草鱼细菌性烂鳃病病原菌的分离及其特异性卵黄抗体的初步研究
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摘要
试验从患典型烂鳃病草鱼病变部位分离到1株柱状黄杆菌(FC-3株),采用0.4%甲醛灭活制成免疫原,接种健康120日龄桃源蛋鸡,收集鸡蛋并提纯卵黄抗体(IgY),并以此IgY为基础建立间接ELISA方法。结果:用FC-3株制备的IgY经初步提纯后电泳分离可见重链和轻链2条蛋白条带,获得了IgY多克隆抗体。建立的间接ELISA方法检测柱状黄杆菌纯培养液的检出限值为1×10~7cfu/mL,抗原包被最佳浓度和抗体最佳工作浓度分别为1∶100和1∶512,辣根过氧化物酶标记兔抗鸡IgY最佳工作浓度为1∶4 000。结论:建立的间接ELISA方法对柱状黄杆菌有明显的检测特异性,与其他菌株无交叉反应,重复性好。
Streak plate method was used to separate the pathogenic strain FC-3 from diseased regions of Ctenopharyngodon idella with gill rot,named Flavobacterium columnare. The strain was inactivated by 0.4% formaldehyde according to the immune plan to stimulate 120 days laying Taoyuan hens' the production of Yolk antibody( IgY). The high level immuned eggs were collected. The purified IgY was used to establish indirect ELISA for detecting the FC-3 strain. Results: the Flavobacterium columnare( FC-3) IgY was preliminary purified,2 protein strips heavy chain and light chain were visible by Electrophoresis separation. This indicate the IgY polyclonal antibody was acquired. Titer of the polystyrene chloride microtitration plates were coated with Flavobacterium columnare antigen were detected by IgY indirect ELISA at the amount of 1×107 cfu/mL. The working concentration of antigen,primary antibody and HRP-labelled rabbit antichicken IgY were 1 ∶100,1 ∶512 and 1 ∶4 000. Conclusion: the detection specificity of the method was also determined,Flavobacterium columnare can be detected,while other bacteria strains can not be detected,good repeatability.
Streak plate method was used to separate the pathogenic strain FC-3 from diseased regions of Ctenopharyngodon idella with gill rot,named Flavobacterium columnare. The strain was inactivated by 0.4% formaldehyde according to the immune plan to stimulate 120 days laying Taoyuan hens' the production of Yolk antibody( IgY). The high level immuned eggs were collected. The purified IgY was used to establish indirect ELISA for detecting the FC-3 strain. Results: the Flavobacterium columnare( FC-3) IgY was preliminary purified,2 protein strips heavy chain and light chain were visible by Electrophoresis separation. This indicate the IgY polyclonal antibody was acquired. Titer of the polystyrene chloride microtitration plates were coated with Flavobacterium columnare antigen were detected by IgY indirect ELISA at the amount of 1×107 cfu/mL. The working concentration of antigen,primary antibody and HRP-labelled rabbit antichicken IgY were 1 ∶100,1 ∶512 and 1 ∶4 000. Conclusion: the detection specificity of the method was also determined,Flavobacterium columnare can be detected,while other bacteria strains can not be detected,good repeatability.
引文
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[3]郭沽,甄宇红,李小宇,等.抗金黄色葡萄球菌卵黄抗体的制备及活性研究[J].现代生物医学进展,2007,7(12):1797-1799.
[4]谢献胜,王峰,周新民,等.抗幽门螺旋杆菌卵黄抗体的制备及其体外抑菌试验[J].中国家禽,2005,27(13):12-17.
[5]钟青萍,王斌,何艳萍.抗大肠杆菌0157:H7的卵黄特异性Ig Y的研究[J].现代食品科技,2008,24(3):230-232.
[6]吕娜,张懋岚,张虹茜.抗柱状黄杆菌单克隆抗体杂交瘤细胞株的建立及其免疫学特性鉴定[J].中国兽医科学,2013,43(7):744-748.
[7]罗玉双,甘灵芝,李娜,等.三种消毒剂对草鱼柱状黄杆菌的抑菌效果及最优组方:中国水产学会学术年会论文摘要集[C].北京:中国水产学会,2013.
[8]张保平,李娜,高惠林,等.抗豪猪大肠埃希菌卵黄抗体的制备[J].江苏农业科学,2017,45(10):133-135.
[9]何爱华,张曦,陶琳丽,等.6种中草药对4种淡水鱼致病菌体外抑菌作用的研究[J].南方水产科学,2011,7(2):73-76.