Expression of biological active VHH camelid single domain antibody in transgenic tobacco

Abstract

Functional VHH single domain antibody lacking light chains occur naturally in Camelidae. The single domain nature of VHH gives rise to several unique features when compared to antigen-binding derivatives of conventional antibodies. The level of expression in Escherichia coli was found to be too low for therapeutic purposes. Therefore, there is a need to examine other production systems such as plants. Several plants are the facile and economic bioreactor for large-scale production of industrial and pharmaceutical agents like proteins and antibodies. Here, we have selected tobacco as the host plant because of large scale production capability and many other advantages such as greater safety and lower production costs when compared to animal-based systems. In this study, we have subcloned VHH gene into pBI121 using phasmid pCANTAB5E. The new construct was used to transform the Agrobacterium strains C58GV3101 and LBA4404. Agrobacterium strain C58GV3101 showed a higher virulence on leaf disks of Nicotiana tabacum (NC25). Transgenic tobacco plants were then developed by introducing VHH gene under the control of CaMV 35S promoter. The presence of the VHH antibody gene in the plant genome was verified by PCR analysis and Southern hybridization experiments. Northern blot analysis showed that the genes coding for the VHH could be expressed in tobacco plants. Three lines of transgenic plant that expresses high levels of mRNA were screened in a further analysis. The expression of VHH was then observed in transgenic plants by ELISA using the specific antibody developed, the results showed three to five folds higher than non-transgenic tobaccos.

Key words: VHH antibody fragment, subcloning, antibodies, transgenic tobacco, bioreactor.