Characterization and Expression of Tomato infectious chlorosis virus Coat Protein Gene in Escherichia coli

Abstract

Tomato infectious chlorosis virus (TICV) is a member of Crinivirus genus. TICV infects tomato crop in Garut and Cipanas, West Java Indonesia. Tomato initially showed bright interveinal yellowing symptoms. The coat protein gene (CP-TICV) was amplified by RT-PCR from total RNA extracted from infected tomato leaves and the amplified fragment was cloned and completely sequenced. The fragment was subsequently subcloned into the pET-21b expression vector. The recombinant plasmid was transformed to Escherichia coli strain BL21(DE3)pLysS to express the coat protein. The coat protein fused to a 6xhistag, was purified by affinity chromatography using a NiNTA spin column. The identity of the purified protein was confirmed by SDS-PAGE. In this experiment, 792 bp of CP-TICV gene of TICV virus has been successfully cloned, sequenced, and expressed in E. coli. Based on nucleotide sequences alignment analysis, TICV-Indonesia strain showed 100% identity to TICV –Spain strain and 99% identity to North America, France, and California strains and based on amino acid sequences alignment analysis, TICV-Indonesia strain showed 100% identity to TICV –Spain, North America, France strains and 99.2% identity to California strain. CP-TICV showed over expressed in E. coli when it is induced with 1 mM IPTG and incubated at 37oC. Purified CP-TICV-Histag recombinants protein sized 29 kDa based on SDSPAGE analysis.

Penyakit klorosis pada tanaman tomat telah ditemukan di Indonesia. Penyebab penyakit klorosis ini adalah Tomato infectious chlorosis virus (TICV) anggota dari genus Crinivirus (famili Closteroviridae). Pada tanaman tomat, infeksi TICV menyebabkan berbagai gejala antara lain: klorosis antar tulang daun (interveinal yellowing), nekrotik, daun rapuh, ukuran buah mengecil, dan proses pemasakan buah terganggu. Deteksi virus dapat dilakukan dengan reverse transcription–polymerase chain reaction (RT-PCR) dan dengan pendekatan serologi yaitu enzyme-linked immunosorbent assay (ELISA), tissue blot immunosorbent assay (TBIA), dan dot blot immunosorbent assay (DIBA). Antiserum merupakan komponen utama dalam uji serologi, namun demikian antiserum terhadap TICV belum tersedia di Indonesia. Usaha penyediaan antiserum bermanfaat sebagai sarana dalam mendeteksi TICV. Ekspresi gen coat protein (CP) TICV pada Escherichia coli menjanjikan tersedianya antigen dalam jumlah yang cukup untuk produksi antiserum.