Growth and Differentiation Factor-8(GDF-8) is a new member of TGF-β super-family.It has been shown that GDF-8 is specifically expressed in skeleton muscle in mouse and its function is to inhibit the growth of muscle ...Growth and Differentiation Factor-8(GDF-8) is a new member of TGF-β super-family.It has been shown that GDF-8 is specifically expressed in skeleton muscle in mouse and its function is to inhibit the growth of muscle cell,so it is named as Myostatin.Here,we amplified 3′half-length GDF-8 cDNA from chicken skeleton muscle by RT-PCR,and cloned it into the prokaryotic expression vector pTrcHisB,which was then transformed into E.coli Top10 cells.The recombinant 6×His-GDF-8 fusion protein expressed in the Top10 cells was purified by Ni+-Affinity Chromatography for future study.展开更多
[Objective] This study was to improve the virus replication efficiency of full length infectious cDNA clones by making use of the ribozyme＇s self incision property.[Method] By employing three-step PCR,HDV ribozyme（H...[Objective] This study was to improve the virus replication efficiency of full length infectious cDNA clones by making use of the ribozyme＇s self incision property.[Method] By employing three-step PCR,HDV ribozyme（HdvRz）cDNA was isolated,and cloned into the downstream flanking the genome of the porcine reproductive and respiratory syndrome virus,and into which the bovine growth hormone polyadenylation sequence（BGH）was inserted via enzyme digestion and ligation,yielding pAPRRS-HB.The newly constructed pAPRRS-HB was used to transfect MARC-145 cells,in which the N protein and non-structural protein（nsp2）were determined by indirect immunofluorescence assay after 72 h of expression;meanwhile the virus titer of cell supernatant was tested using TCID50 assay.[Result] pAPRRS-HB containing complete infectious PRRSV cDNA has been successfully developed,and it performed about 10-fold higher virus rescue rate than pAPRRS without the engineered ribozyme element.[Conclusion] The results laid a foundation for revealing the structure and function of PRRSV gene.展开更多
文摘Growth and Differentiation Factor-8(GDF-8) is a new member of TGF-β super-family.It has been shown that GDF-8 is specifically expressed in skeleton muscle in mouse and its function is to inhibit the growth of muscle cell,so it is named as Myostatin.Here,we amplified 3′half-length GDF-8 cDNA from chicken skeleton muscle by RT-PCR,and cloned it into the prokaryotic expression vector pTrcHisB,which was then transformed into E.coli Top10 cells.The recombinant 6×His-GDF-8 fusion protein expressed in the Top10 cells was purified by Ni+-Affinity Chromatography for future study.
基金Supported by National Science and Technology R&D Program during 11th 5-year Plan Period(2006BAD06A01)~~
文摘[Objective] This study was to improve the virus replication efficiency of full length infectious cDNA clones by making use of the ribozyme＇s self incision property.[Method] By employing three-step PCR,HDV ribozyme（HdvRz）cDNA was isolated,and cloned into the downstream flanking the genome of the porcine reproductive and respiratory syndrome virus,and into which the bovine growth hormone polyadenylation sequence（BGH）was inserted via enzyme digestion and ligation,yielding pAPRRS-HB.The newly constructed pAPRRS-HB was used to transfect MARC-145 cells,in which the N protein and non-structural protein（nsp2）were determined by indirect immunofluorescence assay after 72 h of expression;meanwhile the virus titer of cell supernatant was tested using TCID50 assay.[Result] pAPRRS-HB containing complete infectious PRRSV cDNA has been successfully developed,and it performed about 10-fold higher virus rescue rate than pAPRRS without the engineered ribozyme element.[Conclusion] The results laid a foundation for revealing the structure and function of PRRSV gene.
