In this study, transgenic tobacco plants were generated to express anti-colorectal cancer large single chain (LSC) antibody CO17-1A (LSC CO) and LSC CO tagged with the endoplasmic reticulum (ER) retention signal KDEL (LSC COK). The LSC antibodies were constructed by linking the C-terminus of variable region (VL) of the light chain (LC) to the N-terminus of the heavy chain (HC) of mAb CO17-1A with a linker peptide. Reverse-transcription PCR (RT) and immunoblot analyses showed that the LSC CO17-1AK expression level was higher than LSC CO17-1A in plant. In glycosylation analysis, oligomannose type glycan form was observed in LSC COK and plant-specific α(1,3)-fucose was observed in LSC CO. Binding activity of both LSC CO17-1A and LSC CO17-1AK to human colorectal cancer cell lines SW480 and SW620 were confirmed using indirect cell enzyme-linked immunosorbent assay (ELISA). In indirect cell ELISA, the LSC antibodies had a higher binding activity than full-size mAb CO17-1AK. In surface plasmon resonance (SPR) assay using epidermal cell adhesion molecule (EpCAM) highly expressed on the human colorectal cancer cells, both LSC antibodies showed a similar binding activity to the full-size mAb CO17-1A. These results indicated that LSC antibodies with functional binding activities to human colorectal cancer cells and EpCAM protein were successfully expressed in the transgenic tobacco plant.

中文翻译：

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抗结直肠癌大单链抗体（LSC）在植物中的表达，功能和糖基化

在这项研究中，产生了转基因烟草植物，以表达带有内质网（ER）保留信号KDEL（LSC COK）的抗结直肠癌大单链（LSC）抗体CO17-1A（LSC CO）和LSC CO。通过将mAb CO17-1A的轻链（LC）可变区（VL）的C端与重链（HC）的N端连接，可以构建LSC抗体。逆转录PCR（RT）和免疫印迹分析表明，植物中LSC CO17-1AK的表达水平高于LSC CO17-1A。在糖基化分析中，在LSC COK中观察到寡甘露糖型聚糖形式，在LSC CO中观察到植物特异性α（1,3）-岩藻糖。使用间接细胞酶联免疫吸附测定法（ELISA）证实了LSC CO17-1A和LSC CO17-1AK与人结肠直肠癌细胞系SW480和SW620的结合活性。在间接细胞ELISA中，LSC抗体的结合活性高于全尺寸mAb CO17-1AK。在使用在人大肠癌细胞上高度表达的表皮细胞粘附分子（EpCAM）进行的表面等离振子共振（SPR）分析中，两种LSC抗体均显示出与标准mAb CO17-1A相似的结合活性。这些结果表明在转基因烟草植物中成功表达了具有与人结直肠癌细胞和EpCAM蛋白功能结合活性的LSC抗体。在使用在人大肠癌细胞上高度表达的表皮细胞粘附分子（EpCAM）进行的表面等离振子共振（SPR）分析中，两种LSC抗体均显示出与标准mAb CO17-1A相似的结合活性。这些结果表明在转基因烟草植物中成功表达了具有与人结直肠癌细胞和EpCAM蛋白功能结合活性的LSC抗体。在使用在人大肠癌细胞上高度表达的表皮细胞粘附分子（EpCAM）进行的表面等离振子共振（SPR）分析中，两种LSC抗体均显示出与标准mAb CO17-1A相似的结合活性。这些结果表明在转基因烟草植物中成功表达了具有与人结直肠癌细胞和EpCAM蛋白功能结合活性的LSC抗体。