Full Resistance to Tobacco Mosaic Virus Infection Conferred by the Transgenic Expression of a Recombinant Antibody in Tobacco

Abstract

The objective of this study was to obtain transgenic tobacco (Nicotiana tabacum L. cv. Samsun NN) plants resistant to tobacco mosaic virus, TMV-vulgare strain, by the expression of a single-chain variable fragment ( scFv-anti-TMV) against the TMV coat protein.The scFv-anti-TMV gene was made by combining the coding sequences of the heavy and light chain variable domains of the full antibody molecule with a sequence encoding a. flexible linker. The scFv-anti-TMV was cloned under the control of the 35S promoter in the plant expression vector pBI12I, and used for Agrobcterium-mediated transformation of tobacco. Integration of the scFv-anti-TMV gene into the genome of the transgenic plants and its expression were confirmed using PCR, immunoblot, and ELISA analysis. Transgenic plants expressing scFv-anti-TMV and untransformed control plants transformed with pBII2I only were inoculated with I f-Lg/ml TMV. Constitutive expression of this scFv-anti-TMV resulted in complete immunity against TMV infection in transgenic plants ( 100% reduction of virus infection). In contrast, control plants were heavily infected by TMV. These results confirmed the applicability of antibody expression as a means of protecting plants from virus infection.

Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance

“…CP-specific antibodies were predominantly produced in transgenic plants until recently with results ranging from very low resistance (Fecker et al 1997) to immunity (Bajrovic et al 2001). Another approach has focused on the use of scFvs raised against non-structural viral proteins, as those involved in virus replication (Boonrod et al 2004) or proteases involved in virus polyprotein cleavage (Gargouri-Bouzid et al 2006;Bouaziz et al 2009;Nickel et al 2008), which generally provided strong and wide-spectrum resistance.…”

“…Interestingly, expression in the cytosol of a scFv derived from the same MAb provided similar protection level to virus challenge even when the scFv accumulated 20,000-fold lower than the full-length antibody (Zimmermann et al 1998;Fischer et al 2001). In general, scFvs have been preferred over full-length antibodies for immunomodulation purposes in transgenic plants, because they are easier to engineer, can be expressed in the cytosol, and are effective even at low accumulation levels (Fecker et al 1997;Zimmermann et al 1998;Schillberg et al 2000;Xiao et al 2000;Bajrovic et al 2001;Nölke et al 2009). More recently, the development of phage display approaches and the generation of synthetic libraries of more stable scFvs together with the targeting of non-structural viral proteins, as RNA replicase and movement proteins, have extended the possibilities of this strategy to interfere with virus infections (Esteban et al 2003;Boonrod et al 2004;Prins et al 2005;Villani et al 2005;Zhang et al 2008).…”

Genetic Manipulation

“…Expression of these in the plant lead to complete immunity against TMV (Bajrovic et al, 2001). Expression of these in the plant lead to complete immunity against TMV (Bajrovic et al, 2001).…”

Cloning of anti-HBsAg single-chain variable fragments from hybridoma cells for one-step ELISA

“…The scFvs were prepared according to the RPAS ScFv and Expression Modules protocol (Amersham Biosciences Corp., Piscataway, NJ) and cloned into the SfiI/NotI linearized pDUCK1 phagemid. The ScFv/pDUCK1 phagemid was then introduced into E. coli TG1 cells, and rescued with the helper phage M13KO7 (Amersham Biosceinces Corp., Piscataway, NJ) [18,19].…”