摘要
目的制备在大肠杆菌中高效表达６Ｂ１１卵巢癌抗独特型单链抗体。方法采用聚合酶链反应（ＰＣＲ）克隆技术，将６Ｂ１１ｓｃＦｖ基因克隆到原核高效表达载体ｐＫＰＬ３ａ上，重组子转化大肠杆菌ｐｏｐ２１３６，温度诱导表达；复性蛋白经二乙氨乙基琼脂糖快速离子交换层析纯化，十二烷基硫酸钠聚丙烯酰胺凝胶鉴定蛋白纯度；直接法和竞争抑制法ＥＬＩＳＡ检测其活性。结果６Ｂ１１ｓｃＦｖ以包涵体形式表达获高效表达；包涵体经溶解复性纯化，纯度达９５％以上；表达蛋白能与卵巢癌单抗ＣＯＣ１６６－９特异结合并能有效抑制卵巢癌抗原ＯＣ１６６－９与卵巢癌单抗ＣＯＣ１６６－９的特异结合。结论６Ｂ１１ｓｃＦｖ基因可在原核细胞中高效表达，表达产物经复性纯化后具有较好的免疫活性。
Objective To express high level 6B11 ovarian carcinoma anti idiotypic antibody single chain Fv (scFv) genes in E. Coli. Methods Using PCR cloning technique, We cloned 6B11scFv genes into bacterial expression vector pKPL 3a. Recombinant plasmid vector pL 6B11scFv was transformed into E. Coli pop2136 with temperature control to produce proteins. Renaturated proteins were purified on DEAE Sepharose Fast Flow ion exchange column with salt gradient elution. Immunoactivity was determined with ELISA and inhibition ELISA tests respectively. Results 6B11scFv genes were expressed as inclusion bodies in the cytoplasm. The purity of 6B11scFv turned out by SDS PAGE was more than 95%. The expressed proteins after purification specifically reacted with anti ovarian carcinoma monoclonal antibody COC166 9 and efficiently inhibited the reaction between primary ovarian carcinoma antigen OC166 9 and COC166 9. Conclusion We successfully expressed high level 6B11scFv genes in E. Coli. Expressed proteins showed pretty good immunoactivity.
出处
《中华医学杂志》
CAS
CSCD
北大核心
1999年第3期221-223,共3页
National Medical Journal of China
