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目的 构建荧光素酶短发夹环产生质粒在BHK 2 1细胞中抑制荧光素酶的表达。方法 从人基因组DNA中用PCR方法调出人U6snRNA启动子 ,接以被 9bp序列间隔的 2 1bp荧光素酶靶序列的反向重复序列 ,置于AAV载体质粒pSNAV中 ,构建成荧光素酶短发夹环RNA(shRNA)产生质粒pSNAV U6 Luc。与pMAMneoLuc质粒共转染BHK 2 1细胞 ,检测其对荧光素酶表达的影响。并且单独转染荧光素酶细胞株 ,检测其抑制荧光素酶表达的效果。结果 pSNAV U6 Luc对共转染的pMAMneoLuc中荧光素酶的表达抑制 5 0 % ,而对荧光素酶细胞株中荧光素酶的表达抑制 70 %。结论 实验表明荧光素酶shRNA产生质粒能够有效抑制荧光素酶在BHK 2 1细胞中的表达。
Objective To construct a luciferase short hairpin loop plasmid to inhibit the expression of luciferase in BHK 2 1 cells. Methods The human U6snRNA promoter was transferred from human genomic DNA by PCR and ligated with the inverted sequence of 2 1bp luciferase target sequence separated by 9 bp sequence and placed in AAV vector plasmid pSNAV to construct luciferase short Hairpin loop RNA (shRNA) produces plasmid pSNAV U6 Luc. BHK 2 1 cells were cotransfected with plasmid pMAMneoLuc to determine their effect on luciferase expression. Luciferase was transfected alone to detect the effect of luciferase expression. Results pSNAV U6 Luc inhibited luciferase expression in cotransfected pMAMneoLuc by 50% and inhibited luciferase expression in luciferase cell line by 70%. Conclusion Experiments show that luciferase shRNA plasmid can effectively inhibit luciferase expression in BHK 2 1 cells.