p72 were amplified using the corresponding pairs of primers Pr5-Pr2, Pr7-Pr8, flanking these nucleotide sequences according to the procedure described in example 1.

Analysis of the PCR results was carried out using agarose gel electrophoresis.

381 bp p71 and p72 fragments isolated from agarose and 992 n.p. were subjected to restriction enzyme treatment with EcoM, and then were ligated with each other, which led to the formation of the p7 nucleotide sequence (1353 bp) containing the EcoRI site and encoding a polypeptide with a modified MLKRIQ region.

Example 3. Obtaining a p8 DNA fragment by the method of polymerase chain reaction based on the gene you , encoding the surface of you protein HBV

The p8 DNA fragment was amplified using a pair of Pr5-Pr8 primers flanking this nucleotide sequence according to the procedure described in Example 1.

Analysis of the PCR results was carried out using agarose gel electrophoresis.

In addition to the EcoRI site, the recognition sites of other restriction enzymes were not included in the primer sequence shown in Table 1, since the p6, p7, p8 nucleotide sequences were subsequently ligated with commercial pGEM-T Easy vectors (Promega, USA) that did not require restriction sites.

Example 4. Analysis of the results of PCR using electrophoresis in agarose gel

Analysis of the PCR results was carried out by separation of DNA fragments in a 1% agarose gel using horizontal electrophoresis (figure 10, 11, 12). The selection of the desired amplified DNA sections was carried out using the QIAquick Gel Extraction Kit (Qiagen, USA).

The analysis of the sizes of the obtained DNA fragments was carried out based on a comparison of their electrophoretic mobility with the electrophoretic mobility of the molecular weight marker (DNA fragments Sph I - Pst I phage wild-type lambda).

Figure 10 (Electrophoregram of amplified DNA fragments of p6):

1 - marker, wild-type phage DNA + Sph I + Pst I;

2 - p6 + Sph I + Pst I.

As a result of the polymerase chain reaction with Pr6-Pr3 primers, the expected size of the DNA fragment of you gene with 406 bp 1287 bp amounted to 882 bp, as shown in Fig.10.

11 (Electrophoregram of amplified p7 DNA fragments):

1 - marker, wild-type phage DNA + Sph I + Pst I;

2 - p71 + Sph I + Pst I, p72 + Sph I + Pst I.

As a result of the polymerase chain reaction with primers Pr5-Pr2 and Pr7-Pr8, the expected sizes of DNA fragments of you gene with 301 bp 682 bp each and from 662 bp 1653 bp each amounted to 382 bp and 992 bp respectively, as shown in FIG. 11.

On Fig (Electrophoregram amplified DNA fragments of p8):

1 - marker, wild-type phage DNA + Sph I + Pst I;

2 - p8 + Sph I + Pst I.

As a result of the polymerase chain reaction with Pr5-Pr8 primers, the size of the DNA fragment of you gene with 301 bp 1653 bp each amounted to 1353 bp, as shown in Fig.12.

Example 5. Cloning of the p6 DNA fragment into the pQE30 / 31/32 expression vector system

Plasmids pQE30 / 31/32 (3462 bp, 3462 bp, 3463 bp, The QIAexpress System, Qiagen, USA) into which the p6 insert extracted from agarose will be cloned (882 bp) were pretreated along with the p6 fragment with SphI and PstI enzymes, leading to the formation of sticky ends (0.25 μg of DNA was dissolved in 16 μl of H ₂ O, 2 μl of the corresponding buffer and 1 unit of restrictase were added). Restriction products were separated by horizontal electrophoresis on a 1% agarose gel, and then isolated from agarose using the QIAquick Gel Extraction Kit (Qiagen, USA).

Mixed in a 4: 1 ratio of 30 μl of the prepared p6 DNA fragment and 2 μl of one of the prepared plasmids pQE30 / 31/32, 5 μl of ten-fold ligase buffer and 1 μl of phage T4 ligase were added. Then they were incubated at 4 ° C for 12 hours, after which the volume of the mixture was previously adjusted to 50 μl with water.

As a result of ligation, three variants of recombinant plasmid DNA were obtained that carried the p6 nucleotide sequence and one of the vectors pQE30 / 31/32.

Example 6. Cloning of the p7 DNA fragment into the pQE30 / 31/32 expression vector system

The p7 fragment (1353 bp) isolated from agarose was cloned into the pQE30 / 31/32 expression vector system, as described in Example 5.

As a result of ligation of the p7 DNA fragment and pQE30 / 31/32, three variants of recombinant plasmid DNA were obtained that carried the p6 nucleotide sequence and one of the pQE30 / 31/32 vectors.

Example 7. Cloning of the p8 DNA fragment into the pQE30 / 31/32 expression vector system

The p8 fragment (1353 bp) isolated from agarose was cloned into the pQE30 / 31/32 expression vector system, as described in Example 5.

As a result of ligation of the p8 DNA fragment and pQE30 / 31/32, three variants of recombinant plasmid DNA were obtained that carried the p6 nucleotide sequence and one of the pQE30 / 31/32 vectors.

Example 8. Isolation and purification of recombinant P6 polypeptide

Cultures of the Escherichia coli strain JM109 capable of synthesizing the recombinant P6 polypeptide were grown in a small volume of sterile medium (10 ml each) of Terrific broth with the addition of ampicillin (to a final concentration of 100 μg / ml) and kanamycin (to a final concentration of 25 μg / ml) for 16-18 hours with strong shaking in a thermostat, 37 ° C. Then, the resulting culture was added in a ratio of 1:50 to a fresh sterile medium containing ampicillin (100 μg / ml) and kanamycin (25 μg / ml). Grown under the same conditions for 3-4 hours until the liquid reaches an optical density of OD ₆₀₀ 0.6-0.8. To induce protein expression, a 1M IPTG solution was added to a final concentration of 1 mM and grown under the same conditions for 4 hours. After that, the cells were precipitated by centrifugation (3000 rpm, 15-20 min, 4 ° С) and resuspended in a single binding buffer A (8X = 4 M NaCl; 160 mM Tris-HCl pH 7.9; 40 mM Imidazole, pH 7.9). Cell lysis was carried out on an ultrasonic disintegrator (UZDN-1U4.2, USSR). An ultrasonic pulse applied to the culture three times within 10-15 seconds. Between pulses, cultures were placed in ice for 1-2 minutes. Cell debris was separated from the culture lysate for 15 minutes by centrifugation at 20,000 rpm, 4 ° C. Further, the isolation and purification of the recombinant polypeptide was carried out using affinity chromatography. The filtered lysate was applied to a column of pre-reduced sorbent Ni-NTA agarose washed with buffer A until the OD ₂₈₀ values of the resulting solution were less than 0.01. The polypeptide was eluted with a single buffer B, pH 7.9 (4X = 2M NaCl; 400 mM EDTA; 80 mM Tris-HCl pH 7.9) containing 400 tM EDTA. 4 fractions of eluates were taken, 500 μl each. Analysis of samples for protein content was performed by SDS-polyacrylamide gel electrophoresis. Fractions with the polypeptide were purified from the elution buffer by dialysis against phosphate buffered saline, pH 7.4 (PBS-8g NaCl, 0.2g KCl, 1.44g Na ₂ HPO ₄ , 0.24g KH ₂ PO _{4 were} dissolved in 800 ml of distilled water, adjusted to pH to 7.4 with HCl, add water to 1 l).

13 shows an electrophoregram of recombinant P6 polypeptide at various stages of isolation and purification.

On Fig (Electrophoregram of recombinant P6 polypeptide):

1 - molecular weight marker;

2 - fraction of pure recombinant P6 polypeptide;

3 - sample after washing the sorbent with buffer A, pH 7.9, from unbound proteins;

4 - cell lysate of Escherichia coli JM109 before applying to the column;

5 - lysate after passing through the column.

The results of electrophoresis made it possible to conclude that the purification of the polypeptide is satisfactory, as well as its molecular weight: P6 - (35 ± 0.5) kDa compared to the mileage of proteins of known molecular weight (Precision Plus Protein standards (161-0373), Bio-Rad , USA).

Example 9. Isolation and purification of recombinant P7 polypeptide

The procedure for isolation and purification of recombinant P7 polypeptide is similar to that described in example 8.

On Fig shows the electrophoregram of the recombinant P7 polypeptide at various stages of isolation and purification.

On Fig (Electrophoregram of recombinant P7 polypeptide):

1 - lysate after passing through the column;

2 - cell lysate of Escherichia coli JM109 before applying to the column;

3 - sample after washing the sorbent with buffer A, pH 7.9, from unbound proteins;

4 - fraction of pure recombinant P7 polypeptide;

5 - molecular weight marker.

The results of electrophoresis made it possible to conclude that the purification of the polypeptide is satisfactory, as well as its molecular weight: P7 - (52 ± 0.5) kDa compared to the mileage of proteins of known molecular weight (Precision Plus Protein standards (161-0373), Bio-Rad , USA).

Example 10. Isolation and purification of recombinant P8 polypeptide

The procedure for isolation and purification of recombinant P8 polypeptide is similar to that described in example 8.

On Fig shows an electrophoregram of recombinant P8 polypeptide at various stages of isolation and purification.

On Fig (Electrophoregram of recombinant P8 polypeptide):

1 - molecular weight marker;

2 - fraction of pure recombinant P8 polypeptide;

3 - sample after washing the sorbent with buffer A, pH 7.9, from unbound proteins;

4 - lysate after passing through the column;

5 - lysate of Escherichia coli JM109 cells before application to the column.

The results of electrophoresis made it possible to conclude that the purification of the polypeptide is satisfactory, as well as its molecular weight: P8 - (52 ± 0.5) kDa compared to the mileage of proteins of known molecular weight (Precision Plus Protein standards (161-0373), Bio-Rad , USA).

Example 11. Comparison of the binding efficiency of polypeptides P6, P7, P8 with serum IgA and secretory IgA

Analysis of the IgA receptor ability of polypeptides with respect to various molecular forms of IgA was carried out by indirect ELISA, provided that the concentrations of IgA preparations were equal (weighted). For ELISA, NUNC MaxiSorb (Danemark) tablets with increased sorption activity were used.

The preparation of diluted immunoglobulin preparations (serum immunoglobulin A, dilution: 1/100; purified secretory IgA from colostrum, dilution: 1/50) and detecting antibodies was carried out using 10% fetal bovine serum in a single wash in Tris-buffer solution containing 0 05% tween-20 (RP), pH 7.4-7.5.

Removal of unbound reagents was performed by washing the RP plates three times, pH 7.4-7.5.

Polypeptides P6, P7, P8, diluted in PBS, pH 7.4 to 5 μg / ml, were adsorbed onto the plate for 18 hours at 4 ° C. After washing three times, diluted IgA preparations were added to the plates. Incubation was carried out at 37 ° C for 1 hour. Next, the tablets were washed three times from unbound reagents, the conjugate of antibodies against light chains (λ + κ) with horseradish peroxidase was added and incubated for 37 hours at 37 ° C. Washed from unbound reagents 4 times.

The activity of the enzyme label horseradish peroxidase was determined on a substrate consisting of orthophenylenediamine and a citrate-phosphate buffer solution with pH 5.0, and the reaction was stopped by the addition of 2N sulfuric acid. The optical density was determined at a wavelength of 492 nm on a spectrophotometer.

The data obtained during the work indicate a very weak manifestation of IgA receptor activity by P6, P7, P8 polypeptides in relation to secretory IgA (Fig. 8, B), compared with the binding of serum IgA (Fig. 8, A), respectively, in 2.2, 2.9 and 2.4 times. Probably, the secretory component in SIgA screens part of the surface of the IgA α-chains.

Example 12. Comparison of the binding efficiency of polypeptides P6, P7, P8 with IgA1 and IgA2

When studying the characteristics of the interaction of recombinant polypeptides with myeloma serum IgAl and IgA2 samples, in each case a mixture of sera containing κ- and λ-type paraproteins was used indirect ELISA on adsorbed P6, P7, P8.

Serum myeloma IgA1 (λ + κ) (1: 1 = IgA1λ: IgA1κ) was used in dilutions of 1: 1000, 1: 10000, 1: 100000, 1: 500000, 1: 1,000,000.

Serum myeloma IgA2 (λ + κ) (1: 1 = IgA2λ: IgA2κ) was used in dilutions of 1: 1000, 1: 10000, 1: 100000, 1: 500000, 1: 1,000,000.

The experimental technique is similar to that described in example 11.

It was shown that the myeloma paraproteins of the immunoglobulin A of the first subclass (Fig. 16, E) interact with polypeptides significantly more effectively than the immunoglobulin A of the second subclass (Fig. 16, E).

In Fig.16 (Interaction of polypeptides P6, P7, P8 with IgA1 and IgA2):

D - binding of polypeptides to IgA1;

E - binding of polypeptides to IgA2;

On the abscissa axis, polypeptides P6, P7, P8;

The ordinate is the optical density (OD ₄₉₂ ).

Example 13. The binding of serum-labeled IgA adsorbed polypeptides P6, P7, P8.

Analysis of IgA receptor ability was performed by direct ELISA. For ELISA, NUNC MaxiSorb (Danemark) tablets with increased sorption activity were used.

The preparation of diluted preparations of immunoglobulins and detecting antibodies was carried out using a 10% solution of fetal bovine serum in a single RP, pH 7.4-7.5.

Removal of unbound reagents was performed by washing the RP plates three times, pH 7.4-7.5.

Polypeptides P6, P7, P8, diluted in PBS, pH 7.4 to 5 μg / ml, were adsorbed onto the plate for 18 hours at 4 ° C. After washing three times, diluted labeled IgA was added to the plates. Incubation was carried out at 37 ° C for 1 hour. Next, the tablets were washed 4 times from unbound reagents.

The activity of the enzyme label horseradish peroxidase was determined on a substrate consisting of orthophenylenediamine and a citrate-phosphate buffer solution with pH 5.0, and the reaction was stopped by the addition of 2N sulfuric acid. The optical density was determined at a wavelength of 492 nm on a spectrophotometer.

The studied polypeptides have IgA receptor activity against labeled serum IgA, and the amount of ligand bound by protein P6 is higher than that of P8 (Table 2, the statistical significance of the differences was noted when the IgA preparation was diluted 3000 times). Perhaps the reason lies in the size of the proteins - compared to P8, the P6 protein is shortened at both ends, which probably gives the latter an advantage in terms of spatial availability. The efficiency of the interaction of P7 with the antibody molecule is minimal (Table 2), which may be due to amino acid substitutions in the IgA-binding region.

Example 14. The binding of serum labeled IgA polypeptides P6, P7, P8 in solution

For ELISA, NUNC MaxiSorb (Danemark) tablets with increased sorption activity were used.

The preparation of diluted preparations of immunoglobulins and detecting antibodies was carried out using a 10% solution of fetal bovine serum in a single RP, pH 7.4-7.5.

Removal of unbound reagents was performed by washing the RP plates three times, pH 7.4-7.5.

Polypeptides at a concentration of 5 μg / ml were incubated with labeled IgA diluted 3, 6 and 12 thousand times for 2 hours at room temperature on a shaker. This mixture was introduced into tablets with immobilized recombinant polypeptides (immobilization was carried out according to the standard scheme described in example 11), an hour of incubation at 37 ° C. The plates were washed four times and the horseradish peroxidase tag enzyme activity was determined (as described in Example 11).

During the experiment, the inhibition of polypeptides P6, P7, P8 in solution (pre-incubated for two hours at room temperature with labeled IgA), the interaction of labeled IgA with adsorbed recombinant molecules occurs.

This is most pronounced in P6 and P8 (Fig.17) - the inhibitory effect in the range of 70-90%. The weak IgA receptor activity of the adsorbed P7 polypeptide did not allow an unambiguous conclusion about the inhibitory ability of the protein in solution. Perhaps the reason is a feature of the spatial configuration of the P7 protein, due to the presence of two amino acid substitutions. Thus, the recombinant molecules P6 and P8 have an IgA receptor ability both in the adsorbed state and in solution.

On Fig (Comparison of the efficiency of IgA binding by adsorbed P6, P7, P8 polypeptides and P6, P7, P8 polypeptides in solution):

Triangle - P6;

Lozenge - P8;

Square - P7;

The abscissa shows the dilution of labeled serum IgA, thousand times;

The ordinate axis shows inhibition,%.

Example 15. The study of the position of the binding site of the polypeptides P6, P7, P8 on the IgA molecule using monoclonal antibodies (Mkat)

For ELISA, NUNC MaxiSorb (Danemark) tablets with increased sorption activity were used.

The preparation of diluted preparations of immunoglobulins and detecting antibodies was carried out using a 10% solution of fetal bovine serum in a single RP, pH 7.4-7.5.

Removal of unbound reagents was performed by washing the RP plates three times, pH 7.4-7.5.

In order to determine the position of the binding site, a collection of 9 horseradish peroxidase-labeled MkAt was used (provided by the hybridoma technology laboratory of the Federal Research Center for Chemistry and Technology of Rosmedtechnology). McAb bind various highly conserved antigenic determinants of the IgA heavy chain. The position of the binding site with the polypeptides was evaluated relative to the McAt epitopes, and it was decided to establish whether among the McAb there are reagents competing with proteins for binding to IgA.

In this regard, two versions of the ELISA were carried out:

1. Preliminary binding of labeled IgA with MkAt, followed by transfer to adsorbed polypeptides P6, P7, P8 (inhibitory ELISA).

2. The binding of serum IgA (pool of serum healthy donors) with adsorbed proteins P6, P7, P8 and subsequent interaction with Mkat (indirect ELISA).

The formulation of inhibitory ELISA was as follows: polypeptides at a concentration of 5 μg / ml were adsorbed onto the plate (immobilization was carried out according to the standard scheme described in example 11), and then a pre-incubated mixture was added to them (100 μl per cell) (in the ratio 1: 1, incubation at room temperature (22 ° C) on a shaker for two hours) from peroxidase-labeled IgA horseradish (working dilution 1/3000) and one of MkAt (each MkAt sample was taken in two concentrations of 10 μg / ml and 50 μg / ml). The incubation of immobilized polypeptides with a mixture of MkAt and labeled IgA was carried out for 20 minutes at 37 ° C. In the course of the work, the effect of monoclonal antibodies on the interaction of labeled IgA with proteins P6, P7, P8 was tested. The result was judged by the presence of peroxidase activity of the IgA label enzyme, which was bound to adsorbed protein molecules (as described in Example 11).

Inhibitory ELISA made it possible to identify reagents among MkAt that interfere with polypeptides for binding to IgA - it is MkAt 1B12, 1H9, 3B4, capable of reducing IgA receptor activity of P6, P7, P8 protein derivatives by 60-90% (Fig. 18).

On Fig (Inhibition of MKAT binding of serum labeled IgA with P6, P7, P8):

Triangle - P6;

Lozenge - P8;

Square - P7;

On the abscissa axis are MkAt ciphers (used in two concentrations of 10 and 50 μg / ml);

The ordinate axis shows inhibition,%.

Based on the results obtained, the following assumptions were made (regarding MKat 1B12, 1H9, 3B4).

First, McAb can recognize the same IgA site that the recombinant proteins P6, P7, P8 bind, thus preventing the interaction of recombinant proteins with their antigenic determinants on the immunoglobulin molecule.

Secondly, it is likely that Mabs recognize a ligand region that is topographically close to the binding site P6, P7, P8. When implementing this option, the formation of a complex of one of the antibodies with labeled IgA could lead to steric difficulties for the implementation of fragments of the protein by you IgA-receptron activity.

Thirdly, MkAt, possibly when interacting with the ligand, cause conformational changes in the IgA molecule, which change the binding site. These modifications impede the further interaction of P6, P7, P8 proteins with immunoglobulin.

In order to confirm one of the above assumptions, an indirect ELISA was performed. Identification of unlabeled MkAt (concentration 1 μg / ml) was carried out with labeled rabbit polyclonal antibodies. The experimental procedure is similar to that described in Example 11. This ELISA formulation demonstrated that pre-binding of IgA with P6, P7, P8 did not mainly prevent the binding of McAt to IgA - the inhibition was negligible and amounted to no more than 30-40% (Fig. 19) .

On Fig (Inhibition by the interaction of labeled IgA with adsorbed polypeptides P6, P7, P8 binding of McAt to IgA):

Triangle - P6;

Lozenge - P8;

Square - P7;

On the abscissa axis are MkAt ciphers;

The ordinate axis shows inhibition,%.

The results of inhibitory ELISA, at first glance, contradict the results obtained after the indirect ELISA - preliminary binding of IgA polypeptides did not interfere with the subsequent interaction of immunoglobulin with MkAt.

One of the hypotheses explaining this discrepancy may be the earlier suggestion that the epitopes binding MkAt (3B4, 1H9, 1B12) and the binding sites of polypeptides on IgA are close. The basis was the results of indirect ELISA, indicating the fundamental possibility of the simultaneous binding of any of the three McAt 1B12, 1H9, 3B4 and any of the polypeptides P6, P7, P8 with an IgA molecule.

Thus, the topographic proximity of the regions on the IgA molecule interacting with P6, P7, P8 polypeptides and three McAt is shown.

Example 16. The study of the conservation of the structure of the binding site of the polypeptides P6, P7, P8 on the IgA molecule

Due to possible mutations in the genes of tumor plasma cells, their products may differ significantly from immunoglobulin molecules produced by cells not affected by the tumor. It is likely that if there is a mutation in the genes encoding the IgA heavy chains, the binding site of the recombinant proteins P6, P7, P8 will also be affected.

Indirect ELISA on adsorbed recombinant polypeptides P6, P7, P8 was chosen as the main research method. The identification of immunoglobulins bound to antigens was carried out using labeled McAbs against light chains. A panel of 12 individual myeloma samples was used: 10 IgA1 and 2 IgA2. The size of the M-peak (the color intensity of the band corresponding to the paraprotein) was controlled using native agarose gel electrophoresis. Electrophoresis of IgA1 and IgA2 myeloma sera showed the presence of M-peaks in all sera and differences in physicochemical properties between paraproteins. Since the exact concentration of paraproteins in individual myeloma sera is not known, two dilutions of each sample were used in the course of work: 10 thousand times and 100 thousand times. The experimental technique is similar to that described in example 11.

In 10 presented individual myeloma IgA1 binding to recombinant proteins can be traced quite clearly, with the exception of two samples numbered 9 and 10, in which the interaction with P6, P7, P8 is weaker (Fig. 20) (confidence probability of differences is greater than 0.95). Thus, the binding site to P6, P7, P8 is not so conservative and may be absent or altered in individual paraproteins.

On Fig (Interaction of recombinant proteins P6, P7, P8 with myeloma IgA1 and IgA2):

On the abscissa axis, myeloma IgA (1-10 IgA1 samples, 11-12 IgA2 samples), two dilutions were used: 1: 10000, 1: 100000.

The ordinate is the optical density (OD ₄₉₂ ).

Example 17. The study of the effect of the restoration of disulfide bonds in the IgA molecule on its interaction with polypeptides P6, P8

For ELISA, NUNC MaxiSorb (Danemark) tablets with increased sorption activity were used.

The preparation of diluted immunoglobulin preparations was carried out using a 10% solution of fetal bovine serum in a single RP, pH 7.4-7.5.

Removal of unbound reagents was performed by washing the RP plates three times, pH 7.4-7.5.

To obtain data on the relative stability of the structure of the binding site of the P6, P8 proteins localized on the IgA molecule, the McAt collection was used. A comparison was made of the stability of McAt epitopes on an IgA molecule and the polypeptide binding site under conditions of restoration of disulfide bonds between IgA α chains.

Chigain and serum polyclonal IgA were treated with 0.01 M DTT in 0.4 M Tris-HCl, pH 8.3 at 24 ° C for 2.5 hours, followed by alkylation of the formed SH groups with 0.022 M iodoacetamide at 0 ° C for 30 minutes.

Denaturing treatment of immunoglobulins led to a partial change in the tertiary structure of IgA molecules, which led to a decrease in the ability of some conformational MkAT epitopes to bind MkAt. For MkAt 1H9 and 1B12 epitopes, a decrease in antibody binding was observed after partial restoration of disulfide bonds on IgA. IgA denaturation was not affected by the detection of the 3B4 epitope.

The ability of native or restored IgA to compete with labeled IgA, inhibiting the binding of the latter to adsorbed polypeptides P6, P8, was determined in a competitive inhibitory ELISA. Recombinant polypeptides were adsorbed onto the tablets at a concentration of 5 μg / ml for 1.5 hours at 37 ° C. After washing twice, a mixture of treated or untreated IgA at a concentration of 40 μg / ml and labeled IgA was added, the final dilution was 1:10 thousand. Incubation was carried out for 2 hours at room temperature or 18 hours at 4 ° C. The plates were washed four times and the horseradish peroxidase tag enzyme activity was determined (as described in Example 11).

The restoration of disulfide bonds on an IgA molecule did not impair ligand binding to the studied antigens P6, P7, P8, but led to a slight increase in the ability of P6 and P8 proteins to bind antibodies with altered conformation (Fig. 9 - data for serum IgA, analogous results for SIgA, but not specified). This result demonstrates the stability of the binding site with recombinant polypeptides on the IgA molecule to the restoration of disulfide bonds.

Example 18. The study of the protective properties of P6-specific antibodies in a "mouse" model

To study the protective activity of P6-specific antibodies against infection caused by strain 219/4849 of HBV Ibc serotype, a generalized infection model was used in four groups of laboratory mice.

Saline was administered to two control groups, and the other two were immunized with P6 polypeptide (10 μg / animal). 60 days after the start of immunization with an anti-P6 antibody titer of 1 × 10 ^-5 , intraperitoneal infection of mice with streptococci was performed in sublethal (3.5 × 10 ⁶ CFU / mouse) and lethal (LD ₅₀ ) for 50% of laboratory animals (3.5 × 10 ⁷ CFU / mouse) doses.

In order to control the dynamics of the development of the infectious process and to determine the rate of death of pathogenic bacteria (1, 6, 24, and 48 hours after infection), seeding was performed from peritoneal exudate (obtained by washing the peritoneal cavity with 5 ml PBS), blood and spleen (extracted, homogenized, suspended in 1 ml of PBS, pH 7.2) laboratory animals.

The concentrations of microorganisms at different stages of the infection process are expressed in CFU / ml and are presented in Table 3 (average values for five replicates).

An hour after infection, all four groups of laboratory animals showed an equally high content of streptococci. When infected with a sublethal dose in streptococcus control mice, streptococci were not seeded only by the fifth day, while bacteria were not observed in immune mice after 6 hours.

In the group of control animals, after administration of LD ₅₀ , by the second day, about 50% of laboratory animals died. In mice immunized with the P6 polypeptide, streptococcus elimination occurred after 5 days from the moment of infection, and up to this point pathogenic microorganisms were found in the spleen and blood in small quantities.

Thus, it was shown that the recombinant derivative of you protein P6 is able to induce a protective immune response, accelerating the excretion of HBV from the macroorganism.

Table 3 Change in the concentration of streptococci in the spleen of mice at different stages of infection Aftercare period, h When infecting immune mice with doses (CFU / mouse) When infecting intact mice with doses (CFU / mouse) 3.5 × 10 ⁷ 3.5 × 10 ⁶ 3.5 × 10 ⁷ 3.5 × 10 ⁶ one 3.5 × 10 ⁷ 3.5 × 10 ⁶ 3.5 × 10 ⁷ 3.5 × 10 ⁶ 6 1 × 10 ² 0 6.2 × 10 ⁶ 1.7 × 10 ⁵ 24 3,5 × 10 ³ 0.2 × 10 ² > 10 ⁸ 7 × 10 ⁵ 48 1.4 × 10 ³ 0 50% fallen 7 × 10 ² 120 0 0 50% fallen 0

Example 19. The study of the opsonizing activity of anti-P6 and anti-ScaAB antibodies

The opsonophagocytic reaction is the leading defense mechanism in mammals against infections caused by bacteria, including HBV. Since you protein, on the basis of which the P6 polypeptide was obtained, does not occur in serotype III HBV strains, one more component was chosen to expand the protective spectrum of the vaccine - ScaAB lipoprotein, which is widespread among the GBS and ensures bacterial adhesion to cells. The study of the opsonizing activity of anti-P6 and anti-ScaAB antibodies was carried out on a model of cultured peritoneal macrophages. The monolayer of murine peritoneal macrophages was induced in advance by the administration of 0.5 ml of 10% peptone intraperitoneally to the animals. The obtained peritoneal exudate was suspended in RPMI medium (BioloT, Russia) at a concentration of 1 mln / ml, and then added to 24-well plates for tissue culture with coverslips (Sarstedt, Germany) in a volume of 1.0 ml and to 96-well plates for tissue culture in a volume of 0.2 ml. Incubated 1 hour at 37 ° C. Further, after removal of non-adherent cells, in RPMI medium containing 10% ETS (fetal calf serum, BioloT, Russia) and antibiotics - streptomycin (100 μg / ml) and penicillin (100 IU / ml), the plates were incubated for 24 hours. A suspension of SGV and SGA in RPMI medium was applied to a pre-washed monolayer at a concentration of 1 × 10 ⁷ CFU / ml. Bacteria were pre-incubated for 30 minutes with normal and hyperimmune mouse serum diluted 20 times in RPMI medium. The contact of bacteria with macrophages lasted 30 min, after which the monolayer was washed from streptococci not attached to the surface of the cells. Fresh RPMI medium with 10% ETS was added to some samples and incubated for up to 180 minutes. All the steps described above were carried out at 37 ° C in an atmosphere of 5% CO ₂ . Further, the preparations were fixed for 30 min with alcohol and stained with azide and azure for 30 min. The monolayer was thoroughly washed from paint residues. Microscopy on coverslips examined at least 100 cells in three parallel samples, counting the percentage of macrophages infected with streptococci and the average number of cocci per cell. The degree of infection was evaluated by the index of infection (II), which is a product of both indicators.

Microscopic evaluation showed that hyperimmune serums for the studied polypeptides have opsonizing activity. II during the treatment of HBV and SGA with immune serum exceeded the control value 2.5-4 times (at the adhesion stage). After 180 minutes, a massive elimination of streptococcus anti-P6 and anti-ScaAB antibodies was observed.

Both types of antibodies were shown to have opsonizing activity against the HBV strain 219/4849 Ibc serotype, in addition, ScaAB-specific antibodies were able to opsonize the SGA, which is probably due to the presence of the ScaAB homolog protein - the so-called PsaA.

A sharp decrease in the opsonizing activity of anti-P6 antibodies was noted in relation to the HBV strain subjected to five-fold passaging in mice, in comparison with the initial HBV strain. It has been established that the protective effectiveness of antibodies depends not only on their opsonizing properties, but also on the expression of the polysaccharide capsule, which sharply increased under conditions of passivation and led to screening of you protein, blocking opsonization.

Table 4 The result of the evaluation of the opsonizing activity of anti-P6 and anti-ScaAB antibodies (by infection index) against HBV Monolayer Contact Time Macrophage infection index * HCM ** Anti-P6, titer 1: 12500 Anti-scaAB, titer 1: 1600 30 minutes 238 890 523 180 min 29th 180 22.4 * product of the average number of cocci per cell and the relative proportion of infected macrophages (in%)
** NSM - serum from mice of the control group

Thus, the P6 and ScaAB polypeptides, due to the ability to provide protection at extremely low antibody titers, have opsonizing activity, including against SGA, as well as strains of the third serotype of SGB, are very promising as vaccine components.

Claims (12)

1. A fragment of the chromosomal DNA of strain 219/4849 Ibc serotype of group B streptococci (BHC), which is a portion of you gene with 406 bp 1287 bp with the nucleotide sequence shown in figure 1, encoding a recombinant P6 polypeptide having the ability to selectively bind IgA and protective properties against Streptococcus agalactiae. 2. Recombinant DNA, including a gene fragment of you chromosomal DNA of strain 219/4849 Ibc serotype SGV with 301 bp 1653 bp each relative to the nucleotide sequence of you gene with nucleotide substitutions that allow the creation of a site (GAATTC) recognition by restriction enzyme EcoRI at position 374 bp - 379 bp, and encoding a recombinant polypeptide designated P7, which has the ability to selectively bind IgA, the nucleotide sequence of the chromosomal DNA fragment with substitutions is presented in figure 2. 3. A fragment of the chromosomal DNA of strain 219/4849 Ibc serotype SGV, including a portion of the gene you with 301 bp 1653 bp each and a coding recombinant polypeptide designated P8, which has the ability to selectively bind IgA and whose nucleotide sequence is shown in FIG. 4. Recombinant plasmid DNA, which is a pQE-32 plasmid DNA, carrying the chromosomal DNA fragment according to claim 1, designated as pQE-P6. 5. Recombinant plasmid DNA, which is a pQE-32 plasmid DNA, carrying the recombinant DNA fragment according to claim 2, and designated pQE-p7. 6. Recombinant plasmid DNA, which is a pQE-32 plasmid DNA, carrying the chromosomal DNA fragment according to claim 3, and designated pQE-p8. 7. The strain E. coli JM 109, transformed with the recombinant plasmid DNA according to claim 4 and expressing the recombinant P6 polypeptide. 8. The strain E. coli JM 109, transformed with the recombinant plasmid DNA according to claim 5 and expressing the recombinant P7 polypeptide. 9. The strain E. coli JM 109, transformed with the recombinant plasmid DNA according to claim 6 and expressing the recombinant P8 polypeptide. 10. Recombinant P6 polypeptide, which is a fragment from No. 136 to No. 429 of the amino acid sequence of the surface of you protein of the HBV protein of strain 219/4849 Ibc serotype and characterized by the amino acid sequence shown in figure 4, and the polypeptide has or is not covalently linked to six histidines encoded pQE32 vector. 11. Recombinant P7 polypeptide, characterized by the amino acid sequence shown in figure 5, and which is a fragment from No. 101 to No. 551 of the amino acid sequence of the surface of you protein of the HBV strain 219/4849 Ibc serotype with two amino acid substitutions, where the lysine at position 225 of you protein is replaced arginine, and glutamate at position 227 of you protein per glutamine, and with the ability to selectively bind IgA, moreover, the polypeptide has or is not covalently linked to six histidines encoded by the pQE32 vector. 12. Recombinant P8 polypeptide, characterized by the amino acid sequence shown in Fig.6, and which is a fragment from No. 101 to No. 551 of the amino acid sequence of the surface of you serum HBV protein of strain 219/4849 Ibc serotype, with the ability to selectively bind IgA, and the polypeptide is present or absent covalent bond with six histidines encoded by pQE32 vector.

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