摘要
为探讨一株肝细胞癌特异性鼠源及其人源化单链抗体基因在大肠杆菌中的可溶性表达策略并比较二者对抗原的结合能力,在三种载体中分别以融合、分泌及胞内表达的方式进行了研究,表达产物均以包涵体形式存在;对复性后的单链抗体以细胞ELISA及竞争抑制流式细胞仪法进行检测,表明人源化单链抗体和鼠源单链抗体有相近的抗原结合能力。结论是:在大肠杆菌中表达的基因工程单链抗体的可溶性可能主要由自身氨基酸一级序列决定;先前的设计所采取的人源化方案没有影响到鼠源抗体的CDR的天然构象。
The aim of this research is to exploit the expression strategies for two genes encoding a rodent and its humanized version single chain fragments(scFv)specific for hepatocellular carcinoma (HCC) and compare these two scFves in antigen binding activity.Three vectors were used to express these two genes in different routes of fusion pathway,secrete pathway and intracellular pathway.The refolded single chain antibodies were examined by antigen capture ELISA.Results showed that inclusion bodies were produced in all of the applied vectors despite of variation of IPTG concentration and culture temperature.Cell ELISA and binding competition inhibition indicated that the humanized single chain Fv retained the similar affinity as its rodent counterpart.These results implied that the solubility of genetic antibodies are determined primarily by its amino acids sequence;The adopted humanization strategy in previous design made little effect on the natural conformation of complementarity determining regions(CDR)of the parent antibody.The humanized HCC specific scFv can be further evaluated and developed as therapeutics.
出处
《生物工程学报》
CAS
CSCD
北大核心
2000年第1期86-90,共5页
Chinese Journal of Biotechnology
关键词
单链抗体
肝癌
大肠杆菌
免疫原性
可溶性表达
Humanization,single chain antibody,hepatocellular carcinoma,expression
