摘要
目的克隆、表达、纯化具有免疫原性的重组小龙虾钙结合蛋白(SCP),并制备高效价抗SCP单克隆抗体。方法用亲和层析法获取小龙虾重组SCP,通过Western Blotting和ELISA法进行免疫学活性鉴定;以重组SCP为免疫原,免疫BALB/c小鼠,再取小鼠脾细胞与小鼠骨髓瘤细胞NS-1进行细胞融合;利用ELISA法筛选出能分泌特异性单克隆抗体的杂交瘤细胞,然后利用该杂交瘤细胞株刺激小鼠,取腹水,用蛋白A亲和层析方法纯化获得抗体;通过Ig类/亚类鉴定ELISA试剂盒鉴定该单克隆抗体的Ig亚型;通过Western Blot和间接ELISA法鉴定单克隆抗体的交叉性和特异性。结果经SDS-PAGE鉴定获得了纯度较高的重组SCP,免疫学鉴定结果显示,重组的SCP蛋白具有较好的IgE结合活性,阳性率为40%。通过杂交瘤细胞融合技术,共获得了抗SCP细胞株2株,分别命名为1A12和3H12,效价均高于20万。经抗体亚型鉴定,2株抗体均为IgG1型。Western Blot的结果表明2株抗体能识别重组SCP。在特异性检测实验中,1A12抗体与其他种类食物过敏原无交叉反应,而3H12与鱼粗蛋白过敏原有交叉反应性。结论 SCP是小龙虾的重要过敏原,本实验获得了纯度较高的重组SCP,并制备了2株高效价抗SCP单克隆抗体,为小龙虾SCP、甲壳类食物过敏原的检测及标准化过敏原物质的制备奠定了基础。
Objective To clone,express and purify the sarcoplasmic calcium-binding protein(SCP)from Procambarus clarkii,and to prepare the high titre monoclonal antibodies(McAbs).Methods The recombinant SCP was obtained from Procambarus clarkii by affinity chromatography,and the immunological activity was identified by Western blotting and ELISA.BALB/c mice were immunized with the recombinant SCP,and then splenocytes from the immunized mice were fused with NS-1myeloma cells.The hybridoma cells secreting specific McAbs were screened by ELISA.The antibodies were purified by affinity chromatography from ascitic fluid of mice stimulated with hybridoma cells.The Ig subtype of McAbs was identified by ELISA.The cross-reactivity and specificity of McAbs were identified by Western blot and indirect ELISA.Results SDSPAGE showed that high purity recombinant SCP was obtained by affinity chromatography.Immunological identification showed that the recombinant SCP displayed high IgE-binding activity with a positive rate of 40%.Two hybridoma cell lines secreting McAbs against SCP(1A12and3H12)were obtained by hybridoma fusion.Both McAbs had a titer higher than 1200 000 and were classified as IgG1.Western blot indicated that both McAbs reacted with SCP specifically.The 1A12 antibody had no cross-reaction with other food allergens.However,the 3H12 antibody had cross-reaction with fish allergen.Conclusion The SCP was an important allergen in Procambarus clarkia.High purity recombinant SCP was obtained and two high titer McAbs against SCP were prepared in this study,which laid the foundation for the determination of Procambarus clarkii SCP and shellfish allergens and the desensitization of standard allergens.
出处
《南昌大学学报（医学版）》
CAS
2017年第2期29-33,共5页
Journal of Nanchang University：Medical Sciences
基金
深圳市科技计划基础研究项目(JCYJ20140418095735604)
关键词
小龙虾
钙结合蛋白
免疫学特性
鉴定
单克隆抗体
Procambarus clarkii
sarcoplasmic calcium-binding protein
immunological characteristics
identification
monoclonal antibody
