بیان ژن نوکلئوکپسید ویروس پژمردگی لکه‌ای گوجه فرنگی در در دو سویه BL21(DE3) و BL21(DE3) pLysS باکتری Escherichia coli

Tomato spotted wilt virus is one of the most important virus that infect tomato in tropical and sub-tropical regions. Production of viral proteins in E. coli to raise the antibody is a strategy to overcome the limitation of antigen preparation by classical methods such as virus purification using ultracentrifuge that it is not available in some laboratory. In this research, the TSWV nucleocapsid gene (N) was amplified by a pair of primer (TSWVR and TSWVF) containing restriction sites for BamHI and XhoI enzymes. . The amplified segment was inserted into pTG19 plasmid. Then, the N gene was released from pTG19-TSWVN by digestion with BamHI and XhoI and ligated into the bacterial expression vector, pET32a (Novagen, USA) digested by same enzymes. The new construct (pET32-TSWVN) was transformed into E. coli strains BL21(DE3) and BL21(DE3) pLysS by heat-shock procedure. The integrity of N gene sequence was confirmed by sequencing using T7-promotor and T7-terminator primers (Macrogen, Seoul, South Korea). To induce the protein production, Isopropyl-β-D-thiogalactopyranoside (IPTG) was added into the growth culture at 1 mM. Samples from the incubated culture were analyzed at 4 hours after induction. Analysis of the expressed proteins from both strains by SDS-PAGE (12%) showed an intense band with a size of about 41 kDa, which corresponded to the predicted size of the complete TSWV N plus the fused amino acid tags. In addition, the production of N protein was confirmed in western blot assay using anti-His Tag antibody. The result confirmed that N gene was expressed in both strains of E.coli whereas the expression in BL21 (DE3) pLysS strain was higher than in BL21 (DE3) strain. This protein will be purified and sued for antibody production.