FI95385C - A method for producing a Streptococcus suis adhesin protein and an antibody thereto - Google Patents

Description

1 95385

Method for producing Streptococcus suis adhesin protein and its antibody

Divided separated from patent application FI930413 5

Background - Field of the Invention

The invention relates to a process for the preparation of a therapeutically useful adhesin protein of Streptococcus suis, which adhesin protein has galactose-inhibitory haemagglutination activity, which binds to pigeon ovomucoid and which produces antibodies and has a molecular weight of about 18,000, an electro 4 and the N-terminal sequence is Ala-Ser-Pro-Ala-Glu-Ile-Ala-Ser-Phe-Ser-Pro-15 Ala-Pro-Leu-Ala- (SEQ ID NO: 1) and has the following amino acid composition:

Amino acid nmol amino acid / nmol protein

Asx 13 2 0 Thr 8

Ser 6

Glx 26

Gly 22

Ala 17 • I 25 Vai 10

Cys 0

Met 4

Ile 11

Leu 12 30 Tyr 4 • ·· Phe 6

Ly s 16

His 7

Arg 7 35 Pro 12 2 95385 or a derivative or fragment thereof having the same biological and immunological activity. The invention further relates to a method for producing an antibody against said adhesin protein or a derivative or fragment thereof. The adhesin protein, derivatives and fragments thereof and their antibodies prepared according to the invention can be used therapeutically.

S. suis (Lancefield Group D) is known to cause porcine meningitis, pneumonia, art-10 rites, and sepsis. S. suis type 1 mainly causes septicemia and meningitis in newborn piglets, and type 2 in meningitis of slightly older piglets, about 3 to 10 weeks old, which can also infect humans. As pig farming becomes more concentrated and stocking densities increase, infections become more prevalent and infectious diseases caused by S. suis become more common. For this reason, the development of diagnostics and vaccines is considered very important in the identification and control of swine meningitis and other serious infections. Targeted prevention in pig farmers may also be considered.

Prior art

Bacterial adhesion to the surface of target cells is the first event in the pathogenesis of bacterial disease (Beachey, E.H.

(1981) J. Infect. Dis. 143. 325-345). Bacterial adhesion is often mediated by a protein on the surface of bacteria, adhesin, which specifically adheres to receptor structures on the surface of cells. Thus, adhesin is well suited for vaccine development because the antibodies specifically target a factor necessary for the bacterium, and on the other hand, the specificity avoids adverse side effects.

A wide variety of adhesives are known, but their exact structure and mechanism are often still to be elucidated. They are proteins in nature that specifically recognize the receptors of the target cell, which in turn are generally carbohydrate structures. Different types of bacterial adhesins and the carbohydrate structures they recognize have been presented e.g. in Sharon, N., (1987) FEBS Letters, 217. 145-157.

5 E. coli and many other gram-negative bacteria adhere to specific molecules on the host cell surface with lectin-like bacterial adhesins. Common adhesin receptors include the sugar moieties of glycolipids and glycoproteins. Often, adhesins are attached to the hairy structures of the fimbriae (whistle) on the surface of 10 bacterial cells. There are many types of fimbriae; they vary in both structure and sugar specificity.

Because bacterial re-15 receptor structures are often present on the surface of erythrocytes, bacteria adhere to these structures and agglutinate erythrocytes in vitro. Bacterial cultures may express three or four hemagglutinins (adhesins), each with a different carbohydrate specificity; thus, the bacterium has the ability to infect 20 different cell types.

In enterobacterial studies, adhesion reactions have been divided into two main categories: mannose-sensitive (MS) reactions, in which the hemagglutination reaction is inhibited by α-mannosides, and mannose-resistant (MR) reactions, in which hemagglutination is not inhibited by α-mannosides. E. coli type 1 fimbria (MS construct) consists almost exclusively of 17 kDa identical subunits. Several MR adhesins recognize the α-Gal (1-4) -B-Gal structure (β-specific adhesin) or α-NeuNAc- (2-3) -30 β-Gal (S-specific adhesin). In general, purified fimbriae consist of subunits with molecular weights ranging from 15 to 22 kDa.

The adhesin protein is usually a separate protein attached to the ends of the fimbriae or to the sides of the fimbriae, it may also be attached directly to the outer membrane of the bacterium. In some bacterial strains, adhesin protein is present on the surface of the bacterial cell even though fimbriae are absent. The adhesins may then form an adhesive capsule around the bacterium. The size of E. coli non-5 fimbrial adhesins ranges from 13 to 28 kDa (Jann, K. and Hoschutzky, H. (1990), Current Topics in Microbiology and Immunology 151. 55-70).

Streptococci are known to bind to various soluble proteins and glycoproteins, whereas their oligosaccharide specificity is little known. The specific binding to epithelial cells is also almost unknown. Some strains of Streptococcus sancruis and Streptococcus mitis bind galactose and sialic acid (Murray, P.A., Levine, M.J., Tabak, L.A., and Reddy, M.S. 15 (1982) Biochem. Biophys. Res. Commun. 106, 390-396).

Binding of Streptococcus pneumoniae to epithelial cells is inhibited by the GlcNAcBl-3Gal construct and this bacterium has been reported to bind to glycolipids containing the GalNAcB1-4Gal construct in the lung (Krivan, HC, Roberts, DD and Ginsburg, V. (1988) Proc. Natl. Acad. USA, 85, 6157-6161).

S. mitis adheres to the tooth surface and is a component of plaque formation. It has been possible to isolate sialic acid binding adhesin from this bacterial strain. The sialic acid binding protein had at least two disulfide-linked moieties of 96 kD and 70 kD. Both subunits bound N-acetylneuraminic acid-α2-3-galactose-B1-3-N-acetyl-galactosamine (Murray, PA, Levine, MJ, Reddy, MS, Tabak, LA, Bergey, EJ (1986) Infect. Immun. 53 , 359-. 30 365). The molecular weight of the galactose-binding adhesin of S. sanquis was about 20 kDa and its isoelectric point was 8.5 to 9 (Nagata, K., Nakao, M., Shibata S., Shi-zukuishi, S., Nakamura R. and Tsunemitsu, A. (1983) J. Periodontol. 54, 163-172). In addition, a 36 kDa adhesin protein has been cloned from S. sanauis.

; i m i ami i.i i si. .

5 95385

The carbohydrate specificity of the cloned adhesin is unknown; however, adhesin is known to adhere to saliva-coated hydroxyapatite via a pH-sensitive receptor (Ganeskumar, N., Song, M. and McBride, B.C.

5 (1988) Infect. Immun. 56, 1150-1157).

S. suis is an important porcine pathogen. It spreads to the tonsils or nostrils of piglets and causes serious infections. Capsule polysaccharides and surface proteins of S. suis have been shown to play a role in the pathogenesis of pato-10, but the molecular mechanism of infection is unknown. S. suis bacteria have been shown to bind to sialylated poly-N-acetylactosamine glycans (Liukkonen J., Haataja, S., Tikkanen, K., Kelm, S., and Finne, J. (1992) J. Biol. Chero. 267, 21105-21111). However, hemagglutination by most S. suis is inhibited by galactose, thus galactose-recognizing adhesin is likely to be more common in S. suis than mentioned above (Kuri, D., Haataja S. and Finne, J.

(1989) Infect. Immun. 57, 384-389).

20 Attempts have often been made to remove and insulate adhesives by heat treatment and / or extraction. Proteus mira-bilis adhesin was isolated by heat treatment (65 ° C, 20 min, 2 M urea) and purified by gel filtration (Sepharose CL-4B) (Wray, SK, Hull, SI, Cook, IL 25 RG, Barrish, J. and Hull, RA (1986) Infect. Immun. 54 43-49). The lectin of S. mitis was isolated by extraction of the bacteria with lithium 3,5-diiodosalicylate and the sialic acid binding protein was purified from the extract by gel filtration and affinity chromatography (Murray, PA, Levine, MJ, Reddy, MS, Tabak, LA and Bergey, EJ. ! (1986) Infect. Immun. 53, 359-365). The myocardial tissue of Streptococcus-pyogenes (Lancefield Group A) and the protein adhering to the basement membrane of renal cells could be isolated by treating the bacteria with a base for 35 to 18 h (Stinson, M.W. and Bergey E.J (1982)).

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Immun. 35, 335-342). E. coli nonfimbrial adhesin was extracted by heating the bacterial suspension at 65 ° C for 30 min (Goldhar, 3, Perry, R., Golecki, JR, Hoschutzky, H., Jann B., and Jann, K. (1987) Infect. Immun .

5 55, 1837-1842). Fimbriae were separated from fimbrial E. coli by mechanical homogenization and adhesin protein was removed from the fimbrial homogenate by heat treatment (70 ° C for 1 h, 5 mM EDTA) (Moch, T., Hoschutzky, H., Hacker, J., Kröncke, K.-D and Jann , K. (1987) Proc. Natl. Acad. Sci. 10 USA 84, 3462-3466). Often, after cleavage of the adhesin protein, the adhesin is precipitated by ammonium sulfate precipitation, after which the adhesins are further purified by various methods. However, conventional methods used to isolate adhesins were not suitable for isolating adhesins from S ^ 15 suis bacteria.

Summary of the Invention

The adhesin protein of Streptococcus suis has now been isolated and characterized. The invention therefore relates to a process for the preparation of an adhesin protein or a biologically or immunologically active derivative or fragment thereof having a molecular weight of about 18,000, an isoelectric point of about 6.4 and an N-terminal sequence of Ala-Ser-Pro-Ala- Glu-Ile-Ala-Ser-Phe-Ser-Pro-Ala-Pro-Leu-Ala- (SEQ ID NO: 1). The method according to the invention is characterized in that the S. suis bacterial strain is sonicated and the detached adhesin protein is pre-purified in a conventional manner in protein purification, after which it is purified electrophoretically or chromatographically. Pigeon ovomucoid is preferably used to monitor adhesive protein purification. Kek- * ·. the invention also relates to a method for producing an antibody against an adhesin protein, a derivative or a fragment thereof using said adhesin protein, a derivative or a fragment thereof as an immunogen. The adhesin protein prepared according to the invention, its derivative [| Htt Herring II I fit 7 95385 or a fragment can be used as a vaccine and said antibodies for passive immunization.

DETAILED DESCRIPTION OF THE INVENTION The adhesin protein described herein can be irradiated from the surface of S. suis by sonication. After sonication, a protease inhibitor should be added to prevent proteolysis. The sonicate is then centrifuged and the supernatant is collected. It is a good idea to pre-purify the supernatant containing the adhesin protein in some conventional manner for protein purification, such as e.g.

ultrafiltration, dialysis, gel filtration, salt precipitation. Preferably, ammonium sulfate precipitation is used, and in particular, other proteins can be removed with about 60% saturated ammonium sulfate solution, after which the adhesin protein can be precipitated with about 70 incest saturated ammonium sulfate solution.

The actual purification can be performed electrophoretically, e.g., by gel electrophoresis, or chromatographically, e.g., by affinity chromatography or immunoaffinity chromatography. Preferably, preparative native gel electrophoresis is used. By native electrophoresis is meant electrophoresis without sodium dodecyl sulfate, i.e. SDS. In particular, a continuous elution electrophoresis device is used, which is preferably a second cylinder. Fractions are collected from the device and those protein fractions with adhesin activity are recovered.

The biological activity of the S. suis adhesin protein can be utilized in monitoring the purification of the adhesin protein. The adhesin protein is associated with S. ·. 30 haemagglutination of suis bacteria, which can be inhibited by certain sugar compounds, such as galactose and N-acetylgalactosamine or galactose alone, at millimolar concentrations. In the study of galactose-specific adhesin proteins, sialida-35-treated human erythrocytes are preferably used. Hemagglutination experiments 8 95385 and hemagglutination inhibition experiments are described in Kuri, D.N., Haataja, S. and Finne, J. (1989) Infect. Imxnun. 57, 384-389 and Haataja, S., Tikkanen, K., Liukkonen, J., Francois-Gerard, C. and Finne, J., (1993) Charace-5 rization of a Novel Bacterial Adhesion Specificity of Streptococcus suis Recognizing Blood-Group P Receptor Oligosaccharides. J. Biol. Chem. 268. (in press).

Mono- and oligosaccharide inhibition experiments have shown that the receptor structure in S. suis is Galal-10 4GalB1-4Glc. This forms the oligosaccharide moiety P in the Pk antigen of blood group glycolipids. On the other hand, hemagglutination inhibition experiments and direct binding of glycoproteins and neoglycoproteins have shown that adhesin also recognizes the β-antigen structure, Galal-4GalBl-15 4GlcNac. This is consistent with the finding that adhesin binds strongly to the Galal-4Gal disaccharide structure compared to α1-, α1-6, α-galactose-disaccharide di-derivatives. Inhibition experiments with oligosaccharides show that terminal α-galactose is essential for adhesin binding.

It has now been found that pigeon ovomucoid is a particularly potent inhibitor. 0.06 μg / ml pigeon ovomucoid completely inhibited the hemagglutination induced by S. suis strain 628. The pigeon ovomucoid is a glycoprotein with the terminal sequence of the glycan chains of Galal-4GalB1-4GlcNAc and described in Francois-Gerard C., Gerday C. and Beeley, J.G. (1979) Biochem. J. 117, 679-685. This structure results in the strong binding of S. suis adhesin protein to the pigeon ovomucoid, and consequently, the 30 pigeon ovomucoid is also a blood group Ρ, activity.

The neoglycoprotein Galal-4GalBl-4Glc-0-CETE-BSA has also been shown to be a very potent inhibitor of hemagglutination inhibition.

The purification of the adhesin protein can be easily monitored by a simple pigeon ovomucoid binding assay with t- t * * 95385. The ovomucoid used to monitor purification can be labeled with, for example, a radioactive tracer. The assay may be, for example, a spot test in which a sample containing adhesin protein is pipetted onto nitrocellulose paper, which is then covered with radiolabeled pigeon ovomucoid 11a, incubated and washed, and the radioactivity bound to the paper is determined. The intensity of binding correlates with the hemagglutination activity of the strain.

Preferably, the purification of the adhesin protein is screened by western blot, whereby the samples are subjected to polyacrylamide gel electrophoresis, after which the proteins are transferred by electric current to a membrane, which is then treated as in the paper above. S. suis bacterial isolates yield a single adhesin protein band that moves at different sites on different strains. The intensity of the zone correlates with hemagglutination activity. Hemagglutination-negative strains also distinguish the zone, although usually less well. The reason for this is probably the phase variation associated with the adhesin protein, i.e. the bacteria may express the adhesin at different levels under different conditions, or the inhibitory effect of other surface structures such as the capsule.

By the method described above, an adhesin protein of the bacterium S_j_ suis having a molecular weight of '.Y. 25 about 18,000, the isoelectric point is about 6.4, the N-terminal sequence is Ala-Ser-Pro-Ala-Glu-Ile-Ala-Ser-Phe-Ser-Pro-Ala-Pro-Leu-Ala- (SEQ ID NO: 1) and the amino acid composition is: ♦ 10 95385

The Ser-Pro-Ala-Pro-Leu-Ala- (SEQ ID NO: 1) and amino acid composition is:

Amino acid nmol amino acid / nmol protein 5 Asx = aspartic and / or aspartic acid 13

Thr = threonine 8

Ser = serine 6

Glx = glutamine (Glu) and / or glutamic acid 26 Gly = glycine 22 10 Ala = alanine 17

Vai = valine 10

Cys = cysteine 0

Met = methionine 4

Ile = isoleucine 11 15 Leu = leucine 12

Tyr = tyrosine 4

Phe = phenylalanine 6

Lys = lysine 16

His = histidine 7 Arg = arginine 7

Pro = proline 12

In addition to the biological activity described above, the properties of the adhesin protein further include that it is immunologically active, i.e., it induces the formation of antibodies.

Thus, the adhesin protein to be produced also includes derivatives and fragments of the described protein having substantially the same biological or immunological activity as the described adhesin protein. By derivative is meant a protein in which some of the amino acids have been replaced, deleted or added, but which has retained its essential biological or immunological properties. Substitute or added amino acids include both amino acids present in proteins and their derivatives; in li i i i iti: 11 95385 dannaiset. Possible aggregates are also calculated for said derivatives. By fragment is meant a portion of the described adhesin protein that has retained its essential biological or immunological properties. Derivatives 5 and fragments can be prepared in a conventional manner in protein chemistry. The fragments are preferably prepared by peptide synthesis, e.g. by a solid phase method. The invention further encompasses the preparation of antibodies to an adhesin protein, which antibodies herein also include antibody fragments, such as Fab fragments or phage-expressed portions of antibodies that bind to the adhesin protein, derivative or fragment thereof described herein. .

Due to their immunological properties, the adhesin protein, derivatives and fragments thereof prepared according to the invention may have use as a vaccine against diseases caused by E. coli. Use as a human vaccine may also be possible, for example, for at-risk groups such as pig farmers or slaughterhouse staff. Antibodies could be considered for use in passive immunization.

The following non-limiting examples illustrate the invention.

Example 1 Culture of Streptococcus suis

The following S. suis strains were used in the studies: Haemagglutinating: 628, TEW / 2, R75 / L1, 825 and 752 Non-haemagglutinating: 3027, 1045 and 598 / T5

Strains 628, TEW / 2, R75 / L1 and 825 are described in Kuri, D.N., Haataja, S. and Finne, J., (1989) Infect. Im-: mun. 57, 384–389. The rest of the strains were obtained from Dr. J. Hommez, Regional Veterinary Investigation Laboratory, Torhout, Belgium.

Strains were stored frozen in Todd-Hewitt eluent at -20 ° C. The bacteria were grown anaerobically (Gas Pak system) on fresh sheep blood agar plates at 37 ° C overnight. The bacteria were collected from the plates and suspended in phosphate buffer A (10 mM sodium phosphate drop buffer, 0.15 M NaCl, pH 7.4) to give a concentration of 109 colony forming units / ml (A ^ ^ 5.0).

Example 2

Purification of adhesin protein

The adhesin protein was detached from the bacterial surface by sonication for 5x15 sec. (cooling on ice 2x1 min. and 1x2 min.) in 2.5 ml aliquots to phosphate buffer A. After sonication, the protease inhibitor PMSF (phenylmethylsulfonyl fluoride) was added to a concentration of 2 mM. The sonicates were centrifuged at 15,800 x g, at + 8 ° C, and the supernatants were collected. The adhesin protein was pre-purified from the mixture by fractional ammonium sulfate precipitation. 16 ml of supernatant was used for ammonium sulfate precipitation. Other proteins were removed from the mixture with 60% saturated ammonium sulfate and adhesin was precipitated with 70% saturated ammonium sulfate.

No significant amounts of adhesin protein remained in the 70% supernatant. Cold saturated ammonium sulfate was added dropwise to the solution (0 ° C), allowed to stand for 1 hour in an ice bath, centrifuged at 15,800 x g,

• I

* 25 min. Precipitates from 70% precipitation were collected and dissolved in 16 ml of phosphate buffer B (3.3 mM sodium phosphate buffer, 0.05 M NaCl, pH 7.4). The precipitates were dialyzed overnight at + 8 ° C against H 2 O, lyophilized and dissolved in 4 ml of phosphate buffer A. Purification was monitored by 6% native polyacrylamide gel electrophoresis. radioactive iodine-labeled pigeon ovomucoid on a Bio Rad mini-gel device (see Example 3).

The actual purification was performed using a Bio Rad 491 Prep 35 Cell preparative electrophoresis device. Sylin- 13 95385

a 6% native polyacrylamide gel (bottom gel height 6 cm, top gel 2 cm) was poured into a tin gel device. The total sample volume was 4 ml, of which 3000 μ 3000 of the protein solution prepared above, 920 μΐ of sample buffer-5 (no SDS solution) and 80 μΐ of marker BPB (bromophenol blue). The eluent was 25 mM Tris-192 mM glycine, pH

8.3 and Tris-HCl, pH 8.3 as elution buffer. 4 ml fractions were collected from the samples and analyzed by measuring the absorbance of the fractions at two different wavelengths (214 nm and 280 nm), after which the samples were run on polyacrylamide gel electrophoresis on a Bio Rad mini-gel apparatus as described in Example 3. Adhesin protein was present in fractions 79-83. Fractions 79-81 were pooled, dialyzed overnight at + 8 ° C against phosphate buffer B and lyophilized. The dried precipitate was stored at -20 ° C for later analysis.

Example 3

Purification monitoring

The purification was monitored using the ha-20 finding that S. suis bacteria strongly bound the pigeon ovomucoid, which thus contains the disaccharide structure galactosyl-α-4-galactoside. For this reason, a method for the identification of adhesin protein was developed by radiolabeling pigeon ovomucoid with 125 I marker.

• In addition to the haemagglutinating S. suis 628 bacterial strain in a radiolabeled ovomucoid binding assay, a negative i.e. non-haemagglutinating S. suis strain 598 / T5 and a weakly haemagglutinating S. suis bacterial strain 825 fS were used as control strains. suis 628 strain hemag-30 had a glutination titer of 64, 825 strain titer 4 and 598 /: T5 strain titer 0). The ovomucoid was radiolabeled with the 25I isotope by the iodo-Bead method (Pierce Chemical Co., Rockford II) according to the manufacturer's instructions.

In the first phase, the so-called spot test. In these 35 binding experiments, each bacterial suspension prepared according to Example 1 was diluted (1: 1; 1:10; 1:50) in phosphate buffer A. The suspensions were pipetted onto 1 μΐ squared nitrocellulose paper, after which the additional binding sites were covered by incubating the phosphate for 1.5 h. -5 in C (0.1 M sodium phosphate buffer, 0.5% Tween 20, 150 mM NaCl, pH 5.3). The nitrocellulose was then covered with 125 I-ovomucoid (6x10 5 cpm, specific active 2.5x10 5 cpm ^ g ovomucoid) and incubated at 1, + 8 ° C. The membrane was washed 3x10 min. phosphate buffer C, dried between filter papers and exposed to X-ray film at -80 ° C for 24 h. Binding was observed only in hemagglutinating strains even at low bacterial concentrations, and the intensity of binding correlated with hemagglutination activity.

In a second step, using the method described above, a "western blot" identification method was developed for an adhesin protein by polyacrylamide gel electrophoresis (Laemmli, U.K. (1970) Nature (London) 227. 680-685). Native gel electrophoresis was used; without the addition of SDS-li-20, leaving the proteins in their native form. Bacterial isonates prepared according to Example 2 were separated in several polyacrylamide gel electrophoresis systems containing different concentrations of polyacrylamide (5-9%). Finally, it was decided to use 6%. After separation, the sonicates were transferred by electric current (60 mA, 30 min) to a PVDFp (Millipore) membrane (Burnette, W.N. (1981) Anal. Biochem. 112. 195-203) and the membrane was labeled with 125 I-ovomucoid as above. S. suis strains haemagglutinating strains: 628, TEW / 2, R75 / 30 LI, 825, 752 and non-haemagglutinating strains: 3027 and 1045 were used in the experiments. The intensity of the zone correlated with the hemagglutination activity, but the mobile zone was also distinguished from the nega-35 dense strains at the same site by native 9538385 gel electrophoresis, usually weaker.

Example 4

Molecular weight and purity of the adhesin protein

The purity of the adhesin was confirmed and the molecular weight was determined by electrophoretic mobility in 15% SDS-polyacrylamide gel electrophoresis (Laemmli, U.K. (1970) Nature (London) 227, 680-685). Kan-10 nan 628 adhesin protein (4 μq) was boiled for 5 min. time in a 2% SDS solution containing either 5% mercaptoethanol or 2.5 mM dithiothreitol. One zone separated from the purified adhesin protein. Pharmacia Low Molecular Weight Standards 15 standard proteins were used as molecular weight standards. The molecular weight of the adhesin was 18,000.

Example S Isoelectric point

Isoelectric focusing was performed on a Phast gel-20 electrophoresis device using a Phast isoelectric system (Pharmacia). The gel was Phast Gel 15531 3-9 and the standards were IEF standard 3-10 (Pharmacia). Purified strain 628 adhesin was used for the 0.2 Mg assay. The isoelectric focusing gel was stained using the Silver IEF-Method 6 of the Phast sys-25 theme. The isoelectric point of the adhesin was 6.4.

Example 6 Amino acid analysis

For amino acid analysis, 7 nmol of purified adhesin from strain 628 was dissolved in 100 ® M HCl solution; ta. 60 nmol of norleucine was added as an internal standard. Hydrolysed at 110 ° C for 24 h, lyophilized and analyzed on an LKB 4151 Alpha Plus Aminoacid Analyzer according to the manufacturer's instructions.

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The amino acid composition was as follows:

Amino acid nmol amino acid / __nmol protein_ 1 Asx 13.2 5 2 Thr 8.1 3 Ser 5.8 4 Glx 26.4 5 Gly 22.3 6 Ala 16.9 10 7 Vai 10.3 8 Cys 0.0 9 Met 3.9 10 Ile 11.4 11 Leu 12.3 15 12 Tyr 3.9 13 Phe 6.0 14 Lys 15.9 15 His 6.6 16 Arg 6.8 20 17 Pro 12.4

Example 7

N-terminal amino acid sequence of the adhesin protein The N-terminal amino acid sequence of the adhesin protein was determined using an Applied Biosystems 477A Pulsed Liquid Protein / Peptide Sequencer with 120A Amino Acid Analyzer peptide sequencer according to the manufacturer's instructions.

· Purified adhesin was run on a 6% native polyacrylamide gel, from which the adhesin was electrically transferred to a PVDFp membrane as in Example 3. The membrane was stained with Coomassie Brilliant Blue protein dye (10 min.), The excess dye was removed and the protein band was excised for peptide sequencing.

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The N-terminal sequence of the adhesin protein could also be determined from the supernatants of sonicated bacteria: The mobility of the adhesin protein in native gel electrophoresis is known because the 5-strand of the adhesin protein band can be identified by a 125 I pigeon ovomucoid from the PVDFβ membrane. Five different hemagglutinating S. suis strains (628, TEW / 2, R75 / L1, 825, 752) identified adhesin protein mobility by 6% native polyacrylamide gel electrophoresis; Sonicates from these bacterial 10 strains were run on electrophoresis and the zones were transferred to a PVDFβ membrane. From this, the adhesin bands were excised for amino acid sequencing as above.

The resulting N-terminal sequence was the same for all strains, i.e.:

Ala-Ser-Pro-Ala-Glu-Ile-Ala-Ser-Phe-Ser-Pro-Ala-Pro-Leu-Ala- (SEQ ID NO: 1)

Example 8

Preparation of Adhesin Protein Antibody 20 Adhesin protein antibody of strain 628 was prepared in Balb / c mice. Mice were immunized with either pure adhesin (10 μg / mouse) or a sonicate from S. suis (80 μg / mouse). In the first case, the adhesin was injected twice into Freund's *. 25 in complete adjuvant (F.C.A.) and once in Freund's incomplete adjuvant (F.I.C.A.). In the latter case, the protein mixture was injected into mice once with F.C.A. and mixed twice with F.I.C.A. Immunization was performed subcutaneously. In both cases, antibodies were formed against the adhesin protein; a.

Antibody formation was examined from a PVDFβ membrane to which sonicate supernatants of S. suis had been transferred (see Example 2). Zone 35 of the adhesin protein was identified (cf. Example 3) and the generation of antibody 18 · 38385 against the adhesive protein of S. suis strain 628 was strong. The antibody was able to specifically recognize the adhesin protein at a further dilution of 1: 5x10 5. (Non-specific binding of antibody was prevented by incubating the membrane for 1.5 h, 1.5% milk powder-0.5%

In Tween 20 solution made in buffer D (10 mM Tris, 150 mM NaCl, pH 7.8). The membrane was washed three times with buffer D supplemented with 0.05% Tween 20. Antibody dilutions were added for one hour, after which the membrane was washed 5x with buffer D supplemented with 0.05% Tween 20. The membrane was incubated with alkaline phosphorus. with fatase-labeled mouse antibody for one hour (1: 1000 dilution), washed five times with buffer D, and alkaline phosphatase substrate was added to effect a color reaction (bromochloroindolyl phosphate-nitrosinitrazazolium).

Amino acid sequence No. 1:

Ala Ser Pro Ala Glu Ile Ala Ser Phe Ser Pro Ala Pro Leu Ala 15 10 15 iH irt i 9111! I! 1 teaspoon

Claims (3)

19 95385 A method for bleaching a therapeutically useful adhesin protein of Strepr i-nr.nr.cus suis, which adhesin protein has galactose-inhibitory hemagglutination activity, which binds to a pigeon ovomocoid and which produces antibodies and has a molecular weight of about 18. 000, the isoelectric point is about 6.4 and the N-terminal sequence is Ala-Ser-Pro-Ala- 10 Glu-Ile-Ala-Ser-Phe-Ser-Pro-Ala-Pro-Leu-Ala- (SEQ ID NO: 1) and having the following amino acid composition: Amino acid nmol amino acid / nmol protein Asx 13 15 Thr 8 Ser 6 Glx 26 Gly 22 Ala ^ 7 20 Vai 10 Cys 0 Met 4 Ile H Leu 12 I 25 Tyr 4 Phe 6 Ly s 16 His 7 Arg 7 To prepare Pro 12 «* · or a derivative or fragment thereof having the same biological and immunological activity, characterized in that the s. Suis bacterial strain is sonicated, 35 and the detached adhesin protein is pre-purified in a conventional manner, followed by electrophoretic purification. or chromatographically. Process according to Claim 1, characterized in that preparative native gel electrophoresis is used. A method for producing an antibody, characterized in that it is generated using, as an immunogen, an adhesin protein as defined in claim 1 or a derivative or fragment thereof having the same biological and immunological activity. «Il HU; U HU Ιέ 3-St; :; 21 95385