[二硫键稳定的抗肝癌单链Fv与截短型铜绿假单胞菌外毒素融合蛋白的构建、表达及功能鉴定]
[Construction, expression and functional characterization of disulfide-stabilized anti-hepatocarcinoma single chain Fv fused with truncated Pseudomonas exotoxin].
作者信息
Zhao Jun, Sun Zhi-wei, Liu Yan-fang, Yu Wei-yuan
机构信息
Department of Pathology, Fourth Military Medical University,Xi'an 710032, China.
出版信息
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2003 Nov;19(6):585-7.
AIM
To prepare fusion protein of disulfide-stabilized anti-hepatocarcinoma single chain Fv(dsFv)and truncated Pseudomonas exotoxin(PE38).
METHODS
The gene encoding the fusion protein was cloned into the prokaryotic expression vector pTIG which was then transformed into E.coli origami(DE3). The expressed product was purified through Ni-NTA agarose affinity chromatography column. The cytotoxicity of the purified dsFv-PE38 was detected by MTT colorimetry.
RESULTS
The immunotoxin dsFv PE38 was expressed in E.coli origami(DE3) in a soluble form, which accounted for nearly 21% of the total bacterial soluble proteins. The results of cytotoxic assay showed that the dsFv-PE38 could specifically kill hepatocarcinoma cells, while spared the normal hepatic cells.
CONCLUSION
The immunotoxin dsFv-PE38 may have some potential value in the treatment of hepatocarcinoma.
目的
制备二硫键稳定的抗肝癌单链Fv（dsFv）与截短型铜绿假单胞菌外毒素（PE38）的融合蛋白。
方法
将编码融合蛋白的基因克隆至原核表达载体pTIG，随后转化至大肠杆菌Origami（DE3）。表达产物经镍-亚氨基二乙酸琼脂糖亲和层析柱纯化。采用MTT比色法检测纯化后的dsFv-PE38的细胞毒性。
结果
免疫毒素dsFv-PE38在大肠杆菌Origami（DE3）中以可溶形式表达，占细菌总可溶性蛋白的近21%。细胞毒性试验结果表明，dsFv-PE38可特异性杀伤肝癌细胞，而对正常肝细胞无损伤。
结论
免疫毒素dsFv-PE38在肝癌治疗中可能具有一定的潜在价值。
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