Expression Of The Oral SaRS Candidate Vaccine Antigen S-RBD In Tobacco Chloroplasts

Chloroplast transformation offers a number of unique advantages, including high level oftransgene expression, multi-gene expression in single transformation event and transgenecontainment due to maternal inheritance though the targeted integration of the foreign genes inthe chloroplast genome. Chloroplast engineering is becoming a hot spot in the area of transgnicresearch, providing an ideal platform for recombinant pharmaceutical proteins and oral vaccineantigens production in a cost-effective, safe and scalable manner. Severe acute respiratorysyndrome (SARS), caused by a SARS-associated coronavirus (SARS-CoV) is a newly emerginginfectious disease which is characteristic of rapid spread, short latency high mortality rate. Theglobal outbreak of SARS seriously threatened public health and socioeconomic stabilityworldwide. Although this outbreak was eventually brought under control in2003, theSARS-CoV is still likely to remain in some wild animal hosts and under some suitableconditions a SARS epidemic may recur at any time in the future. Until now, no drugs have beenutilized to cure the disease, the development of effective and safe vaccine is urgently needed toprevent a new SARS epidemic. In this paper, the receptor-binding domain (RBD) in the S1subunit of SARS–CoV S protein, which contains multiple conformational neutralizing epitopes,fused with the chlora toxin B subunit, was expressed in tobacco chloroplasts. Our resuilts canpave the way to scalable production of the oral SARS vaccine antigens and adjuvants throughtransplastomic plants. The main results are obtained as below.1. Based on the codon bias of the most represented gene psbA in tobacco chloroplasts, theDNA sequence of the S-RBD is optimized and constructed into the vector pLD-CTB. Thefidelity of the new vector pLD-CTB-mRBD was confirmed by sequencing.2. The fusion gene was introduced into tobacco chloroplasts by microprojectilebombardment method and total of35regenerated tobacco plantlets were obtained aftermultiple-round selection with500mg/L spectinomycin.3. Totally nine homoplasmic transgenic plants were obtained, as confirmed by PCR andSouthern blot analysis.4. SDS-PAGE showed that the foreign fusion protein CTB-RBD (～34KD) was correctly expressed in transplastomic tobaoo plants.