| In recent years,majority of scientists tend to focus on avian oviduct bioreactors instead of mammary gland ones because of the latter's drawbacks such as long generation interval,complicated biological constituents of lactoprotein and fat in milk,and sophisticated purification procedures of recombinant protein and so on.Ovalbumin gene is tissue-specific gene,and it drives to production of ovalbumin in oviduct,which account for 50%of egg-white.Therefore,transgenic chicken could be used to produce some pharmaceutical protein through oviduct-specific expression of exogenous genes.Method:(1).Cloning of six different truncated ovalbumin promoters(OVP).Based on the online analysis of chicken ovalbumin gene with Ensemble,the sequences of 5-'end flanking chain was predicted,which include the sequence of introns and exons,and primers was designed subsequently.Genomic DNA was extracted from the oviduct of laying hens,six truncated ovalbumin promoters were amplified,and their cloning vectors were constructed,which include pMD18-T-OVP944,pMD18-T-OVP1548,pMD18-T-OVP 1667?pMD18-T-OVP 1999,pMD18-T-OVP 2556 and pMD18-T-OVP 3606.(2).Construction of pGL3-OVP vector.Each OVP fragment was removed from its double digested pMD18T-OVP vector,and subcloned to pGL3-Basic to construct a series of pGL3-OVP vectors.(3).Transcriptional activity determination of OVP through dual luciferase reporter assay system.The endotoxin-free plasmid pGL3-OVP and pRL-TK were combined to transfect 293FT cells with FuGENE-HD transfection reagent.After 48 hours,culture medium was harvested,dual luciferase activity was detected by a 96-well plate chemilumineescence detector.(4).Construction of recombinant lentivirus vectors.With the advanced MultiSite Gateway techniques,OVP1548,OVP 1999 and OVP 3606 were respectively cloned to pENTR 5'-TOPO to construct entry vectors including pENTR 5'-TOPO-OVP 1548?pENTR 5'-TOPO-OVP 1999 and pENTR 5'-TOPO-OVP 3606.Subsequently,each entry vector was ligated to lentivirus vector pLenti 6.4/R4R2/V5-DEST to construct lentiviral expression vectors.The positive clones were respectively named as pLenti-OVP 1548-EGFP?pLenti-OVP 1999-EGFP?pLenti-OVP 3606-EGFP.(5).Lentivirus package.The three recombinant lentivirus vectors were respectively transfected into 293 FT cells by FuGENE-HD transfection reagent.After 48 h,culture medium was harvested,new culture medium was supplemented and EGFP expression was observed under a fluorescence microscope.(6).Infection of 293 FT cells.The viral particles ware used to infect 293 FT cells and chicken oviduct epithelial cells.The results showed that:(1)six ovalbumin promoters with different length,or OVP 944,OVP 1548,OVP 1667,OVP 1999,OVP 2556 and OVP 3606,were successfully cloned.(2)Dual luciferase reporter assay indicated that OVP 1548 enjoyed the highest transcription activity among the above 6 ovalbumin promoters.(3)Observation of EGFP expression in 293 FT cells illustrated that pLenti-OVP1548-EGFP had the highest transfection efficiency among the above 3 recombinant lentivirus vectors,which was consistent with the result of dual luciferase reporter assay.(4)Although it enjoyed the highest transcription activity amoung the 6 cloned ovalbumin promoters,compared with CMV promoter,transcription activity of OVP 1548 is far away from our expectation,which was verified by infection assay of pLenti-OVP 1548-EGFP and pLenti-CMV-EGFP in 293 FT cells and chicken oviduct epithelial cells.In this study,with different length and component parts,a series of ovalbumin promoters were designed and cloned,and their transcription activities were determined by dual luciferase assay system,which was subsequently verified by the lentivirus infection of 293 FT cells.This paper will lead to construction of more powerful specific ovalbumin promoters,and then contribute to production of transgenic chicken that produce pharmaceutical proteins in egg-white. |
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