血清14型副猪嗜血杆菌的分离与多克隆抗体制备

Isolation of Haemophilus parasuis Serotype 14 and Preparation of Its Polyclonal Antibody

【目的】探明引起猪副猪嗜血杆菌(Haemophilus parasuis,HPS)病发病的病原菌,并制备分离病原菌的多克隆抗体,为该病的治疗及疫苗的研发提供技术支撑。【方法】从送检病料分离出保育猪发病病原菌,并对其进行形态观察、生化鉴定与PCR鉴定,明确分离病原菌的类型及血清型;同时使用甲醛对分离病原菌进行灭活后再与弗氏佐剂乳化制备灭活菌体抗原,经肌肉和皮下免疫BALB/C小鼠,制备该分离病原菌的鼠源多克隆抗体;采用饱和硫酸铵沉淀法对制备的多克隆抗体进行纯化,利用SDS-PAGE方法鉴定纯化抗体的纯度,通过免疫荧光进一步检测抗体与抗原的结合效果。【结果】从病料中分离得到1株革兰氏阴性短小杆菌,经形态学观察、生化鉴定与PCR鉴定,分离到的病原菌为血清14型副猪嗜血杆菌(Haemophilusparasuis,HPS);灭活后的HPS与佐剂乳化后免疫BALB/C小鼠制备得到分离病原菌的多克隆抗体,阳性菌液与纯化后稀释500倍和2000倍的多克隆抗体结合并加入荧光二抗后于显微镜下观察,病原菌发出红色荧光;经Western blot检测,制备多克隆抗体在约13 ku、30 ku和50 ku处有3条清晰条带,Western blot和免疫荧光试验均证实制备的多克隆抗体可与分离病原菌发生特异性反应。【结论】制备的血清14型HPS多克隆抗体可与分离病原菌发生特异性反应。

【Objective】The pathogen that caused Haemophilus parasuis(HPS)in pig was explored,and its polyclonal antibody for isolating the pathogen was prepared,so as to provide technical support for curing H.parasuis and studying its vaccinum.【Method】The pathogen was isolated from the diseased materials collected from nursery pigs,then it was studied through morphology observation,biochemical identification and PCR identification,so that the types and serotypes of the isolated pathogen was determined.Formaldehyde was used to inactivate the pathogen,and then was emulsified and combined with Freund’s adjuvant to prepare the antigen.The antigen was injected subcutaneously and intramuscularly into Balb/c mice for immunization,then the murine polyclonal antibody against the pathogen was prepared.The prepared polyclonal antibody was purified by method of saturated ammonium sulfate precipitation.The purity of purified antibody was identified through SDS-PAGE.The combining effects of antibody and antigen were detected further through immunofluorescence.【Result】A Gram-negative short bacillus was successfully isolated from the diseased material,and the isolated pathogen was confirmed to be H.parasuis(HPS)serotype 14 by morphology observation,biochemical identification and PCR identification.The polyclonal antibody was prepared from Balb/c mice injected with antigen which was made from emulsified Freund’s adjuvant and HPS after inactivation,then it was purified and diluted 500-fold and 2000-fold respectively,combined with positive bacterial fluid and fluorescent secondary antibodies,and observed by microscope,finding that the pathogen fluoresced red.Through Western blot detection,the prepared polyclonal antibody had three clear bands at 13 ku,30 ku and 50 ku.Western blot and immunofluorescence both approved that the prepared polyclonal antibody could react specifically with isolated pathogen.【Conclusion】The prepared polyclonal antibody of HPS serotype 14 had specific reaction to isolated pathogen.