Effect of leaf position and days post-infiltration on transient expression of colorectal cancer vaccine candidate proteins GA733-Fc and GA733-FcK in Nicotiana benthamiana plant

PeerJ

article

Kibum Kim¹  Yang Joo Kang¹  Se Ra Park¹  Do-Sun Kim²  Seung-Won Lee¹  Kinarm Ko³  Daniel Ponndorf⁴  Kisung Ko¹ 

[1] Department of Medicine, Therapeutic Protein Engineering Lab, College of Medicine, Chung-Ang University;Vegetable Research Division, National Institute of Horticultural and Herbal Science, Rural Development Administration;Department of Stem Cell Biology, Konkuk University School of Medicine, Konkuk University;Department of Biological Chemistry, John Innes Centre, Norwich Research Park

关键词: Agroinfiltration;    Fusion protein;    KDEL;    Molecular biopharming;    Recombinant vaccine;   

DOI  :  10.7717/peerj.10851

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

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-HT. Agrobacterium tumefaciens (LBA4404) transformed with the vectors pEAQ-HT-GA733-Fc and pEAQ-HT-GA733-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 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 N. benthamiana 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.

【 授权许可】

CC BY