摘要
目的:研究VacA-HpaA融合蛋白的鸡卵黄抗体(IgY)的理化特性和生物学活性,为制备集预防与治疗为一体的抗H.pylori感染的制剂奠定基础。方法:以纯化的VacA-HpaA融合蛋白免疫产蛋鸡,水稀释法获取蛋黄抗体(VacA-HpaAIgY)。(1)用硫酸铵沉淀法纯化IgY后,用SDS-PAGE分析其纯度,Bradford法测定蛋白含量。(2)用间接ELISA法检测该IgY的耐热性、耐酸性及其对酶消化的耐受作用。(3)采用Western blot分析IgY的特异性并检测其效价。(4)用MTT法检测不同浓度VacA-HpaAIgY对细胞毒活性的中和作用。(5)用扫描电镜观察VacA-HpaA IgY对AGS细胞粘附的中和作用。结果:(1)该IgY纯度为60%,蛋白浓度为22g/L;(2)具有一定的耐热性、耐酸性和耐胃蛋白酶能力;(3)Western blot分析显示IgY的特异性,在相对分子量约27000和30000处出现在相应的条带;(4)该IgY对VacA细胞毒活性有中和作用,且具有量效性和时效性;(5)IgY可阻止H.pylori菌体蛋白的粘附作用。结论:该IgY具有良好的理化和生物学特性,可用于制备抗H.pylori感染的防治制剂。
Objective：Researching physical-chemic and biologic speciality of VacA-HpaA IgY,which provide a solid experimental basis for the preparation of vaccine against H.pylori infection.Methods：The purified antigen of fusion protein VacA-HpaA was used to immunize hens and the VacA-HpaA IgY was extracted from the yolk by water dilution methods.（1）After the VacA-HpaA IgY was purified by deposition technique with ammonium sulfate,the purity of IgY was analyzed by SDS-PAGE,and protein content of IgY was analyzed by Bradford method.（2）The indirect ELISA was used to detect its heat-stability,acid-stability and pepsin-stability.（3）The specificity of VacA-HpaA IgY to the antigens was identified by Western blot.（4）MTT was applied to assay the neutralization of VacA-HpaA IgY to The cytotoxity of H.pylori.（5）The neutralization of IgY to AGS cells was observed with scanning electron microscopy（SEM）.Results：（1）The purity of VacA-HpaA IgY was about 60%,and the protein content of IgY was 22g/L;（2）The IgY showed a good heat-stability,a favorable acid-stability,and a well ability of anti-pepsin digest;（3）Western blot exhibited the protein bands with molecular weight of 27 000 and 30 000.The titer of VacA-HpaA IgY to VacA and HpaA were 1∶3 200 and 1∶6 400;（4）A dose and time dependent correlation of the IgY to counteract the cytotoxic activity of VacA;（5）VacA-HpaA IgY could interrupt the adhesive attraction of H.pylori.Conclusion：The VacA-HpaA IgY has good stabilities and well biologic speciality,which imply that the IgY could be used to prepare treatmental antibody against H.pylori infection.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2008年第2期114-118,共5页
Chinese Journal of Immunology
