中文摘要：

目的通过构建基因SOX10及其突变体表达质粒初步研究其外源性表达和定位表达，为研究Waardenburg综合征（Waardenburgsyndrome，WS）发病机制提供实验基础。方法通过分子克隆技术双酶切pECE-SOX10和pCMV—Flag后连接构建SOX10基因重组真核细胞表达质粒pCMV-SOX10-Flag，以其为模板分别构建SOX10基因新发突变G37fs、G38fs和E248fs表达质粒，DNA测序鉴定。SOX10、G37fs、G38fs和E248fs表达质粒分别瞬时转染NIH3T3细胞，Western印迹和细胞免疫荧光分别检测和观察野生和突变SOX10蛋白在NIH3T3细胞中的外源性表达和定位表达。结果SOX10及其突变体G37fs、G38fs和E248fs表达质粒经DNA测序鉴定序列正确，三者在NIH3T3细胞中正确表达，E248fs与SOX10仅在细胞核中分布，G37fs和G38fs在细胞质与细胞核中均有分布。结论成功构建了SOX10基因及其突变体真核细胞表达质粒，突变对SOX10蛋白的亚细胞定位产生影响，为在体外实验进一步研究中国人SOX10基因突变致WS发病的分子机制奠定了实验基础。

英文摘要：

Objective To study the exogenous expression and subcellular localization of wild type （WT） and mutant SOX10 proteins in vitro through generation of expression plasmids in order to reveal the pathogenesis of Waardenburg syndrome （WS）. Methods The plasmids pECE-SOX10 and pCMV-Flag were ligated after they were subjected to double enzyme digestion using molecular cloning technique to generate recombinant eukaryotic expression plasmid pCMV-SOX10-Flag, which was as a template to generate expression plasmids for novel mutations G37fs, G38fs and E248fs of the SOX10 gene. The constructs were verified by direct sequencing. NIH3T3 cells were transiently transfected with the expression plasmids of wide type SOX10, G37fs, G38fs and E248fs, respectively. The exogenous expression of WT SOX10 protein and mutant G37fs, G38fs and E248fs proteins were analyzed using Western blot assay, while their subcellular distribution were observed with an immunofluorescence assay. Results The DNA sequences of expression plasmids for SOX10 and its mutant G37fs, G38fs and E248f were all correct. Both WT and mutant SOX10 proteins were detected at the expected site. WT SOX10 and E248fs proteins have only localized in the nucleus, whereas G37fs and G38fs proteins showed aberrant localization in both cytoplasm and nucleus. Conclusion Recombinant eukaryotic expression plasmids for the SOX10 gene and its mutants were successfully constructed. Preliminary analysis showed that the mutations have affected the subcellular distribution of WT SOX10 proteins, which has laid a basis for further study of the molecular mechanism of WS caused by SOX10 gene mutations.