title: Recombinant avian adeno-associated virus-mediated oviduct-specific expression of recombinant human tissue kallikrein.
abstract: Human tissue kallikrein (hK1) plays an important role in regulation of blood pressure, electrolyte and glucose transport, and renal function. To evaluate the feasibility of viral vector-mediated expression of recombinant human tissue kallikrein (rhK1) in the egg white of laying hens, human tissue kallikrein gene (hKLK1) cDNA-expression cassette was subcloned into avian adeno-associated virus (AAAV) transfer vector pAITR and transfected into AAV-293 cells with AAAV helper vector pcDNA-ARC and adenovirus helper vector pHelper. The recombinant viral particles with a typical AAAV morphology and relatively high titer were generated and identified by PCR and electron microscopy. After 1 intravenous injection of each laying hen with 2 x 10(10) viral particles, oviduct-specific expression of hKLK1 cDNA was demonstrated by reverse transcription-PCR. Secretion of rhK1 into the egg white was detected by enzymatic assay from d 2, reaching the highest level of 107 U/mL in wk 3, and lasted for more than 6 wk after injection. Western blotting showed that the oviduct-expressed rhK1 had the same molecular mass with the natural enzyme. These data suggest that rAAAV can mediate high level and long-lasting transgene expression in oviduct cells, and the established expression system is useful for production of other recombinant proteins.
authors.affiliation: College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, People's Republic of China.
authors.forename: A P
authors.initials: AP
authors.lastname: Wang
authors.forename: H C
authors.initials: HC
authors.lastname: Sun
authors.forename: J Y
authors.initials: JY
authors.lastname: Wang
authors.forename: Y J
authors.initials: YJ
authors.lastname: Wang
authors.forename: W F
authors.initials: WF
authors.lastname: Yuan
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D002645:Chickens / Q000235:genetics / Q000378:metabolism*; D000229:Dependovirus / Q000235:genetics*; D004529:Egg White / Q000737:chemistry; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008854:Microscopy, Electron / Q000662:veterinary; D016254:Mutagenesis, Insertional; D010057:Oviducts / Q000378:metabolism* / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D020840:Tissue Kallikreins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins; D020840:Tissue Kallikreins