JP2000350587A - Novel h gene of canine distemper virus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a novel H gene of canine distemper virus which possesses the base sequence encoding H protein containing a specific amino acid sequence or the base sequence giving a specific restriction enzyme digestive fragment and is effective in the prophylaxis, treatment, examination or the like of canine distemper virus infectious diseases. SOLUTION: This novel H gene of canine distemper virus possesses the base sequence encoding the amino acid sequence of H protein represented by the formula or the base sequence which encodes a polypeptide showing the amino acid sequence homology of more than 95% to this amino acid sequence and gives two kinds of fragments when digested with restriction enzyme NdeI or PshBI and is effective in the prophylaxis, treatment, examination or the like of distemper virus infectious diseases on a mammal, e.g. dog or the like. This H gene is obtained by isolating whole RNA from distemper virus by a widely known method, synthesizing cDNA by using the RNA as a template and cloning the cDNA by PCR method using a primer consisting of the partial sequence.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel H gene and H protein effective for prevention / treatment / test of distemper virus infection in mammals such as dogs, and a novel canine distemper virus containing the same.

[0002]

2. Description of the Related Art Paramyxovirida
e) Department of morbillivirus (Morbillivirus) genus canine distemper virus (hereinafter referred to as a member of the, also referred to as "CDV") is a rinderpest virus and near the edge of the human measles virus and bovine. The disease caused by CDV has been known for centuries and is still present worldwide as one of the important infectious diseases in carnivores such as dogs and large cats (Refs. 3, 34: below ()). Numbers represent literature numbers). Infection of dogs without protective immunity with CDV causes acute to subacute systemic illness with high mortality. The target of infection for CDV is primarily the mucosal and lymphoid tissues throughout the body. The disease is typically characterized by the appearance of fever, anorexia, runny nose, conjunctivitis, and diarrhea. In some cases, there are signs of pustules on the skin, keratosis, and diseases of the central nervous system. However, the disease has subsided in recent decades since the introduction of the live attenuated CDV vaccine (commented 1, 2, and 13).

[0003] CDV is composed of a membrane protein named M and H (hemagglutinin, hemagglutinin: C).
It has an envelope consisting of two glycoproteins, termed the DV attachment protein) and F (fusion), the latter two H and F proteins being major target antigens of the host immune system. is there. High frequency antigenic mutations were found in the H protein (5), and this H protein was considered to be optimal for monitoring genetic changes in the virus as pointed out in the literature (15, 26). Have been.

Accordingly, the H gene (gene encoding H protein) of a recent field CDV isolate (herein referred to as "new CDV") and currently available live attenuated CDV vaccine strains such as Onderstepoort, Fromm, Rock
Significant genetic diversity when compared to the H gene of born, attenuated Snyder Hill, and Convac was noted in European countries (6, 14, 15), the United States (18) and Japan (2).
0). These vaccine strains are 195
Made in the 0's and 1960's (here "Old CD
V "). Antigenic differences between new and old CDVs were found in neutralization studies (12, 19), while the belief that current vaccines are not necessarily ineffective at protecting dogs from new CDV infection (14, 15).

[0005] A further noteworthy feature of the new CDV is the presence of geographically different strains as revealed by phylogenetic analysis of the H gene, as was also found for other measles viruses (8, 28, 31, 33). (6,
7, 15, 18, 20). Although these strains or genotypes are not generally limited by the type of virus host (6, 7, 18), there may be strains specific to the type of host (15). In Japan, the existence of a new CDV of one genotype has been recognized since the 1980s, and that genotype has been
It was found to be different from that of DV (20, 25).

[0006]

DISCLOSURE OF THE INVENTION The present invention can provide a novel canine distemper virus H gene, and can prevent distemper virus infection in mammals such as dogs.
A novel H gene effective for therapy / test, an H protein encoded by the gene, and a novel canine distemper virus containing the same can be provided.

[0007]

Means for Solving the Problems As a result of intensive studies, the present inventors have found a novel CDV genotype, and have achieved the above object of the present invention. The configuration of the present invention will be described below. (1) a nucleotide sequence encoding the amino acid sequence of the H protein described in SEQ ID NO: 1 in the sequence listing, or a polypeptide having an amino acid sequence homology of 95% or more with the amino acid sequence, and a restriction enzyme Nde I or Psh An H gene of canine distemper virus, which has a nucleotide sequence from which two fragments can be obtained by digestion with BI. (2) No. 19 to the amino acid sequence of the H protein
21st, 149th to 151st, 309th to 3rd
11th, 391st to 393rd, 422th to 42nd
4th, 456th to 458th, 587th to 589th
The H gene of canine distemper virus according to (1), wherein the H gene has a sugar chain binding site at No. 603 to 605. (3) A canine distemper virus comprising the H gene according to (1) or (2). (4) The amino acid sequence of the H protein described in SEQ ID NO: 1 in the sequence listing, or has an amino acid sequence homology of 95% or more with the amino acid sequence. No. 151, No. 309-No. 31
No. 1, No. 391 to No. 393, No. 422 to No. 424
No., 456th to 458th, 587th to 589th
No. 603 to No. 605, wherein the H protein of canine distemper virus has an amino acid sequence having a sugar chain binding site.

[0008] The present inventors have proposed that the culture conditions for CDV in vitro ( in vitro ) are difficult, so that instead of the cell culture method, a reverse transcriptase polymerase chain reaction (RT-PC) was used.
CDV RNA in clinical test materials has been routinely tested by the R) method. When a positive diagnosis is made by RT-PCR, restriction fragment length polymorphism (RFLP; restriction fragment) is used for easy genotyping of the H gene.
ment length polymorphism) assay was also applied to the RT-PCR product. The present inventors have examined the genotype characteristics of CDV recently widely recognized among dogs in Japan by the above-mentioned method. A new CDV genotype was obtained from a sample collected from a puppy clinically diagnosed with canine distemper. Detected during. Phylogenetic analysis of the H gene amino acid sequence revealed that its genotype was quite different from that previously known.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The H gene of the canine distemper virus of the present invention has a nucleotide sequence encoding the amino acid sequence of the H protein shown in SEQ ID NO: 1 in the sequence listing, or has at least 95% amino acid sequence homology with the amino acid sequence. Encodes a potential polypeptide and contains restriction enzymes Nde I and PshB
It has a nucleotide sequence from which two fragments can be obtained by digestion with I. Here, conventionally known or commercially available restriction enzymes Nde I and Psh BI can be used. Restriction enzyme
The two types of fragments obtained by digestion with Nde I include:
About 1900 bp and 750 bp combination, Psh B
For I, the combination is about 1650 bp and 950 bp. In addition, the homology test of the amino acid sequence was performed by RT-PCR.
Amino acid sequence deduced from the nucleotide sequence of the H gene obtained by the method such as the method described above, and comparing it with the amino acid sequence described in SEQ ID NO: 1 in the sequence listing using predetermined known personal computer software. Can be implemented. It is preferable that the H gene of the present invention has a sugar chain binding site at the specific place as described above in the amino acid sequence encoded by the H gene. Here, the sugar chain binding site is NX-
A site having an amino acid sequence represented by S / T, which can be determined by estimating the amino acid from the base sequence. Here, N represents Asn (asparagine),
S / T represents Ser (serine) or Thr (threonine). X represents an arbitrary amino acid. Also,
It is preferable that there is no sugar chain binding site at positions 584 to 586 of the amino acid sequence.

[0010] The H gene can be isolated from a field CDV strain and a host animal (eg, a dog) that has developed CDV infection.
Can be isolated from the secretions of the organs, the oral cavity and the rectum, and can also be obtained by chemical synthesis based on the base sequence and amino acid sequence described in SEQ ID NO: 1 in the sequence listing. Here, in order to examine the presence of the H gene of the present invention and the presence or absence of CDV having the H gene in an outdoor CDV strain or host animal, an RT-PCR assay, a fluorescent antibody direct or indirect method, A body binding reaction, a neutralization test, an ELISA method and the like can be used.

In the RT-PCR assay, a virus obtained from a host animal is cultured, total RNA is isolated from a culture supernatant or from a secretory sample, and the RNA is used as a template to synthesize cDNA by a known method. Combine. Its cD
D by PCR method with appropriate primers selected from NA
NA is amplified, and CDVR is performed by agarose electrophoresis.
It can be implemented by detecting NA. Here, the primer used for PCR may be a known H gene primer, that is, a primer described in the literature (18, 20, 32, 35) or the like. It can also be obtained by chemical synthesis based on the base sequence or amino acid sequence described in SEQ ID NO: 1 in the sequence listing. Specifically, primers described in Table 2 below can be used.

The H gene of the canine distemper virus of the present invention is amplified by the RT-PCR method as described above, purified by a conventionally known method, inserted into a known cloning vector, and inserted into a predetermined cloning vector. It can be transformed into host cells and cloned. In addition, the canine distemper virus having the H gene of the present invention can be obtained from an isolate or host tissue and secretion having the H gene, and the H gene obtained as described above can be transformed into another gene by a gene recombination technique. It can also be introduced into a type CDV virus strain.

The H protein of the canine distemper virus of the present invention is obtained by incorporating the H gene obtained as described above into a predetermined expression vector, introducing it into a predetermined host cell to express the gene, and culturing the gene. It can be obtained by recovering / purifying from a product. The H gene of the present invention can be effectively used for vaccine design. For example, it can be used for a recombinant subunit vaccine and a DNA vaccine as described in Reference (10), a recombinant vectorized vaccine as described in Reference (21, 27, 30), and the like.
Further, the CDV having the H gene of the present invention can also be used for a live attenuated vaccine and the like. In addition, the H protein of the present invention itself can be used for vaccines and the like. It is expected that these vaccines can also induce cell-mediated immunity either alone or in combination with an appropriate adjuvant. The H gene, virus and H protein of the present invention are also useful for distemper diagnosis and test. Specifically, the H gene, virus and H protein of the present invention can be obtained by RT-PCR assay, fluorescent antibody direct or indirect method, complement fixation reaction, neutralization test, ELISA
It can be used for diagnosis and inspection of distemper by being applied to the method.

(Literature List) In the specification of the present application, the documents with the numbers in parentheses are shown below. 1.Appel, M. (1987) Canine distemper virus, p.133
-159. In MJ Appel (ed.), Vi rus infections o
f carnivores. Elsevier Science Publishers BV,
Amsterdam.2.Appel, MJG, and BA Summers.
(1955) Vet. Microbiol. 44: 187-191. 3.Appel, MJG, et.al. (1994) J. Vet. Diagn.
Invest. 6: 277-288. 4.Barrett, T., et.al. (1987) Virus Res. 8: 373-
386. 5. Blixenkrone-Mller, M., et.al. (1992) Arch. Vir
ol. 123: 279-294. 6. Bolt, G., et.al. (1997) J. Gen. virol. 78: 36
7-372. 7. Carpenter, MA, et.al. (1998) Vet. Immunol.
Immunopathol. 65: 259-266. 8. Chamberlain, RW, et.al. (1993) J. Gen. Viro.
l. 72: 443-447. 9. Curran, MD, et.al. (1991) J. Gen. Virol. 72
: 443-447.10.De Vries, p., Et.al. (1988) J. Gen. Virol. 69:
2071-2083.

11. Felsenstein, J. (1993) PHYLIP: Ph
ylogeny inference package, version3.5. De partm
ent of Genetics, University of Washington, Seattl
e. 12. Gemma, T., et.al. (1996) J. Vet. Med. Sci. 58
: 791-794. 13. Greene, CE, and MJ Appel (1998) Canine di
stemper, p.9-22.In CE Gre ene (ed.), Infec
tious diseases of the dog and cat.2nd ed.WB S
aunders C ompany, Philadelphia, Pa. 14.Haas, L., et.al. (1997) Zur Situation der Hund
estaupe in Deutschland. Kleint ierpraxis 42: 6
13-620. 15. Haas, L., et.al. (1997) Virus Res. 48: 165-17
1. 16. Hagiwara, S., et.al. (1994) Unpublished data. 17. Harder, TC, et.al. (1995) Vaccine 13: 521-
523. 18. Harder, TC, et.al. (1996) J. Gen. Virol. 77
: 397-405. 19. Harder, TC, et.al. (1993) J. Virol. Methods
41: 77-92. 20.Iwatsuki, K., et.al. (1997) J. Gen. Virol. 78
: 373-380.

21. Jones, L., et. Al. (1997) Amer. J.
Vet. Res. 58: 590-593 22.Kovamees, J., et.al. (1991) Virus Res. 19: 22
3-234. 23. Mamaev, LV, et.al. (1995) Vet. Microbiol. 4
4: Mochizuki, M., et. Al. (1998) Unpublished data. 25. Ohashi, K., et. Al. (1998) J. Vet. Med. Sci. 60
: 1209-1212. 26. Orvell, C., et.al. (1990) J. Gen. Virol. 71:
2085-2092. 27. Pardo, MC, et.al. (1997) Amer. J. Vet. Res.
58: 833-836.28.Rima, BK, et.al. (1995) J. Gen. Virol. 76:
1173-1180.29.Sixt, N., et.al. (1998) J. Virol. 72: 8472-84
76. 30.Stephensen, CB, et.al. (1997) J. Virol. 71
: 1506-1513.

31. Taylor, MJ, et. Al. (1991) J. Ge
n. Virol. 72: 83-88. 32. Thompson, JD, et.al. (1994) Nucleic Acid Re.
s. 22: 4673-4680. 33.Wamwayi, HM, et.al. (1995) Vet. Microbiol.
44: 151-163. 34. Wood, SL, et.al. (1995) Can.Vet. J. 36: 3
4-35. 35. Yoshida, E., et.al. (1998) Vet. Microbiol. 59
: 237-244

[0018]

EXAMPLES Hereinafter, the contents of the present invention will be specifically described using examples, but the contents of the present invention are not limited thereto. (Virus and clinical samples) Seven CDV strains were used for reference; six were: Onderstepoort, Fromm, DFE-H
C, Lederle VR-128, Rockborn and FXNO strains are vaccine strains currently used in Japan. As the remaining one, a laboratory wild-type Snyder Hill strain was used. These reference CDVs appear to be old CDVs.
RT-P with vaccine and cell culture solution dissolved in sterile distilled water
Used for CR. The KDK-1 strain isolated from a 1991 field outbreak has been passaged in Vero cell culture. The culture at passage 6 was used for H gene sequencing and RFLP analysis. The Vero cells contain 10
The cells were cultured in Eagle's Minimum Essential Medium (MEM) supplemented with 5% tryptose phosphate broth and 8% fetal bovine serum. In 1994, four additional CDV isolates, C710D, C714D, C717D, and C720D, were also used for 10 passages in Vero cell culture. 11 oral swabs and 90 rectal swabs for a total of 1
One-ten clinical samples were collected from dogs showing signs of disease in the respiratory or intestines. These samples were 1997/19
Submitted by animal hospitals in various locations in Japan in both years of 1998, of which three oral samples and four rectal samples were diagnosed as CDV RNA positive by RT-PCR assay. The characteristics of these field CDV isolates and CDV RNA positive clinical samples are summarized in Table 1 below.

[0019]

[Table 1]

(Selection of primers) Four sets of oligonucleotide primers were used in this study according to the sequence information of the Ondertepoort strain (FIG. 1; Table 2). H for daily diagnosis
A set of primers targeting the inside of the gene (CDVH
13 and CDVH18). H gene RF
To obtain PCR products for LP analysis and sequencing,
Two sets of primers were used: one between the F gene and the H gene (primer RH-3) and one between the H gene and the large (L) protein gene (primer RH-
4) Designed from the sequence of each untranslated region of (18), and the other is F gene region (primer CDV-F
8) and the L gene region (primer CDV-R8). For detection and sequencing of the nucleocapsid (N) gene, a set of previously described primers (NF1287 and p2) (35) was utilized. The sequence of each primer is shown in Table 2 below.

FIG. 1 is a diagram showing an outline of an RT-PCR assay. In FIG. 1, the structure of the CDV genome is shown in the center. The various CDV genes are represented by boxes and indicate nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), hemagglutinin protein (H) and large protein (L). At the bottom, F
The lengths of primer pairs and RT-PCR products set between genes, L genes and H genes are shown. nt: nucleotide.

[0022]

[Table 2]

(Reverse Transcription and PCR Amplification) From 250 μl of the supernatant of the infected cell culture or swab extract,
Total RNA was obtained by using n-LS (manufactured by Nippon Gene Co., Ltd., Toyama) according to the manufacturer's instructions.
Dissolved in μl of TE buffer (pH 8.0). cDNA
Was synthesized using a random 9-mer primer (Takara Shuzo, Tokyo). That is, 1 μl of random primer (50 pmol / μl) was added to 9 μl of RNA
After addition to the solution, the mixture was incubated at 70 ° C. for 10 minutes and cooled on ice. To this mixture, add 4 μl of 5 × re
action buffer (Takara Shuzo, 250 mM Tris-
HCl: pH 8.3, 500 mM KCl, 20 mM dithiothreitol and 5 mM MgCl ₂ ), 1 μl of 1
33 U / μl of ribonuclease inhibitor
inhibitor) (Takara Shuzo) and 4 μl of 2.5 mM
dNTPmixture (Takara Shuzo) was added and the mixture was incubated at 25 ° C. for 5 minutes, then 1 μl of 3
7 U / μl of AMV reverse transcriptase
e) XL (Takara Shuzo) was added last. Next, a total of 2
0 μl of this reaction mixture was incubated at 25 ° C. for 10 minutes, 42 ° C. for 50 minutes and 70 ° C. for 10 minutes.

For routine diagnosis of clinical samples, PCR
Was performed as follows. As a total of 50 μl reaction mixture, 0.5 μl cDNA, 0.25 μl Taq polymerase (5 U / μl) (Takara Shuzo), 5 μl of 10
× PCR buffer (Takara Shuzo, 100 mM Tris-
HCl: pH 8.3, 500 mM KCl), 3 μl 2
5 mM MgCl ₂ , 4 μl of 2.5 mM dNTP mi
xture, 36.25 μl of distilled water (dH ₂ O), and primers CDVH13 and CDVH18 (50
pmol / μl) were prepared by mixing 0.5 μl each. Trio-Thermoblo
ck (Biometra, Tampa, FL, USA). The temperature cycling protocol is 9
Denaturation at 4 ° C, annealing of primers at 55 ° C,
And 30 cycles of extension at 72 ° C. for 30 seconds each. Primers NF1287 and p2 (50 pm
ol / μl) was used to amplify the N gene portion. The temperature cycling protocol at this time consisted of 30 cycles of denaturation at 94 ° C. for 1 minute, primer annealing at 55 ° C. for 2 minutes, and extension at 72 ° C. for 2 minutes.

In order to obtain a PCR product for sequencing the H gene of the KDK-1 strain, primers RH-3 and R
0.5 μl of each of H-4 (50 pmol / μl) for a total of 4
Added to 9 μl of the above reaction mixture. Denaturation at 94 ° C (3
0 sec), annealing of primer at 55 ° C. (1
The amplification was carried out by a temperature cycling protocol consisting of 25 cycles of the reaction of extension at 25 ° C., and 72 ° C. (1 minute) followed by a final extension step at 72 ° C. for 10 minutes.

To obtain PCR products for RFLP analysis of the rectal sample 98-002 and sequencing of the H gene, primers CDV-F8 and CDV-R8 (50 pm
ol / μl) with 0.5 μl of cDN
A, 0.25 μl ExTaq polymerase (5 μl /
μl) (Takara Shuzo) and 5 μl of 10 × Ex Taq b
uffer (Takara Shuzo, 100 mM Tris-HCl: p
H8.3, 500 mM KCl and 20 mM MgC
l ₂ ), consisting of 4 μl of 2.5 mM dNTP mixture and 39.75 μl of distilled water (dH ₂ O) for a total of 49
Added to μl reaction mixture. Denaturation at 94 ° C. for 1 minute,
35 cycles of primer annealing at 50 ° C. for 2 minutes and extension at 72 ° C. for 2 minutes followed by 72 cycles
Amplification was performed by a temperature cycling protocol consisting of a final extension step at 2 ° C. for 2 minutes.

(Sequence Determination) The PCR products prepared from the KDK-1 strain and the rectal sample 98-002 were subjected to Wizard PCR.
Purification was performed using Preps DNA Purification system (Promega, Madison, WI, USA). Next, the purified PCR product was transferred to Regular pT7Blue
(R) T-Vector (Promega, Madison, Wis., USA) and inserted into Epicurian Coli XL2-Blue
MRF 'Competent Cell (Stratagene, La Jolla, CA, USA) was transformed and cloned. For each sample, 10 plasmids containing the PCR product were added to the Wizard MiniPreps DNA Purificat
Purified using the Ion System (Promega, Madison, Wis., USA) and AutoRead Sequencin
g Kit (AmershamPharmaciaBiot
ech Co., Tokyo) using Cy5 labeled m13 primer, and Cy5 designed from the obtained sequence.
Was sequenced using primers labeled with.

(RFLP analysis of PCR amplification product) CDV
-F8 and PC obtained using CDV-R8 primer
R product using WizardPCR Preps DNA Purification System
And concentrate the resulting product with restriction enzymes. FbaI andNd
eI (Takara Shuzo). Of them
8 μl for 2 hours at 37 ° C. under the conditions recommended by the manufacturer
1.2 unitsFbaI and 1.0 unitsNdeDigest with I
Was. The obtained restriction fragments are subjected to 2% agarose gel electrophoresis.
And separate the bands with ethidium bromide.
Visualization was performed after staining.

(Phylogenetic Classification Analysis)
The amino acid sequence was translated using tyx-Mac Ver. 8.5 (manufactured by Software Development Co., Ltd., Tokyo). Clustal W Ver. 1.74 (34)
, Alignment preparation and bootstrap analysis were performed. This data was input to PHYLIP Ver. 3.5 (11), and a phylogenetic tree was created without specifying an out group.

Although the term "genotype" has often been used in the classification of CDV (6, 15), there is no formal definition yet. Here, the genotype was defined by the phylogenetic nature of the amino acid sequence of the H gene. That is, 95%
Strains in the same clade exhibiting more than 300 amino acid homologies are considered to belong to the same genotype.

(DDBJ / EMBL / GenBank of nucleotide sequence
The accession numbers for the H gene sequences and the data used here are as follows: Snyder Hill (15), Vaccine / Ond
erstepoort (4, 9) [AF014953, D00758], vaccine / Convac (22) [Z35493], Chinese leopard / A92-27 / 4
(18) [Z54156], black leopard / A92-6 (18) [Z5416
6], dog / US89 (6) [Z47762], leopard / US91
(6) [Z47763], Raccoon / US89 (6) [Z4776
5], Peccary / US89 (6) [Z47765], Dog / DK9
1, B + C (6) [Z477661], mink / DK86 (6) [Z4775
9], dog / GR88 (6) [Z47760], dog / 404 (15)
[Z77671], dog / 2544 (15) [Z77672], dog / 45
13 (15) [77673], Siberian seal / PDV-2 (2
3) [X84998], ferret / 1493 / Han89 (23) [X
84999], dog / 5804 / Han90 (23) [X85000], dog / Ueno (20) [D85753], dog / Hamamatsu (20)
[D85754], dog / Yanaka (20) [D85755], and tanuki / Tanu96 [AB016776].

(Results of Example) (RFLP analysis of H gene derived from CDV strain for reference and CDV RNA-positive clinical sample)
The strain and the KDK-1 strain were compared and examined. In FIG. 2, Lane M, 100 bp step size markers; Lanes 1-5, KDK-1, C710 of field isolates.
D, C714D, C717D and C720D; lanes 6-10, 98/001, 98/003, 9 of clinical samples.
8/005, 98-001, and 98-002; Lanes 11-17, reference strains Onderstepoort, Fromm, DFE-
HC, Lederle VR-128, Rockborn, FXNO and Wild-type Snyd
er Hill.

As shown in the results of FIG. 2, digestion of all seven reference CDV strains with FbaI resulted in three fragments approximately 1,800, 600 and 200 bp in size. However, only two fragments, approximately 2,400 and 200 bp in size, were obtained for the KDK-1 strain. Nde
I (FIG. 3; FIG.
), KDK-1 is about 1,500, 300 and 7
Although digested into 4 fragments of 50 and 100 bp in size, the reference CDV strain was not digested at all. These results indicated that the KDK-1 strain isolated in 1991 was genetically different from the old CDV for reference.

From a total of seven clinical samples diagnosed as CDVRNA positive by RT-PCR, three oral samples and two rectal samples were submitted for RFLP analysis of the H gene region. In the case of two rectal samples (97-050 and 97-063), no suitable PCR product was obtained for analysis due to the interference background of non-specific PCR products. Therefore, four CDV isolates and five clinical samples were analyzed (Table 1, and FIGS. 2 and 3). CDV
Isolates C710D, C714D, C717D, and C720D, 98/002 and 98/00 of oral samples
3, and the rectal sample 98-001 showed an RFLP pattern similar to that of the KDK-1 strain. Oral sample 98/001 and rectal sample 98-002 collected from the same dog
The RFLP pattern (Table 1) is similar to strain KDK-1 when it is digested with Fba I, unlike when it is digested with Nde I, 2 kinds of fragments observed about 1,900 and 750bp (Figure 3).

(H of KDK-1 and rectal sample 98-002)
Genetic Amino Acid Sequence Analysis) KDK-1: As a representative of a new Japanese CDV isolate, the sequence of the H gene of the KDK-1 strain was determined, and its deduced amino acid sequence was determined. The H gene consisted of a single open reading frame (ORF) capable of encoding 1,824 bp, and 607 amino acids. Amino acid homology to the published vaccine strains Onderstepoort and Convac, and to the wild-type Snyder Hill strain (15) are 89.6%, 90.3% and 90.9, respectively.
%Met. On the other hand, for the new CDV Ueno, Yanaka, and Hamamatsu (20) in Japan, the maximum homology (9
8.5% to 98.7%), new CDV reported from other areas, and Siberian seal morbil
The homology to the livirus PDV-2 strain was rather low (9
3.1% to 95.9%). A total of eight sugar chain binding sites are 149th to 151th, 309th to 311th,
391st to 393rd, 422th to 424th, 456th to 458th, 584th to 586th, 5th
It was recognized at positions 87-589 and 603-605. Of the nine sugar chain binding sites shared by all CDVs in Japan reported to date (20), the KDK-1 strain lacks the sugar chain binding sites at positions 19 to 21. Had been, but the rest had been saved.

98-002: 98-002 was used for H gene sequencing as RFLP analysis showed that oral sample 98/001 and rectal sample 98-002 were almost identical. The resulting nucleotide sequence and amino acid sequence were compared with SEQ ID NO: 1 to SEQ ID NO: 1 in the sequence listing.
No. 1824 (base sequence).

The nucleotide length of the 98-002 H gene fragment (No. 1 to No. 1824 of SEQ ID NO: 1 in the sequence listing) is also 1,824 bp, and is a single nucleotide capable of encoding 607 amino acids. The ORF has been identified. Old CDV
Amino acid homology to strains Onderstepoort and Convac and Snyder Hill was low (89.2% -90.
1%). However, homology to other Japanese CDVs such as the KDK-1 strain (93.2% -93.4%) as well as homology to new CDVs from other geographic regions (9
(2.6% -93.9%) was lower than that seen in the analysis of strain KDK-1. Unlike other Japanese CDVs (20), a total of 8 sugar chain binding sites are located at positions 19 to 21, 149 to 151, and 149 to 151 in the H gene amino acid sequence.
No. 309 to No. 311, No. 391 to No. 393, No. 422 to No. 424, No. 456 to No. 458, No. 5
Amino acid substitutions were found at positions 87-589 and 603-605, indicating that the amino acid substitutions were scattered throughout the gene. Although not shared by CDVs from other regions, such as older CDVs, KDK-
The sugar chain binding site at positions 584 to 586 shared by all Japanese CDVs such as one strain is 98-0.
02 was not.

In both strain KDK-1 and sample 98-002, the cysteine residue is the first in the amino acid sequence.
No. 39, No. 154, No. 188, No. 283, No. 29
No. 6, No. 377, No. 382, No. 390, No. 490
No. 566, No. 575 and No. 607 are completely stored in twelve positions, and these positions are the same for all CDVs in the database. The homology (%) of the H gene base sequence and amino acid sequence between each strain and each sample is shown in Table 3 below.

[0039]

[Table 3]

[0040]

[Table 4]

(Hygene Amino Acid Sequence Phylogenetic Analysis) FIG. 4 shows the estimated phylogenetic relationship between the old and new CDVs of sample 98-002 and the like based on the amino acid alignment of the H gene. This phylogenetic tree was created using Clustal W version 1.74 (22) and PHYLIP version 3.5 module (23). KDK- of CDV strain
The sequences 1 and 98-002 were first revealed in this study. Vaccine strain, Onderstepoort [AF014953, D0075
8], Convac [Z35493]; American isolate, Chinese leopard / A92-27 / 4 [Z54156], Black leopard / A92-6 [Z5416]
6], dog / US89 [Z47762], leopard / US91 [Z4776
3], Raccoon / US89 [Z47765], Peccary / US8
9 [Z47765]; European isolate, dog / DK91, B + C
[Z477661], mink / DK86 [Z47759], dog / 404 [Z7
7671], dog / 2544 [Z77672], dog / 4513 [7767]
3], ferret / 1493 / Han89 [X84999], dog / 55804
/ Han90 [X85000]; Asian isolate, Siberian seal / PDV-2 [X84998]; Greenland isolate, dog / GR88 [Z47760]; Japanese origin, dog / Ueno [D85753].
Dog / Hamamatsu [D85754], Dog / Yanaka [D8575
5] and the sequence of Tanuki96 [AB016776] were extracted from the database. Data for the Snyder Hill strain is provided from reference (15).

Referring to FIG. 4, old CDVs, such as Onderstepoort, Convac and Snyder Hill strains, form independent clades, while new CDVs are classified into several separate clades independent of the old CDV. Was done. K
The DK-1 strain is an independent clade of Japan's new C
Joined the group of DV isolates. The American isolates seem to be in one clade, and several strains exist among European isolates, as described previously (6, 15, 18). This phylogenetic tree showing high bootstrap values indicates that 98-002 diverges from Japanese clade ancestry and does not match any known CDV clade.

(Clinical characteristics of dogs from which 98-002 samples were obtained) A two-month-old male Shih Tzu with no vaccination history and a clinical history of diarrhea, coughing and depression was from Chiba, Japan. Veterinarian diagnosed. The male dog was purchased from a pet shop 10 days before and had watery diarrhea two days after purchase. Fever, nasal discharge, tonsillitis, and excessive salivation were also observed during hospitalization, and signs of the central nervous system appeared on the last day. The male dog was clinically diagnosed with canine distemper and hospitalized, but died three days later without any improvement despite active treatment. No necropsy was performed, but oral (98/001) and rectal (98-002) swabs were submitted for virus testing (Table 1). No dog parvovirus, rotavirus, coronavirus was detected in the rectal swab samples, and no virus was recovered from the oral swab samples. However, both samples were determined to be CDV RNA positive by the RT-PCR assay, and both samples were considered to be contaminated by the same virus, though not conclusively, by RFLP analysis (FIGS. 2 and 3).

(Detection and sequence of N protein gene from rectal sample 98-002)
p was amplified and the nucleotide sequence was obtained. The amplified region was composed of 419 bp, and was 372 bp excluding the primer region with respect to the known sequence of the Onderstepoort strain.
The length homology was 92.2%.

It has long been difficult to attempt a comparative study between CDV strains because only a few types of cultivable strains could be applied to in vitro experiments. However, in 1990
During the ages several papers appeared describing the particularly genetic nature of CDV from dogs as well as wildlife (6,
7, 12, 14, 15, 17-20, 23). In such studies, the virus is usually passaged by cell culture,
This can result in changes in viral genes. However, in this study and other studies (7, 17, 2)
5) Molecular studies on clinical samples were directly performed, and a new CDV genotype represented by sample 98-002 was unexpectedly discovered in the present invention.
Indeed, we attempted to isolate the virus from the swab extract without success (24). Therefore, this direct method seems to be more appropriate for elucidating the exact epidemiology and molecular virology of CDV, especially when fresh and sufficient samples are not available for virus isolation.

The current Japanese CDV isolate is present in one similar clade (FIG. 4), and as described previously (20), new and old Cs of European and American origin.
Clearly away from DV. The CDV strain KDK-1 shows the closest relationship to this clade, with 98.5%
The above amino acids show homology, indicating that they belong to the same genotype. H genes amplified from other CDV isolates and from all clinical samples except 98/001 and 98-002 were analyzed for KDK- by RFLP analysis.
It was also found that they belonged to the same genotype as 1. These results support the findings of a recent study in Japan that most CDVs spread among dogs in Japan since the 1980s belonged to the same genotype as the KDK-1 strain of this study (25). ing. However, the new 98-002
By finding the different H gene properties of the samples,
In Japan, CDV of another origin or genotype
It turned out that there was.

The H gene in the 98-002 sample was CD
V origin, or whether the virus was not recovered from the extract and whether CDV was actually present in our swab extract because the electron microscopy did not show it in the extract. It may be doubtful. FA for homology to 98-002 sequence
As a result of a homology search using the STA program, it was found that the top 20 sequences in descending order of similarity were of CDV origin, strongly suggesting that the 98-002 sample was of CDV origin. In addition, we have shown that 419 represents the N protein gene portion.
The bp product was successfully amplified from rectal swab extract 98-002. This means not only the H gene,
This indicates that the actual CDV virus was most likely present in the sample.

Recombinant subunit (10), experimental DN
A (29), in order to make a new type of CDV vaccine as recently described, such as experimental and commercial recombinant vectorized vaccines (21, 27, 30), it was theoretically cloned with at least cDNA. 98-002
H gene is sufficient. These vaccines are expected to induce cell-mediated immunity essential for canine distemper prevention. In order to examine the effectiveness of the H gene, the H protein of the present invention and the CDV having the H gene as a vaccine, for example, immunity against a H protein of the present invention is imparted to a dog (for example, a vaccine containing the H protein). Was administered parenterally to dogs), and then
Performing a forced infection experiment (a so-called attack test) with a DV is mentioned. However, an in vivo system for producing the virulent CDV has not yet been developed, and it is difficult to experimentally reproduce clinical canine distemper. This is well known (10, 18). Therefore, at present, such forced infection experiments cannot be performed in this field. However, it is clear that the effectiveness of the present invention is remarkable as described above.

[0049]

INDUSTRIAL APPLICABILITY According to the present invention, a novel H gene of canine distemper virus can be provided, and a novel H gene which is effective for prevention / treatment / test of distemper virus infection in mammals such as dogs, and the H gene encoded by the same, can be provided. A protein and a novel canine distemper virus containing the same can be provided.

[0050]

[Sequence List] SEQUENCE LISTING <110> Kyoritu Shoji Company <120> A New H gene of Canine Disptemper Virus <130> P-31995 <140> <141> <160> 2 <170> PatentIn Ver. 2.0

<210> 1 <211> 1824 <212> DNA <213> canine distemper virus <220> <221> CDS <400> 1 atg ctc tcc tac caa gac aag gtg ggt gcc ttc tat aag gat aat gca 48 Met Leu Ser Tyr Gln Asp Lys Val Gly Ala Phe Tyr Lys Asp Asn Ala 1 5 10 15 aga gct aat tca tcc aag ctg tcc tta gtg aca gaa gaa caa ggg ggc 96 Arg Ala Asn Ser Ser Lys Leu Ser Leu Val Thr Glu Glu Gln Gly Gly 20 25 30 agg aga cca ccc tac ttg ctg ttt gtc ctt ctc atc cta ctg gtt gga 144 Arg Arg Pro Pro Tyr Leu Leu Phe Val Leu Leu Ile Leu Leu Val Gly 35 40 45 atc ctg gcc ttg ctt gct atc gct gga gtt cga ttt cgc cag gtg tca 192 Ile Leu Ala Leu Leu Ala Ile Ala Gly Val Arg Phe Arg Gln Val Ser 50 55 60 act agc aat gtg gaa ttt ggc aga ttg ctg aag gat gat ttg gag aaa 240 Thr Ser Asn Val Glu Plu Gly Arg Leu Leu Lys Asp Asp Leu Glu Lys 65 70 75 80 tcg gag gcc gtg cat cac caa gtc atg gat gtc ttg aca ccg ctc ttc 288 Ser Glu Ala Val His His Gln Val Met Asp Val Leu Thr Pro Leu Phe 85 90 95 aaa att att gga gat gag att ggg tta cgg ttg cca caa aaa cta aac 336 Lys Ile Ile Gly Asp Glu Ile Gly Leu Arg Leu Pro Gln Lys Leu Asn 100 105 110 gag atc aaa cag ttt atc ctt caa aag aca aac ttc ttc aat ccg aac 384 Glu Ile Lys Gln Phe Ile Leu Gln Lys Thr Asn Phe Phe Asn Pro Asn 115 120 125 aga gaa ttc gac ttc cgc gat ctc cac tgg tgc att aac ccg cct agt 432 Arg Glu Phe Asp Phe Arg Asp Leu His Trp Cys Ile Asn Pro Pro Ser 130 135 140 aag atc aag gtg aat ttt acc aat tac tgt gat gca att ggg gtc aga 480 Lys Ile Lys Val Asn Phe Thr Asn Tyr Cys Asp Ala Ile Gly Val Arg 145 150 155 160 aaa tcc att gca tcg gca gca aat ccc atc ctt tta tca gca ctc tcc 528 Lys Ser Ile Ala Ser Ala Ala Asn Pro Ile Leu Leu Ser Ala Leu Ser 165 170 175 gga ggc aga ggt gac ata ttc cca cca tac aga tgc agt gga gct act 576 Gly Gly Arg Gly Asp Ile Phe Pro Pro Tyr Arg Cys Ser Gly Ala Thr 180 185 190 act tca gta ggc aga gtt ttc ccc cta tca gta tca tta tcc atg tct 624 Thr Ser Val Gly Arg Val Phe Pro Leu Ser Val Ser Leu Ser Met Ser 195 200 205 ttg atc tca aaa aca tca gag ata atc aat atg ctg acc gcc atc tca 672 Leu Ile Ser Lys Thr Ser Glu Ile Ile Asn Met Leu Thr Ala Ile Ser 210 215 220 gac gga gtg tat ggt aaa act tac ttg tta gtg cct gat tat att gaa 720 Asp Gly Val Tyr Gly Lys Thr Tyr Leu Leu Val Pro Asp Tyr Ile Glu 225 230 235 240 agg gag ttc gac aca caa aaa att cga gtc ttt gag ata ggg ttc atc 768 Arg Glu Phe Asp Thr Gln Lys Ile Arg Val Phe Glu Ile Gly Phe Ile 245 250 255 aaa cgg tgg ctg aat gac atg cca tta ctc cag aca acc aac tat atg 816 Lys Arg Trp Leu Asn Asp Met Pro Leu Leu Gln Thr Thr Asn Tyr Met 260 265 270 gtc ctc ccg gag aat tcc aaa gct aag gtg tgt act ataca ggc 864 Val Leu Pro Glu Asn Ser Lys Ala Lys Val Cys Thr Ile Ala Val Gly 275 280 285 gag ctg aca ctg gct tcc ttg tgt gta gat gag agc acc gta tta tta 912 Glu Leu Thr Leu Ala Ser Leu Cys Val Asp Glu Ser Thr Val Leu Leu 290 295 300 tat cat gac agc aat ggt tcg caa gac agt att cta gta gtg acg ctg 960 Tyr His Asp Ser Asn Gly Ser Gln Asp Ser Ile Leu Val Val Thr Leu 305 310 315 320 gga ata ttt ggg gca aca ccg atg aat caa gta gaa gag gtg ata cct 1008 Gly Ile Phe Gly Ala Thr Pro Met Asn Gln Val Glu Glu Val Ile Pro 325 330 335 gtg gct cat cca tca gta gaa agg ata cat atc aca aat cac cgt ggt 1056 Val Ala His Pro Ser Val Glu Arg Ile His Ile Thr Asn His Arg Gly 340 345 350 ttc ata aaa gat tca gta gca acc tgg atg gtg cct gca ttg gtc tct 1104 Phe Ile Lys Asp Ser Val Ala Thr Trp Met Val Pro Ala Leu Val Ser 355 360 365 gag caa caa gaa gga caa aaa aat tgt ctg gag tcg gct tgt caa aga 1152 Glu Gln Gln Glu Gly Gln Lys Asn Cys Leu Glu Ser Ala Cys Gln Arg 370 375 380 380 aaa tcc tac cct atg tgc aac caa aca tca ccc ttc gga gga 1200 Lys Ser Tyr Pro Met Cys Asn Gln Thr Ser Trp Glu Pro Phe Gly Gly 385 390 395 400 gta cag ttg cca tct tac ggg cgg ttg aca tta cct cta gat gca agc 1248 Val Gln Leu Pro Ser Tyr Gly Arg Leu Thr Leu Pro Leu Asp Ala Ser 405 410 415 att gac ctt caa ctt aac ata tcg ttt aca tac ggt ccg gtg ata ctg 1296 Ile Asp Leu Gln Leu Asn Ile Ser Phe Thr Tyr Gly Pro Val Ile Leu 420 425 430 aat gga gat ggt atg gat tat tat gaa aac cca ctt ttg gac tcc gga 1344 Asn Gly Asp Gly Met Asp Tyr Tyr Glu Asn Pro Leu Leu Asp Ser Gly 435 440 445 tgg ctt acc att cct ccc aag aac gga aca ata ctt ggatta aat 1392 Trp Leu Thr Ile Pro Pro Lys Asn Gly Thr Ile Leu Gly Leu Ile Asn 450 455 460 aaa gca agt aga gga gac cag ttc act gta acc ccc cat gta ttg aca 1440 Lys Ala Ser Arg Gly Asp Gln Phe Thr Val Thr Pro His Val Leu Thr 465 470 475 480 ttt gcg ccc agg gag tcg agt gga aat tgt tat cta cct atc caa aca 1488 Phe Ala Pro Arg Glu Ser Ser Gly Asn Cys Tyr Leu Pro Ile Gln Thr 485 490 495 tcc cag att atg gat aaa gat gtc ctt act gag tcc aat tta gtg gtg 1536 Ser Gln Ile Met Asp Lys Asp Val Leu Thr Glu Ser Asn Leu Val Val 500 505 510 ttg cct aca cag aat ttt gga tat gtc gta gca aca tat gat ata tcc 1584 Leu Pro Thr Gln Asn Phe Gly Tyr Val Val Ala Thr Tyr Asp Ile Ser 515 520 525 cgg gaaat cat gcg att gtt tat tat gtt tat gac cca atc cgg acg 1632 Arg Glu Asn His Ala Ile Val Tyr Tyr Val Tyr Asp Pro Ile Arg T hr 530 535 540 att tct tat acg tac cca ttt aga cta act acc aag ggt aga cct gat 1680 Ile Ser Tyr Thr Tyr Pro Phe Arg Leu Thr Thr Lys Gly Arg Pro Asp 545 550 555 560 ttc cta agg att gaa tgt ttt gtg tgg gat gac gat ttg tgg tgt cac 1728 Phe Leu Arg Ile Glu Cys Phe Val Trp Asp Asp Asp Leu Trp Cys His 565 570 575 caa ttt tac aga ttt gag tct gat atc acc aac tct aca acc agt gtc 1776 Gln Phe Tyr Arg Phe Glu Ser Asp Ile Thr Asn Ser Thr Thr Ser Val 580 585 590 gaa gat tta gtc cgt ata aga ttc tca tgt aat cgt tca aaa cct tga 1824 Glu Asp Leu Val Arg Ile Arg Phe Ser Cys Asn Arg Ser Lys Pro 595 600 605

<210> 2 <211> 607 <212> PRT <213> canine distemper virus <400> 2 Met Leu Ser Tyr Gln Asp Lys Val Gly Ala Phe Tyr Lys Asp Asn Ala 1 5 10 15 Arg Ala Asn Ser Ser Lys Leu Ser Leu Val Thr Glu Glu Gln Gly Gly 20 25 30 Arg Arg Pro Pro Tyr Leu Leu Phe Val Leu Leu Ile Leu Leu Val Gly 35 40 45 Ile Leu Ala Leu Leu Ala Ile Ala Gly Val Arg Phe Arg Gln Val Ser 50 55 60 Thr Ser Asn Val Glu Phe Gly Arg Leu Leu Lys Asp Asp Leu Glu Lys 65 70 75 80 Ser Glu Ala Val His His Gln Val Met Asp Val Leu Thr Pro Leu Phe 85 90 95 Lys Ile Ile Gly Asp Glu Ile Gly Leu Arg Leu Pro Gln Lys Leu Asn 100 105 110 Glu Ile Lys Gln Phe Ile Leu Gln Lys Thr Asn Phe Phe Asn Pro Asn 115 120 125 Arg Glu Phe Asp Phe Arg Asp Leu His Trp Cys Ile Asn Pro Pro Ser 130 135 140 Lys Ile Lys Val Asn Phe Thr Asn Tyr Cys Asp Ala Ile Gly Val Arg 145 150 155 160 Lys Ser Ile Ala Ser Ala Ala Asn Pro Ile Leu Leu Ser Ala Leu Ser 165 170 175 Gly Gly Arg Gly Asp Ile Phe Pro Pro Tyr Arg Cys Ser Gly Ala Thr 180 185 190 Thr Ser Val Gl y Arg Val Phe Pro Leu Ser Val Ser Leu Ser Met Ser 195 200 205 Leu Ile Ser Lys Thr Ser Glu Ile Ile Asn Met Leu Thr Ala Ile Ser 210 215 220 Asp Gly Val Tyr Gly Lys Thr Tyr Leu Leu Val Pro Asp Tyr Ile Glu 225 230 235 240 Arg Glu Phe Asp Thr Gln Lys Ile Arg Val Phe Glu Ile Gly Phe Ile 245 250 255 Lys Arg Trp Leu Asn Asp Met Pro Leu Leu Gln Thr Thr Asn Tyr Met 260 265 270 270 Val Leu Pro Glu Asn Ser Lys Ala Lys Val Cys Thr Ile Ala Val Gly 275 280 285 285 Glu Leu Thr Leu Ala Ser Leu Cys Val Asp Glu Ser Thr Val Leu Leu 290 295 300 Tyr His Asp Ser Asn Gly Ser Gln Asp Ser Ile Leu Val Val Thr Leu 305 310 315 320 Gly Ile Phe Gly Ala Thr Pro Met Asn Gln Val Glu Glu Val Ile Pro 325 330 335 Val Ala His Pro Ser Val Glu Arg Ile His Ile Thr Asn His Arg Gly 340 345 350 Phe Ile Lys Asp Ser Val Ala Thr Trp Met Val Pro Ala Leu Val Ser 355 360 365 Glu Gln Gln Glu Gly Gln Lys Asn Cys Leu Glu Ser Ala Cys Gln Arg 370 375 380 Lys Ser Tyr Pro Met Cys Asn Gln Thr Ser Trp Glu Pro Phe Gly Gly 385 390 395 400 Val Gln Leu Pr o Ser Tyr Gly Arg Leu Thr Leu Pro Leu Asp Ala Ser 405 410 415 Ile Asp Leu Gln Leu Asn Ile Ser Phe Thr Tyr Gly Pro Val Ile Leu 420 425 430 Asn Gly Asp Gly Met Asp Tyr Tyr Glu Asn Pro Leu Leu Asp Ser Gly 435 440 445 Trp Leu Thr Ile Pro Pro Lys Asn Gly Thr Ile Leu Gly Leu Ile Asn 450 455 460 Lys Ala Ser Arg Gly Asp Gln Phe Thr Val Thr Pro His Val Leu Thr 465 470 475 475 480 Phe Ala Pro Arg Glu Ser Ser Gly Asn Cys Tyr Leu Pro Ile Gln Thr 485 490 495 Ser Gln Ile Met Asp Lys Asp Val Leu Thr Glu Ser Asn Leu Val Val 500 505 510 Leu Pro Thr Gln Asn Phe Gly Tyr Val Val Ala Thr Tyr Asp Ile Ser 515 520 525 Arg Glu Asn His Ala Ile Val Tyr Tyr Val Tyr Asp Pro Ile Arg Thr 530 535 540 Ile Ser Tyr Thr Tyr Pro Phe Arg Leu Thr Thr Lys Gly Arg Pro Asp 545 550 555 555 560 Phe Leu Arg Ile Glu Cys Phe Val Trp Asp Asp Asp Asp Leu Trp Cys His 565 570 575 Gln Phe Tyr Arg Phe Glu Ser Asp Ile Thr Asn Ser Thr Thr Ser Val 580 585 590 590 Glu Asp Leu Val Arg Ile Arg Phe Ser Cys Asn Arg Ser Lys Pro 595 600 605

[Brief description of the drawings]

FIG. 1 is a diagram showing an outline of an RT-PCR assay.

FIG. 2 is a view showing the results of electrophoresis showing a restriction enzyme cleavage pattern of a CDVH gene digested with Fba I.

3 is a diagram showing the results of electrophoresis showing the restriction enzyme cleavage pattern of CDVH gene digested with Nde I.

FIG. 4 is a diagram showing a phylogenetic tree obtained as a result of phylogenetic analysis of the amino acid sequence of the coding region of the CDVH protein.

Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat II (reference) (C12N 15/09 ZNA C12R 1:92) F term (reference) 4B024 AA01 AA14 BA32 CA02 CA12 DA02 DA06 EA04 GA11 4B065 AA26X AA95Y AB01 BA02 CA24 CA43 CA46 4C085 AA03 BA60 CC08 DD62 EE01 4H045 AA10 BA10 CA10 DA86 EA20 EA31 EA53 FA74

Claims (4)

[Claims] 1. A nucleotide sequence encoding the amino acid sequence of the H protein shown in SEQ ID NO: 1 in the sequence listing, or a polypeptide having an amino acid sequence homology of 95% or more with the amino acid sequence, and a restriction enzyme Nde I Or Psh B
An H gene of canine distemper virus, which has a nucleotide sequence from which two fragments can be obtained by digestion with I. 2. The first amino acid sequence of the H protein.
No. 9 to No. 21, No. 149 to No. 151, No. 309 to No. 311, No. 391 to No. 393, No. 422 to No.
The canine distemper virus according to claim 1, which has a sugar chain binding site at positions 424, 456 to 458, 587 to 589, and 603 to 605. H gene. 3. A canine distemper virus comprising the H gene according to claim 1 or 2. 4. The amino acid sequence of the H protein shown in SEQ ID NO: 1 in the sequence listing, or has an amino acid sequence homology of 95% or more with the amino acid sequence,
No.-No. 21, No. 149-No. 151, No. 309-
No. 311, No. 391 to No. 393, No. 422 to No. 424, No. 456 to No. 458, No. 587 to No. 5
An H protein of canine distemper virus, which has an amino acid sequence having a sugar chain binding site at positions 89 and 603 to 605.