Title: Fused protein, gene
therefor, recombinant vector, recombinant virus, and its use
United States Patent: 7,348,422
Issued: March 25, 2008
Inventors: Saitoh; Shuji
(Kawasaki, JP), Tsuzaki; Yoshinari (Kawasaki, JP), Yanagida; Noboru
Assignee: Zeon Corporation
Appl. No.: 09/147,052
Filed: March 28, 1997
PCT Filed: March 28, 1997
PCT No.: PCT/JP97/01084
April 05, 1999
PCT Pub. No.: WO97/36924
PCT Pub. Date: October 09,
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A DNA coding for a fusion protein
comprising a polypeptide having the antigenicity of Mycoplasma
gallisepticum and a polypeptide derived from Herpesvirus outer membrane
protein, in which the polypeptide derived from the outer membrane protein
has been ligated with the polypeptide having the antigenicity of
Mycoplasma gallisepticum at the N terminus thereof, is prepared. The DNA
is inserted into a region non-essential to growth of Avipox virus and the
resulting recombinant Avipox virus is provided as a more potent
recombinant virus as an anti-Mycoplasma vaccine.
Description of the
DISCLOSURE OF THE INVENTION
Under the situation of the prior art stated above, the present inventors
have made extensive studies to provide a recombinant virus capable of
expressing a Mycoplasma antigenic protein having an enhanced infection
prevention activity in large quantities, which allows a host to recognize
the antigen highly efficiently. As a result, it has been found that by
infecting to a host a recombinant Avipox virus, in which a hybrid DNA
obtained by ligating a DNA of the outer membrane protein of the genus
Herpesvirus with a DNA of the antigenic protein of Mycoplasma has been
inserted, the antigen recognizing ability of the host can be markedly
improved. The present invention has thus been accomplished.
Accordingly, the present invention provides:
a fusion protein comprising a polypeptide having the antigenicity of
Mycoplasma gallisepticum (hereinafter sometimes referred to as Mycoplasma-derived
polypeptide) and a polypeptide derived from the outer membrane protein of a
herpes virus (hereinafter sometimes referred to as Herpesvirus-derived
polypeptide) characterized in that the polypeptide derived from outer
membrane protein is ligated with the polypeptide having the antigenicity of
Mycoplasma gallisepticum at the N terminus thereof;
a hybrid DNA coding for the fusion protein;
a recombinant Avipox virus in which the hybrid DNA has been incorporated;
a live vaccine comprising the recombinant Avipox virus as an effective
BEST MODE FOR PRACTICING THE INVENTION
Mycoplasma-derived Polypeptides and Genes Therefor
In the present invention, the term Mycoplasma-derived polypeptides is used
to mean the antigenic proteins that cause an antigen-antibody reaction with
MG immune serum or MG infected serum and that are derived from MG. These
polypeptides are not restricted to proteins per se that native Mycoplasma
gallisepticum expresses, and may include modified polypeptides. For example,
one or more amino acids of the polypeptides may be modified naturally or
artificially in a conventional manner such as site-specific mutation, etc. (JPB
6-16709, etc.) through loss, addition, insertion, deletion, substitution,
etc. Of course, the proteins, even after such modification, should contain
the epitope showing the antigenicity. For determination of the epitope
region, there are available known methods based on the peptide scanning
technique such as the method of Geysen et al. (J. Immunol. Meth., 102,
259-274 (1987)), the method of Hopp et al., (Proc. Natl. Acad. USA, 78,
3824-3828 (1981)), the method Chou et al. (Advances in Enzymology, 47,
145-148 (1987)), etc.
Specific examples of the peptides having the antigenicity include antigenic
proteins disclosed in JPA 2-111795 (U.S. patent application Ser. Nos.
359,779, 07/888,320 and 08/299,662), JPA 5-824646 (U.S. Pat. No. 5,489,430),
WO 94/23019 (U.S. patent application Ser. No. 08/525,742, JPA 6-521927) and
proteins of Mycoplasma gallisepticum containing the amino acid sequences of
those proteins. Of course, so long as the epitope is contained therein, a
part of the peptides described above may also be usable.
Of these peptides, preferred are the polypeptide of about 40 killodaltons (kd)
described in JPA 5-824646, the polypeptide of about 66 kd encoded by TM-66
gene and the polypeptide of about 67 kd encoded by TM-67 gene described in
JPA 5-521927, which are designated as SEQ NO: 16 and SEQ NO: 27 therein.
In the present invention, genes of the Mycoplasma-derived polypeptides bear
DNA sequences coding for the polypeptide having the antigenicity of
Mycoplasma gallisepticum described above. Such DNA can be obtained by
synthesis or acquired from wild bacteria belonging to Mycoplasma
gallisepticum. Specific examples of such bacteria are strains R, S6, KP-13,
PG31, etc. DNA may also be derived from MG isolated from wild strains. These
genes can also be modified by loss, addition, insertion, deletion,
substitution, etc. in a conventional manner as described in Methods in
Herpesvirus-derived Polypeptides and Genes Thereof
The Herpesvirus-derived polypeptides in the present invention refer to
polypeptides derived from proteins that construct an envelope of viruses
belonging to the genus Herpesvirus. The Herpesvirus-derived polypeptides may
not always be the full length of the proteins. Where the polypeptides are
used solely to be expressed on the surface of cell membranes function as
fusion proteins, it is sufficient for the polypeptide to contain a membrane
anchor and a signal sequence therein, and where the polypeptides are
employed for secretion, the polypeptides may contain only a signal sequence
for that purpose. The outer membrane proteins may be either type I or type
II of the outer membrane proteins. The signal sequence and the membrane
anchoring sequence are both readily detectable by analyzing the amino acid
sequence in the hydrophobic peptide region at the carboxyl terminus or amino
Specific examples of the outer membrane protein include gB, gC, gD, gH and
gI which are glycoproteins of herpes simplex viruses, and gBh, gCh, gDh, gHh
and gIh of MDV corresponding to herpes simplex viruses glycoproteins gB, gC,
gD, gH and gI, and proteins of the genus Herpesvirus homologous to the
proteins described above.
Of course, polypeptides bearing the epitope other than the signal sequence
of the outer membrane proteins may also be ligated with the aforesaid
polypeptides having the antigenicity. By the ligation it is expected that
the epitope will give the immunity to the living body in vivo.
In the present invention, the genes for the Mycoplasma-derived polypeptides
contain DNA sequences coding for the Herpesvirus-derived polypeptides
described above and such DNAs can be synthesized or acquired from naturally
occurring herpes viruses. These genes may also be modified by loss,
addition, insertion, deletion, substitution, etc. in a conventional manner
as described in Methods in Enzymology, etc.
Fusion Protein and Hybrid DNA
The fusion proteins of the present invention are obtained by incubating a
recombinant Avipox virus inserted hydrid DNA, which will be later described,
in culture cells such as chick embryo fibroblast cells (hereinafter referred
to as CEF cells) or embryonated chorioallantoic membrane cells, etc.
The thus obtained fusion proteins can be employed as a component vaccine.
The hybrid DNA of the present invention comprises the gene for the
Mycoplasma-derived polypeptide and the gene for the Herpesvirus-derived
polypeptide, which are ligated with each other directly or via an optional
The hybrid DNA of the present invention can be produced in a conventional
manner, for example, by a method in which the outer membrane protein and the
antigenic protein of Mycoplasma gallisepticum are digested with restriction
enzymes, respectively, and the resulting ligatable DNA fragment coding for
the outer membrane protein of herpes viruses or for the signal sequence of
the outer membrane protein is ligated with the resulting ligatable DNA
fragment coding for the antigenic protein of Mycoplasma gallisepticum, using
a ligase directly or via an appropriate linker.
Specific examples of the amino acid sequences for the fusion proteins of the
present invention include SEQ ID NO: 2 and SEQ ID NO: 4. The sequence of the
antigenic protein of 40 killodaltons derived from Mycoplasma gallisepticum
is found in amino acids 64-456 of SEQ ID NO: 2 and in amino acids 693-1086
of SEQ ID NO: 4. The signal sequence of outer membrane protein gB derived
from MDV is found in amino acids 1-63 of SEQ ID NO: 2. In SEQ ID NO: 4,
amino acids 1-672 correspond to almost the full length of outer membrane
protein gB derived from MDV. Specific examples of nucleotide sequences of
the hybrid DNAs coding for these fusion proteins are those shown by SEQ ID
NO: 1 and SEQ ID NO: 3.
These fusion proteins and hybrid DNAs are given by way of examples but are
not deemed to be limited thereto.
Recombinant Avipox Virus
The recombinant Avipox virus of the present invention is a recombinant
Avipox virus in which the aforesaid DNA or hybrid DNA has been inserted in
the non-essential region. The recombinant Avipox virus of the present
invention can be constructed in a conventional manner, e.g., by the method
described in Japanese Patent Application Laid-Open No. 1-168279. That is,
the non-essential region of Avipox virus is incorporated into a DNA fragment
to construct a first recombinant vector.
As the non-essential region of Avipox virus which is used in the present
invention, there are a TK gene region of quail pox virus, a TK region of
turkey pox virus and DNA fragments described in JPA 1-168279, preferably a
region which causes homologous recombination with EcoRI fragment of about
7.3 Kb, HindIII fragment of about 5.2 Kb, EcoRI-HindIII fragment of about
5.0 Kb, BamHI fragment of about 4.0 Kb, described in the patent
Examples of the vector used in the present invention include plasmids such
as pBR322, pBR325, pBR327, pBR328, pUC7, pUC8, pUC9, pUC18, pUC19, and the
like; phages such as .lamda. phage, M13 phage, etc.; cosmid such as pHC79
and the like.
The Avipox virus used in the present invention is not particularly limited
so long as it is a virus infected to avian. Specific examples of such a
virus include pigeon pox virus, fowl pox virus (hereafter abbreviated as FPV),
canary pox virus, turkey pox virus, preferably pigeon pox virus, FPV and
turkey pox virus, more preferably pigeon pox virus and FPV. Specific
examples of the most preferred Avipox virus include FPVs such as ATCC
VR-251, ATCC VR-249, ATCC VR-250, ATCC VR-229, ATCC VR-288, Nishigahara
strain, Shisui strain, CEVA strain and a viral strain among CEVA
strain-derived viruses which forms a large plaque when infected to chick
embryo fibroblast, and a virus such as NP strain (chick embryo-attenuated
pigeon pox virus Nakano strain), etc. which is akin to FPV and used as a
fowlpox live vaccine strain. These strains are commercially available and
Next, the hybrid DNA of the present invention is inserted into the
non-essential region of the first recombinant vector described above to
construct a second recombinant vector. In general, the hybrid DNA employed
may have any nucleotide sequence, irrespective of synthetic or natural one,
so long as the hybrid DNA effectively functions as a promoter in the system
of transcription possessed by Avipox viruses. Accordingly, not only
promoters inherent to Avipox viruses such as promoters for Avipox
virus-derived genes coding for thymidine kinase but also DNAs derived from
viruses other than Avipox viruses and DNAs derived from eukaryotes or
prokaryotes may also be employed in the present invention, insofar as these
substances meet the requirements described above. Specific examples of such
promoters include promoters for vaccinia viruses (hereinafter often referred
to as VV) as described in Journal of Virology, 51, 662-669 (1984), more
specifically, a promoter of VV gene coding for 7.5 K polypeptide, a promoter
of VV gene coding for 19 K polypeptide, a promoter of VV gene coding for 42
K polypeptide, a promoter of VV gene coding for thymidine kinase, a promoter
of VV gene coding for 28 K polypeptide, etc. Furthermore, there may be used
a synthetic promoter obtained by modification of the Moss et al. method (J.
Mol. Biol., 210, 49-76 and 771-784, 1989), Davidson's synthetic promoter, a
promoter obtained by modifying a part of the Davidson's promoter through
deletion or change in such a range that the promoter activity is not lost
TTGAAAAACTATTCTAATTTATTGCACTC SEQ ID NO: 5,
AAAAATTGAAAAACTATTCTAATTTATTGCACTC SEQ ID NO. 6 etc.).
Further in view of easy detection of the recombinant virus, a marker gene
such as a DNA coding for .beta.-galactosidase may also be inserted.
The recombinant Avipox virus can be constructed by transfecting the second
recombinant vector described above to animal culture cells, which has been
previously infected with Avipox virus, and causing homologous recombination
between the vector DNA and the viral genome DNA. The animal culture cells
used herein can be any cells, so long as Avipox can grow in the cells.
Specific examples of such animal culture cells are CEF cells, embryonated
egg chorioallantoic membrane cells, and the like.
The objective recombinant Avipox virus is isolated from the virus infected
to host cells by plaque hybridization, etc.
The recombinant virus of the present invention constructed by the method
described above can be inoculated to avian as a live vaccine for Mycoplasma
The live vaccine of the present invention is prepared by, e.g., the
following method, though the process is not particularly limited thereto.
The recombinant virus of the present invention is infected to cells in which
the virus can grow (hereafter referred to as host cells). After the
recombinant virus grows, the cells are recovered and homogenated. The
homogenate is centrifuged to separate into the precipitates and the high
titer supernatant containing the recombinant virus. The resulting
supernatant is substantially free of host cells but contains the cell
culture medium and the recombinant virus and hence can be used as a live
vaccine. The supernatant may be diluted by adding a pharmacologically inert
carrier, e.g., physiological saline, etc. The supernatant may be
freeze-dried to be provided for use as a live vaccine. A method for
administration of the live vaccine of the present invention to fowl is not
particularly limited and examples of the administration include a method for
scratching the skin and inoculating the live vaccine on the scratch,
effecting the inoculation through injection, oral administration by mixing
the live vaccine with feed or drinking water, inhalation by aerosol or
spray, etc. In order to use as the live vaccine, the dosage may be the same
as ordinary live vaccine; for example, approximately 10.sup.2 to 10.sup.8
plaque forming unit (hereinafter abbreviated as PFU) is inoculated per
chick. Where the inoculation is effected by injection, the recombinant virus
of the present invention is generally suspended in about 0.1 ml of an
isotonic solvent such as physiological saline and the resulting suspension
is provided for use. The live vaccine of the present invention can be
lyophilized under ordinary conditions and can be stored at room temperature.
It is also possible to freeze the virus suspension at -20 to -70.degree. C.
and store the frozen suspension.
Particularly where the genes coding for the polypeptides derived from the
outer membrane proteins of herpes viruses described above are those coding
for polypeptides having more than one epitope of herpes viruses, preferably
having at least 90% homology to native outer membrane proteins, the live
vaccine of the present invention functions as a vaccine for both Mycoplasma
gallisepticum infection and Avipox viral infection. In addition, the live
vaccine of the present invention can also function as an effective vaccine
for infection with herpes virus originating from outer membrane proteins.
That is, the live vaccine of the present invention can be used as a
so-called trivalent vaccine.
Claim 1 of 6 Claims
1. A DNA molecule having a nucleotide
sequence coding for a polypeptide having the amino acid sequence set forth
in SEQ ID NO: 2.
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