Syndecan-1高表达载体及不脱落突变体的构建及表达鉴定

Construction and identification of mouse wildtype-syndecan-1 and unshedding-syndecan-1 expressing vector

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。

AIM： To construct the expressing vector pcDNA3.0- wildtype-syndecan-1 （ WT-sdc1 ） and pcDNA3.0- unshedding-syndecan-1 （uS-sdct）, and to explore the expression in vitro. METHODS： （1） The mouse WT-sdc1 DNA was successfully amplified by PCR and then cloned into pcDNA3.0, uS-sdc1 was construct by Gene splicing by overlap extension- PCR on the basis of WT-sdc1. The two vectors confirmed by DNA sequencing. （2）there are 4 groups in our research, control group, pcDNA3.0 transfected group, WT-sdcl transfected group and uS-sdc1 transfected group, each vecter was transfected into IEC-6 cells by Lipofectamine^TM 2000. RT-PCR, Western blot and Dot blot were performed to detect the expression of syndecan-1 before and after stimulation of phorbol 12-myristate 13-acetate （PMA） for 15 min. RESULTS： The vector WT-sdc1l and uS-sdcl were successfully constructed although an nonsense mutation was in uS-sdc1. Compared to control and pcDNA3. 0 transfected groups, W T-sdcl and uS-sdcl groups showed a significant increase in the expression of syndecan-1 in both mRNA and protein levels. In response to the stimulation of PMA, the expression of syndecan-1 was down-regulated at the protein levels but not mRNA levels. CONCLUSION： The WT-sdcl and uS-sdcl are successfully constructed, which lays thefoundation for further studying of syndecan-] in gastrointestinal inflammation.