EP0975757A2

EP0975757A2 - Dictyocaulus viviparus antigen for the diagnosis of lungworm disease and for vaccination

Info

Publication number: EP0975757A2
Application number: EP98924107A
Authority: EP
Inventors: Joachim Hofmann; Karlheinrich Schmid; Annette Pauli
Original assignee: Hoechst AG
Current assignee: Akzo Nobel NV
Priority date: 1997-04-15
Filing date: 1998-04-09
Publication date: 2000-02-02
Also published as: ZA983098B; WO1998046742A3; AR012404A1; WO1998046742A2; AU7642698A; DE19715586A1

Abstract

An immunogenic protein (DV17) from the lungworm Dictyocaulus viviparus having a protective effect is disclosed, as well as a process for isolating the same, a process for isolating the corresponding DNA sequence, the production of the protein by genetic engineering and the use of the protein in diagnostic kits and as vaccines.

Description

Dictyocaulus viviparus antigen for the diagnosis of lung worm infestation and for vaccination.

The invention relates to an antigen from the adult stages of the lung worm Dictyocaulus viviparus (hereinafter also called D. viviparus or Dictyocaulus) of the bovine, with which one can detect lung worm infestation in cattle immunodiagnostically. In a vaccine, the antigen can cause immune protection against D. viviparus.

Lungworms are particularly pathogenic and economically important for small and large ruminants. Dictyocaulus is the only lung worm that reaches sexual maturity in cattle. It occurs worldwide where temperate temperatures of 15-20 ° C prevail at least at times. Endivically, D. viviparus is common in Europe in the large floodplains, rainy coastal areas, but also on alpine pastures (R. Jörgensen (1980) Vet. Parasitol. 7, 153-167; H. Pfeiffer (1976) Vienna. Tierärztl. Mschr. 63 , 54-55). In the Netherlands e.g. Clinical dictyocaulosis was found in over 77% of the calf groups kept on pastures (J. Boch, R. Supperer (1992) Veterinary Parasitology. 4th ed., Parey, Berlin, pp. 294-301).

The uptake of the third larvae with the pasture grass causes the disease (dictyocaulosis) in the calf exposed for the first time. The larvae reach the alveoli of the lungs through the bloodstream, which they break through to reach the air-carrying parts of the lungs. This creates lesions that serve as an entry point for bacterial secondary infections; the multiplication of bacteria and other microbial pathogens leads to limited or generalized pneumonia with all possible consequences such as pulmonary edema and heart failure (T. Schnieder, A. Bellmer, F.-J. Kaup (1989) Vienna. Tierärztl. Mschr. 76: 372- 476). Breathing is made considerably more difficult, also by the adult stages leading to obstructions in the upper respiratory tract. Visible consequences of the severe impairment of the general condition reduced weight gain or even weight loss combined with growth retardation. Sometimes the clinical symptoms worsen dramatically and quickly lead to death.

Lungworm disease in cattle can be diagnosed on the basis of clinical symptoms (G. Gräfner (1987) Monthly Vet. Med. 42: 178-181) or on the basis of the larvae excreted with the faeces (J. Boch, R. Supperer (1992). These options are particularly suitable for diagnosis on single animals if there is a strong infection, but modern mass animal husbandry requires epidemiological predictions and risk assessments regarding the outbreak of dictyocaulosis in the advanced grazing season based on appropriate diagnostics, ie in surveys many may still weak infected calves are tested with a safe, sensitive method. For this purpose, serological methods suitable (A. Bellmer, T. Schnieder, AM Tenter (1989) Proc. 13 ^th Conf. Wrld Ass. Adv. Vet. parasite. , P. 33, Berlin, August 7-11, 1989. Antigens identified in Dictyocaulus viviparus, isolated and partially shown recombinantly become serods diagnostics used. Medications effective against adult and juvenile stages can be used for the healing treatment of dictyocaulosis (e.g. Levamisol ^® , (Pro) Benzimidazole, Netomin ^® , Ivermectin ^® ). These preparations are highly effective and can therefore usually prevent failures caused by acute lungworm diseases (H. Mehlhom, D. Düwel, W. Raether (1993) Diagnosis and therapy of parasitosis of domestic, domestic and domestic animals. 2nd ed. Gustav Fischer Verlag , Pp. 223-227). In the case of prophylactic / metaphylactic treatment, the active ingredients, owing to their radical effectiveness, may not allow the parasite to grapple with the host's immune system and thus the development and maintenance of resilient (partial) immunity. The animals are then exposed to infection in the second year of pasture protection (COBS, DE, SR Pitt, J. Förster, MT Fox (1987) Res. Vet. Sci. 43: 273-275). Therefore, from an epidemiological point of view, immunization of the calves of the first pasture season has been increasingly requested, either by minor subclinical infection or by vaccination. Currently only stands a live vaccine in the form of X-ray-attenuated larvae is available which induces basic immunity which must be maintained by further natural infection (Mehlhorn H., et al., (1993)). In the event of insufficient immunization via natural infection, breakthroughs with cough and disease sometimes occur in the event of sudden, high exposure. Since the vaccine can only be kept in the refrigerator for about 3 weeks, it must be stored carefully and used quickly. This procedure prohibits "area-wide"use; the vaccine is therefore primarily reserved for special endemic areas. Due to the lack of stability and quality, the development of defined vaccines (subunit vaccine) is necessary. The task was now to overcome the disadvantages of the current vaccination method To provide a new, advantageous vaccine, which has been achieved by the present invention.

The invention relates to a new, immunogenic, native protein, called DV 17, which was isolated from adult worms of Dictyocaulus viviparus. Its immunogenicity is mainly due to the fact that, after subcutaneous application, it induces an antibody response in cattle, which gives the animal immune protection. This protein can also be used in the ELISA for retrospective immunodiagnosis of dictyocaulosis in cattle. DV 17 is characterized by the following physical properties. The protein is stable in all buffers used. A reduction in the immunoreactivity after freezing (-85 ° C) of the purified antigen could not be determined. A retention time of 14 min was determined for antigen DV 17 using an HPLC system and a Nucleosil C 18 column (150 mm × 4.6 mm; 5 u). measured (gradient elution consisting of aqua dest / 0.1% TFA (= 0% B) and acetonitrile / 0.1% TFA (= 100% B)). DV 17 has an estimated molecular weight of approx. 16500 daltons in SDS polyacrylamide gel (Phastgel 8-25%). The isoelectric point of DV 17 is in the range of 5.3-5.9. Finally, after proteolysis with endopeptidase Lys C, the partial amino acid sequences according to Table 1 were determined. As a biological property, the inhibition of development of Dictyocaulus viviparus in cattle is outstanding after vaccination.

The object of the invention is therefore an immunogenic protein with a protective effect, which is isolated from adult worms of the lung worm Dictyocaulus viviparus and which preferably has a molecular weight of 15000-18000 Da, an isoelectric point between 5.3-5.9 and partial amino acid sequences according to Table 1.

A preferred object of the invention is a protein which has a molecular weight of 16500 ± 1500 Da and / or an isoelectric point of 5.6.

Another object of the invention is a protein, characterized in that it contains the amino acid sequence according to Table 6 (SEQ ID NO .: 30) or parts thereof.

Another object of the invention is a method for isolating a protein in which the isolation is carried out with the aid of extraction methods and chromatographic methods known to the person skilled in the art.

Another object of the invention is a DNA coding for a protein as described above, preferably a DNA which contains a DNA sequence according to Table 1.

Another object of the invention is a DNA, characterized in that it

(a) contains a DNA sequence according to Table 6 (SEQ ID NO .: 29) or parts thereof or

(b) hybridized under stringent conditions with a DNA sequence according to (a). The invention further relates to a method for isolating said DNA, in which

a) degenerate oligonucleotides containing a DNA sequence according to Table 1 or parts thereof are produced,

b) the oligonucleotides produced according to a) are radioactively or non-radioactively labeled and c) from a cDNA bank, produced from Dictyocaulus viviparus, cDNA clones are isolated which hybridize with the hybridization samples produced according to b) under stringent conditions.

Another object of the invention is a method for isolating said DNA, in which

a) PCR primers containing a DNA sequence according to Table 1 or parts thereof or containing an oligo-dT sequence are prepared,

b) PCR fragments generated in this way from a cDNA bank, produced from Dictyocaulus viviparus, PCR fragments,

c) which are cloned and analyzed according to common methods and

d) to complete the cDNA sequence by hybridization methods as described above in place of the degenerate oligonucleotides.

The method described last can also be modified in such a way that RNA is used as the template for the PCR reaction, which is first reverse-transcribed in an additional step and the resulting cDNA first strand is used for PCR. Another object of the invention is a recombinant protein containing partial amino acid sequences according to Table 1, which is preferably obtainable by expression of a cDNA obtained as described above in prokaryotes or eukaryotes and purification by methods known to those skilled in the art.

The invention also relates to an immunochemical method using the above-described protein from D. viviparus, with which the amount of DV 17-specific antibodies in the blood of cattle is determined by incubating EL 17 plates coated with DV 17 with bovine serum to be examined and any DV 17 / antibody complexes formed by peroxidase-conjugated, polyclonal antibodies and a corresponding color reaction known to the person skilled in the art.

Another object of the invention is the use of the above-described protein from D. viviparus as a vaccine, combined with a carrier or adjuvant and, if appropriate, auxiliaries for immunizing cattle against dictyocaulosis.

Another object of the invention is a diagnostic kit containing the protein of D. viviparus described above.

Finally, an object of the invention is a vaccine containing the protein of D. viviparus described above as well as a carrier, an adjuvant and optionally auxiliary substances.

The invention will now be explained in more detail with the aid of examples, without being restricted thereto. The tables are described as follows:

Table 1: Partial amino acid sequences of the isolated and enriched DV 17 protein from Dictyocaulus viviparus and the derived degenerate nucleotide sequence. Abbreviations: N = A, G, C, T; Y = T, C; H = A, C, T; R = A, G; M = A, C Table 2: Degenerate primers used for the amplification of DV17 DNA, abbreviations see description for Table 1

Table 3: PCR fragments obtained from DV RACE cDNA using the degenerate primers from Table 2

Table 4: Gene-specific primers from RACE experiments to generate DV17 cDNA with the complete 3 'end of the molecule

Table 5: DV17 cDNA fragments obtained by RACE

Table 6: cDNA and protein sequence of DV17.

For the identification of protein DV 17, normal sera and infection sera from cattle were used which were infected with gastrointestinal nematodes such as Ostertagia ostertagi and Cooperia oncophora as well as the lung worm Dictyocaulus viviparus.

Chromatographically separated protein fractions obtained from homogenized, adult lungworms were further separated using SDS polyacrylamide gel electrophoresis and immobilized on Immobilon P membranes (semidry blotting). The lung worm-specific protein DV 17 was then detected with the specific infection sera and further purified using a reverse phase HPLC column. The purity of the protein fraction was checked in silver-colored SDS polyacrylamide gels (phast gels). The BCA protein assay was used to determine the protein concentration in electrophoretically pure DV 17 fractions and to freeze them at -85 ° C. Helminthennaive cattle were vaccinated twice with a defined amount of purified DV 17. The cattle were challenged with L3 larvae of Dictyocaulus viviparus 1 week after the second vaccination. Animals that were not vaccinated served as controls. 4 weeks after the challenge, the cattle were slaughtered, the number of adult worms determined in the lungs and the length of the male and female worms measured. As The reduction in the number of adult worms compared to the non-vaccinated control was defined as a measure of immune protection.

"Stringent conditions" in connection with DNA hybridization means 6xSSC, 68 ° C. in the present application.

The PCR conditions are to be determined in preliminary tests using any method known to those skilled in the art.

Example 1: Preparation of infection sera

Helminthennaive cattle at the age of 6 months were infected with different doses of third larvae of different nematode species (Dictyocaulus viviparus, Ostertagia ostertagi, Cooperia oncophora). The infection doses in the Dictyocaulus group were 2500, 1250 and 500 larvae / cattle; 3 animals were used per dose. Infection doses of 70,000, 30,000 and 15,000 larvae / cattle were selected for the Ostertagia group. In addition to an uninfected group (= negative control), a mixed group was also carried in which each cattle was infected with 2,500 Dictyocaulus larvae, 10,000 Ostertagia larvae and 10,000 Cooperia larvae. On days DO (= day of infection), D +21, D +40, D +56, D +70, D +84, D +98 and D +112, serum samples were obtained from each bovine, which were aliquoted at -25 ° C were stored. The sera were used to identify the Dictyocaulus antigen DV 17 in electrophoretically, chromatographically separated protein fractions and to assess the specificity.

Example 2: Obtaining adult lungworms

6-month-old, helminthennaive cattle were orally infected with 5000 third larvae of Dictyocaulus viviparus; the following day, the animals received the same dose of infection. The cattle were slaughtered 28 days after infection and after the section adult worms are collected from the lungs. The worms were then washed 3 times with phosphate-buffered saline, weighed and stored at -85 ° C until working up.

Example 3: Extraction of DV 17 from adult lungworms

10 g of frozen worm mass was thawed at room temperature and then homogenized in a tissue homogenizer with 40 ml of 0.025 M Tris-HCl solution pH 7.4 + 2 mM Pefabloc ^® . To remove coarse tissue components, the homogenate was centrifuged at 3010 g and 4 ° C. for 15 minutes and the pellet was discarded. The supernatant was centrifuged at 4 ° C for 20 min at 39,800 g and the supernatant was recentrifuged under the same conditions for 10 min. The clear supernatant was dialyzed after filtration with 1.2 μm filters in 1 liter of phosphate-buffered saline (PBS) overnight at 4 ° C. (cut off of the dialysis membrane 8000).

Example 4: Preparative Gel Filtration

The dialyzed supernatant was centrifuged at 39,800 g for 15 min and the clear supernatant was separated in the Pharmacia FPLC system using a preparative gel filtration column (column type XK 16/60; separation medium: Superdex 75 prep grade, column volume: 124 ml). PBS pH 7.4 was used for elution. The fractions with the retention volumes 65-75 ml were collected and concentrated with ultrafiltration modules (cut off 3000). Protein DV 17 was detected using an amplified Western blot. Example 5: Western blot analysis

The concentrated Superdex 75 prep grade fraction was mixed in a 1: 2 ratio with reduced SDS buffer; 40 μl of each / sample well was applied to an SDS Excel gel (Pharmacia). The electrophoresis was carried out in the Multiphor II chamber (from Pharmacia) under standardized running conditions (600 V, 50 mA, 30 W, running time: 90 min). The electrophoretically separated proteins were transferred to Immobilon P membranes by means of "semidry blotting" (Tovey ER, Baldo BA. Electrophoresis. 8, 1987, 384-387) (transfer conditions: 45 min, constant current strength 0.8 mA / cm ² ) and after a 24-hour block phase with 3% bovine serum albumin in Tris buffered saline (TBS) with a Dictyocaulus-specific immune serum (obtained on D +40, see Example 1) in a dilution of 1:20 for 1 hour. As a negative control, bovine normal serum was incubated (Dilution 1:20 used) After washing 3 times with TBS + 0.05% Tween 20, the blot film was incubated with a biotin-labeled goat anti-bovine IgG (H + L) antibody (1: 500; Pierce) for 1 hour After washing three times (TBS + 0.05% Tween 20), the mixture was incubated for 1 hour with the enzyme conjugate biotin-streptavidin-alkaline phosphatase (1: 2500; Pierce). The substrate development was carried out using the Biorad substrate kit leads.

Example 6: Reverse phase HPLC

After the immunological identification of DV 17 in the preparative gel filtration fractions, the protein was further purified by means of HPLC. For this purpose, a Nucleosil C 18 5U column from Alltech was used (150 mm x 4.6 mm). The protein was eluted using a linear buffer gradient (buffer A: ultrapure water + 0.1% trifluoroacetic acid (TFA); buffer B: acetonitrile + 0.1% TFA). 500 μl of the fraction concentrated in Example 4 were diluted 1: 2 with buffer A and then injected into the column. The flow rate was 0.5 ml / min. The gradient elution was started 5 minutes after injection and ended 10 minutes later. A retention time of 14 min was measured for DV 17. Example 7: Purity detection and molecular weight determination

The fraction purified by HPLC with a retention time of 14 min was treated with an SDS-polyacrylamide gel (Phast SDS-Gel 8-25%) in the phast system (Fa. Pharmacia) analyzed under standardized conditions. The "Silver Stain SDS-PAGE Standards, low range" kit from Biorad was used as the molecular weight marker. DV 17 was visualized by means of silver staining after electrophoresis (Silverstain Kit, from Pharmacia). The molecular weight determination was carried out using a video densitometer (Molecular analyst, from Biorad) using the "Profile analyst II" evaluation program. A molecular weight of 16,500 ± 1,500 daltons was calculated for DV 17.

Example 8: Determination of the isoelectric point

DV 17 isolated according to Example 6 was diluted with ultrapure water and applied to prefabricated focusing gels (IEF Phastgels pH 3-10, Pharmacia). The focus in the phast system was carried out under standardized conditions. In order to determine the isoelectric point of DV 17, marker proteins with a defined, isoelectric point (pH 3.5 - 9.3 from Pharmacia) were carried along. The result was an isoelectric point from 5.3 to 5.9.

Example 9: Amino acid sequence analysis

DV 17 (40 μg) isolated and enriched according to Example 6 was cleaved with the aid of endopeptidase Lys C and the resulting peptides were purified using a C 18 reserve phase HPLC. 7 peptides were sequenced at the N-terminal. The seven partial amino acid sequences according to Table 1 were identified.

Example 10: Evidence of a protective effect of DV 17

5-month-old, helminthennaive bull calves (barn rearing, coproscopic evidence negative) were vaccinated subcutaneously with 50 μg each of purified, native DV 17. 4 weeks later there was a booster vaccination with 45 μg antigen / bovine. The animals were 1 week after the 2nd vaccination with 2 x 1000 L3- Larvae of Dictyocaulus viviparus contaminated (= challenge). Animals that were not vaccinated served as controls. The animals were slaughtered 35 days after the challenge and the number of adult worms was determined in the lungs; at the same time the length of intact male and female worms was measured. A reduced number of adult worms was found in the vaccinated group by 80%. Adult worms from the vaccinated group were significantly smaller compared to the control group (reduction 33%).

Example 11: ELISA for the serological detection of a dictyocaulosis

ELISA plates (Maxisorb from Nunc) were coated with a concentration of 5 μg DV 17 / ml PBS; the reaction volume was 100 ul per well. After incubation for 1 hour at 37 ° C., the plates were washed 3 times in an ELISA washer, the washing volume per well was 200 μl. Ultrapure water + 0.1% Tween 20 was used as the washing solution. Unspecific binding sites were blocked by incubation (3 hours at room temperature) with a proteolytic mixture of gelatin (Boehringer Mannheim). The incubation was carried out on a microtiter plate shaker at a shaking frequency of 300 rpm. After washing three times, the wells were loaded with a 1: 200 dilution of specific infection and control sera and incubated for 1 hour at room temperature. After washing three times, peroxidase-conjugated, polyclonal rabbit antibodies against bovine IgG (Fc fragment specific; Dianova) were used as detection antibodies in a dilution of 1: 10,000; the incubation lasted 30 min. The plates were then washed 4 times and then incubated with substrate solution (composition: 5 ml 10-fold ABTS buffer + 45 ml ultrapure water + 1 tablet ABTS (* 50 mg ABTS). The substrate development was carried out at room temperature and was carried out in an ELISA reader monitored every 10 min at 414 nm Dictyocaulus-specific antibodies were detected in the ELISA no earlier than 28 days after a lung worm infection. Example 12: Cloning and sequencing of the DV17 antigen

D. viriparus total RNA was isolated by means of "DYNABEADS" (DYNABEADS mRNAdirekt hit, DYNAL). 400 ng of these were precipitated with ethanol and used as a template for a RACE (re-amplification of cDNA ends; Frohmann et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8998-9002) cDNA synthesis, the "Marathon cDNA Amplification Kit" (Clontech) was used. The cDNA adapter-specific primers from the kit mentioned and the "Expand Long Template PCR System" (Boehringer Mannheim) were used. Conditions: 600 nM from each primer; 1, 75mM MgCI ₂ , 400mM dNTPs, 392ng Taq Start Antibody (Clontech), 0.35U DNA Polymerase Mix. The temperature profile was as follows:

Cycle 1 92 ° C 120 s

60 ° C 60 s

68 ° C 360 s

Cycles 2-30 92 ° C 60 s

60 ° C 60 s

68 ° C 360 s

The amplified DV17 RACE cDNA was diluted 1:20 in Tris-Glycine buffer (from the Marathon cDNA Amplification Kit) for use as a template in PCR reactions.

A region of the DV17 cDNA sequence was PCR amplified from the RACE cDNA using degenerate primers whose sequences were obtained from the peptide sequences of the purified antigen. The primers were provided with restriction enzyme sites in such a way that the subsequent subcloning of the PCR fragments obtained was facilitated. The primers used are listed in Table 2. Five DNA fragments were obtained after 2 rounds of PCR in a PE 9600 Thermal Cycler (Perkin Elmer) (500 nM of each primer; 0.1 vol PCR buffer; 200 mM dNTP's; 1.25 units of the Ampli Taq Gold (Perkin Elmer The following temperature profile was available:

Cycle 1 92 ° C 600 s

Cycles 2-31 94 ° C 40 s

55 ° C 40 s

72 ° C 60 s

Four of the fragments (see Table 3) were obtained in high enough yields to be gel purified and sequenced using the primers used to prepare them.

Alignment of the sequence data obtained using the "Geneworks" program (IntelliGenetics) would result in two contigs. Taken together, these contained 5 or 6 unambiguous peptide sequences obtained from the native antigen. Non-degenerate (gene specific) primers (see Table 4) obtained from this sequence data were used in RACE experiments with the marathon cDNA adapter-specific primers to obtain DNA fragments that correspond to the 3 'end of the represent cDNA molecule.

The RACE reactions contained concentrations of 600 nM of each primer; 0.2 ul of diluted, amplified DV17 RACE cDNA; 0.1 vol of PCR buffer I; 200 µM dNTPs and 1.25 U Ampli Taq Gold. The temperature profile was the same as in the PCR reaction with the non-degenerate primers except that the annealing temperature was 60 ° C and the samples were left at 72 ° C for 360 s in the last cycle. With the gene-specific primers, 2 PCR fragments were obtained (Table 5). After RACE, the fragments were gel purified, subcloned and sequenced using a T7 promoter sequencing primer (Promega; sequence: TAATACGACTCACTATAGGG, SEQ ID NO .: 31).

Alignment with the sequences of these fragments and from the fragments obtained with the non-degenerate primers was carried out (Geneworks) and is shown in Table 6.

Table 1

1 5

Ser Glu Ser Leu Tyr Glu Lys (SEQ ID NO .: 1)

TCN GAR UCN YTN TAY GAR AAR (SEQ ID NO .: 8)

1 5

Met Met Asp Asn Phe Val Lys (SEQ ID NO .: 2)

ATG ATG GAY AAY TTY GTN AAR (SEQ ID NO .: 9)

1 5 10

Tyr Lys Asp Glu Asn Glu Phe Met Asp Ala

TAY AAR GAY GAR AAY GAR TTY ATG GAY GCN

14

Leu Lys Gin Lys (SEQ ID NO .: 3)

YTN AAR CAR AAR (SEQ ID NO .: 10) 1 5 10

4. Tyr Asp lle Pro Glu Gin Tyr Arg Glu lle TAY GAY ATH CCN GAR CAR TAY MGN GAR ATH

15 20 lle Pro Gin Asn Val Ala Glu His Leu Lys (SEQ ID NO .: 4)

ATH CCN CAR AAY GTN GCN GAR CAY YTN AAR (SEQIDNO .: 11)

1 5 10

5. Asp Ala lle Glu Lys Tyr Glu Asp lle Pro GAY GCN ATH GAR AAR TAY GAR GAY ATH CCN

15 20

Glu Gin Tyr Arg Glu lle lle Pro Gin Asn

GAR CAR TAY MGN GAR ATH ATH CCN CAR AAY

25

Val Ala Glu His Leu Lys (SEQ ID NO.5)

GTN GCN GAR CAY YTN AAR (SEQ ID NO .: 12)

1 5 10

6. Phe His Ala Glu Leu Leu Ala Gly lle Lys TTY CAY GCN GAR YTN YTN GCN GGN ATH AAR

15

Pro Ser Leu Glu Glu Leu Lys Lys (SEQ ID NO .: 6)

CCN TCN YTN GAR GAR YTN AAR AAR (SEQ ID NO: 13) 1 5 10

Gin Phe Pro le Leu Thr Ser Val Phe Ser CAR TTY CCN ATH YTN ACN TCN GTN TTY TCN

14 (SEQ ID NO .: 7) Asn Glu Glu Lys (SEQIDNO .: 14) AAY GAR GAR AAR

Table 2

Oligo orientation DNA / amino acid sequence

A055 / 1001 sense CCG GAA TTC GAY GCN ATN GAR AAR TAY GA

(SEQIDNO .: 15) EcoRI D A I E K Y E

(SEQ ID NO .: 16)

A055 / 1002 sense CGC GGA TCC GAR ATN ATN CCN CAR AAY GT

(SEQIDNO .: 17) BamHI E l I P Q N V

(SEQIDNO .: 18)

A055 / 1003 sense CCG GAA TTC TAY AAR GAY GAR AAY GAR TT

(SEQIDNO .: 19) EcoRI Y K D E N E F

(SEQ ID NO .: 20) A055 / 1004 antisense AAAA CTG CAG NGC RTC CAT RAA YTC RTT YTC

(SEQIDNO .: 21) Pstl A D M F E N E

(SEQ ID NO .: 22) A055 / 1005 antisense AAAA CTG CAG YTTYTC YTC RTT RCT RAA NAC

(SEQ ID NO .: 23) Pstl K E E N S F V

(SEQ ID NO .: 24)

Table 3

Primer combination approximate fragment size (bp) ^* sequenced

A055 / 1001: A055 / 1004 180 Yes

A055 / 1001: A055 / 1005 480 No.

A055 / 1002: A055 / 1004 150 Yes

A055 / 1002: A055 / 1005 470 Yes

A055 / 1003: A055 / 1005 400 Yes * determined by gel electrophoresis

Table 4

A075 / 1001 GAC ATG CAT GTA GAC GCA CTT GGA GAA GAG GC

(SEQ ID NO .: 25) Nsil V D A L G E E A

(SEQ ID NO .: 26) A108 / 1001 CCG GAA TTC CCT GAA CAG TAC AGA GAG ATC ATT CCA

(SEQ ID NO .: 27) EcoRI P E Q Y R E I I P

(SEQI ID NO .: 28) Table 5

Primer combination ^* fragment size (bp) cloning vector (Promega) A075 / 1001.AP1 342 pGEM7Zf-A108 / 1001: AP 1 541 pGEM3Z

* AP1 is the Marothon cDNA adapter specific primer

Table 6

TGGAGAARTA CGAAGATATT CCTGAACAGT ACAGAGAGAT CATTCCACAA

E K Y E D I P E Q Y R E I I P Q AACGTGGCCG AGCATTTAAA ATCGATAACT GAGGAAGAGA AAAAAGTGCT N V A E H L K S I T E E E K K V L CAAAGAATTT GTTAAAGACT ATGCAAAATA CAAAGATGAA AATGAGTTCA N K E FAA TAGA

D A L K Q K S E S L Y E K A K K CTTCAAGATT TGCTGAAATC AAAAGTAGAC GCACTTGGAG AAGAGGCAAA L Q D L L K S K V D A L G E E A K ACAATTTGTG ATGAAGCTTA TCGCTGAGGC TCGTAAATTC CACGCAGAGCAA TCGTA TAG

LAGIKPSLEELKAVAK AAGCATATTG AAGAGTTTGA GAAGTTATCA GATGCAGCTA AAGATGATTT KHIEEFEKLSDAAKDDF CAAAAAGCAA TTCCCTATCC TCACATCCGT GTTCAGCAAT GAAAAAGCAA KKQFPILTSVFSNEKAK GTGAAAGATTAG GAGAAAGTATGAG

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02 Z0 / 86da / XOd ZfrZ, 9fr / 86 OΛV SEQUENCE LOG

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Hoechst Aktiengesellschaft

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(ii) APPLICATION TITLE: Dictyocaulus viviparus antigen for diagnosis of lung worm infestation and vaccination

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(D) SOFTWARE: Patentin Release # 1.0, Version # 1.25 (EPA)

(2) INFORMATION ABOUT SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Ser Glu Ser Leu Tyr Glu Lys

1 5

(2) INFORMATION ABOUT SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..7

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Met Asp Asn Phe Val Lys 1 5 (2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..14

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Tyr Lys Asp Glu Asn Glu Phe Met Asp Ala Leu Lys Gin Lys 1 5 10

(2) INFORMATION ABOUT SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..20

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Tyr Asp lle Pro Glu Gin Tyr Arg Glu lle lle Pro Gin Asn Val Ala 1 5 10 15

Glu His Leu Lys 20

(2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..26

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Asp Ala lle Glu Lys Tyr Glu Asp lle Pro Glu Gin Tyr Arg Glu lle 1 5 10 15 lle Pro Gin Asn Val Ala Glu His Leu Lys 20 25 (2) INFORMATION ON SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..18

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Phe His Ala Glu Leu Leu Ala Gly lle Lys Pro Ser Leu Glu Glu Leu 1 5 10 15

Lys Lys

(2) INFORMATION ABOUT SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..14

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Gin Phe Pro le Leu Thr Ser Val Phe Ser Asn Glu Glu Lys 1 5 10

(2) INFORMATION ABOUT SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..21

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TCNGARUCNY TNTAYGARAA R 21

(2) INFORMATION ON SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..21

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: ATGATGGAYA AYTTYGTNAA R 21

(2) INFORMATION ABOUT SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..42

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TAYAARGAYG ARAAYGARTT YATGGAYGCN YTNAARCARA AR 42

(2) INFORMATION ABOUT SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 60 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..60

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TAYGAYATHC CNGARCARTA YMGNGARATH ATHCCNCARA AYGTNGCNGA RCAYYTNAAR 60

(2) INFORMATION ON SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 78 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..78

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GAYGCNATHG ARAARTAYGA RGAYATHCCN GARCARTAYM GNGARATHAT HCCNCARAAY 60 GTNGCNGARC AYYTNAAR 78

(2) INFORMATION ON SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..54

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TTYCAYGCNG ARYTNYTNGC NGGNATHAAR CCNTCNYTNG ARGARYTNAA RAAR 54

(2) INFORMATION ABOUT SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..42

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CARTTYCCNA THYTNACNTC NGTNTTYTCN AAYGARGARA AR 42

(2) INFORMATION ON SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon (B) LOCATION: 1. .29

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CCGGAATTCG AYGCNATNGA RAARTAYGA 29

(2) INFORMATION ON SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..7

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

Asp Ala lle Glu Lys Tyr Glu 1 5

(2) INFORMATION ON SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..29

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CGCGGATCCG ARATNATNCC NCARAAYGT 29

(2) INFORMATION ON SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..7

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: Glu lle Pro Gin Asn Val 1 5

(2) INFORMATION ABOUT SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..29

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: CCGGAATTCT AYAARGAYGA RAAYGARTT 29

(2) INFORMATION ABOUT SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..7

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

Tyr Lys Asp Glu Asn Glu Phe 1 5

(2) INFORMATION ON SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..31

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: AAAACTGCAG NGCRTCCATR AAYTCRTTYT C 31 (2) INFORMATION ABOUT SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..7

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

Ala Asp Met Phe Glu Asn Glu 1 5

(2) INFORMATION ABOUT SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..31

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: AAAACTGCAG YTTYTCYTCR TTRCTRAANA C 31

(2) INFORMATION ON SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein (ix) CHARACTERISTICS:

(A) NAME / KEY: Protein

(B) LOCATION: 1..7

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

Lys Glu Glu Asn Ser Phe Val 1 5

(2) INFORMATION ON SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid (C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..32

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: GACATGCATG TAGACGCACT TGGAGAAGAG GC 32

(2) INFORMATION ON SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..8

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

Val Asp Ala Leu Gly Glu Glu Ala 1 5

(2) INFORMATION ABOUT SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..36

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: CCGGAATTCC CTGAACAGTA CAGAGAGATC ATTCCA 36

(2) INFORMATION ON SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein (ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..9

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

Pro Glu Gin Tyr Arg Glu lle Pro 1 5

(2) INFORMATION ON SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 560 base pairs

(B) TYPE: nucleic acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURES:

(A) NAME / KEY: exon

(B) LOCATION: 1..562

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

TGGAGAARTA CGAAGATATT CCTGAACAGT ACAGAGAGAT CATTCCACAA AACGTGGCCG 60

AGCATTTAAA ATCGATAACT CAGGAAGAGA AAAAAGTGCT CAAAGAATTT GTTAAAGACT 120

ATGCAAAATA CAAAGATGAA AATGAGTTCA TGGACGCATT AAAGCAAAAA TCTGAAAGCC 180

TTTATGAGAA AGCTAAAAAA CTTCAAGATT TGCTGAAATC AAAAGTAGAC GCACTTGGAG 240

AAGAGGCAAA ACAATTTGTG ATGAAGCTTA TCGCTGAGGC TCGTAAATTC CACGCAGAGC 300

TACTGGCCGG CATCAAACCA TCGCTAGAAG AACTAAAAGC CGTCGCTAAA AAGCATATTG 360

AAGAGTTTGA GAAGTTATCA GATGCAGCTA AAGATGATTT CAAAAAGCAA TTCCCTATCC 420

TCACATCCGT GTTCAGCAAT GAAAAAGCAA AGAAAATGAT GGACAACTTT GTGAAAAATT 480

AAAGTTGTAT GATTTGCAGG ATATGAAATA AATGTTAAAT TGAAAAAAAA AAAAAAAAAA 540

AAAAAAAAAA AAAAAAAAAA AG 562

(2) INFORMATION ABOUT SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 159 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(ix) FEATURES:

(A) NAME / KEY: Protein

(B) LOCATION: 1..159 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:

Glu Lys Tyr Glu Asp lle Pro Glu Gin Tyr Arg Glu lle lle Pro Gin 1 5 10 15

Asn Val Ala Glu His Leu Lys Ser lle Thr Glu Glu Glu Lys Lys Val 20 25 30

Leu Lys Glu Phe Val Lys Asp Tyr Ala Lys Tyr Lys Asp Glu Asn Glu 35 40 45

Phe Met Asp Ala Leu Lys Gin Lys Ser Glu Ser Leu Tyr Glu Lys Ala 50 55 60

Lys Lys Leu Gin Asp Leu Leu Lys Ser Lys Val Asp Ala Leu Gly Glu 65 70 75 80

Glu Ala Lys Gin Phe Val Met Lys Leu lle Ala Glu Ala Arg Lys Phe 85 90 95

His Ala Glu Leu Leu Ala Gly lle Lys Pro Ser Leu Glu Glu Leu Lys 100 105 110

Ala Val Ala Lys Lys His lle Glu Glu Phe Glu Lys Leu Ser Asp Ala 115 120 125

Ala Lys Asp Asp Phe Lys Lys Gin Phe Pro lele Leu Thr Ser Val Phe 130 135 140

Ser Asn Glu Lys Ala Lys Lys Met Met Asp Asn Phe Val Lys Asn 145 150 155

Claims

claims

1. Immunogenic protein DV 17 with a protective effect, characterized in that the protein is isolated from adult worms of the lung worm Dictyocaulus viviparus.

2. Protein, according to claim 1, characterized in that said protein has a molecular weight of 15,000 - 18,000 daltons, an isoelectric point between 5.3 and 5.9 and partial amino acid sequences according to Table 1.

3. Protein, according to one of claims 1 to 2, characterized in that said protein has a molecular weight of 16 500 ± 1 500 Daltons and has an isoelectric point of 5.6.

4. Protein, characterized in that it contains the amino acid sequence according to Table 6 (SEQ ID NO .: 30) or parts thereof.

5. A method for isolating a protein, according to one of claims 1 to 4, characterized in that the isolation is carried out with the aid of extraction methods and chromatographic methods.

6. DNA coding for a protein according to any one of claims 1 to 4.

7. DNA, according to claim 6, characterized in that it contains at least one DNA sequence according to Table 1.

8. DNA according to claim 6, characterized in that it

(a) contains a DNA sequence according to Table 6 (SEQ ID NO .: 29) or parts thereof, or (b) hybridized under stringent conditions with a DNA sequence according to (a).

9. A method for isolating a DNA according to claim 6, 7 or 8, characterized in that

b) the oligonucleotides produced are radioactive or non-radioactive labeled and

c) isolated from a cDNA bank, produced from Dictyocaulus viviparus, cDNA clones which hybridize with the hybridization samples produced according to b) under stringent conditions.

10. A method for isolating a DNA according to claim 7 or 8, characterized in that

c) which are cloned and analyzed according to common methods and

d) to complete the cDNA sequence by hybridization method according to claim 8, instead of the degenerate oligonucleotides, the PCR fragment obtained according to point b) is used.

11. The method according to claim 10, characterized in that RNA is used as the template for the PCR reaction, which is first reverse transcribed in an additional step and the resulting first strand is used for PCR.

12. Protein obtainable by expression of a cDNA obtained according to one of claims 9 to 11 in prokaryotes or eukaryotes and subsequent purification.

13. Immunochemical method for determining the amount of DV 17-specific antibodies in the blood of cattle using a protein, according to one of claims 1 to 4 or 12, characterized in that ELISA plates coated with DV 17 are incubated with bovine serum to be examined and any DV 17 / antibody complexes formed with peroxidase-conjugated, polyclonal antibodies and a color reaction can be detected.

14. Use of a protein according to one or more of claims 1 to 4 or 12 as a vaccine combined with a carrier or adjuvant and optionally auxiliaries for immunizing cattle against dictyocaulosis.

15. Diagnostic kit containing a protein according to any one of claims 1-4 or 12.

16. Vaccine containing a protein according to one of claims 1-4 or 12 and a carrier, an adjuvant and optionally auxiliary substances.