鸭疫里默氏杆菌不同血清型的外膜蛋白特性比较及免疫原性的研究
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摘要
本文对4种血清型的(1型RA、2型RA、4型RA、5型RA)的外膜蛋白及其免疫原性进行了探讨。
结果表明采用超速离心法提取细菌的外膜蛋白，在电镜下观察都呈典型的双层泡状结构，但是形态有一定差异，有的呈“O”形，有的呈牙齿状。
所提取的4种血清型RA外膜蛋白经SDS-PAGE凝胶电泳分析结果表明，其外膜蛋白电泳图谱基本相同，但是分子量范围有一定差异，并且他们之间的主要条带也有差异。1型RA的分子量范围在25-143KD之间，其中主要蛋白带分子量分别是31KD，44KD，69KD，77KD，82KD，91KD和107KD，另外还有一些轻染的次要蛋白带；2型RA的分子量范围是26KD-135KD之间，其中主要蛋白带分子量分别是31KD，33KD，75KD，101KD和116KD；4型RA的分子量范围是26KD-124KD之间，其中主要蛋白带分子量是32KD，38KD，80KD，114KD；5型RA的分子量范围是28-121KD之间，其中主要蛋白带分子量是31KD，43KD，70KD，89KD，92KD，111KD。
经过免疫印迹证实，1型RA的44KD外膜蛋白与免疫鸭血清出现较强的阳性反应，是1型RA的主要免疫源；2型RA的40KD外膜蛋白与免疫鸭血清出现较强的阳性反应，是2型RA的主要免疫源；4型RA的75KD外膜蛋白与免疫鸭血清出现较强的阳性反应，是4型RA的主要免疫源；5型RA的39KD外膜蛋白与免疫鸭血清出现较强的阳性反应，是5型RA的主要免疫源。
4种血清型的外膜蛋白免疫小鸭后都能诱导机体产生较强的抗体反应，对同源细菌攻击的保护率为100％。
Four major serotypes of Riemerella anatipestifer-serotype 1,2,4,5 are chosed for the test, we extract their outer membrane protein and study their immunogenicity.
Ultracentrifugationg-Sarkosyl extraction was chosen for the isolation of the outer membrane proteins (OMP) of Riemerella antipestifer (RA). Under the electron microscopy, the ultra structure of RA was observed. The OMP of RA showed the typical double round vesicules with negative stain under electronic microscopy; however, there are some differences among the morphologic. Some are O form, some are tooth form.
The electrophoretogram of the four serotypes is some similarity after SDS-PAGE, but there are many differences among them. The molecular weight of OMP of serotype 1 RA ranges from 25kd to 143kd, among them, 31KD,44KD,69KD,77KD,83KD, 91KD and 107KD proteins are more heavily stained by Commassie blue; serotype 2 RA ranges from 26kd to 135kd, among them, 31kd,33kd,75kd, 101kd and 116kd are more heavily stained by Commassie blue; serotype 4 RA ranges from 26kd to 124kd, among them, 32kd,28kd,80kd,114kd are more heavily stained by Commassie blue; serotype 5 ranges from 28kd to 121kd,among them, 31kd, 43kd,70kd,89kd,92kd,lllkd are more heavily stained by Commassie blue.
The 44KD of OMP of serotype 1 RA have strong masculine with duck serum reaction by WESTERN-BLOTTING; the 40KD of OMP of serotype 2 RA appears obvious reaction with duck serum by WESTERN-BLOTTING; the 75KD of OMP of serotype 4 RA appears obvious reaction with duck serum by WESTERN-BLOTTING; the 39KD of OMP of serotype 5 RA appears obvious reaction with duck serum by WESTERN-BLOTTING;
From the result of duckling immured by outer membrane protein, we know that OMP of all the four serotypes can induce obvious antibody reaction, and the protective ratio of the attack from the RA of the same bacteria was 100%.
结果表明采用超速离心法提取细菌的外膜蛋白，在电镜下观察都呈典型的双层泡状结构，但是形态有一定差异，有的呈“O”形，有的呈牙齿状。
所提取的4种血清型RA外膜蛋白经SDS-PAGE凝胶电泳分析结果表明，其外膜蛋白电泳图谱基本相同，但是分子量范围有一定差异，并且他们之间的主要条带也有差异。1型RA的分子量范围在25-143KD之间，其中主要蛋白带分子量分别是31KD，44KD，69KD，77KD，82KD，91KD和107KD，另外还有一些轻染的次要蛋白带；2型RA的分子量范围是26KD-135KD之间，其中主要蛋白带分子量分别是31KD，33KD，75KD，101KD和116KD；4型RA的分子量范围是26KD-124KD之间，其中主要蛋白带分子量是32KD，38KD，80KD，114KD；5型RA的分子量范围是28-121KD之间，其中主要蛋白带分子量是31KD，43KD，70KD，89KD，92KD，111KD。
经过免疫印迹证实，1型RA的44KD外膜蛋白与免疫鸭血清出现较强的阳性反应，是1型RA的主要免疫源；2型RA的40KD外膜蛋白与免疫鸭血清出现较强的阳性反应，是2型RA的主要免疫源；4型RA的75KD外膜蛋白与免疫鸭血清出现较强的阳性反应，是4型RA的主要免疫源；5型RA的39KD外膜蛋白与免疫鸭血清出现较强的阳性反应，是5型RA的主要免疫源。
4种血清型的外膜蛋白免疫小鸭后都能诱导机体产生较强的抗体反应，对同源细菌攻击的保护率为100％。
Four major serotypes of Riemerella anatipestifer-serotype 1,2,4,5 are chosed for the test, we extract their outer membrane protein and study their immunogenicity.
Ultracentrifugationg-Sarkosyl extraction was chosen for the isolation of the outer membrane proteins (OMP) of Riemerella antipestifer (RA). Under the electron microscopy, the ultra structure of RA was observed. The OMP of RA showed the typical double round vesicules with negative stain under electronic microscopy; however, there are some differences among the morphologic. Some are O form, some are tooth form.
The electrophoretogram of the four serotypes is some similarity after SDS-PAGE, but there are many differences among them. The molecular weight of OMP of serotype 1 RA ranges from 25kd to 143kd, among them, 31KD,44KD,69KD,77KD,83KD, 91KD and 107KD proteins are more heavily stained by Commassie blue; serotype 2 RA ranges from 26kd to 135kd, among them, 31kd,33kd,75kd, 101kd and 116kd are more heavily stained by Commassie blue; serotype 4 RA ranges from 26kd to 124kd, among them, 32kd,28kd,80kd,114kd are more heavily stained by Commassie blue; serotype 5 ranges from 28kd to 121kd,among them, 31kd, 43kd,70kd,89kd,92kd,lllkd are more heavily stained by Commassie blue.
The 44KD of OMP of serotype 1 RA have strong masculine with duck serum reaction by WESTERN-BLOTTING; the 40KD of OMP of serotype 2 RA appears obvious reaction with duck serum by WESTERN-BLOTTING; the 75KD of OMP of serotype 4 RA appears obvious reaction with duck serum by WESTERN-BLOTTING; the 39KD of OMP of serotype 5 RA appears obvious reaction with duck serum by WESTERN-BLOTTING;
From the result of duckling immured by outer membrane protein, we know that OMP of all the four serotypes can induce obvious antibody reaction, and the protective ratio of the attack from the RA of the same bacteria was 100%.
引文
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[2] Rimer. Kurze. Mitteilung (?)iber eine bei G(?)nsen beobachtete exsudative septik(?)mie und deren Erreger[J]. Zentralbl B akteriol I Aot I Orig 37:641～-648
[3] Hendrickson, J. M.,and K.F.Hilbert. A new and serious septicemic disease of young ducks with a description of the causative organism, Pfeifferella anatipestifer,N.S[J]. Cornell Vet, 1932,22:239～252
[4] Dougherty, E., L.Z.Saunders.and E.H.Parsons. The pathology of infectious serositis of ducks[J]. Am J Pathol, 1955,31:475～487
[5] 郭玉璞，陈德威，范国雄，等。北京小鸭传染性浆膜炎的调查研究[J]。畜牧兽医学报，1982，13(2)：107～112
[6] 郭予强。鸭疫综合症的研究[J]。华南农学院学报。1984，2：11～21
[7] 高福，郭玉璞．小鸭传染性浆膜炎疫苗的研究Ⅰ．鸭疫巴氏杆菌的血清型鉴定[J]．中国兽医杂志，1987，13(4)：47～48
[8] 林业杰，林禧龄，曾颜钦．福州首次从康贝尔鸭分离出鸭疫巴氏杆菌[J]．中国兽医科技，1989，2：26～27
[9] 张大丙等．北京地区鸭病毒性肝炎与传染性浆膜炎的流行情况与防制现状[J]．中国兽医杂志，1993，19(3)：11～13
[10] 程安春，汪铭书，陈孝跃．鸭疫巴氏杆菌的分离和鉴定[J]．四川畜牧兽医，1996，(2)：7～9
[11] 朱家新，徐昌法．肉鸭鸭疫巴氏杆菌与大肠杆菌混合感染的诊治[J]．上海畜牧兽医通讯，1996，3：27
[12] 钱忠明，田惠芳，蒋志伟等．肉鸭鸭疫巴氏杆菌病抗原型的研究及其防治：Ⅰ．肉鸭鸭疫巴氏杆菌病的流行病学调查及诊断[J]．中国家禽，1997，(10)：1～2
[13] 陈兴生，李布勇．海南省鸭疫里默氏杆菌病流行病学调查[J]．中国预防兽医学报，1999，21(3)：231～233
[14] 李继祥，徐刚．重庆地区鸭传染性浆膜炎病原分离鉴定[J]．中国预防兽医学报，2000，19～21
[15] 张保华，周福东，秦淑美，等．一起肉鸭暴发传染性浆膜炎的报道[J]．畜牧与兽医，2000，32(3)：28
[16] 程安春等主编．现代禽病诊断和防治全书[M]．成都四川大学出版社，1997．
[17] B.W.卡尔尼克主编．禽病学[M](第十版)．高福、苏敬良等译．中国
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