title: Production and characterization of anti-(mucin MUC1) single-domain antibody in tobacco (Nicotiana tabacum cultivar Xanthi).
abstract: 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.
authors.affiliation: Department of Plant Breeding, School of Agriculture, Tarbiat Modarres University, Jalale-Ale-Ahmad High Way, Tehran 14115-336, Iran.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Ismaili
authors.forename: Mokhtar
authors.initials: M
authors.lastname: Jalali-Javaran
authors.forename: Mohammad J
authors.initials: MJ
authors.lastname: Rasaee
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Rahbarizadeh
authors.forename: Mehdi
authors.initials: M
authors.lastname: Forouzandeh-Moghadam
authors.forename: Hamid Rajabi
authors.initials: HR
authors.lastname: Memari
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000276:immunology; D016466:CHO Cells; D002162:Camelus; D018288:Carcinoma, Small Cell / Q000276:immunology; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis* / Q000276:immunology; D018396:Mucin-1 / Q000276:immunology*; D010455:Peptides / Q000276:immunology; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000096:biosynthesis; D012231:Rhizobium / Q000235:genetics; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D000906:Antibodies; D000951:Antigens, Neoplasm; D007143:Immunoglobulin Heavy Chains; D018396:Mucin-1; D010455:Peptides; D011994:Recombinant Proteins