【摘要】 目的探讨人高密度脂蛋白结合蛋白(HDLBP)-VIGILIN蛋白与其他蛋白质相互作用的机制。方法根据VIGILIN基因全长编码区的结构域,将VIGILIN基因全长编码区分为N端、KH1-7、KH8-12、KH13-14、C端5段,以pDsred2-N1/VIGILIN为模板分别扩增5个片段及全长cDNA后克隆至pGEX 5X3原核表达载体,测序,然后将鉴定后的重组质粒转化到表达宿主菌E.coli BL21中进行诱导表达GST-VIGILIN融合蛋白,并用SDSPAGE电泳及Western blot检测蛋白表达结果。结果成功扩增了人VIGILIN基因编码区分段片段,并且构建到原核表达载体中,经酶切、测序鉴定证实序列完全正确,重组载体构建成功,并且在pGEX原核表达系统中诱导表达GST-VIGILIN融合蛋白,经SDS-PAGE电泳及Western blot检测初步证实表达成功。结论本实验首次根据VIGILIN蛋白不同的结构域成功构建了GST-VIGILIN分段克隆原核表达载体,并且对融合蛋白表达条件进行了优化,成功表达了GST融合蛋白。更多还原

【Abstract】 Objective To investigate the mechanisms of interaction between high-density lipoprotein binding protein(HDLBP)-VIGILIN with other proteins,we cloned VIGILIN cDNA N,KH1-7,KH8-12,KH13-14,and C fragments separately into expression vector,and identify the expressed proteins.Methods The recombinant plasmid pDsred2-N1/VIGILIN was used as template to amplify VIGILINfull length,VIGILIN N terminal,KH1-7,KH8-12,KH13-14,C terminal and recombinated them with pGEX 5X3.After transformed into E.coli BL21 cells,the recombinants were confirmed by enzyme digestion and sequence analysis.After optimizing the IPTG inducing condition,we induced GST-VIGILIN fusion proteins on the appropriate conditions.Results The recombinant plasmids of pGEX 5X3/VIGILIN FL,pGEX 5X3/VIGILIN N terminal,pGEX 5X3/VIGILIN KH1-7,pGEX 5X3/VIGILIN KH8-12,pGEX 5X3/VIGILIN KH13-14,pGEX 5X3/VIGILIN C terminal were constructed successfully,and induced the GST-VIGILIN fusion proteins.Conclusion pGEX 5X3/VIGILIN FL,pGEX 5X3/VIGILIN N terminal,pGEX 5X3/VIGILIN KH1-7,pGEX 5X3/VIGILIN KH8-12,pGEX 5X3/VIGILIN KH13-14,pGEX 5X3/VIGILIN C terminal recombinant plasmids were constructed successfully,and their corresponding fusion proteins were successfully expressed.更多还原