摘要
目的:构建小鼠FGF21(fibroblast growth factor 21,FGF21)重组表达载体pGEX-4T-3-MFGF21,并在大肠杆菌BL21(DE3)中表达,纯化后获得小鼠FGF21融合蛋白。方法:提取小鼠肝脏总RNA后,经RT-PCR扩增获得目的片段,将其克隆至pMD19-T进行保存。以T载体为模板,扩增MFGF21 mat-peptide,构建重组原核表达载体pGEX-4T-3-MFGF21。将重组质粒转化至大肠杆菌菌株BL21(DE3)中,在不同IPTG浓度、温度及转速下对表达载体进行诱导表达,优化表达条件,表达产物经SDS-PAGE电泳以鉴定是否为可溶性表达,采用亲和层析法纯化各表达产物后,进行Western blotting鉴定。结果:成功构建重组表达载体pGEX-4T-3-MFGF21,对其进行可溶性表达后成功纯化出GST-MFGF21,经Western-blotting鉴定为目的蛋白。结论:成功构建pGEX-4T-3-MFGF21,得到纯化的GST-MFGF21蛋白。
Objective：To construct the recombinant expression vector of mouse FGF21 （fibroblast growth factor 21, FGF21 ）, express it in the E. coli BL21 （DE3）, and obtain the fushion protein of mouse FGF21 after purification. Method： The total RNA was extracted from mouse liver,and the target gene fragment was obtained after RT -PCR. The amplified target gene fragment was cloned into pMD19 -T for preservation. The mat -peptide of MFGF21 gene fragment was ligased with the expression vector of pGEX -4T -3. The pGEX -4T -3 - MFGF21 recombinant was transformed into BI21 （ DE3 ） for expression. The recombination strain was induced in different concentrations of IPTG, kinds of termperature and in various rotate speed. The fusion protein was induced in a best condition, and the product was a soluble one analyzed by SDS - PAGE. The fusion protein was identified by Western blot after being purified with glutathione - agarose. Result： The amplified fragment of mouse FGF21 gene was cloned into pMD19 -T for preservation. The recombinant plasmid of pGEX -4T -3 - MF- GF21 was successfully constructed and expressed in a soluble form, and the GST - MFGF21 protein was also purified. Western blot analy- sis showed that the fusion protein had specific reaction with FGF21 antibody. Conclusion：The pGEX -4T- 3 -MFGF21 recombinant was successfully constructed, and the GST- HFGF21 protein was also purified and obtained.
出处
《生物技术》
CAS
CSCD
北大核心
2014年第2期18-24,共7页
Biotechnology
基金
贵州省科技计划项目("FGF21在原发性高血压中的作用机制研究"
编号:LG2011012)
贵阳医学院研究生教育创新计划("FGF21与原发性高血压相关性的研究"
编号:S201109)资助
