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.
Members of the Camelidae (camels, dromedaries, llamas, alpacas, guanacos and vicunas) are known to produce Igs (immunoglobulins) devoid of light chains and CH1s (constant heavy-chain domains). The antigen-specific binding fragments of these heavy-chain antibodies therefore comprise one single domain (the so-called 'VHH') and are of great importance in biotechnological applications. To evaluate the expression and biological activity of sdAbs (single-domain antibodies) in plants, which, on account of their small size and antigen-recognition properties, would have a major impact on antibody-engineering strategies, we constructed a pBI121-VHH gene encoding the recombinant sdAb fragments with specificity for a cancer-associated mucin, MUC1. Analysis of transgenic tobacco (Nicotiana tabacum cultivar Xanthi) plants by PCR and Western blotting demonstrated the expression of sdAb, while ELISA results with various MUC1 antigens and immunocytochemistry with cancerous cell lines confirmed that the activity of these molecules compared favourably with that of the parent recombinant antibodies. Protein purification was achieved by using sequential (NH4)2SO4 precipitation, gel filtration and immunoaffinity chromatography. Analysis of the purified VHH by ELISA indicated that the purified antibody fragments were able to react successfully with a MUC1-related peptide. These results reaffirm that the tobacco plant is a suitable host for the production of correctly folded VHH antibody fragments with diagnostic and therapeutic applications.
Electrophoretic pattern and quantitative changes in soluble proteins were determined in the leaves of spring and winter cultivars of barley (Hordeum vulgare L., cv. Makouei and cv. Reyhan, respectively) exposed to 4 degrees C for 14 d. Seedlings were grown in a controlled growth chamber for 2 weeks at a constant air temperature of 20 degrees C and then transferred to constant 4 degrees C for 14 d followed by returning to 20 degrees C (cold treatment), or they were maintained throughout at 20 degrees C during the experimental period of 40 d (control treatment). Plants were sampled every 48 h for leaf fresh weight measurements. Total leaf soluble proteins were extracted and their concentration was either determined by a colorimetric method, or size-fractionated on SDS-PAGE. Low temperature-induced increases in protein amount occurred over the second week of exposure to cold treatment irrespective of cultivar: the winter cultivar was 2 d prior in this response. The protein patterns and their density showed differences between-cultivars and between-temperature treatments. A new cold-induced polypeptide was recognized in the leaves of winter barley cultivar on day 22 (8 d at 4 degrees C) compared to the control. This polypeptide was produced earlier over the first 48 h of low temperature in the winter cultivar compared with the spring one, recognizing in the leaves of cold-treated seedling until day 26. This more rapid response to a low temperature by the winter barley cultivar indicates a more sensitive response compared with the spring barley, probably cold-shock protein is a component of this cold-induced response.
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