RPE细胞光损伤相关新基因s-lap编码区克隆及s-lap/GFP融合蛋白在B16黑色素瘤细胞中的表达与定位

Clone of s-lap gene coding area,a novel light injury-related gene from RPE cell of pig and expression and localization of s-lap/GFP fusion protein in B16 melanoma cell lines

目的 克隆新基因s lap编码区序列 ,研究其编码的蛋白质在B16黑色素瘤细胞内的表达与定位。方法 分析s lapcDNA序列 ,建立视网膜色素上皮 (retinalpigmentep ithelium ,RPE)细胞光损伤模型 ,RT PCR克隆其编码区序列 ,构建了带有绿色荧光蛋白的目的基因真核表达重组质粒 pcDNA3 .1 GFP/s lap ,转染B16黑色素瘤细胞 ,观察s lap/GFP融合蛋白在B16黑色素瘤细胞内的表达与定位。结果 s lapcDNA序列含有编码10 1个氨基酸的开放读码框架 ,有 2个可能的N 糖基化位点 ,1个可能的caseinkinaseII磷酸化位点和 2个可能的PKC磷酸化位点 ;成功地克隆了s lap蛋白编码区序列 ,构建了其真核表达载体 ,荧光显微镜观察 ,在转染 pcDNA3 .1 GFP质粒的B16黑色素瘤细胞中 ,荧光呈网状分布于细胞浆内 ,而细胞核内低表达 ;在转染s lap/GFP融合基因的B16黑色素瘤细胞中 ,荧光均匀分布于整个细胞 ,尤其以细胞核内高表达。结论 新基因s lap编码的蛋白质可在B16黑色素瘤细胞中获得表达 ,并以细胞核内高表达。

Objective To clone the sequence of the cDNA of s-lap novel gene coding area and to study the expression and localization of s-lap/GFP fusion protein in B16 melanoma cells lines.Methods To analyze s-lap cDNA sequence and using visible light damaged cultured retinal pigment epithelial cell ds cDNA as template to obtain s-lap novel gene coding area sequence with technique of RT-PCR and to construct its recombinant plasmid pcDNA 3.1-GFP/s-lap with recombinant DNA technique.Then the s-lap/GFP fusion gene was transfected into B16 melanoma cells.The expression and localization of s-lap/GFP fusion protein was observed under fluorescence microscope.Results s-lap cDNA sequence revealed an open reading frame encoding 101 amino acids,two potential N-glycosylation sites,one case in kinase II phosphorytion site,and two potential PKC phosphorytion sites;To establish visible light damaged model of cultured RPE cells of pig and to clone coding area sequence of RPE cells visible light damaged related gene s-lap;s-lap/GFP recombinant vector was constructed successfully and its fusion protein was expressed in B16 melanoma cells.The observation under fluorescence microscope,s-lap/GFP fusion protein was spread in entire cells,but was high expressed in the nucleus.Conclusion The s-lap/GFP fusion protein could be expressed in B16 melanoma cells and located in entire cells,it is high expressed in the nucleus.