Recombinant avian adeno-associated virus-mediated oviduct-specific expression of recombinant human tissue kallikrein.
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.
Wang
A P
AP
College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, People's Republic of China.
Sun
H C
HC
Wang
J Y
JY
Wang
Y J
YJ
Yuan
W F
WF
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
D012333:RNA, Messenger; D011994:Recombinant Proteins; D020840:Tissue Kallikreins