Cloning and expression of coat protein gene of the most prevailing citrus tristeza virus isolate in Northern Iran

Citrus tristeza is one of the most destructive diseases of citrus and its causing agent CTV was introduced from Japan to northern Iran in the late 1960s. After about three decades of restriction to originally infected Satsuma trees, natural spread of CTV has being undertaken and melon aphid (Aphis gossypii Glov.) was confirmed as a potent vector in these areas. Regarding the specificity of aphidvirus interaction, in this research we aimed to characterize the most dominating strain of CTV in northern Iran and accordingly express their CP25 gene as a source of antigen for antibody production. Ninety CTV isolates were recovered from originally infected satsuma trees and some other neighboring or distant citrus growing orchards and their coat protein gene was amplified using RT-PCR method. SSCP profile analysis of the amplified CP25 genes showed that all isolates are classified into 3 groups each with unique profile. However, CP25 gene nucleotide sequence of 3 representative isolates of each SSCP group showed high sequence identity (up to 98%) and close relationship to NUAgA severe seedling yellows strain of Japan. Biological properties of these representative isolates studied through graft inoculation onto known indicator plants confirmed that they produce seedling yellows-type symptoms. CP25 gene of a representative isolate was ligated to pET28a(+) expression vector and transformed into Bl21 competent cells to express recombinant coat protein. Protein expression was induced using 1 mM IPTG for 6 h and recombinant protein purified through Ni-NTA affinity chromatography. SDS-PAGE analysis confirmed the presence of a recombinant 25 KD protein.