Title 透明帯糖蛋白質合成ペプチドを抗原としたエゾシカ避妊ワ クチン

Title
透明帯糖蛋白質合成ペプチドを抗原としたエゾシカ避妊ワ
クチン開発に関する研究( 要約版(Digest) )
Author(s)
小林, 恒平
Report No.(Doctoral
Degree)
博士(獣医学) 甲第414号
Issue Date
2014-03-13
Type
博士論文
Version
none
URL
http://repository.lib.gifu-u.ac.jp/handle/123456789/49037
※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
Ꮫ ఩ ㄽ ᩥ せ ⣙
Ặ ྡ ᑠᯘ ᜏᖹ
㢟 ┠㏱᫂ᖏ⢾⺮ⓑ㉁ྜᡂ࣌ࣉࢳࢻࢆᢠཎ࡜ࡋࡓ࢚ࢰࢩ࢝㑊ዷ࣡
ࢡࢳࣥ㛤Ⓨ࡟㛵ࡍࡿ◊✲
㏆ᖺ໭ᾏ㐨࡟࠾࠸࡚㸪࢚ࢰࢩ࢝ಶయᩘࡢቑຍࡀ♫఍ⓗၥ㢟࡜࡞ࡗ࡚࠸ࡿࠋ࢚
ࢰࢩ࢝ၥ㢟ࡣ㎰ᯘᴗ⿕ᐖ㸪⏕ែᏛⓗ⿕ᐖ㸪஺㏻஦ᨾ࡞࡝ከᵝ࡞ഃ㠃ࢆᣢࡗ࡚࠾
ࡾ㸪࠸ࡎࢀࡶ῝้࡞≧ែ࡛࠶ࡿࠋ⾜ᨻࡣ㥑㝖ࢆ୺య࡜ࡋࡓᑐ⟇ࢆㅮࡌ࡚࠸ࡿࡀ㸪
ࣁࣥࢱ࣮ࡢ୙㊊ࡸ࢚ࢰࢩ࢝ࡢᏛ⩦࡟ࡼࡿ㥑㝖ຠ⋡ࡢపୗ࡞࡝࠿ࡽ┠ᶆࡢᤕ⋓㢌
ᩘ࡟⮳ࡽࡎ㸪౫↛࡜ࡋ࡚῝้࡞⿕ᐖࡀᣢ⥆ࡋ࡚࠸ࡿࠋ࢚ࢰࢩ࢝ࡣ෤Ꮨࡢ኱㔞Ṛ
࡜㸪ࡑࢀ࡟ぢྜ࠺᪲┒࡞⦾Ṫຊࡢ㸰⪅ࡢࣂࣛࣥࢫࡀಖࡓࢀࡿ஦࡛ಶయᩘࡀಖࡓ
ࢀ࡚ࡁࡓࠋࡋ࠿ࡋ㏆ᖺ㸪෤Ꮨࡢ㣵ࡸ㉺෤ᆅࡀቑ࠼㸪Ṛஸᩘࡀῶࡗࡓࡇ࡜࡟ࡼࡾ㸪
⌧ᅾࡢࡼ࠺࡞⇿Ⓨⓗ࡞ቑຍ࡟⮳ࡗ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋ⌧ᅾ⾜ࢃࢀ࡚࠸ࡿᤕ⋓
ࡣ⎔ቃせᅉ࡟ᑐࡍࡿᑐ⟇࡛࠶ࡿࡀ㸪ᢪ࠼࡚࠸ࡿㄢ㢟ࡶከ࠸ࠋࡑࡇ࡛⌧⾜ࡢᑐ⟇
࡜㑊ዷ࣡ࢡࢳࣥࢆ⏝࠸ࡓ⦾Ṫᢚไ࡟ࡼࡿ⏕⌮ⓗせᅉᑐ⟇࡜ྜࢃࡏࡿ஦࡛ࡼࡾຠ
⋡ⓗ࡞ಶయᩘ⟶⌮ࡀྍ⬟࡟࡞ࡿ࡜⪃࠼ࡽࢀࡿࠋ
㑊ዷ࣡ࢡࢳࣥ࡟㛵ࡍࡿ◊✲ࡣᾏእ࡟࠾࠸࡚ࡣከࡃ⾜ࢃࢀ࡚࠾ࡾ㸪⢭〇ࡋࡓࣈ
ࢱ㏱᫂ᖏ⢾ࢱࣥࣃࢡ㉁ࡣከࡃࡢື≀✀࡟࠾࠸࡚㑊ዷຠᯝࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡋ
࠿ࡋ㸪ᮏ◊✲࡛ࡣ㸪ಶయᩘ⟶⌮࡟ྥࡅࡓ㔝እ㐺⏝ࢆ๓ᥦ࡜ࡋ࡚࠸ࡿࡓࡵ㸪ᤕ⋓
ࡋ࡚ᢞ୚ࡍࡿ࣡ࢡࢳ࡛ࣥࡣ࡞ࡃ⢓⭷ᢞ୚➼࡛ከᩘࡢಶయ࡟ຠ⋡ⓗ࡟ᢞ୚ࡍࡿᚲ
せࡀ࠶ࡾ㸪ࡑࢀࡽࡢ◊✲ࢆࡑࡢࡲࡲ㐺⏝ࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋࡉࡽ࡟ከᩘಶయ
࡬ࡢᢞ୚࡟㝿ࡋ࡚ᑐ㇟࡜ࡋ࡞࠸ື≀࡬ࡢᙳ㡪ࢆ᭱ᑠ㝈࡟ࡋ࡞ࡃ࡚ࡣ࡞ࡽࡎ㸪✀
≉␗ᛶࡢ㧗࠸ᢠཎࢆ⏝࠸ࡿᚲせࡀ࠶ࡿࠋࡑࡇ࡛ᮏ◊✲࡛ࡣ㸪ᢠཎࡢ⠊ᅖࢆᑠࡉ
ࡃࡍࡿࡇ࡜࡟ࡼࡿ✀≉␗ᛶࡢ☜ಖ࠾ࡼࡧ⢓⭷ᢞ୚ᆺ࣡ࢡࢳࣥ࡬ࡢⓎᒎᛶࢆ⪃៖
ࡋ࡚ྜᡂ࣌ࣉࢳࢻࢆᢠཎ࡜ࡋ࡚㸪㏱᫂ᖏ⢾ࢱࣥࣃࢡ㉁ࡢ୰࡛㧗࠸✀≉␗ᛶࢆ᭷
ࡋ࠿ࡘ㑊ዷຠᯝࢆࡶࡓࡽࡍ㡿ᇦࢆ㑅ᐃࡋ㸪㑊ዷ࣡ࢡࢳࣥ࡜ࡋ࡚ࡢ᭷⏝ᛶࢆ᳨ウ
ࡍࡿࡇ࡜࡜ࡋࡓࠋ
➨㸰❶࡛ࡣ㸪ࣈࢱ㏱᫂ᖏ⢾⺮ⓑ㉁ =3% ⣽⬊࢚ࣆࢺ࣮ࣉ࡟ᑐࡍࡿ࢚ࢰࢩ࢝࡟࠾
ࡅࡿᢠయ⏘⏕࠾ࡼࡧᢠయࡢᶵ⬟ࢆ᳨ウࡋࡓࠋࣈࢱ㏱᫂ᖏ⢾⺮ⓑ㉁ࡣ =3㸪=3㸪
=3 ࡟ศࡅࡽࢀࡑࡢ࠺ࡕ =3 ࡢ % ⣽⬊࢚ࣆࢺ࣮ࣉ㓄ิࡀ᫂ࡽ࠿࡟࡞ࡗ࡚࠾ࡾ㸪࢜
ࢪࣟࢪ࢝࡟࠾࠸࡚ࡑࡢ㓄ิ࡟ᇶ࡙࠸ࡓྜᡂ࣌ࣉࢳࢻ࡛ᢠయ౯ࡢୖ᪼ࡀㄆࡵࡽࢀ
ࡓሗ࿌ࡀ࠶ࡿࠋࡋ࠿ࡋࡑࡢ◊✲࡛ࡣᢠయࡢ⤖ྜ࡞࡝ࡑࡢᚋࡢゎᯒࡣࡋ࡚࠾ࡽࡎ㸪
ྜᡂ࣌ࣉࢳࢻࡢ㑊ዷຠᯝࡣ☜࠿ࡵࡽࢀ࡚࠸࡞࠸ࠋࡑࡇ࡛ᮏ◊✲࡛ࡣ㸪ࣈࢱࡢ =3%
⣽⬊࢚ࣆࢺ࣮ࣉ㓄ิ࡟ᇶ࡙ࡃྜᡂ࣌ࣉࢳࢻࡢ࢚ࢰࢩ࢝࡟ᑐࡍࡿᢠཎ࡜ࡋ࡚ࡢ᭷
⏝ᛶࢆ᳨ウࡍࡿࡓࡵ㸪㣫⫱ୗࡢ࢚ࢰࢩ࢝㸰㢌࡟࢔ࢪࣗࣂࣥࢻ࡜ඹ࡟ᢞ୚ࡋ㸪ᢠ
య౯ࡢ᥎⛣㸪ᚓࡽࢀࡓᢠయࡢᶵ⬟᳨ᰝ㸪஺㓄࠾ࡼࡧዷፎデ᩿ࢆ⾜ࡗࡓࠋࡑࡢ⤖
ᯝ㸪ᢠయ౯ࡢⴭࡋ࠸ୖ᪼ࡀㄆࡵࡽࢀࡓࡀ㸪ࡑࡢᢠయࡣ㸪ࣈࢱ㏱᫂ᖏ࡟ࡣ⤖ྜࡋ
ࡓࡀ㸪࢚ࢰࢩ࢝㏱᫂࡟ࡣ⤖ྜࡋ࡞࠿ࡗࡓࠋࡲࡓ㸪 㢌ࡢᢞ୚ಶయࡢ࠺ࡕ 㢌࡛ዷ
ፎࡀㄆࡵࡽࢀࡓࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࣈࢱ =3㸯% ⣽⬊࢚ࣆࢺ࣮ࣉ࡜࢚ࢰࢩ࢝ࡢ࢚
ࣆࢺ࣮ࣉࡢ㓄ิࡀ␗࡞ࡗ࡚࠸ࡿ࡜⪃࠼ࡽࢀ㸪✀≉␗ᛶࡀ♧၀ࡉࢀࡓࠋ
➨㸱❶࡛ࡣ㸪࢚ࢰࢩ࢝㏱᫂ᖏ⢾⺮ⓑ㉁ % ⣽⬊࢚ࣆࢺ࣮ࣉ࡟ᑐࡍࡿᢠయࡢ㏱᫂
ᖏ࡬ࡢ⤖ྜ࡜ࡑࡢ✀≉␗ᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ࢚ࢰࢩ࢝ࡢ =3 ࡢ㑇ఏᏊ㓄ิࡢ
ྠᐃࢆ⾜࠸㸪࢚ࢰࢩ࢝࡟࠾ࡅࡿ =3% ⣽⬊࢚ࣆࢺ࣮ࣉ㡿ᇦࡢ࢔࣑ࣀ㓟㓄ิ࡟ᇶ࡙
ࡃྜᡂ࣌ࣉࢳࢻࢆసᡂࡋࡓࠋྜᡂ࣌ࣉࢳࢻࢆ࢘ࢧࢠ࡟ච␿ࡋ࡚సᡂࡉࢀࡓᢠయ
ࢆ⏝࠸࡚ච␿ᰁⰍ࠾ࡼࡧయእཷ⢭㜼ᐖヨ㦂ࢆ⾜࠸㸪ᢠయࡢᶵ⬟࠾ࡼࡧ✀≉␗ᛶ
ࢆㄪ࡭ࡓࠋࡑࡢ⤖ᯝ㸪ᢠయࡣ࢚ࢰࢩ࢝㏱᫂ᖏࢆ✀≉␗ⓗ࡟ㄆ㆑ࡋ㸪ࣈࢱ࠾ࡼࡧ
࢘ࢩࡢ㏱᫂ᖏࡣㄆ㆑ࡋ࡞࠿ࡗࡓࠋࡑࡢࡇ࡜࠿ࡽྜᡂ࣌ࣉࢳࢻࢆᢠཎ࡜ࡋ࡚ᶆⓗ
ࢆ୍ࡘࡢ࢚ࣆࢺ࣮ࣉ࡟ࡲ࡛⤠ࡾ㎸ࡴࡇ࡜࡛㸪✀≉␗ⓗ࡟㏱᫂ᖏࢆㄆ㆑ࡍࡿᢠయ
ࡀᚓࡽࢀࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋ
➨㸲❶࡛ࡣ㸪࢚ࢰࢩ࢝࡟ᑐࡍࡿ࢚ࢰࢩ࢝㏱᫂ᖏ⢾⺮ⓑ㉁ % ⣽⬊࢚ࣆࢺ࣮ࣉྜ
ᡂ࣌ࣉࢳࢻᢞ୚࡟ࡼࡿᢠయ⏘⏕࠾ࡼࡧᢠయࡢᶵ⬟ࢆ᳨ウࡋࡓࠋࡑࡇ࡛㸪⮬ᕫᢠ
ཎ࡛࠶ࡿ࢚ࢰࢩ࢝ =3% ⣽⬊࢚ࣆࢺ࣮ࣉྜᡂ࣌ࣉࢳࢻࢆᢞ୚ࡍࡿࡇ࡜࡛࢚ࢰࢩ࢝
࡟࠾࠸࡚ࡶ᭷ຠ࡞ᢠయ⏘⏕ࡀᚓࡽࢀࡿࡇ࡜ࢆ☜࠿ࡵࡿࡓࡵ㸪㣫⫱ୗ࢚ࢰࢩ࢝ࢆ
⏝࠸ࡓච␿ヨ㦂࠾ࡼࡧᚓࡽࢀࡓᢠయࢆ⏝࠸ࡓච␿ᰁⰍࢆ⾜ࡗࡓࠋࡑࡢ⤖ᯝ㸪ᢠ
యࡣ࢚ࢰࢩ࢝㏱᫂ᖏࢆ✀≉␗ⓗ࡟ㄆ㆑ࡋ㸪ࣈࢱ࠾ࡼࡧ࢘ࢩࡢ㏱᫂ᖏࡣㄆ㆑ࡋ࡞
࠿ࡗࡓࠋࡑࡢࡇ࡜࠿ࡽ㸪⮬ᕫᢠཎࡢ࢔࣑ࣀ㓟㓄ิ࡟ᇶ࡙ࡃྜᡂ࣌ࣉࢳࢻ࡛࠶ࡗ
࡚ࡶ㸪࢚ࢰࢩ࢝࡟࠾ࡅࡿᢠయ⏘⏕ࢆㄏᑟࡋ㸪ࡑࡢᢠయࡀ࢚ࢰࢩ࢝㏱᫂ᖏࢆ✀≉
␗ⓗ࡟ㄆ㆑ࡍࡿࡇ࡜ࡀ᫂ࡽ࠿࡟࡞ࡗࡓࠋ
ᮏ◊✲࡟ࡼࡾ㸪࢚ࢰࢩ࢝࡟≉␗ⓗ࡞㑊ዷ࣡ࢡࢳࣥᢠཎࡢೃ⿵ࡀ㑅ᐃࡉࢀࡓࠋ
ᮏ◊✲࡛⏝࠸ࡓ᪉ἲ㸪ࡍ࡞ࢃࡕ㸪㸯㸧㏱᫂ᖏ⢾⺮ⓑ㉁ࡢ㑇ఏᏊ㓄ิࢆゎㄞࡋ㸪
ࡑࡢ୰࡛࢚ࣆࢺ࣮ࣉ࡜࡞ࡗ࡚࠸ࡿ㡿ᇦࡢ㓄ิࢆ௚✀ື≀࡟࠾ࡅࡿ┦ྠ㡿ᇦࡢ㓄
ิ࡜ẚ㍑ࡋ㸪✀≉␗ⓗ࡞࢚ࣆࢺ࣮ࣉ㡿ᇦࢆ᥎ᐃࡍࡿ㸪㸰㸧ࡑࡢ࢚ࣆࢺ࣮ࣉ㡿ᇦ
ࡢ࢔࣑ࣀ㓟㓄ิ࡟ᇶ࡙࠸࡚ྜᡂ࣌ࣉࢳࢻࢆసᡂࡋච␿ࡍࡿࡇ࡜࡛ᚓࡽࢀࡓᢠయ
ࢆ⏝࠸࡚㸪ᑐ㇟✀ࡸ௚✀ࡢ༸ᕢࢆ⏝࠸ࡓච␿ᰁⰍ࠾ࡼࡧయእཷ⢭㜼ᐖヨ㦂ࢆ⾜
࠸㸪ᢠయࡢ✀≉␗ᛶ࠾ࡼࡧᶵ⬟ࢆ᳨ᰝࡍࡿ㸪࡜࠸࠺ᢠཎ㡿ᇦࡢ㑅ᐃἲࡣ㸪㑊ዷ
࣡ࢡࢳࣥ㛤Ⓨࡀᮃࡲࢀࡿ௚ࡢື≀✀࡟ࡶᛂ⏝ྍ⬟࡞᪉ἲ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ
Ꮫ ఩ ㄽ ᩥ せ ⣙
Ặ ྡ
KOBAYASHI, Kohei
㢟 ┠
Studies on Development of Immunocontraceptive Vaccine
using Zona Pellucida Synthetic Peputide Antigen in
the Hokkaido Sika Deer (Cervus nippon yesoensis)
㸦㏱᫂ᖏ⢾⺮ⓑ㉁ྜᡂ࣌ࣉࢳࢻࢆᢠཎ࡜ࡋࡓ࢚ࢰࢩ࢝
㑊ዷ࣡ࢡࢳࣥ㛤Ⓨ࡟㛵ࡍࡿ◊✲㸧
Overabundance of the Hokkaido sika deer (Cervus nippon yesoensis) causes many
problems, including agricultural, forestry, and ecological damage. The Japanese
government has implemented deer management plans focused on shooting, but the
efficiency of this method is inadequate and the problem remains serious.
The
development of new methods for deer population control is imperative. Thus, a form of
management that incorporates both shooting and reproductive control should be
considered as an effective tool for population control.
Immunocontraception is a method of inhibiting reproduction through the animal’s own
immune system. In previous studies, anesthesia or immobilization was required when
the immunocontraceptive vaccine was administered to wildlife. Since capture and
immobilization for administration of immunocontraceptive vaccines in free-ranging
populations requires much effort and high cost, the development for an effective method,
that allows administration without anesthesia, is necessary for population control of
wildlife. When an oral immunocontraceptive vaccine is used, both target species and
non-target species can ingest the vaccine and become vaccinated.
So, non-target
species may vaccinated with oral vaccine. Therefore, ensuring species specificity of the
vaccine to avoid affecting non-target species is a key priority in developing a
immunocontraceptive vaccine in free-ranging wildlife.
The synthetic peptide antigen is candidate of immunocontraceptive antigen, because it
is easy to make, and has the potential for oral administration. Antibody production
against a synthetic peptide based on the B-cell epitope of porcine zona pellucida (PZP),
which has the potential to inhibit fertilization, has been reported in white-tailed deer.
However, its ability to bind to zona pellucida and its species specificity remain
unknown. In this study, the potential of synthetic peptide antigen for species specific
immuncontraception is tested.
In Chater 2, vaccine trial with the synthetic peptide based on the amino acid sequence
of the B-cell epitope of PZP was conducted to elucidate antibody production against the
synthetic peptide in the Hokkaido sika deer and the ability of the antibody to bind to the
ZP. Production of antibodies that bind to PZP was observed. However, the antibody
produced in the Hokkaido sika deer could bind to porcine ZP but not bind to deer ZP. In
addition, the pregnancy of immunized deer was observed. Thus, the present results
suggest that the B-cell epitope of ZP that has a role in fertilization is different between
porcine and the Hokkaido sika deer. This finding suggests the potential for the
development of species specific immunocontraceptive vaccine using the synthetic
peptide based on the amino acid sequence of B-cell epitope.
In Chapter 3, the DNA sequence of the Hokkaido sika deer zona pellucida and the
difference between porcine and sika deer was revealed. Sequence of deer DNA of the
area in which used for synthetic peptide in vaccine trial showed 88% homology to
porcine. The synthetic peptide based on amino acid sequence of B-cell epitope of sika
deer ZP1 was vaccinated in rabbit. The ability of the antibody to bind zona pellucida
and species specificity was examined by immunohistochemistry using porcine, bovine,
and deer ovary. Antibody against the synthetic peptide based on the B-cell epitope in
sika deer ZP was not able to bind porcine and bovine ZP. In contrast, the antibody was
able to bind sika deer ZP. These results suggest the species specificity of the antibody
against the synthetic peptide based on the amino acid sequence of the B-cell epitope of
ZP. Therefore, the synthetic peptide based on the amino acid sequence of the B-cell
epitope which is different between species seems to be a target of species specific
immunocontraceptive vaccine.
In Chapter 4, the synthetic peptide based on the amino acid sequence of sika deer ZP
B-cell epitope was vaccinated on three captive sika deer to examine the production and
function of the antibody against the synthetic peptide based on auto-antigen. In the
result, the antibody production was observed. The ability to bind sika deer ZP and
species specificity was same as antibody from rabbit immunized in Chapter 3. Therefore,
the synthetic peptide based on the amino acid sequence of sika deer ZP B-cell epitope
was evaluated as candidate antigen of sika deer specific immunocontraceptive vaccine.
In addition, the finding that the synthetic peptide based on specific auto-antigen is able
to induce the production of the antibody which has ability to bind own ZP is showing
the method of antigen selection for develop species specific immunocontraceptive
vaccine. The function and species specificity of the antibody which is produced in the
Hokkaido sika deer is similar to the antibody which is produced in rabbit. Therefore,
functioning of the antibody obtained from Hokkaido sika deer could be evaluated using
antibody obtained from rabbits immunized with the synthetic peptide.
The present study found that the synthetic peptide based on amino acid sequence of
own ZP seems to be the species-specific antigen using for immunocontraception of the
Hokkaido sika deer. On the bases of the present results, the following is proposed as an
effective method for the generation of a species-specific antigen for the
immunocontraception: 1) designing the synthetic peptide based on the amino acids
sequence of a target antigen that is critical for fertilization; 2) immunization of rabbits
for preliminary evaluation of antibody function and immunohistochemical evaluation of
the ability of the antibody to bind to the zona pellucida, using ovaries of the target
species.