硒蛋白S截短和全长重组质粒的构建与表达

摘要

目的 构建截短和全长硒蛋白 S(SelS)重组体,以期在细胞系中或动物体内高表达并观察其生物学效应.方法 用基因重组技术构建SelS的截短体和全长的重组质粒,截短体基因只包含mRNA的编码序列(CDS),全长包括mRNA的CDS和3′非翻译区(UTR)序列,此序列中含硒代半胱氨酸的插入序列(SECIS).将重组质粒送公司测序并比对.用脂质体2000将硒蛋白SelS质粒转染至细胞中,24 h后观察绿色荧光蛋白的表达,流式细胞技术检测细胞的转染效率;收集细胞用Trizol法提取总RNA,将mRNA反转录为cDNA,以此为模板用PCR扩增以观察目的基因的表达.结果 测序结果显示重组序列与目的基因完全一致,质粒构建成功.显微镜下可观察到细胞高表达绿色荧光蛋白,经流式细胞技术验证细胞的转染效率达到40% 以上.PCR产物的凝胶电泳结果显示:与对照组及其他实验组相比,只有转染了目的基因的实验组高表达截短和全长的SelS基因.结论 成功构建SelS截短的和全长的重组质粒并转染入细胞中高表达,对观察硒蛋白截短体和完整形式在细胞系或动物体内的生物学效应提供了基础.%Objective To construct the recombinant plasmids of normal and truncated selenoprotein S genes so as to observe their biological function in vitro or in vivo.Methods We constructed the recombinant plasmids of normal and truncated selenoprotein genes by gene recombinant technology.The gene of truncated selenoprotein was coding domain sequence(CDS)fragment of mRNA;the gene of normal selenoprotein was CDS and 3'untranslated region(including Sec insertion sequence)fragment of mRNA.We confirmed the sequence of recombinant genes by sending them to a company for comparison.The recombinant plasmids of normal and truncated genes of SelS were transfected into cells by Lipofectamine 2000.After 24 hours,the expression of green fluorescent protein was observed and transfection efficiency was detected by FACS analysis.We collected the cells to isolate the total RNA by TRIzol method,and then cDNA was obtained by mRNA reverse transcription and amplified by PCR.Results The sequencing results showed that the recombinant genes were completely the same as the target genes,indicating that we constructed the plasmids successfully.The expression of green fluorescent protein could be observed and transfection efficiency was detected up to 40% by FACS analysis.PCR results showed that the target selenoprotein gene was highly expressed in the experimental group than in control group.Conclusion The truncated and normal selenoprotein S genes were successfully constructed and transfected into cells where they were highly expressed.It lays foundation for observing the biological effect of truncated and normal selenoprotein in cell line or animal body.