Construction and characterization of the recombinant immunotoxin RTA-4D5-KDEL targeting HER2/neu-positive cancer cells and locating the endoplasmic reticulum
Construction and characterization of the recombinant immunotoxin RTA-4D5-KDEL targeting HER2/neu-positive cancer cells and locating the endoplasmic reticulum
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靶向 HER2/neu 阳性癌细胞并定位内质网的重组免疫毒素 RTA-4D5-KDEL 的构建和表征
DOI：
10.1007/s00253-018-9291-z
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发表时间：
2018
影响因子：
5
通讯作者：
Ren Yuhong
中科院分区：
文献类型：
--
作者：
Jiao Pengyan;Zhang Jian;Dong Yuguo;Wei Dongzhi;Ren Yuhong
免疫毒素的特异性靶向性使其在癌症治疗中得到广泛应用。蓖麻毒素蛋白的A链(RTA)是一种N-糖苷酶，催化从28S rRNA上去除腺嘌呤，阻止蛋白质翻译并导致细胞死亡。蓖麻毒素毒性很强，但只能在细胞质内发挥其毒性作用。在本研究中，我们将抗HER2单链可变区4D5单链抗体和内质网靶向肽KDEL连接到RTA的C末端，构建了免疫毒素RTA-4D5-KDEL。体外实验表明，RTA-4D5-KDEL对卵巢癌细胞SKOV-3的抑制作用分别是RTA和RTA-4D5的440倍和28倍。RTA-4D5-KDEL对HER2过表达的SKOV-3细胞有较强的抑制作用，对正常HEK-293细胞和H460肺癌细胞的损伤较小。免疫荧光实验表明，免疫毒素RTA-4D5能与SKOV-3细胞特异性结合，但不能与正常细胞HEK-293结合。免疫毒素RTA-4D5-KDEL能迅速将重组蛋白定位于内质网。这些结果表明，重组免疫毒素RTA-4D5-KDEL对高表达HER2的卵巢癌细胞有较强的抑制作用，而对正常细胞的损伤较小。
The specific targeting of immunotoxins enables their wide application in cancer therapy. The A-chain of the ricin protein (RTA) is an N-glycosidase that catalyzes the removal of adenine from the 28S rRNA, preventing protein translation and leading to cell death. Ricin is highly toxic but can only exert its toxic effects from within the cytoplasm. In this study, we linked the anti-HER2 single-chain variable fragment 4D5 scFv and the endoplasmic reticulum-targeting peptide KDEL to the C-terminal of the RTA to construct immunotoxin RTA-4D5-KDEL. In vitro experiments showed that the anticancer effect of RTA-4D5-KDEL towards ovarian cancer cells SKOV-3 increased 440-fold and 28-fold relative to RTA and RTA-4D5, respectively. RTA-4D5-KDEL had a strong inhibitory effect on HER2-overexpressing SKOV-3 cells and caused little damage to normal HEK-293 cells and H460 lung cancer cells. Immunofluorescence experiments showed that the immunotoxin RTA-4D5 could specifically bind to SKOV-3 cells, but not to normal cells HEK-293. The immunotoxin RTA-4D5-KDEL could rapidly localize the recombinant protein to the endoplasmic reticulum. These results suggest that the recombinant immunotoxin RTA-4D5-KDEL has a strong inhibitory effect on ovarian cancer cells that overexpress HER2 but little harm to the normal cells.