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目的:建立小鼠syndecan-1的pcDNA3.0高表达和不脱落的真核表达载体并进行表达鉴定。方法:(1)PCR扩增syndecan-1编码序列,构建载体pcDNA3.0-widetype-synde-can-1(WT-sdc1),经定点突变技术构建载体pcDNA3.0-unshedding-syndecan-1(uS-sdc1);(2)设立阴性对照组、转染组(分别为pcDNA3.0转染组、WT-sdc1转染组及uS-sdc1转染组,转染48 h),予1μmol/L的佛波酯(PMA)刺激15 min后经RT-PCR、Western blot及Dot blot鉴定刺激前后syndecan-1表达的情况。结果:(1)真核表达载体WT-sdc1测序完全正确;载体uS-sdc1目的突变点已成功突变,除了一处无义突变外,其余序列与GenBank中的序列完全一致;(2)转染48 h后,WT-sdc1和uS-sdc1转染组细胞均强表达syndecan-1mRMA和蛋白,细胞上清中仅检测出少量脱落的syndecan-1。PMA刺激后,各组mRMA表达无显著改变;WT-sdc1转染组syndecan-1蛋白表达量较uS-sdc1转染组显著减弱,上清中脱落的syndecan-1含量显著增加。结论:成功构建了WT-sdc1载体和uS-sdc1载体,为深入研究syndecan-1及其脱落在胃肠道疾病中的具体作用及基因治疗奠定了基础。
OBJECTIVE: To construct and express eukaryotic expression vector pcDNA3.0 in mouse syndecan-1 with high expression and non-shedding. Methods: (1) The pcDNA3.0-widetype-syndecan-1 (WT-sdc1) vector was amplified by PCR and the vector pcDNA3.0-unshedding-syndecan-1 -sdc1). (2) A negative control group was established and transfected (pcDNA3.0 transfected group, WT-sdc1 transfected group and uS-sdc1 transfected group, transfected 48 h) After 15 min stimulation with phorbol ester (PMA), the expression of syndecan-1 was detected by RT-PCR, Western blot and Dot blot. Results: (1) The eukaryotic expression vector WT-sdc1 was correctly sequenced. The mutation point of the target vector uS-sdc1 was successfully mutated. Except for a nonsense mutation, the rest of the sequences were exactly the same as those in GenBank. (2) After 48 h, both syndecan-1 mRNA and protein were strongly expressed in WT-sdc1 and uS-sdc1 transfected cells, and only a small amount of syndecan-1 was detected in the supernatant. The expression of syndecan-1 protein in WT-sdc1 transfection group was significantly weaker than that in uS-sdc1 transfection group, and the content of syndecan-1 in the supernatant was significantly increased after PMA stimulation. Conclusion: The vectors of WT-sdc1 and uS-sdc1 were successfully constructed, which laid the foundation for further study on the specific role of syndecan-1 and its shedding in gastrointestinal diseases and gene therapy.