肝癌特异性鼠源及人源化单链抗体基因的克隆与表达

为探讨肝癌特异性鼠源及其人源化单链抗体基因的表达策略并比较二者的结合能力,在3种载体中分别以融合、分泌及胞内表达的方式进行了研究;对复性后的单链抗体以抗原捕获ELISA法进行检测。结果表明,在3种载体中表达的鼠源及人源化单链抗体都是包含体,诱导物浓度及培养温度不影响表达形式;抗原捕获细胞ELISA表明人源化的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CDRs的天然构象,表达的人源化单链抗体提供了免疫原性评价及临床应用的基础。

Cloning and expression of two genes encoding murine single-chain antibody and its humanization one specific for hepatocellular carcinoma

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. Antigen capture ELISA indicated that the humanized single-chain antibody retained the similar binding property as its rodent counterpart. These implied that the solubility of genetic antibodies are determined primarily by its amino acids sequences The adopted humanization strategy in previous design made little effect on the natural conformation of complementarity-determining regions (CDRs) of the parent antibody. The humanized HCC specific scFv can be further evaluated and developed as therapeutics.