[Objective] To investigate the possibilities of expressing bovine PrP27-30 gene in CHO-K1 cells.[Method] The purified PCR products of PrP27-30 were digested and ligated to the pCI-neo vector to yield pCI-neo-PrP27-30 ...[Objective] To investigate the possibilities of expressing bovine PrP27-30 gene in CHO-K1 cells.[Method] The purified PCR products of PrP27-30 were digested and ligated to the pCI-neo vector to yield pCI-neo-PrP27-30 that was used as an expression vector.Then CHO-K1 cells were transfected by pCI-neo-PrP27-30,and stable expression clone cells were screened by methotrexate(MTX) at a concentration of 0.1 and 1.0 μmol/L.The transient expression was detected by indirect immunofluorescence assay and western blot.[Result] After drug selection with MTX,the expression of PrP27-30 gene was detected in CHO-K1 cells.[Conclusion] Recombinant protein PrP27-30 expressed in CHO-K1 cells has better immunoreactivity and can be used to study secondary structure and regulation mechanism of pathological isoform of prion protein(PrPC).展开更多
基金supported by grants from the National Natural Science Foundation (30671563 and 30700597)Key Project of Science and Technology of GanSu Province (0801NKDA034)the Development Planning Project of Science and Technology of Lanzhou (07-2-12 and 2008-1-167)
文摘[Objective] To investigate the possibilities of expressing bovine PrP27-30 gene in CHO-K1 cells.[Method] The purified PCR products of PrP27-30 were digested and ligated to the pCI-neo vector to yield pCI-neo-PrP27-30 that was used as an expression vector.Then CHO-K1 cells were transfected by pCI-neo-PrP27-30,and stable expression clone cells were screened by methotrexate(MTX) at a concentration of 0.1 and 1.0 μmol/L.The transient expression was detected by indirect immunofluorescence assay and western blot.[Result] After drug selection with MTX,the expression of PrP27-30 gene was detected in CHO-K1 cells.[Conclusion] Recombinant protein PrP27-30 expressed in CHO-K1 cells has better immunoreactivity and can be used to study secondary structure and regulation mechanism of pathological isoform of prion protein(PrPC).
