JPH0770184A - Peptides effective in preventing small Piroplasma infections in cattle - Google Patents

Abstract

(57) [Summary] [Objective] To provide a novel peptide capable of preventing the infection of small Piroplasma pathogens in cattle and a method for using the same. A peptide comprising a partial amino acid sequence of the amino acid sequence of the 33 kd peptide of bovine small Piroplasm pathogen, which contains the Lys-Glu-Lys amino acid sequence. Peptide effective for prevention of insect infection; small bovine pyroplasma vaccine for preventing pathogen infection of the bovine, which comprises the peptide and a substance having an adjuvant activity; and small bovine pyroplasma using the peptide as a measuring antigen Method for measuring antibodies to pathogens.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a peptide useful for controlling infectious diseases of cattle. More specifically, the small Piroplasma pathogen of cattle, namely Theileria Sergentii (T
A peptide useful for the prevention of infectious diseases caused by heileria sergenti), a vaccine comprising this peptide, and a diagnostic assay method for the infectious diseases.

[0002]

2. Description of the Related Art Bovine small piroplasmosis is a protozoan disease transmitted by ticks, and its main symptoms are anemia and slimming. This disease occurs in pastures all over the world, and the high mortality rate is the biggest obstacle in pasture management. As a countermeasure, acaricide administration of protozoa and application of acaricide to cattle and mast fields for the purpose of killing mites that act as vectors are mainly performed, but the therapeutic effect of protozoa administration is, Environmental pollution due to drug residues in milk and the use of acaricides is a problem.

As a preventive measure, a method of inoculating a small P. gondii infected blood has been tried so far.
This method has now been banned due to the risk of spreading various infectious diseases including bovine leukemia due to direct administration of bovine blood. In addition, recently, sporozoites of small Pyroplasma protozoa were isolated and purified from the salivary glands of vector mites infected with small Pyroplasma protozoa, and by inoculating a fixed amount of the purified and isolated sporozoites to cattle before grazing, It has been reported that the incidence of small-sized Piroplasmosis is reduced [Kamio et al .: 113th Annual Meeting of the Japanese Society of Veterinary Medicine, p. 156 (1992)]. However, this has not yet been put to practical use.

According to a recent study, the molecular weight of the major protein of the small Pyroplasma protozoan piroplasm (developing Pyroplasma parasite parasitizing bovine erythrocytes) is 32 to 33.
In kd (kilodalton), it was also revealed that this major protein is a major target antigen for humoral immunity of the host cow [Kawazu et al .: 111th Annual Meeting of the Japanese Society of Veterinary Medicine, 135. Page (1991) and Sugimoto et al .: No. 11
5th Annual Meeting of the Japanese Society of Veterinary Medicine, 143 pages (199
3)]. In addition, the present inventors have revealed the nucleotide sequence from the isolation and purification of a gene encoding a major protein of the pyroplasm of P. pyogenes which is one of the small Pyroplasma protozoa and the analysis result of the gene. [Kawazu et al .: Proceedings of the 113th Annual Meeting of the Japanese Society of Veterinary Medicine, p. 155 (1992)]. On the other hand, in malaria parasite, which is the same schistosome as the small Pyroplasma parasite, the surface antigen of the malaria parasite has a repeating sequence consisting of several amino acids, and the surface antigen protein of the malaria parasite at the erythrocyte stage is lysine-glutamic acid ( KE) possesses several charged amino acid pairs in the molecule, and among them, the amino acid sequence of lysine-glutamic acid-lysine (KEK), lysine-glutamic acid-valine (KEV) or lysine-glutamic acid-leucine (KEL). Regions have been shown to function in binding to the human erythrocyte receptor, and prevention of malaria by synthetic peptides containing KEK and KE sequences has been studied [Parasite Immunolog
y, 14, P111-124 (1992)].

[0005]

However, no effective vaccine capable of preventing small Piroplasmosis in cattle has been developed so far, and the present countermeasures have various problems as described above. Therefore, it is required to develop a new countermeasure method that is useful for the prevention of small P. gondii infection in cattle.

[0006]

As described above, the present inventors have revealed the amino acid sequence of the major protein of Theileria sergentii, which is one of the small Pyroplasma protozoa. It was found that there is a KEK sequence as found in the above-mentioned malaria parasite. The present inventors have paid attention to this fact, and as a result of conducting an immunity test of cattle with a peptide containing this sequence,
It was found that a peptide having this sequence has immunogenicity related to the 33 kd peptide, which is a major protein of bovine Theileria sergenti, and is extremely effective in preventing small P. pyloriplasma infection in cattle containing Theileria sergenti. The invention was completed. That is, according to the present invention, there is provided a novel peptide capable of preventing a small Piroplasma disease caused by Theileria cergenti infection.

As described above, the peptide provided by the present invention is an immunogenic peptide related to the 33 kd peptide, which is the major protein of Theileria cergenty, and can be synthesized by a known solid-phase peptide synthesis method. . The peptide of the present invention is a peptide having a lysine-glutamic acid-lysine (KEK) sequence in its amino acid sequence and consisting of a partial amino acid sequence of the amino acid sequence of the 33 kd peptide of Theileria sergentii. Such a lysine-glutamic acid-lysine (KEK) sequence corresponds to the amino acid sequence from the 139th position to the 141st position in the amino acid sequence of Sequence Listing: SEQ ID NO: 2, and the peptide of the present invention refers to Tyleria sergeti containing this region. Of 33 kd peptide of the above. The peptide of the present invention, even if it is a peptide consisting of 3 amino acids, which is the minimum unit of lysine-glutamic acid-lysine (KEK), is immunogenic against bovine small pyroplasma disease by devising binding to a carrier and the like. Since it can be prepared as a peptide compound having, it can be used for the prevention of small Piroplasmosis in cattle. Further, as a preferred form of the peptide of the present invention, from the viewpoint of sufficient immunogenicity, by further preparing a peptide in which this peptide is extended to both ends of the N-terminus and the C-terminus, the peptide of the present invention having a higher effect can be obtained. Peptides can be prepared. The length of the peptide is K
It is desirable to use a peptide having 30 amino acids or less containing the EK sequence, preferably a peptide having 20 amino acids or less containing the KEK sequence. An example of such a preferred form of peptide is a peptide consisting of the following amino acid sequence (A). (A) Glu-Val-Val-Trp-Lys-Glu-Lys-Ly
s-Glu-Val-Lys-Asp-Leu-Asp-Ala

In preparing the peptide of the present invention,
Using the range of the above amino acid sequence (A) as an index, L
Peptides of suitable length with ys-Glu-Lys (KEK) can be prepared. Further, a peptide longer than the peptide of (A) above can be prepared, but in that case, the T.
According to the amino acid sequence of the major protein of 33kd of Sergenty (sequence listing: SEQ ID NO: 2), the above peptide is provided so as to have the original amino acid sequence of the 33kd protein.
A peptide in which the region of (A) is extended to the N-terminal side and the C-terminal side can be prepared (the above-mentioned peptide (A) is from the 134th position to the 14th position in the amino acid sequence of SEQ ID NO: 2).
It corresponds to the 8th amino acid sequence).

Furthermore, in order to facilitate the binding between the peptide of the present sequence and the carrier protein support, it is possible to additionally add 1 or 2 amino acids to the N-terminal or C-terminal of the peptide of the present invention. Is. Amino acids useful for such purposes include tyrosine, lysine,
Glutamic acid, aspartic acid, cysteine and their derivatives are mentioned. Furthermore, it is also possible to facilitate the attachment of the peptide or carrier protein support of the present invention by a conventional protein modification method such as acetylation at the amino terminus or acidation at the carboxy terminus. In addition, MAP (multiple antigen pepti) was prepared by repeatedly binding lysine to β-alanine and further binding lysine to both the α and ε amino groups.
It is also possible to attach the peptide of the present invention to the amino group of the de) skeleton.

The peptide of the present invention thus prepared can be treated with a suitable adjuvant such as aluminum gel,
After adding an oil adjuvant or the like, it can be used as an immunogen. These immunogens produce an antibody against the peptide of the present invention in the bovine body by injecting into cows, and suppress the development of small Piroplasma disease even when infected by the blood-sucking of ticks infected with Theileria Sergenty. It is possible to obtain the effect or the effect of remarkably reducing the symptoms.

Further, since the peptide of the present invention plays an important role in the prevention of small pyroplasma disease in cattle as described above, by measuring the antibody against the peptide of the present invention in the bovine body, It becomes possible to investigate the immune status against Piroplasmosis. That is,
By using the peptide of the present invention as a measuring antigen, it becomes possible to examine the immune status of cattle by examining the antibody specific to this peptide present in the serum of cattle. In constructing such an antibody measurement system, it is possible to construct various antibody measurement systems using a known method. One example thereof is enzyme-linked immunosorbent assay (ELIS).
A simple measuring method is a measuring method using A).

[0012]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited thereto. Example 1 KEK sequence characteristic of the present invention (lysine-glutamic acid-
A peptide having the following amino acid sequence (hereinafter referred to as "KEK peptide") having lysine) was synthesized according to the solid phase peptide synthesis method. Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-G
lu-Val-Lys-Asp-Leu-Asp-Ala Using the above-prepared KEK peptide, a peptide in the form of MAP is prepared in which five KEK peptides are bound as one molecule via lysine. did. Freund's complete adjuvant was added to the peptide thus prepared, and the peptide dose was 1 mg / head.
The injections were carried out three times at an interval of 4 weeks into the gluteal muscles of four Holsteins of about 4 months old. 3 after the third injection
On week one, a small piroplasm-infected adult mite was attacked by sucking blood. Also, 4 cows were used as non-inoculation control cows. 60 days after the attack, clinical observation, temperature measurement, blood test (red blood cell count,
Hematocrit) and parasitemia were tested. Blood tests were performed using Cell Duck manufactured by Nippon Denko Co., Ltd.
It was determined by the ratio of parasitized red blood cells per 000.

As a result, by injecting the KEK peptide as an immunogen into cattle, the antibody in the bovine body against the KEK peptide was increased, and the measurement value (absorbance) was 2 in the ELISA for measuring the KEK antibody at the time of attack. .00
The above is shown. On the other hand, the control group showed the same ELISA value of 0.1 or less. Further, similarly, precipitation in an agar gel for measuring an antibody against the KEK peptide (hereinafter,
"AGP"), the antibody titer in the peptide-administered group of the present invention was 4 to 8 times. On the other hand, the AGP antibody in the control group was negative.

The infection profile after the challenge was as shown in Table 1 below. The post-challenge Pyroplasma protozoa began to be observed 9 to 15 days after the start of blood feeding, and the maximum parasitism rate was 9 in 1 of 4 animals in the peptide-immunized group of the present invention.
%, But the maximum parasitism rate of the remaining three was 3.6.
It was ~ 6.9%. On the other hand, in the control group, the maximum parasitism rate of 10.4 to 14.2% was shown in 3 out of 4 heads, and the parasitism rate in the immunization group was obviously lower than that in the control group.

[0015]

[Table 1]

The red blood cell count in the peptide-immunized group of the present invention decreases with an increase in the parasitism ratio, and the lowest red blood cell counts are 409, 460, 464 and 2.36 million / mm ^3.
However, there was a tendency for recovery immediately, and at the end of observation, 6
It was recovered up to 80 to 8 million pieces / mm ³ . on the other hand,
The lowest red blood cell count in the control group is 189 to 388,000 / mm
³ was recovered, and at the end, it was recovered to 3.5 to 6 million cells / mm ³ , but the recovery of erythrocytes tended to be earlier in the immunized group than in the control group.

It was also confirmed that the hematocrit value in the immunized group was similar to that of the red blood cell count and was milder than that in the control group. From the above results, K of the present invention
It has been confirmed that immunization of cattle with EK peptide alleviates symptoms against infection with small Pyroplasmosis.

Example 2 The antibody reactivity to the KEK peptide of the present invention in the field-resistant small cows infected with Pyroplasma gondii was compared with experimentally infected bovine serum. Regarding the reactivity, the ELIS using the KEK peptide of the present invention as a measurement antigen
Method A was used. The field-resistant hyperserum to be tested was 11 serum samples that had been grazing for more than 2 years, and the experimentally infected serum was 18 serum samples 3-4 months after infection. The results are shown in Table 2. Although the ELISA antibody titer against the KEK peptide of the present invention in the serum of field-infected cow was low, it was observed in 8 of the 11 animals, and the absorbance was 0.
It was 15 to 0.32. On the other hand, all of the experimentally infected bovine sera showed 0.1 or less. From the above, it was confirmed that the antibody against the KEK peptide of the present invention was present in field-resistant hyperserum.

[0019]

[Table 2]

Using the serum immunized with the KEK peptide of the present invention, the small Pyroplasma protozoan, Theileria sergenti Ikeda (hereinafter referred to as "TSI"), Theileria bufferi Warwick (Theileria bufferi) Warwick (hereinafter "TBW") and Theileria orientalis Esse
x) (hereinafter, referred to as "TOE") was analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with purified pyroplasm.
SDS-polyacrylamide gel electrophoresis and Western blotting were carried out by the method of Lamley [L.
eammli et al., Nature, 227, p680-685, (19
70)] and Dan's method [Dunn, Anal. Biochem. 1
57, p 144-153 (1986)]. The result is shown in FIG. The KEK peptide-immunized bovine serum of the present invention strongly recognized the major protein of TSI, but did not react with the major proteins of TBW and TOE, confirming that the KEK peptide of the present invention is specific to TSI. Was done. In FIG. 1, 1, 2 and 3 represent TSI-infected bovine serum, 4, 5 and 6 represent anti-peptide bovine serum,
1 and 4 are TSI, 2 and 5 are TBW, 3 and 6
Means TOE.

[0021]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-Glu-Val-Lys-Asp-Leu -Asp-Ala 1 5 10 15

SEQ ID NO: 2 Sequence length: 852 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Theileria sergentii
genti) Sequence ATG TTG TCC AAG AGA ACG TTC AAC GTA CTT TGC CTA GGA TAC TTC CTT 48 Met Leu Ser Lys Arg Thr Phe Asn Val Leu Cys Leu Gly Tyr Phe Leu 1 5 10 15 ATC GTC TCT GCT ACC GCC GCA GAG GAA AAA AAA GAT GCA AAG GCT GAA 96 Ile Val Ser Ala Thr Ala Ala Glu Glu Lys Lys Asp Ala Lys Ala Glu 20 25 30 GAG AAG AAG GAC TTA ACT CTC GAA GTT AAC GCC ACC GCA GCC GAA CAT 144 Glu Lys Lys Asp Leu Thr Leu Glu Val Asn Ala Thr Ala Ala Glu His 35 40 45 TTT AAA GTC GAC GCC TCA AAC GCC AAC GAC GTC GTT TTT ACT GCC GAA 192 Phe Lys Val Asp Ala Ser Asn Ala Asn Asp Val Val Phe Thr Ala Glu 50 55 60 GAG GGA TAC CGC ATC AAG ACA CTC AAG GTC GGA GAT AAG AAC CTG TAT 240 Glu Gly Tyr Arg Ile Lys Thr Leu Lys Val Gly Asp Lys Asn Leu Tyr 65 70 75 80 ACC GTA GAT ACT TCC AAG TTC ACC CCA ACT GTC GCC CAC AGA CTG AAG 288 Thr Val Asp Thr Ser Lys Phe Thr Pro Thr Val Ala His Arg Leu Lys 85 90 95 CAT GCT GAC GAC CTG TTC TTC AAG CTC AAC CTG TCC CAC GCA AAG CCA 336 His Ala Asp Asp Leu Phe Phe Lys Leu Asn Leu Ser His Ala Lys Pro 100 105 110 TTG CTG TTC AAG AAG AAG ACT GAC AAG GAT TGG GTT CAA TTC AGC TTC 384 Leu Leu Phe Lys Lys Lys Thr Asp Lys Asp Trp Val Gln Phe Ser Phe 115 120 125 GCC CAG TAC CTC GAT GAA GTT GTA TGG AAG GAG AAG AAG GAA GTA AAA 432 Ala Gln Tyr Leu Asp Glu Val Val Trp Lys Glu Lys Lys Glu Val Lys 130 135 140 GAC CTC GAC GCA TCC AAG TTC GCA GAC GCA GGT CTT TTC GCC GCT GAG 480 Asp Leu Asp Ala Ser Lys Phe Ala Asp Ala Gly Leu Phe Ala Ala Glu 145 150 155 160 GCT TTC GGT ACC GGA AAG CTG TAC AAC TTC ATT GGA AAC TTC AAG GTC 528 Ala Phe Gly Thr Gly Lys Leu Tyr Asn Phe Ile Gly Asn Phe Lys Val 165 170 175 AAG AAG GTC ATG TTC GAG GAG AAG GAC GTT GGA GAT TCA AAC AAG GCC 576 Lys Lys Val Met Phe Glu Glu Lys Asp Val Gly Asp Ser Asn Lys Ala 180 185 190 AAA TAC ACC GCT GTC AAA GTT TAC GTC GGT TCC GAT GAG AAG AAA GTC 624 Lys Tyr Thr Ala Val Lys Val Tyr Val Gly Ser Asp Glu Lys Lys Val 195 200 205 GTA AGA CTC GAC TAC TTC TAC ACT GGT GAT GAG AGA TTC AAG GAG GTT 672 Val Arg Leu Asp Tyr Phe Tyr Thr Gly Asp Glu Arg Phe Lys Glu Val 210 215 220 TAC TTC AAA TTG GTA GAC GGA AAA TGG AAG AAG GTT GAG CAG AGC GAG 720 Tyr Phe Lys Leu Val Asp Gly Lys Trp Lys Lys Val Glu Gln Ser Glu 225 230 235 240 GCA AAC AAG GAT TTG CAC GCC ATG AAC AGT GCT TGG CCT TCG GAC TAC 768 Ala Asn Lys Asp Leu His Ala Met Asn Ser Ala Trp Pro Ser Asp Tyr 245 250 255 AAG CCT CTT GTC GAC AAG TTC TCA CCA CTT GCC GTT CTC AGC GCG GTT 816 Lys Pro Leu Val Asp Lys Phe Ser Pro Leu Ala Val Leu Ser Ala Val 260 265 270 CTC ATC GCC TCC CTC GCA GTA TTC TAT TAT CTC TAG 852 Leu Ile Ala Ser Leu Ala Val Phe Tyr Tyr Leu 275 280

[Brief description of drawings]

FIG. 1 is a photograph showing the results of electrophoresis showing the reaction between serum immunized with the KEK peptide of the present invention and various pyroplasms in the analysis by Western blotting of Example 2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location // (A61K 39/295 39: 002 39:12) (A61K 39/295 39: 002 39: 155 ) (A61K 39/295 39: 002 39: 235) (A61K 39/295 39: 002 39: 102) (A61K 39/295 39: 002 39:08) (72) Inventor Mamoru Kashiwazaki Takanodai, Tsukuba City, Ibaraki Prefecture 3-9-6 (72) Inventor Tsuguhiko Kamio 3-106-201 Takezono, Tsukuba-shi, Ibaraki (72) Inventor Akira Taneno 1360-2 Shimohirakawa-cho, Kumamoto-shi, Kumamoto (72) Inventor Fujio Nonaka Kumamoto-ken 7-1752-47 Hanazono, Kumamoto City (72) Inventor Tokuji Miyahara 3566-22-13-5 Tsukure, Kikuyo-cho, Kikuchi-gun, Kumamoto Prefecture Inventor Shinji Yamada 49-3 (72) Neryo-cho, Kumamoto City, Kumamoto Prefecture Inventor Hidefumi Sakai 2-12-17 Takahira, Kumamoto City, Kumamoto Prefecture

Claims (6)

[Claims] 1. A 33 kd small bovine piroplasm pathogen.
A peptide comprising a partial amino acid sequence of the amino acid sequence of the peptide and containing the amino acid sequence of Lys-Glu-Lys, which is effective for the prevention of infection of small Piroplasma pathogens in cattle. 2. The peptide according to claim 1, which comprises the following amino acid sequence. Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-G
lu-Val-Lys-Asp-Leu-Asp-Ala 3. The peptide according to claim 2, comprising the following amino acid sequence. Glu-Val-Val-Trp-Lys-Glu-Lys-Lys-G
lu-Val-Lys-Asp-Leu-Asp-Ala 4. A vaccine for preventing the infection of small bovine Piroplasma pathogenic infections, which comprises the peptide according to any one of claims 1 to 3 and a substance having an adjuvant activity. 5. A bovine mixed vaccine comprising the vaccine according to claim 4 and one or more vaccines selected from the following. Bovine infectious rhinotracheitis vaccine, mucosal bovine infectious diarrhea vaccine, bovine parainfluenza vaccine, bovine adenovirus vaccine, hemophilus vaccine, Clostridium vaccine 6. A method for measuring an antibody against a small bovine piroplasma pathogen, which comprises using the peptide according to any one of claims 1 to 3 as an antigen for measurement.