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

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-lap cDNA序列，建立视网膜色素上皮(retinal pigment epithelium，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-lap cDNA序列含有编码101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casein kinase Ⅱ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。

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

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.