SI9500155A - Novel synthetic peptides and their antibodies, their prepatation and use in diagnostics and therapy of m. gallisepticum - Google Patents

Abstract

The invention describes novel synthetic peptides imitating natural epitopes on the membrane of pathogenic mycoplasma M. gallisepticum and a process for the preparation thereof. By the mentioned peptides native antigens in serological tests can be substituted and they can be used as vaccines or as antigens for the preparation of antibodies. Antibodies prepared by means of these peptides recognize native mycoplasma epitopes and are useful in various diagnostic tests for determination of poultry infections with M. gallisepticum, they prevent the growth of the mentioned mycoplasma and are also suitable for the isolation of native or recombinant main immunogenic protein pMGA or its subunits.

Description

FIELD OF THE INVENTION

The present invention relates to the fields of biochemistry, immunochemistry and veterinary medicine. Refers to new synthetic peptides derived from the amino acid sequence of individual regions of pMGA ah its multigenic variant pMGA 1.2, the major immunogenic protein in M. gallisepticum, the process for their preparation, to antibodies against these peptides and to the use of these antibodies and peptides in diagnostic tests and vaccines in the diagnosis and therapy of poultry infected with Mycoplasma gallisepticum (MG).

A technical problem

Mycoplasma gallisepticum causes considerable economic damage when breeding poultry such as chickens and turkeys. In addition to direct damage caused by death, lower production and poor quality of poultry products, there are also indirect costs, which include the costs of treatment, possible vaccination and mandatory diagnostic tests. Diagnosing poultry infections with M. gallisepticum remains problematic, as the procedures are nonspecific, time consuming and expensive.

The state of the art

The most reliable method of diagnosing a disease - M. gallisepticum infection - is the method of isolation and identification of M. gallisepticum (Yoder, HW, (1991). Mycoplasma gallisepticum infection. In: Poultry diseases (BW Calnek, H J. Bames, CW Beard, WM Reid and HW Yoder eds)) p. 198-212, Iowa State University Press), which can only be performed by highly specialized laboratories, and isolation of M. gallisepticum is very time consuming, expensive and even impossible for individual M. gallisepticum strains. For mass testing, serological tests are used, e.g. HSA (rapid serum agglutination) and IHA (hemagglutination inhibition), and more recently immunoenzyme assays (ELISA). All of these tests have a number of disadvantages, e.g. nonspecific interactions, and their downside is that they demonstrate specific antibodies to M. gallisepticum, which makes them impossible to diagnose in an acute state of infection when treatment would be most effective, and does not allow the diagnosis of latent infections.

An alternative to these tests would be immuno-enzymatic assays to prove M. gallisepticum antigens. In this way, the presence of M. gallisepticum could be detected already in the acute phase. Appropriate avid and selective antibodies against M. gallisepticum surface antigens should be prepared for this purpose. Native antigens can be used for immunization by treating cell membranes appropriately

M. gallisepticum. Considering that the amino acid sequence of hemagglutinin pMGA is known (Markham, PF, Glew, MD, Whithear, KG, Walker, ID (1993) Molecular cloning of a member of the gene known to encode pMGA but hemagglutinin of Mycoplasma gallisepticum, Infection and Immunity 61, pp. 903-909), i a major immunogenic protein on the membrane of M. gallisepticum, synthetic or recombinant peptides similar to individual segments of the pMGA protein can be used as antigens.

Peptide synthesis is already an established method for various studies of the biological activities of protein molecules. The Merrifield solid-state synthesis technique is mainly used (Merrifield, R.B. (1963) Automated Synthesis of Peptides, Science 150, 178-184). By this method, the first amino acid is bound from the C-terminal end of the peptide via a carboxyl group to a solid support, and new amino acids are added at the N-terminal end of the peptide chain. The use of suitable protecting groups for the N-terminal end and the side chains of individual amino acids, and the introduction of less aggressive peptide bond synthesis methods, enabled the automation of the process. The synthesized peptides require further purification as they contain impurities of reagents as well as a certain proportion of peptides with irregular sequences. The most useful method for this purpose is high-performance liquid chromatography (HPLC). The composition and purity of peptides can be verified by determining the composition or sequence in the amino acid chain.

Shorter peptides are commonly used as haptens as they only produce a strong immune response when bound to a carrier molecule. Various proteins can be used as carrier molecules, e.g. bovine serum albumin, ovalbumin or hemocyanin (KLH). Covalent binding can be via the -SH group of cysteines, using GMBS (gamamaleimido butyryloxy succinimide) as a heterobifunctional reagent (Geysen, HM, Bartheling, SJ, Meloen, RH (1985) Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein, etc. Natl Acad. Sci. USA 82, 178-182} Also used is lysine binding via glutaraldehyde or a carboxyl group by forming a carbodiimide bond (Erlanger, BF (1980 Preparation of antigenic hapten-camer conjugates: A survey Meth. Enzymol 70, 85-104} The conjugated peptides are suspended in complete Freund's adjuvant and injected subcutaneously into the experimental animals After 2, 3, 7, 8. , Weeks 9 and 10, the procedure is repeated with booster injections.

Polyclonal antibodies can be isolated from serum in several ways, e.g. by precipitation (ammonium sulfate, caprylic acid), glaze filtration, ion exchange chromatography or affinity chromatography. In affinity chromatography, immobilized antigen or staphylococcal protein A and streptococcal protein G can be used as ligand, which specifically binds immunoglobulins G mainly via Fc receptors.

In order to prepare direct immuno-enzymatic assays, isolated antibodies need to be conjugated with a marker enzyme that allows the visualization of the result by degradation of the added chromogen. Horseradish peroxidase, alkaline phosphatase and beta-galactosidase are the most used as marker enzymes, and the methods of covalent bonding are similar to those for the preparation of conjugates between peptides and carrier molecules.

Description of solution to a technical problem

By determining the cDNA sequence and by analyzing the partial amino acid sequence of p67 protein purified by immunoaffinity chromatography on mobilized Mab, we determined the amino acid sequence of pMGA, a major immunogenic protein, with Mr 67000 located in the membrane of M. gallisepticum. This protein has been found to be responsible for hemagglutination as well as pathogen adhesion to host epithelial cells. Studies of Related Microorganisms - M. pneumoniae, (Jacobs, E., Fuchte,

K., Bredt, W., (1987) Amino acid sequence and antigenicity of the amino-terminus of the 168 kDa adherence protein of Mycoplasma pneumoniae. Journal of General Microbiology 133, 2233-2236, Gerstenecker, B., Jacobs, E. (1990) Topological mapping of the Pl-adhesin of Mycoplasma pneumoniae with adherence-inhibiting monoclonal antibodies. Journal of General Microbiology 136, 471-476) have shown that the N-terminal end of Pl is adhesin, i.e. protein that has a similar function to pMGA, the exposed part of the molecule by stimulating the immune response.

A peptide similar to the N-terminal end of a pMGA molecule that elicits an immune response could be used to prepare antibodies and thus to develop direct immunodiagnostic assays for M. gallisepticum, to prepare vaccines, and as an antigen (pMGA subunit) to demonstrate specific antibodies in poultry such as chickens and turkeys.

The present invention therefore relates to synthetic peptides that mimic natural epitopes on the membrane of the pathogenic Mycoplasma gallisepticum mycoplasma. The peptides mentioned can replace the native antigen in serological tests, can be used as vaccines or as antigens for the production of antibodies. Antibodies derived from these peptides recognize native mycoplasma epitopes and are useful in various diagnostic tests for the detection of M. gallisepticum (MG) poultry infections, inhibit the growth of said mycoplasma, and are also suitable for the isolation of native or recombinant major immunogenic protein pMGA or . its multigenic variants of pMGA 1.2 or subunits thereof.

The first object of the present invention are synthetic peptides characterized in that they mimic individual segments of the pMGA or pMGA 1.2 molecule or its N-terminal end is modified to have cysteine bound at one or both ends of the peptide chain. Binding of cysteine to one or both ends of the peptide chain enables efficient binding of the peptides to the carrier molecule and increases their immunogenicity. Since it has been shown that the N-terminal end of the pMGA molecule can also be variable, it is important that these peptides are more mutually distinct and mimic different parts of the N-terminal end.

In particular, the present invention relates to synthetic peptides obtained in the manner described above, which are defined by the following gene sequences:

Pl C-T-T-P-T-P-S-P-A-P-N-P-N-P-P-S-N-C pMGA P2 C-T-T-P-T-P-N-P-T-P-N-P-N-P-P-S-G-C pMGA 1.2 P3 C-T-T-P-T-P-S-P pMGA (Part One) P4 P-N-P-N-P-P-S-N-C pMGA (rear) Q5 P-S-P-A-P-N-P-N-C pMGA (middle)

A further object of the present invention is a process for the preparation of the above synthetic peptides characterized in that the N-terminal end of the pMGA or pMGA 1.2 molecule is modified by binding cysteine to one or both ends of the peptide chain.

A fourth object of the invention is therapeutic and diagnostic agents for infections of poultry with M. ga / foepricum containing the above peptides as an active ingredient.

Preferably, the present invention relates to vaccines characterized in that they contain, as an active substance, single or a combination of the above peptides.

The sixth object of protection is the use of the above peptides, in different combinations or individually, for the preparation of vaccines.

The use of the above synthetic peptides for the detection of serum (systemic) and topical antibodies in M gallisepticum infections is also novel.

An eighth object of the invention is the synthetic peptides mentioned above for use in the diagnosis and therapy of poultry infected with M gallisepticum.

The ninth object of the invention is novel antibodies, or portions thereof, characterized by the recognition of native antigens by M. gallisepticum. They are obtained by conventional immunization of animals with the above peptides.

Tenth objects of the invention are vaccines, characterized in that they contain the antibodies mentioned above or portions thereof as the active substance.

A further object of the invention is antibodies or portions thereof, as described above, for use in in vitro or in vivo inhibition of M gallisepticum growth.

A further object of the invention is the use of the abovementioned antibodies or portions thereof for the preparation of immunodiagnostic assays for the detection of M. gallisepticum. in poultry and in any other specimen.

The subject of the invention is also the use of the above antibodies in the analysis of antigenic determinants on pMGA or pMGA 1.2.

It is also an object of the invention to use antibodies to obtain and purify pMGA or pMGA 1.2 ah subunits thereof from a fermet broth or from cell suspensions by affinity chromatography. The antigen thus prepared can be used in diagnostic tests and in the preparation of vaccines.

The present invention is illustrated by the following non-limiting embodiment.

Ί

An implementation example

Using an automated peptic synthesizer (Applied Biosystems), we prepared five differently long peptides corresponding to the N-terminal sequences of pMGA or its multigenic variants pMGA 1.2 (Markham, PF, Glew, MD, Brandon, MR, Walker, ID, Whithear, KG ( 1992) Characterization of a major hemgglutinin protein from Mycoplasma gallisepticum, Infection and Immunity 60, 3885-3891, Markham, PF, Glew, MD, Whithear, KG Walker, ID (1993) Molecular cloning of a member of the gene family that encodes pMGA. and hemagglutinin of Mycoplasma gallisepticum. Infection andlmmunity 61, 903-909.):

Pl C-T-T-P-T-P-S-P-A-P-N-P-N-P-P-S-N-C pMGA P2 C-T-T-P-T-P-N-P-T-P-N-P-N-P-P-S-G-C pMGA 1.2 P3 C-T-T-P-T-P-S-P pMGA (Part One) P4 P-N-P-N-P-P-S-N-C pMGA (rear) Q5 P-S-P-A-P-N-P-N-C pMGA (middle)

The peptides Pl and P2 cut into the first 17 amino acids in the pMGA and pMGA 1.2 genes. They differ in three amino acids: S7 - N7, A9 - T9, N16 - G16. For both peptides, cysteine was added at the N-terminal end to allow better binding to the carrier molecule. The added cysteine also allows the formation of peptide loops attached to the carrier molecule via two thiol groups. In this way, greater peptide exposure and a better immune response are achieved. The shorter peptides P3, P4 and P5 overlap the first, last and central regions of the Pl peptide. They can more precisely locate the epitope at the N-terminal end of the pMGA or Pl peptide. Cysteine was added to the peptides P4 and P5 due to the possibility of binding to the carrier molecule via the -SH bond.

HOBT / TBTU / DIEA chemistry was used for the synthesis, and the F-mo (fluorenylmethoxycarbonyl) group was used to protect the N-terminal end (Carpino, L.A. (1987) The

9-fluorenylmethyloxycarbonyl family of base-sensitive amino-protecting groups, Acc. Chem. Really. 20, 401-407). The synthesized peptides were cleaved from the vehicle with 96% trifluoroacetic acid. The relaxed protecting groups were trapped in anisole and ethanediol and the protein precipitated in diethyl ether. Reverse phase liquid chromatography (RP HPLC) was used to analyze and purify the peptides. We worked on a Milton Roy, USA machine with a Nucleosil 300 08 column from Machery-Nagel, Germany. The CH ₃ CN / H ₂ O / 0, 1% TFA system was used as the mobile phase. The solvent fractions were removed by rotavapor evaporation (Devarot, Electromedicine) and then lyophilized to dryness (Hetossic, Heto Lab.,

Denmark). The dried peptides were stored at -20 ° C.

For the preparation of polyclonal antibodies, peptides were first conjugated to GMBS (Ν-γmaleimidobutyryloxy succinimide) via the thiol group of cysteine on hemocyanin (KLH) (Kim, S.J., Hirose, S., Miyazaki, H., Ueno, N., Higashimori, K., Morinaga, N., Kimura, T., Sahakibara, S., Murakami, K. (1985) Identification of plasma inactive renin as prorenin with a site directed antibody. Biochem, Biophys. Res. Commun. 126, 641-645). A suspension of hemocyanin in 65% (NH ₄ ) ₂ SO ₄ was centrifuged for 20 min. At 10,000 rpm. The precipitate was dissolved in 0.1 M phosphate buffer, pH 7.0 and desalted by gel filtration on Sephadex G-25 (PD 10 column, Pharmacia, Sweden). Then 0.7 mg of GMBS was added to 4 mg of KLH in 0.1 M phosphate buffer and stirred for 30 minutes at room temperature. Hemocyanin-GMBS was separated from unreacted GMBS by re-gel filtration on Sephadex G-25. To the eluted KLH-GMBS complex was added 5 mg of each peptide dissolved in 0.1 M phosphate buffer, pH

7.5 and stirred for 3 hours at room temperature. The conjugates were stored at -20 ° C.

After 0.1 ml of individual conjugates were suspended in an equal volume of complete Freund's adjuvant and rabbits were immunized intracutaneously. After two and three weeks, the immunization was repeated with incomplete Freund's adjuvant and after eight, nine and 10 weeks with peptide solution (0.5 mg) in PBS. The rabbits were bled one week after the last booster injection. Serums were concentrated to a quarter of the volume on an ultrafilter (Amicon, ΥΜ5 membrane) and applied to protein A-Sepharose balanced with 0.1 M phosphate buffer, pH 8.3 + 0.3 M NaCl. The bound immunoglobulins were eluted with citrate buffer, pH pH 3.5 was immediately adjusted to 7.5 with 1 M Tris buffer pH 10.5. A two-step glutaraldehyde method was used to bind horseradish peroxidase to immunoglobulins G (Avrameas, S., Ternyck, T. (1971) Immunochemistiy 8, 1175-1179). 10 mg of horseradish peroxidase (Sigma, type VI, USA) was dissolved in 0.4 ml of 1.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, and incubated for 20 hours at 22 ° C. Then 0.6 ml of phosphate buffer was added to the reaction mixture and dialyzed against 0.1 M carbonate buffer at pH 9.2.

5 ml of rabbit immunoglobulin G. were also dialyzed against carbonate buffer. After dialysis, both solutions were combined and incubated for 20 hours at 4 ° C. Subsequently, the remaining reactive groups were blocked with the addition of 0.1 ml of 0.2 M lysine.

The prepared antibodies and conjugates were tested by the following procedures:

1. Indirect dot-immunobinding assay (DIBA) (Kotani, H., McGarrity, G.J. (1986) Identification of mycoplasma colonies by immunobinding. J. Ciin. Microbiology 23, 783-785.

2. Indirect Immunoperoxidase Test (UPA) (Bencina, D., Bradbury, J.M., Varga, S.S., Dovc, P., Stipkovits, L. (1992) Variation of immunodominant surface antigens in unclassified mycoplasma strains isolated from chickens. 9th International Congress of the International Organization for Mycoplasmology, Ames, Iowa, USA, IOM Letters 2, 130.

3. Hemagglutination inhibition - (IHA), hemadsorbtion inhibition (IHAD) (Benčina, D., Bradbury, J.M. (1991) Indirect immunoperoxidase assayforthe detection ofantibody in chick mycoplasma infection. Avian Pathology 20,113-124.

4. MG (IR) growth inhibition on agar and liquid media.

5. Immunoblot (IBL) (Benchina, D., Kleven, S.H., Elfaki, M.G., Snoj, A., Dovch, P., Dorrer, D., Russ, I. (1994) Variable expression ofepitops on the surface of mycoplasma gallisepticum demonstrated with monoclonal antibodies Avian Pathology 23: 19-36.

6. Rapid serum agglutination (HSA).

7. Dual gel diffusion precipitation (GDP).

Peptides (Pl, P2, P3, P4, P5) were tested using the following methods:

1. Indirect dot immunobinding test (DIBA)

2. Indirect immunoperoxidase assay (UPA) - to neutralize binding of rabbit anti-Pl antibodies to M gallisepticum colonies.

3. Immunoblot (IBL) - Pl bound to KLH.

4. Dual gel diffusion precipitation (GDP) -Pl and P2 as antigen.

Test description

Indirect dot immunobinding test f DIB Al:

2-3 μ [peptides at concentrations of 10 / tg / ml to 1 mg / ml were applied to Immobilon P membrane strips (Millipore). After blocking in phosphate buffer with 0.5% Tween® 20, for 30 min, the bands were incubated with peptide antibodies (dilutions 1: 100 to 1:

10 ^-4 ) 45-60 min. at room temperature. After washing with PBS-T buffer (0.05% Tween® 20), 3 x 15 min, the bands were incubated for 40 min. at room temperature with conjugate (goat anti-rabbit IgG-HRP, Sigma, USA, A-6154) diluted 1: 1000. After rinsing, 2 x PBS-T 0.05%, 1 x PBS (pH 7.4), DAB (diamino benzidine) was added, 0.25 mg / ml in 0.03% H ₂ O ₂ - When sufficient reaction became intensive, the bands were washed in distilled water and dried. The rate of reactions was evaluated in terms of signal intensity and negative controls (pre-immunization rabbit serum + conjugate). The test was also performed with conjugated rabbit antibodies against peptides.

All peptides (Pl - P5), affinity purified (with Mab 86) pMGA obtained from PFMarkham, MG lectin (Sigma, USA, L5884), MG p64 in ISCOM obtained from G. Czifre (Czifra, G., Tuboly, T., Sundquist, B.G., Stipkovits, L. (1993) Monocolonal antibodies to Mycoplasma gallisepticum proteins. Avian Diseases 37, 689-696), MG cell suspensions (1: 1000), M. synoviae strain cell suspensions ,

M. iowae strains, M. meleagridis (strain 17529) and M. pneumoniae (FH strain).

Long peptides (Pl and P2) responded better than short peptides (P3, P4, P5), and antibody titers (whole serum, IgG without KLH Ab, affinity M. gallisepticum purified antibodies) were high (1: 10 ⁴ ). Antibodies were also reacted with pMGA (0.01 mg / ml), M. gallisepticum lectin, p64-ISCOM, and whole cells of different M. gallisepticum strains (more than 10 strains tested).

The longer peptides in the DIBA test react well with antibodies from sera (and with topical antibodies in trachial mucus of the upper respiratory tract) of hens naturally infected with MG.

Indirect immunoperoxidase assay (UPA)

UPA was performed with colonies of 20 different M. gallisepticum strains. We used the same procedure as described in the article by Benchin et al. (1994) Avian Path., Except that a goat conjugate (goat anti-rabbit IgG-HRP) diluted 1: 200 was used in the second stage. The antibody titers were as follows:

a) whole serum

b) IgG (after KHL sepharose)

1: 2560 1: 2560

c) affinity purified anti-MG 1: 1280 antibody

d) control (IgG bound to KLH) <1: 20

The positive UPA reaction could be blocked - neutralized by synthetic peptides Pl and P2 at a concentration of <10 Mg / ml (incubation with antibodies for 30 min., 37 ° C). Peptides P3, P4 and P5 were less effective.

The sera of naturally and experimentally infected chickens blocked the UPA reaction against peptides Pl and P2 with a titer of 1: 8.

Haemagglutination (HA), Hemagglutination Inhibition (IHA), Hemadsorption Inhibition (IHAD)

Only the whole anti-Pl peptide serum, but not the purified antibodies, demonstrated the ability of IHA and IHAD. IHA assays were performed in microtiter plates (see Benchina et al. (1994) Avian Path.). We used clones of different strains of M. gallisepticum and also M. synoviae to haemagglutinate chicken erythrocytes. It turns out that not even the whole serum produces a consistent reaction with all M. gallisepticum clones or clones. strains. At least a partial correlation was shown by a strong reaction in the IIPA test. We observed that in some cases there was no IHA reaction at low dilution of anti-Pl antibodies (e.g. 1: 5,1: 10), and higher serum dilutions (1: 80 -1: 320) gave positive IHA. Such reactions are not unusual, because unlike normal antibodies against M. gallisepticum, antibodies to the Pl peptide are narrowly specific, and several factors and at least two different receptors on erythrocytes are certainly involved in the HA reaction.

Positive reactions were observed in IHAD if the total serum was diluted to 1: 10 (inhibition of erythrocyte hemadsorption on M. gallisepticum 'colonies). In this test, inconsistent reactions are also possible if the strain HAD has a property but lacks epitopes for antibodies against Pl.

Growth inhibition

Whole serum against peptide Pl inhibited the growth of M. gallisepticum positive clones in the IR assay. The growth inhibition zone on agar media was 2 - 4 mm (with 50 μϊ antiserum). Serum antibodies inhibit the growth of M. gallisepticum clones having expressed surface epitopes, which means that they are positive in UPA and react with the p67 protein in IBL.

In one case, the ΑΑΥ30 strain also inhibited the growth of M. synoviae partially (1–2 mm) antiserum against Pl peptide, whereas no growth inhibition was observed in other M. synoviae strains.

In cases of positive IR, a precipitation line was observed indicating the precipitation of pMGA (probably soluble p67 protein released into the medium by M. gallisepticum colonies) with antibodies against Pl. This indicates the possibility of obtaining pMGA by immunoprecipitation with antibodies to the Pl peptide.

Immunoblot (IBL)

IBL analyzes were performed with PHAST-SYSTEM apparatus (Pharmacia, Sweden). Antibodies against peptide Pl recognized the epitope on M. gallisepticum p67 that was associated with surface expression on colonies (correlation with UPA reactivity). The epitope has been demonstrated in various M. gallisepticum strains (including variant strain K 703), and in no case was IBL reacted with other types of mycoplasma, e.g. M. synoviae, M. iowae or e.g. M. pneumoniae. Surface proteolysis with V8 St. endoprotease. whole cell aureus (Glu-C, Sigma, USA) confirmed that the Pl antibody antibody epitope was located at the N-terminal end of p67. M. gallisepticum strains that did not infect experimental chickens (ALEX, A5969, clone K4 / 4) also did not show sp 67 in IBL. Conversely, strains that promptly infected chicks (S6 Lp and TS 102) had IBL reaction with p67. Partially, the IBL reaction with p67 was also associated with the ability of M. gallisepticum strains to haemagglutinate chicken erythrocytes, so HA-negative clones of M. gallisepticum strains F (K9 / 4/16) and A5969 (K4 / 4) did not have IBL reactions s p67. There is a strong positive correlation between recognition of M. gallisepticum p67 antigen by anti-Pl antibodies and antibodies from infected chickens. Clones of M. gallisepticum strains (S6, Χ95, R) that were inhibited by Pl antibodies in growth (IR test) had IB67 reaction with p67, whereas those clones of M. gallisepticum strains that were not inhibited by antibodies against Pl, also did not react with p67 in IBL. We used antibodies in the 1: 1000 classics in IBL analyzes.

Properties of synthetic peptides:

Pl (Cl - N17 + C) mimics the conformation of the epitope located at the N-terminal part of pMGA and at native M. gallisepticum p67 (hemagglutinin). Rabbit anti-Pl antibodies suggest that this is a variable surface epitope (UPA) closely related to p67 (IBL). With less than 10 µg / ml Pl (and P2), it is possible to neutralize the antibody response to Pl in the UPA assay (30 min., 37 ° C). Serum and local (upper respiratory) antibodies of hens that are experimentally or naturally infected with M. gallisepticum in DIBA respond well to Pl and P2 even in relatively high serum dilutions (1: 100 -1: 500).

In UPA, hen sera block the binding of rabbit antibodies to M. gallisepticum colonies at the early stage of infection (or as soon as specific antibodies against MG appear). This indicates the potential of Pl and P2 to be used as antigens (for serology) or. as a narrowly defined (subunit) vaccine.

Antibody properties:

Rabbit antibodies against Pl recognize a variable epitope located on the surface of M. gallisepticum membranes (pMGA). Therefore, antibodies react in assays where antibodies bind to surface antigenic determinants, eg: DIBA, UPA, and whole serum also reacts in IR, IHA and solubilic MG p67 precipitates.

The antibodies are suitable for the analysis of M. gallisepticum cultures according to the expression of immunodominant surface epitopes (with UPA and IBL), which are important in the preparation of M. gallisepticum antigens and M. gallisepticum vaccines. In our analyzes, M. gallispetucum vaccine strains used in the US (F strain K810) and (6/85 strain) respectively in Australia and the USA (TS 11 strain) and live vaccine strains of M. gallisepticum (temperature-sensitive mutants of strain S6 , i.e., strain TS 100 and TS 102) all had a variable surface epitope on p67 recognized by said antibodies. These live M. gallisepticum strains on the market are used for the vaccination of broilers and laying hens.

Antibodies against the longer peptide Pl show a much stronger reaction with the surface epitopes of M. gallisepticum in the assays than antibodies against the shorter P3, P4 and P5 peptides.

After binding of antibodies to protein A covalently bound to CNBr activated sepharose (Pharmacia, Sweden) and after crosslinking with DMP (dimethyl pimelimidate, Pierce, USA), pMGA or fragments thereof from fermentation broth or from cellular cells can be isolated on affinity chromatography columns. suspensions.

Claims (15)

Patent claims Synthetic peptides characterized in that they mimic individual segments of pMGA or pMGA 1.2 molecules and are modified to have cysteine bound at one or both ends of the peptide chain. Peptides according to claim 1, characterized in that they contain the following peptide sequences: C-T-T-P-T-P-S-P-A-P-N-P-N-P-P-S-N-C C-T-T-P-T-P-N-P-T-P-N-P-N-P-P-S-G-C C-T-T-P-T-P-S-P P-N-P-N-P-P-S-N-C P-S-P-A-P-N-P-N-C pMGA pMGA 1.2 pMGA (first part) pMGA (last part) pMGA (middle part) A method for preparing peptides according to claim 1 ah 2, characterized in that individual segments of the pMGA or pMGA 1.2 molecule are modified by binding of cysteine to one or both ends of the molecule. Diagnostic ah therapeutic agents for infections of poultry with M. gattisepticum, characterized in that they contain the peptides as claimed in claim 1 according to claim 1 ah 2. Vaccines characterized in that they contain, as an active substance, the individual or combination of peptides according to claim 1 or 2. The peptides of claim 1 or 2 for use in the diagnosis and therapy of poultry infected with M. gallisepticum. Use of peptides according to claim 1 ah 2 for the detection of serum and local antibodies in infections with M. gallisepticum. Use of peptides according to claim 1 or 2, in different combinations ah individually, for the preparation of vaccines. 24774-05! 95-D7-Re. Antibodies or portions thereof obtained against peptides according to claim 1 or 2, characterized in that they recognize native antigens to M. gallisepticum. Vaccines characterized in that they contain, as active ingredient, the antibodies or portions thereof according to claim 9. The antibodies or portions thereof according to claim 9, for use in in vitro or in vivo inhibition of M gallisepticum growth. Use of the antibodies or portions thereof according to claim 9, for the preparation of immunodiagnostic assays for the detection of M. gallisepticum in poultry and other specimens. Use of the antibodies or portions thereof according to claim 9 in the analysis of antigenic determinants on pMGA or pMGA 1.2. The use of the antibodies or portions thereof according to claim 9, in the production and purification of pMGA or pMGA 1.2 or their subunits by affinity chromatography. Use of antibodies or portions thereof according to claim 9, for the preparation of vaccines. For