Development of biopharmaceuticals in plant expression systems: cloning, expression and immunological reactivity of human cytomegalovirus glycoprotein B (UL55) in seeds of transgenic tobacco.
Plant seeds offer unique opportunities for the production and delivery of oral subunit vaccines. We have used the immunodominant glycoprotein B complex of human cytomegalovirus (HCMV), introduced into tobacco plants, as a model system for studying the merit of this promising approach. Given the advantages of expressing proteins in seeds, a novel expression vector was developed incorporating regulatory sequences of glutelin, the major rice seed storage protein, to direct synthesis of recombinant glycoprotein B. Analysis of genomic DNA of 28 selected tobacco transformants by PCR amplification showed that 71% harboured the gB cDNA, a finding further documented by Southern blotting. Specific immunoassays of protein extracts from seeds of positive plants showed that all were producing antigenic glycoprotein B at levels ranging from 70-146 ng/mg extracted protein. In addition, similarity with native glycoprotein B produced in HCMV-infected cells was also demonstrated by inhibition of immunofluorescence on HCMV-infected human fibroblasts. These data are the first to report the expression of an immunodominant antigen of HCMV in plant tissues, indicating the fidelity with which this very large heterologous viral glycoprotein can be synthesized in this model system.
Tackaberry
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Bureau of Biologics and Radiopharmaceuticals, Therapeutic Products Programme, Health Canada, Ottawa, Ont. eilleen_tackaberry@hc-sc.gc.ca
Dudani
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Prior
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Tocchi
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Sardana
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Altosaar
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Ganz
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PR
D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics* / Q000276:immunology; D006801:Humans; D016056:Immunodominant Epitopes / Q000096:biosynthesis / Q000235:genetics; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012639:Seeds / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D014026:Nicotiana / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes; D014614:Vaccines, Synthetic / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*
D016056:Immunodominant Epitopes; D011994:Recombinant Proteins; D014614:Vaccines, Synthetic; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus