Liu Y, Qian H, Huang H
Gynecologic Oncology Center, People's Hospital Beijing Medical University, Beijing 100034.
Zhonghua Yi Xue Za Zhi. 1999 Mar;79(3):221-3.
To express high-level 6B11 ovarian carcinoma anti-idiotypic antibody single chain Fv (scFv) genes in E. Coli.
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.
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.
We successfully expressed high-level 6B11scFv genes in E. Coli. Expressed proteins showed pretty good immunoactivity.
在大肠杆菌中高效表达6B11卵巢癌抗独特型抗体单链Fv（scFv）基因。
采用PCR克隆技术，将6B11scFv基因克隆至细菌表达载体pKPL-3a。将重组质粒载体pL-6B11scFv转化至温控大肠杆菌pop2136中以表达蛋白。复性后的蛋白经DEAE-琼脂糖快速流动离子交换柱以盐梯度洗脱法进行纯化。分别通过ELISA和抑制ELISA试验测定免疫活性。
6B11scFv基因在细胞质中以包涵体形式表达。经SDS-PAGE检测，6B11scFv的纯度超过95%。纯化后的表达蛋白能与抗卵巢癌单克隆抗体COC166-9特异性反应，并有效抑制原发性卵巢癌抗原OC166-9与COC166-9之间的反应。
我们成功在大肠杆菌中高效表达了6B11scFv基因。表达的蛋白显示出良好的免疫活性。
