Expression of the human igf-1 in transgenic plastids

Abstract

A plastid transformation vector for a stably transforming a plastid genome, comprising, as operably-linked components, a first flanking sequence, a DNA sequence coding for synthetic insulin-like growth factor-1 (IGF-1s) (seek ID NO. 1) or a substantially homologous DNA sequence of IGF-1s, which is capable of expression in the plastid genome, and a second flanking sequence.

1 . A plastid transformation vector for a stably transforming a plastid, comprising, as operably-linked components, a first flanking sequence, a DNA sequence coding for an insulin-like growth factor-1 (IGF-1) or, which is capable of expression in said plastid genome, and a second flanking sequence.  
     
     
         2 . The vector of  claim 1 , wherein the DNA sequence coding for the IGF-1 is a synthetic IGF-1 (IGF-1s) and consists of about 60% adenine and thymine nucleotides.  
     
     
         3 . The vector of  claim 1  further comprising a regulatory sequence.  
     
     
         4 . The vector of  claim 3 , wherein said regulatory sequence comprises a promoter operative in said plastid genome.  
     
     
         5 . The vector of  claim 4 , wherein said promoter is 16 sRNA.  
     
     
         6 . The vector of  claim 3 , wherein said regulatory sequence comprises psbA 5′ and psbA 3′ elements.  
     
     
         7 . The vector of  claim 3 , wherein said regulatory sequences further comprise a 5′ UTR capable of providing transcription and translation enhancement of said DNA sequence coding for IGF-1.  
     
     
         8 . The vector of  claim 3 , wherein said regulatory sequences further comprise a 3′ untranslated region (UTR) capable of conferring transcript stability to said IGF-1.  
     
     
         9 . The vector of  claim 1 , wherein said first flanking sequence is trnI, and wherein said second flanking sequence is trnA.  
     
     
         10 . The vector of  claim 1 , wherein the vector is component for stably integrating into a plastid genome of a plant, and wherein said first and second flanking DNA sequences are substantially homologous to sequences in a spacer region of said plastid genome, and wherein said first and second flanking sequences are conserved in the plastid genome of said higher plant species.  
     
     
         11 . The vector of  claim 10 , wherein said spacer region is a transcriptionally active spacer region.  
     
     
         12 . The vector of  claim 10 , wherein said trnI and trnA provide for homologous recombination to insert an IGF-1 or region of an inverted repeat region of a chloroplast genome.  
     
     
         13 . The vector of  claim 1 , wherein said DNA sequence coding for IGF-1 or a substantially homologous DNA sequence of IGF-1 is located in a single copy region of said plastid genome.  
     
     
         14 . The vector of  claim 7 , wherein said 5′ UTR is a 5′ UTR of psbA.  
     
     
         15 . The vector of  claim 8 , wherein said 3′ UTR is a 3′ UTR of psbA.  
     
     
         16 . The vector of  claim 1 , further comprising a DNA sequence encoding a selectable marker.  
     
     
         17 . The vector of  claim 16 , wherein said selectable marker is an antibiotic-free selectable marker.  
     
     
         18 . The vector of  claim 17 , wherein said antibiotic-free selectable marker is Betaine aldehyde dehydrogenase (BADH).  
     
     
         19 . The vector of  claim 16 , wherein said DNA sequence encoding is selectable marker encodes an antibiotic resistance selectable marker.  
     
     
         20 . The vector of  claim 19 , wherein said antibiotic resistance selectable marker is aadA.  
     
     
         21 . A method for producing IGF-1 comprising: 
 integrating the plastid transformation vector of  claim 1  into the plastid genome of a plant cell; and    growing said plant cell to thereby express said IFN.    
     
     
         22 . The method of  claim 21 , wherein said IGF-1 is competent to produce an immunogenic response in a mammal.  
     
     
         23 . The method of  claim 22 , wherein said immunogenic response is substantially free of negative side effects associated with injected IGF-1.  
     
     
         24 . An isolated and purified IGF-1, competent to produce and immunogenic response in a mammal.  
     
     
         25 . The isolated and purified IGF-1 of  claim 24 , wherein said IGF-1 is a synthetic IGF-1 having an adenine and thymine DNA content of about 60%.  
     
     
         26 . An orally administerable therapeutic human interferon IGF-1, suitable for oral administration to a mammal.  
     
     
         27 . A method for variable-expressing IGF-1 comprising: 
 integrating a plastid transformation vector according to  claim 1  into a plastid genome of a plant cell; and    growing said plant cell to express said IGF-1.    
     
     
         28 . A plant stably transformed with the transformation vector of  claim 1 .  
     
     
         29 . A progeny of the plant of  claim 28 .  
     
     
         30 . A seed of the plant of  claim 28 .  
     
     
         31 . A part of the plant of  claim 28 , comprising a plastid including said DNA sequence coding for therapeutic human interferon IFN.  
     
     
         32 . The plant of  claim 28 , wherein said plant is an edible plant suitable for mammal consumption.  
     
     
         33 . The plant of  claim 28 , wherein said plant further comprises at least one chloroplast transformed with the vector of  claim 1 .  
     
     
         34 . The plant of  claim 28 , wherein said plant further comprises mature leaves transformed with the vector of  claim 1 .  
     
     
         35 . The plant of  claim 28 , wherein said plant further comprises young leaves transformed with the vector of  claim 1 .  
     
     
         36 . The plant of  claim 28 , wherein said plant further comprises old leaves transformed with the vector of  claim 1 .  
     
     
         37 . The plant of  claim 28 , wherein the expression of IGF-1 is at least about 6.0 percent total soluble protein.  
     
     
         38 . The plant of  claim 28 , wherein said expression of IGF-1 in said edible plant is between about 9.5-32.5% total soluble protein.