earticle

영어

Immunization with the highly expressed tumor-associated antigen GA733 glycoprotein in colorectal cancer is considered to 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-HT. Agrobacterium tumefaciens (LBA4404) transformed with the vectors pEAQ-HT-GA733-Fc and pEAQ-HTGA733- FcK was infiltrated into the leaves of Nicotiana benthamiana 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 protein expression was confirmed by western blot, and GA733-Fc and GA733-FcK proteins were successfully purified from infiltrated N. benthamiana leaves. The binding affinity of purified GA733-Fc and GA733-FcK with Recombinant Human Fcγ RI/CD64 protein (R&D Systems, MN) were measured by SPR spectrometry. Therefore, 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 after penetration. This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ0162662021)” Rural Development Administration, Republic of Korea.