Construction of a tetracycline-induced expression system for recombinant protein expression in large yellow croaker(Larimichthys crocea)cells
In order to construct a tetracycline-induced expression system for recombinant protein expression in large yellow croaker(Larimichthys crocea)cells to study the protein function of large yellow croaker,a Tet-On system recombinant plasmid,designated as pTRE3G-CMV,was constructed by replacing the EF-1α promoter and its downstream 5'-long terminal repeat(5'-LTR)sequence of transcription activator(rtTA)of a Tet-On 3G inducible expression plasmid pTRE3G-MCS-EGFP-3×FLAG-Tetone-Puro with the CMV enhancer/promoter of pEGFP-N1 plasmid.When transfected YCK-hk(large yellow croaker head kidney cell line)cells with pTRE3G-CMV plasmid DNA by lipofection reagent,the expression of EGFP positive cells was observed post doxycycline induction for 3 h,but the percentages of EGFP positive cells was low(<1%).After screened with purinomycin,the EGFP positive ratio of cells can be increased to about 30%,suggesting that the pTRE3G-CMV stable cell lines can be established by combined with purinomycin screening technique and single-cell clonal lines screening technique.These results indicated that the Tet-On plasmid pTRE3G-CMV constructed in this study can normally express foreign genes in large yellow croaker cells and can be used to study the protein function of large yellow croaker in vitro.
tetracycline-induced expression systemLarimichthys crocearecombinant protein expression
万方数据
维普
