THE CONSTRUCTION AAV SYSTEM OF SYNTHETIC GENE RBD AND SPIKE FROM INDONESIAN SARS-COV2

Abstract

Since the advanced technology in medical sector, AAV (Adeno Associated Virus) is one new breakthrough in treatment of genetic diseases. This vector offers many advantages such as efficient gene transfer, a few of toxicity, good in gene expression, and stable of pH. High number of severe acute respiratory syndrome (SARS) case that caused of SARS coronavirus (SARS-CoV), attracted the scientist to develop an effective vaccine. Previously, receptor-binding domain (RBD) and spike of SARS-CoV (S) protein has reported as main target of antibodies. These were recommended as ideal vaccine candidates. Recombinant of adeno associated virus (AAV) has been proven as an effective vector for gene delivery in these vaccine program. This study constructed a new recombinant of receptor-binding domain (RBD) and spike based on adeno associated virus (AAV) system. Genes that encoded of RBD (825 bp) and spike (3930 bp) were designed by SARS Coronavirus that infected Indonesian patient These genes were inserted in pAAV-IRES-hrGFP plasmid using KpnI and XhoI as restriction enzyme. The recombinant also cloned in E. coli DH5α and screened by antibiotic selection. The recombinat sequences were confirmed by sequencing and have a good expression in E. coli DH5α. The immunogenicity of recombinant AAV were evaluated in mice, ELISA, and neutralization assay to know the effectivity doses that could be produced enough antibodies. Thus, these result show that the constructions were suggested that AAV-RBD and AAV-Spike may be promising as SARS COV-2 vaccine candidates.