摘要
目的:对野生型成纤维细胞生长因子-21(FGF21)进行突变改造及表达与纯化,为后续蛋白质体外偶联(Pull-down)实验及建立人源肝癌裸鼠模型奠定基础。方法:采用PCR定点突变方法,将FGF21cDNA第59位赖氨酸密码子突变为精氨酸密码子,所得的突变体片段与SUMO分子伴侣相连后插入表达载体pET20b,转化至大肠杆菌BL21(DE3)中,IPTG诱导表达。对表达产物进行Ni-NTA柱亲和层析、酶切和除盐等纯化分析。结果:PCR扩增出约546bp的特异性片段,测序分析表明已成功引入突变;所构建的pET20b-SUMO-FGF21[Arg59]重组阳性克隆经PCR与双酶切鉴定及测序分析,与预期结果一致;SDS-PAGE显示表达产物相对分子质量约31500,且为可溶性;Western blotting分析证实:纯化后产物是成熟的FGF21[Arg59]突变体蛋白。结论:成功构建FGF21[Arg59]突变体基因,并经表达纯化得到成熟的FGF21[Arg59]突变体蛋白。
Objective To clone fibroblast growth factor-21（FGF21）[Arg59] mutant gene,express it in E.coli and purify the expression products,and provide basis for Pull-down test and further study on human hepatoma in mouse models. Methods The codon of the 59th Lysine of FGF21 cDNA was replaced by Arginine codon by PCR site-directed mutation. Then the mutant fragment and SUMO fragment were fused by PCR and subcloned into pET20b expression vector to obtain recombinant plasmid pET20b-SUMO-FGF21[Arg59]. The recombinant plasmid was transformed into E.coil BL21 （DE3） and the expression was induced by IPTG. The expressed protein was purified by Ni-NTA Agarose,SUMO protease cutting,molecular sieve chromatography and so on. Results The 546 bp gene fragment was amplified by PCR,and the sequencing result showed there was an aim mutation. The positive clones of pET20b-SUMO-FGF21[Arg59] was identified by PCR and digestion identification. A fusion protein whose relative molecular mass was 31 500 was soluably expressed after induction.The Western blotting result indicated that the purified product was mature FGF21[Arg59] mutant protein. Conclusion FGF21[Arg59] mutant gene is cloned and the fusion gene is expressed in E.coil and purified successfully.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2010年第3期491-495,共5页
Journal of Jilin University：Medicine Edition
基金
吉林省科技厅科技发展计划项目资助课题(20080712)
吉林省长春市净月科技三项费用项目资助课题(2008B003)
浙江省温州市科技计划项目资助课题(Y20090015)
