产气荚膜梭菌α毒素突变体构建及其卵黄抗体制备  

Construction of Clostridium perfringens alpha toxin mutant and preparation of egg yolk IgY antibody

作　　者：李箐[1,2] 辛文文[2] 康琳[2] 高姗[2] 王景林[2] 

机构地区：[1]安徽医科大学研究生院,安徽230032 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学》2013年第9期671-675,共5页Military Medical Sciences

基　　金：全军医学科技"十二五"重大专项资助项目(AWS11C001)

摘　　要：目的制备特异性的产气荚膜梭菌α毒素(Clostridium perfringens alpha-toxin,CPA)卵黄抗体(yolk immunoglobulin,IgY)。方法利用定点突变技术,将CPA第56位天冬氨酸和第68位组氨酸分别突变为丝氨酸,构建了重组表达载体pTIG-mCPAD56S和pTIG-mCPAH68S,将其转化入E.coli Origami中进行诱导表达。用亲和层析的方法对突变体蛋白进行纯化并对其活性及抗原性进行检测,将获得的突变体蛋白分别免疫健康母鸡,收集鸡蛋;经水稀释法纯化卵黄中IgY;用酶联免疫吸附试验检测IgY效价;通过筛选保护剂而选择最佳的IgY冻干条件。结果与结论pTIG-mCPA在Origami表达菌株中得到高效表达,经验证,mCPAD56S和mCPAH68S均完全失去生物学活性同时保留抗原性;纯化卵黄后得到较高纯度的特异性IgY,其效价可达到1∶200 000;经过IgY冻干条件的筛选,最终选择不添加保护剂大量冻干IgY作为抗体的储备。该研究为研制基于IgY抗体的检测方法奠定了基础。Objective To prepare the egg yolk IgY antibody against Clostridium perfringens alpha-toxin（CPA）.Methods The pTIG vector containing mutant gene fragments constructed by site-directed mutagenesis was transformed into Escherichia coli strain Origami for expression.The resulting proteins were purified using Ni2 ＋-chelating chromatography.After the bioactivity and immunogencity test,the mutant proteins were used as an immunogen to immunize healthy hens for yolk immunoglobulin（IgY） production.IgY was obtained by the water dilution method under acidic conditions.An indirect enzyme linked immunosorbent assay（ELISA） was used to detect the titer of IgY against alpha toxin.Protective agents were selected for freeze-drying IgY.Results and Conclusion The two mCPAs expressed in the E.coli strain Origami has been confirmed with no bioactivity but retained immunogenicity.After immunization of the hens,the titer of the purified IgY antibody against alpha toxin can reach 1∶ 200 000.Freeze-drying IgY without any protective agent has been proved to possess better bioactivity for storage.The high titer IgY can be used for seeking methods for detecting CPA.

关 键 词：产气荚膜梭菌α毒素 突变 IGY 重组表达 

分 类 号：R392.1[医药卫生—免疫学]