Cloning of d1 domain of coxsackie B and adenovirus receptor (CAR) into an expression vector

Human adenovirus (Ads) has been one of the therapeutic systems used for treating cancers and other diseases. Attachment of an adenovirus (Ad) to a cell is mediated by the capsid fiber protein known as a receptor. To date, only the cellular fibre receptor for subgroup C serotypes 2 and 5, the coxsackievirus-adenovirus receptor (CAR) protein, has been identified and cloned. The coxsackie B and adenovirus receptor (CAR) is a member of the immunoglobulin superfamily consisting two domain, domain1 (D1) and domain 2 (D2). It has been suggested that CAR extracellular domain, D1 is sufficient to allow adenovirus attachment and infection, whilst the transmembrane domains, including D2 of CAR are not essential for attachment of adenovirus. Therefore, in this study, investigation was carried out to clone CAR D1 into an expression vector. Full length CAR was used to construct a CAR D1 fusion protein. The CAR D1 fusion protein was fused to an enhanced green fluorescent protein (EGFP) vector. Restriction enzymes digestion analysis and PCR screening were carried out to prove the correct insert was obtained. The construct was sent for sequencing and the sequences were checked using the BLAST software and were proven to match with CAR D1. Hence, CAR D1 fusion protein was successfully constructed and cloned into the EGFP vector.