title: Synthesis and expression of recombinant interferon alpha-5 gene in tobacco chloroplasts, a non-edible plant.
abstract: The production of interferon alpha from microbial to mammalian expression system, have certain precincts in terms of cost, scalability, safety and authenticity. Modern biotechnology exploits transgenic crops to get large quantities of complex proteins in a cost-effective way. In order to overcome several challenges from biosafety point of view, the chloroplast transformation strategy is one of the best approaches since plastids are strictly maternally inherited in most of the cultivated species. In the present study the interferon alpha 5 gene was synthesized by using complex set of oligos. After sequence confirmation of the synthesized gene, the histidine residues along with the thrombin protease site were engineered upstream to the synthetic interferon alpha 5 gene. The recombinant fragment was then tethered with chloroplast light inducible promoter, rbcl followed by sequential cloning to develop chloroplast transformation vector to target the cassette into the inverted repeat region of plastome through two events of homologous recombination. The putative transgenic plants obtained through biolistic delivery method and as a result of antibiotic selection of bombarded leaves, were subjected to different rounds of selection and regeneration for homoplasmicity. The spectinomycin-resistant shoots were analyzed through Polymerase Chain Reaction and Sothern blotting. The expression of introduced synthetic genes was recorded using Enzyme Linked ImmunoSorbant Assay technique. It was experienced that mature leaves contained comparatively high levels of interferon compared to young and senescence leaves.
authors.affiliation: National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, P. O. Box 577, Faisalabad, 38000, Pakistan. sarwarkhan_40@hotmail.com
authors.forename: Muhammad Sarwar
authors.initials: MS
authors.lastname: Khan
authors.forename: Farwa
authors.initials: F
authors.lastname: Nurjis
mesh_terms: D000595:Amino Acid Sequence; D019470:Biolistics; D015139:Blotting, Southern; D002736:Chloroplasts / Q000378:metabolism*; D018744:DNA, Plant / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010936:Plant Extracts / Q000378:metabolism; D010945:Plants, Edible / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D018744:DNA, Plant; D016898:Interferon-alpha; D010936:Plant Extracts; D011994:Recombinant Proteins