摘要
用DNA重组技术构建CT-B表达性质粒pXB1及进一步修饰的pXB2,使CT-B基因在大肠杆菌中得到较高水平的表达(280～350ng/ml),且有71.5％分泌率。表达动力学研究阐明,克隆子培养24h产物收率较高。一定浓度的Mg^(2+)、L-组氨酸和天冬酰胺能刺激CT-B的表达。GM1-ELISA、CHO细胞测毒结合间接免疫荧光检测和家兔肠段结扎试验证实,表达的CT-B能与游离的或活细胞膜上的GM1受体结合,但无细胞和肠段毒性。这种细胞测毒结合免疫荧光序贯试验,为客观证实CT-B的生物学活性开拓了新途径。
The CT-B gene-expressing plasmid pXB1 and modified pXB2 were constructed using recombinant DNA techniques.These resul ed in high CT-B gene expression in E. coli (280-350 ng/ml) , with CT-B accounting for 71.5% of total extracellular protein. The resul.s of CT-B expression kinetics showed lhat the CT-B product levels reached a peak after 24 h in culture. Certain concentrations (100μg/ml) of MgCl2, L-histidine and asparagine can increase the CT-B product level by 10%-25% .The binding of the expressed CT-B product to GM1 recep or on free or live cell membranes was identified by GM1-ELISA, CHO cytotoxicity test, indirect immunofluorescence, and the rabbit ileal loop test. No toxic response to cells or rabbits was detected.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
1991年第5期332-337,共6页
Acta Academiae Medicinae Sinicae
基金
863"计划课题
关键词
霍乱毒素
B亚单位
基因克隆
cholera toxin B subunit gene clone gene expression
