鹿腐蹄病C型节瘤拟杆菌纤毛蛋白基因的克隆及表达产物免疫原性研究
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摘要
纤毛蛋白（pili）是鹿及其它反刍动物腐蹄病主要保护性
抗原。C型节瘤拟杆菌是引起我国鹿、牛、羊等反刍动物腐蹄
病的主要菌型之一。本项研究通过克隆C型节瘤拟杆菌纤毛蛋
白基因，构建了C型节瘤拟杆菌纤毛蛋白基因表达载体，并在
实验动物上初步评价了该基因表达产物纤毛蛋白的免疫原性，
从而为研制生产简便、成本低廉的鹿腐蹄病C型节瘤拟杆菌纤
毛蛋白基因工程疫苗打下了基础，试验结果如下。
从C型节瘤拟杆菌菌株中提取染色体DNA。用PCR技术扩
增出具有免疫保护性抗原基因0.85kb纤毛蛋白基因（pili基
因），利用该基因构建了纤毛蛋白基因表达载体。先将C型节瘤
拟杆菌在厌氧条件下培养72小时；菌数达到1×10~8个/ml以
上时，依据革兰氏阴性杆菌DNA快速提取方法，提DNA；利用
所设计的专一性引物进行PCR，从DNA中扩增出pili基因；将
该基因克隆于T Easy载体系统并进行序列测定，所扩增的pili
基因序列正确；用EcoR1酶切，低熔点胶回收，klenow补平后，
用T_4DNA连接酶将其与经Hpal酶切后去磷酸化的PPLλ中间载
体连接，将 pill 基因克隆于 PPL入载体；经 BamHI，BamHI＋Hind
Ill，Dra酶切鉴定，鉴定出正向插入 PPL入载体的克隆质粒；
扩增 PPL入－Pi i重组质粒，用 B。＊ 切出 2．Ikb大小的片断，
回收该片段，与经BamHI酶切去磷酸化的PME290表达质粒连
接，转化宿主细胞 PAK乃 巳 在营养肉汤中进行 Pi i基因的
表达。培养 18刁 4小时后，培养上清液中加入 0．IM MgC；，离
心提取纤毛蛋白。用羊抗兔C型节瘤拟杆菌抗血清与提取的表
达纤毛蛋白进行对流免疫电泳试验，证明了表达纤毛蛋白的特
异性。用 PAGE电泳和 W既 t盯nhi川证明其为表达的纤毛蛋白。
从而构建了腐蹄病 C型节瘤拟杆菌 Pil基因的表达质粒。
用基因表达的C型节瘤拟杆菌纤毛蛋白与等量弗氏完全佐
剂混合乳化制备C型节瘤拟杆菌纤毛蛋白基因工程疫苗，接种
3只健康家兔；进行了免疫原性试验；并同时用节瘤拟杆菌灭
活菌苗及菌体纤毛蛋白疫苗免疫家兔，进行了免疫比较试验。
将 C型节瘤拟杆菌纤毛蛋白基因工程疫苗接种于含 5 00Pg州
控苇青霉素的营养肉汤培养基中培养；MgC；沉淀法提取纤毛
蛋白，等量弗氏完全佐剂混合免疫健康家兔，分2次进行，问
隔一周，每周采血检测免疫家兔血清中抗体水平。结果纤毛蛋
白基因工程疫苗于7天开始出现血清凝集反应，14天后可达
－2－
．u2000”以上，21天后可达到8000”以上，30天后可达到32000
”以上，而且其高滴度抗体可维持到1N天以上。与前期研究
工作用全菌苗和菌体纤毛蛋白疫苗免疫试验兔相比，C型节瘤
拟杆菌纤毛蛋白基因工程疫苗比其它两种疫苗具有更好的免疫
效果。同时利用C型节瘤拟杆菌菌体裂解抗原与纤毛蛋白基因
工程疫苗免疫家兔抗血清进行对流免疫电泳试验；结果从第7
天开始直到第120天都有明显沉淀线。试验证明C型节瘤拟杆
菌纤毛蛋白基因工程疫苗有良好的免疫原性。
Master Degree Candidate: Miao Liguang
Supervisor: Wang Kejian
D.nodosus pili is the main protective immumogen against footrot
in ruminants.
A plasmid expressing pili gene of D.nodosus serotype C was
constructed by which a
recombinant phi vaccine was developed. The immumogenicity of the
recombinant
pili vaccine was evaluated through experimental animals.
The pili gene that dominates the main protective immunogen was
amplified
and cloned from D.nodosus serorype C by PCR. An expression
plasmid was
constructed by cloning the pili gene into PME29O.The plasmas
harbouring the pili
sequence was designated PME29O-Pili.The PME29O-pili was
transformed into the
host competent cell PAKI2pfs and the recombinant pill was
expressed in the
supernatant of the cultures of the transformant cell PAK]2pfs.
The recombinant pill
was purified by Mgcl2 from the supernatant of the culture of the
transformed
PAKI2pfs. The specific reaction of the recombinant pili and
antiserum of D.nodosus
serotype C pili was demonstrated by cross electrophoresis. The
recombinant pili was
expressed at high level in PAKI2pfs.
Healthy rabbits were inoculated each with vaccine of the
recombinant pill
protein at 250ug per dose to potentiate the immunogenicity.
Rabbit inoculated two
times at one week interval. The blood of each rabbit was
collected at days 0,7, 14,21,
30, 60, 90, 120. The antibody titers were evaluated by
k-agglutination test. The
results showed that the lower agglutination titers were observed
at day 7 and up to
higher levels over 8000xat day 21 induced by the vaccine of
recombinant phi.
Above results suggested that the good immunogenicity of the
recombinant pill
vaccine could match with that previously measured of either the
D.nodosus pill or the killed D.nodosus vaccine.
抗原。C型节瘤拟杆菌是引起我国鹿、牛、羊等反刍动物腐蹄
病的主要菌型之一。本项研究通过克隆C型节瘤拟杆菌纤毛蛋
白基因，构建了C型节瘤拟杆菌纤毛蛋白基因表达载体，并在
实验动物上初步评价了该基因表达产物纤毛蛋白的免疫原性，
从而为研制生产简便、成本低廉的鹿腐蹄病C型节瘤拟杆菌纤
毛蛋白基因工程疫苗打下了基础，试验结果如下。
从C型节瘤拟杆菌菌株中提取染色体DNA。用PCR技术扩
增出具有免疫保护性抗原基因0.85kb纤毛蛋白基因（pili基
因），利用该基因构建了纤毛蛋白基因表达载体。先将C型节瘤
拟杆菌在厌氧条件下培养72小时；菌数达到1×10~8个/ml以
上时，依据革兰氏阴性杆菌DNA快速提取方法，提DNA；利用
所设计的专一性引物进行PCR，从DNA中扩增出pili基因；将
该基因克隆于T Easy载体系统并进行序列测定，所扩增的pili
基因序列正确；用EcoR1酶切，低熔点胶回收，klenow补平后，
用T_4DNA连接酶将其与经Hpal酶切后去磷酸化的PPLλ中间载
体连接，将 pill 基因克隆于 PPL入载体；经 BamHI，BamHI＋Hind
Ill，Dra酶切鉴定，鉴定出正向插入 PPL入载体的克隆质粒；
扩增 PPL入－Pi i重组质粒，用 B。＊ 切出 2．Ikb大小的片断，
回收该片段，与经BamHI酶切去磷酸化的PME290表达质粒连
接，转化宿主细胞 PAK乃 巳 在营养肉汤中进行 Pi i基因的
表达。培养 18刁 4小时后，培养上清液中加入 0．IM MgC；，离
心提取纤毛蛋白。用羊抗兔C型节瘤拟杆菌抗血清与提取的表
达纤毛蛋白进行对流免疫电泳试验，证明了表达纤毛蛋白的特
异性。用 PAGE电泳和 W既 t盯nhi川证明其为表达的纤毛蛋白。
从而构建了腐蹄病 C型节瘤拟杆菌 Pil基因的表达质粒。
用基因表达的C型节瘤拟杆菌纤毛蛋白与等量弗氏完全佐
剂混合乳化制备C型节瘤拟杆菌纤毛蛋白基因工程疫苗，接种
3只健康家兔；进行了免疫原性试验；并同时用节瘤拟杆菌灭
活菌苗及菌体纤毛蛋白疫苗免疫家兔，进行了免疫比较试验。
将 C型节瘤拟杆菌纤毛蛋白基因工程疫苗接种于含 5 00Pg州
控苇青霉素的营养肉汤培养基中培养；MgC；沉淀法提取纤毛
蛋白，等量弗氏完全佐剂混合免疫健康家兔，分2次进行，问
隔一周，每周采血检测免疫家兔血清中抗体水平。结果纤毛蛋
白基因工程疫苗于7天开始出现血清凝集反应，14天后可达
－2－
．u2000”以上，21天后可达到8000”以上，30天后可达到32000
”以上，而且其高滴度抗体可维持到1N天以上。与前期研究
工作用全菌苗和菌体纤毛蛋白疫苗免疫试验兔相比，C型节瘤
拟杆菌纤毛蛋白基因工程疫苗比其它两种疫苗具有更好的免疫
效果。同时利用C型节瘤拟杆菌菌体裂解抗原与纤毛蛋白基因
工程疫苗免疫家兔抗血清进行对流免疫电泳试验；结果从第7
天开始直到第120天都有明显沉淀线。试验证明C型节瘤拟杆
菌纤毛蛋白基因工程疫苗有良好的免疫原性。
Master Degree Candidate: Miao Liguang
Supervisor: Wang Kejian
D.nodosus pili is the main protective immumogen against footrot
in ruminants.
A plasmid expressing pili gene of D.nodosus serotype C was
constructed by which a
recombinant phi vaccine was developed. The immumogenicity of the
recombinant
pili vaccine was evaluated through experimental animals.
The pili gene that dominates the main protective immunogen was
amplified
and cloned from D.nodosus serorype C by PCR. An expression
plasmid was
constructed by cloning the pili gene into PME29O.The plasmas
harbouring the pili
sequence was designated PME29O-Pili.The PME29O-pili was
transformed into the
host competent cell PAKI2pfs and the recombinant pill was
expressed in the
supernatant of the cultures of the transformant cell PAK]2pfs.
The recombinant pill
was purified by Mgcl2 from the supernatant of the culture of the
transformed
PAKI2pfs. The specific reaction of the recombinant pili and
antiserum of D.nodosus
serotype C pili was demonstrated by cross electrophoresis. The
recombinant pili was
expressed at high level in PAKI2pfs.
Healthy rabbits were inoculated each with vaccine of the
recombinant pill
protein at 250ug per dose to potentiate the immunogenicity.
Rabbit inoculated two
times at one week interval. The blood of each rabbit was
collected at days 0,7, 14,21,
30, 60, 90, 120. The antibody titers were evaluated by
k-agglutination test. The
results showed that the lower agglutination titers were observed
at day 7 and up to
higher levels over 8000xat day 21 induced by the vaccine of
recombinant phi.
Above results suggested that the good immunogenicity of the
recombinant pill
vaccine could match with that previously measured of either the
D.nodosus pill or the killed D.nodosus vaccine.
引文
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78. Lambell,R.G., A field trial with a commercial vaccine ayainst foot-rot in sheep, Aust. Vet. J.,63,415,1986
79．王克坚等，奶牛腐蹄病灭活菌苗的研制及动物试验，“九五”国家攻关验收材料。
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