Kim Kibum, Kang Yang Joo, Park Se Ra, Kim Do-Sun, Lee Seung-Won, Ko Kinarm, Ponndorf Daniel, Ko Kisung
Department of Medicine, Therapeutic Protein Engineering Lab, College of Medicine, Chung-Ang University, Seoul, South Korea.
Vegetable Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration, Wanju-gun, South Korea.
PeerJ. 2021 Apr 7;9:e10851. doi: 10.7717/peerj.10851. eCollection 2021.
Immunization with thetumor-associated antigen GA733 glycoprotein, which is highly expressed in colorectal cancer, is considered to be a promising strategy for cancer prevention and treatment. We cloned a fusion gene of GA733 and immunoglobulin Fc fragment (GA733-Fc), and that of GA733-Fc and an endoplasmic reticulum retention motif (GA733-FcK) into the Cowpea mosaic virus (CPMV)-based transient plant expression vector, pEAQ-. (LBA4404) transformed with the vectors pEAQ--GA733-Fc and pEAQ--GA733-FcK was infiltrated into the leaves of plants. To optimize harvesting of leaf to express therapeutic glycoproteins both spatially and temporally, protein expression levels at various leaf positions (top, middle, and base) and days post-infiltration (dpi) were investigated. The GA733-Fc and GA733-FcK genes were detected in leaves at 1-10 dpi using PCR. As assessed by western blot, GA733-Fc and GA733-FcK were expressed at the highest levels in the top leaf position at 5 dpi, and GA733-FcK was expressed more than GA733-Fc. The proteins were successfully purified from infiltrated leaves using protein A affinity chromatography. ELISA verified that an anti-GA733 antibody recognized both purified proteins. Thus, a functional GA733-Fc colorectal cancer vaccine protein can be transiently expressed using a CPMV virus-based vector, with an optimized expression time and leaf position post-infiltration.
用在结直肠癌中高表达的肿瘤相关抗原GA733糖蛋白进行免疫被认为是一种很有前景的癌症预防和治疗策略。我们将GA733与免疫球蛋白Fc片段的融合基因（GA733-Fc）以及GA733-Fc与内质网滞留基序的融合基因（GA733-FcK）克隆到基于豇豆花叶病毒（CPMV）的瞬时植物表达载体pEAQ-中。用载体pEAQ--GA733-Fc和pEAQ--GA733-FcK转化的（LBA4404）被浸润到植物叶片中。为了在空间和时间上优化收获叶片以表达治疗性糖蛋白，研究了不同叶位（顶部、中部和基部）以及浸润后天数（dpi）的蛋白质表达水平。使用PCR在1至10 dpi时在叶片中检测到GA733-Fc和GA733-FcK基因。通过蛋白质印迹评估，GA733-Fc和GA733-FcK在5 dpi时在顶部叶位表达水平最高，并且GA733-FcK的表达量高于GA733-Fc。使用蛋白A亲和层析从浸润的叶片中成功纯化出了这些蛋白质。ELISA证实抗GA733抗体识别这两种纯化的蛋白质。因此，使用基于CPMV病毒的载体可以瞬时表达功能性GA733-Fc结直肠癌疫苗蛋白，并在浸润后有优化的表达时间和叶位。
