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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 a specific Clostridium perfringens alpha-toxin (CPA) yolk immunoglobulin (IgY). Methods Site-directed mutagenesis was used to mutate the 56th aspartate and 68th histidine of CPA into serine. The recombinant expression vectors pTIG-mCPAD56S and pTIG-mCPAH68S were constructed and transformed into E. coli Origami Induction of expression. The mutant protein was purified by affinity chromatography and its activity and antigenicity were tested. The obtained mutant protein were respectively immunized healthy hens to collect eggs; IgY was purified by water dilution method; IgY titer was detected by immunoadsorption assay; the best conditions of IgY lyophilization were selected by screening protective agent. RESULTS AND CONCLUSION pTIG-mCPA was highly expressed in Origami-expressing strains. The results showed that both mCPAD56S and mCPAH68S completely lost their biological activity and retained their antigenicity. After purification of yolk, a higher purity of specific IgY was obtained and its titer reached 1 : 200 000; after IgY lyophilization screening, the final choice without adding a large amount of protective agent lyophilized IgY antibody as a reserve. The research laid the foundation for the development of IgY antibody-based detection methods.