摘要
目的制备高纯度、具有生物活性的CagA蛋白,建立间接ELISA法检测CagA抗体,并对该方法进行评估。方法构建重组表达质粒CagA-PET28a,转化至大肠杆菌BL21(DE3)中诱导表达,利用蛋白纯化液相色谱仪进行镍亲和层析纯化后再进行离子交换层析纯化。利用验证后的蛋白建立了间接ELISA法,通过与市售试剂盒进行比对,对间接ELISA法进行评价。结果成功表达并纯化出了高纯度的CagA蛋白,分子量为130 kD,纯度为99.87%。利用该抗原建立了间接ELISA法,并对204例待测血清进行检测。与Mikrogen公司血清学检测试剂盒的结果比较,阳性符合率为86.5%,阴性符合率为91%,总符合率为88.7%。对伯劳特公司提供的参考品进行检测,表明该间接ELISA法具有较好的重复性且最低检测限检测结果符合预期。结论利用重组CagA蛋白建立的检测患者血清中CagA抗体的方法具有较好的检测性能,且重复性较好,最低检测限与市售试剂盒相当。
Objective To prepare high-purity and bio-active CagA protein,and establish an indirect ELISA method for detecting the CagA antibody,and evaluate this method.Methods The recombinant expression plasmid CagA-PET28a was constructed and transformed into E.coli BL21 to induce expression.Protein purification liquid chromatography was used to purify target protein by Ni affinity chromatography and then by ion exchange chromatography.An indirect ELISA method was established using the validated proteins.The indirect ELISA method was evaluated by comparing with commercial kits.Results High-purity CagA protein was successfully expressed and purified in this study,with a molecular weight of 130 kD and a purity of 99.87%.An indirect ELISA method was established using the antigen.Two hundred four samples of serum were detected by this method.Compared with the Mikrogen test kit results,the positive coincidence rate was 86.5%,the negative coincidence rate was 91%,and the total coincidence rate was 88.7%.Testing the reference product provided by Blot Biotech showed that the indirect ELISA method had good repeatability and the detection limit met expectations.Conclusion An indirect ELISA method is established to detect CagA antibody in the serum of patients using this protein.The method has satisfactory detection performance and good repeatability;and the detection limit is comparable to the commercial kit.
作者
李玉萍
成蕾
王洪涛
乔苗苗
熊丹
林骏
LI Yu-ping;CHENG Lei;WANG Hong-tao;QIAO Miao-miao;XIONG Dan;LIN Jun(Shenzhen Institute for Drug Control/Shenzhen Testing Center of Medical Devices,Shenzhen 518000,China;Shenzhen Blot Biotech Co.,Ltd,Shenzhen 518000,China;Clinical Laboratory,Shenzhen Luohu People's Hospital,Shenzhen 518001,China)
出处
《中国医药生物技术》
2023年第4期304-310,共7页
Chinese Medicinal Biotechnology
基金
广东省药品监督管理局2022年科技创新项目(2022TDB54)。
