SEDT-PA致病基因WISP3突变体的构建及在COS-7细胞中的表达定位

目的通过构建晚发型脊柱骨骺发育不良伴进行性骨关节病（SEDT-PA）致病型（1000 T-C，840deIT）Wnt诱导分泌蛋白3（WISP3）与绿色荧光蛋白融合表达的真核表达载体，观察其在COS-7细胞中的表达和亚细胞定位，为研究WISP3突变致SEDT-PA的机理奠定基础。方法从正常人软骨细胞获得野生型WISP3基因的全长cDNA（WT-WISP3）；定点突变方法构建携带WISP3基因致病型的重组真核表达质粒MUT^1000T-C／pEGFP-C2和MUT^840deIT／pEGFP-C2；以空白载体pEGFP-C2为对照，脂质体转染法将重组质粒瞬时转染COS-7细胞，48h后用荧光显微镜检测绿色荧光蛋白表达；半定量RT-PCR法检测WISP3基因的表达。结果测序和酶切鉴定证实插入片段大小和序列的正确；突变体在COS-7细胞中均高效表达；荧光显微镜观察发现，转染野生型WISP3基因的COS-7细胞中绿色荧光均匀分布在细胞浆和细胞膜，转染突变体的COS-7细胞中荧光呈斑点状染色和聚集现象。结论成功构建了SEDT-PA致病基因WISP3突变体，且发现野生型WISP3蛋白定位于细胞浆和细胞膜，而突变型的WISP3蛋白在细胞浆内异常聚集，为进一步探讨SEDT-PA的发病机制创造条件。

Construction and expression of the mutants of SEDT-PA''''s pathogenic gene WISP3 in COS-7

Objective To construct two types of WISP3 gene's mutants(1000T-C,840delT) found in SEDT-PA patients and green fluorescence protein fusion gene, and to observe their expression and subcellular localization in COS-7 cells. Methods Full-length cDNAs of wild type WISP3 gene(WT-WISP3) was amplified from human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated WISP3 genes(MUT 1000T/C and MUT 840delT ). The recombined plasmids WT-WISP3/pEGFP-C2, MUT 1000T/C /pEGFP-C2 and MUT 840delT /pEGFP-C2 were transfected transiently into COS-7 cells through liposome-mediated method, and pEGFP-C2 vector was used as control. The green fluorescence protein expression and localization of plasmids were observed using fluorescence microscope after 48 hours of transfection. Results By restriction endonuclease analysis and sequencing, the sequences of MUT 1000T/C and MUT 840delT were consistent with that mutated in SEDT-PA, and the open reading frames were matched with the vector sequence. The recombined plasmids were highly expressed in COS-7 cells. Green fluorescence signal was distributed uniformly in cytoplasm and cell membrane transfected with WT-WISP3/pEGFP-C2, however, the fluorescence signal aggregated to speckles or agglomerates in cytoplasm transfected with mutants. Conclusions WISP3 gene's mutants of SEDT-PA are successfully constructed using genetic recombination, and Wild type WISP3 protein is localized in cytoplasm and cell membrane, and the mutated WISP3 protein aggregated abnormally in cytoplasm, which provide a basis for future studies on its molecular functions in SEDT-PA.