A promising alternative to conventional antibodies is the single-domain antibody fragment of the Camelidae (V(HH)), which (because of features such as small length, high expression, solubility, and stability) is preferred to other antibody derivatives. In this report, a recombinant single-domain antibody (V(HH)) against MUC1 mucin in the tobacco plant, which may be considered as a suitable and economical alternative expression system, was produced. This antibody was expressed under the control of a strong constitutive promoter, CaMV35S, and NOS terminator. A plant high-expression sequence (Kozak sequence) was linked at the 5' end for overexpression of the V(HH) gene. The constructed cassette (pBIV(HH)) was transferred to agrobacterium, and the VHH gene was inserted into the plant genome by agrobacterium-mediated transformation. Transgenic lines were selected on kanamycin (100 mg/L) and maintained in soil, and subsequent generations were obtained. The presence and expression of the transgene was confirmed in the transformants by polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot. Tobacco transgenic lines leave expressed V(HH) at levels varying from 1.12% to 1.63% of the total soluble protein. This report examines the transformation and expression of recombinant single-domain antibody (V(HH)) against antigen-associated tumor in tobacco plants.

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BackgroundMolecular farming is the production of important recombinant proteins in transgenic organisms on an agricultural scale. Interferons are proteins with antiviral and antitumor activities and can be used for viral infections and cancers treatments.ObjectivesThis study reports the transformation of INF α2b gene in tobacco plant for the first time in Iran.Materials and MethodsInterferon α2b gene was amplified by PCR using specific primers containing appropriate restriction enzymes, plant highly expression sequence and Histidine tag sequence. Target sequence was cloned in plant expression vector pCAMBIA1304 and the construct named pCAMINFα. pCAMINFα was transferred to E. coli strain DH5α and plated on LB agar medium containing kanamycin 50 mgl-1. The colonies were confirmed by colony PCR and sequencing. The construct was transferred into Agrobacterium tumefaciens by freeze-thaw method and transformed colonies were confirmed by colony PCR. Tobacco plants (cultivar xanthi) were inoculated with A. tumefaciens strain LBA4404 by leaf disc method. Inoculated explants were cultured on MSII (MS + BAP 1mgl-1 + NAA 0.1 mgl-1) at 28°C and darkness for 48 hours. Then explants were transferred to selection medium containing cephotaxime (250 mgl-1) and hygromycin (15 mgl-1) in a 16/8 (day/night) h photoperiod in growth room with an irradiance of 5000 lux. Transgenic plants were regenerated and transferred to perlite. Genomic DNA was extracted from regenerated plants by Dellaporta method at 5-leaf step and transgenic lines were confirmed by PCR with specific primers. Expression of Interferon α2b gene was confirmed by dot blotting.ConclusionsSince no report of interferon alpha production in plants in Iran has been expressed yet, this research could create a field of producing this drug in tobacco, in Iran.

Background: Plants are among promising and suitable platform systems for production of recombinant biopharmaceutical proteins due to several features such as safety, no need for fermentation, inexpensive investment, and fast and easy scale-up. Human insulin is one of the most widely used medicines in the world. Up to now different expression systems including Escherichia coli, yeast and CHO have been exploited for producing recombinant human insulin and a variety of different recombinant insulin are extensively used. Objectives: This study reports on the transformation and expression of proinsulin gene in tomato plants for the first time in Iran. Materials and Methods: This study reports the cloning, transformation and expression of proinsulin gene in tomato plants. Specific primers were designed and used for PCR amplification and cloning of the proinsulin gene in the plant expression vector pCAMBIA1304. The recombinant construct was transferred into Agrobacterium tumefaciens strain LBA4404, and used for Agrobacterium mediated stable transformation of tomato plants. Presence of the desired gene in transgenic lines was confirmed through colony PCR and sequencing. The expression of the protein in transgenic lines was confirmed by immunodot blot assay. Results: The presence of the proinsulin gene in the genomic DNA of transgenic tomato was confirmed by PCR. Also total protein of transgenic tomato was extracted and the expression of proinsulin was detected using dotblot assay. Conclusions: This survey addresses the possibility of proinsulin gene transfer and expression in tomato transgenic lines. This study can be used as a basis for future researches to produce human proinsulin in tomato and other candidate plants.