血清2型鸭疫里默氏杆菌脂多糖单克隆抗体的研制及特性鉴定

Preparation and characterization of monoclonal antibodies against lipopolysaccharide from Riemerella anatipestifer serotype 2 strain

【背景】鸭疫里默氏杆菌(Riemerella anatipestifer,RA)可感染雏鸭、鹅、火鸡等多种禽类,引起急性或慢性传染病。RA血清型众多且各血清型之间缺乏有效的交叉保护。细菌脂多糖(Lipopolysaccharide,LPS)位于革兰氏阴性菌细胞壁的外侧,其组成和结构变化决定了细菌表面抗原决定簇的多样性。【目的】制备血清2型鸭疫里默氏杆菌LPS单克隆抗体并对其特性进行研究。【方法】以血清2型RA NJ3株免疫BALB/c小鼠,细胞融合后以RA NJ3株LPS作为包被抗原,筛选出能够稳定分泌单克隆抗体的杂交瘤细胞株,通过体内诱生腹水法制备抗体,采用酶联免疫吸附试验(ELISA)检测单克隆抗体效价,玻片凝集试验和Westernblot检测单抗特异性。【结果】获得两株能稳定分泌抗血清2型鸭疫里默氏杆菌LPS单克隆抗体的杂交瘤细胞株,分别命名为8G5和8G10;两株单抗的亚型均为IgM/κ链。ELISA结果表明,8G5和8G10腹水效价分别为1:32 000和1:16 000。Western blot结果显示,两株单抗仅与血清2型RA菌株发生特异性反应,而与其他血清型RA菌株和禽源致病菌无反应性。【结论】研究获得的单克隆抗体具有良好的反应性和血清型特异性,可用于RA致病机制的基础研究和进一步建立RA血清型快速检测方法。

[Background]Riemerella anatipestifer causes septicemic and exudative diseases of domestic ducks,gooses and turkeys,leading to economically devastating to poultry industries.A total of 21 serotypes of R.anatipestifer have been identified.Moreover,there is poor cross-protection among these serotypes.Lipopolysaccharide is the important component of outer membrane of Gram-negative bacteria,and provides the diversity of bacterial surface antigen determinants.[Objective]To prepare and characterize monoclonal antibodies(McAbs)against lipopolysaccharide of R.anatipestifer serotype 2 strain.[Methods]BALB/c mice were immunized with inactivated R.anatipestifer serotype 2 strain NJ3.Spleen cells from immunized mice were fused with murine myeloma SP2/0 cells.The hybridoma cell line that stably secret McAb against LPS of R.anatipestifer serotype 2 strain,was obtained through clone selection and screening by indirect enzyme linked immunosorbent assay(ELISA).The mouse ascites was prepared and identified by Western blot analysis and ELISA.The specificity of these McAbs was tested by slide agglutination assay and Western blot.[Results]We successfully obtained 2 hybridoma cell lines named 8 G5 and 8 G10 that could stably produce anti-LPS McAbs.The isotype of both McAbs was identified as IgM withκlight chain.The titers of 8 G5 and 8 G10 were 1:32 000 and 1:16 000,respectively.Western blot results showed that both McAbs specifically reacted with R.anatipestifer serotype 2 strains but did not react with R.anatipestifer other serotypes strains,avian pathogenic Escherichia coli,Salmonella enterica and Pasteurella multocida strains.[Conclusion]We prepared two specific anti-LPS McAbs that can be used for pathogenic mechanism research and development of rapid detection of R.anatipestifer serotype 2 strains.