PP2A B56α调节亚基原核表达载体的构建及表达  

Construction of Prokaryotic Expression Vector Conjugated with B56α Regulatory Subunit of PP2A

作　　者：赵亚平[1] 程晓清[1] 孙晓莉[1] 李良渊 张朝[1] 

机构地区：[1]南京师范大学生命科学学院,江苏省分子医学生物技术重点实验室,江苏南京210023

出　　处：《南京师大学报（自然科学版）》2016年第3期74-78,共5页Journal of Nanjing Normal University(Natural Science Edition)

基　　金：国家自然科学基金(30570662、30871228、31171302)

摘　　要：为了构建PP2A B56α调节亚基原核表达载体,以p CEP-4HA-B56α质粒为模板,设计引物克隆人源PP2A B56αc DNA,连入p GEX-4T-1载体中,测序正确后,转化E.coli BL21(DE3),IPTG诱导表达,并将诱导表达重组蛋白的菌体超声破碎后,进行可溶性分析,对可溶性蛋白进行纯化.SDS-PAGE电泳及Western Blot分析鉴定重组蛋白.结果表明,经测序和酶切鉴定后成功构建重组质粒p GEX-4T-1-B56α,表达大小约79 k D的重组蛋白,可溶性表达的重组蛋白为菌体总蛋白质量的8.6%,经GST纯化系统纯化得到纯度约为78.9%的重组蛋白,回收率达到52.2%.因此,本研究成功构建了PP2A B56α原核表达体系,获得重组蛋白,为研究PP2A B56α的生物学功能奠定了基础.pCEP-4HA-B56α was used as a template to construct the prokaryotic expression vector of PP2A B56α, primers were designed according to cDNA sequence to clone the gene. The amplified cDNA fragment was inserted into pGEX-4T-1 vector. Positive clones were verified via sequencing. The recombinant plasmid was transformed into BL21 E.coli. Fragmentation of IPTG-indueed E.coli culture was achieved by sonication on ice. Soluble and insoluble fractions were separated and analyzed. Lastly, soluble fraction was purified. Results showed that, the constructed plasmid contained the fragment of PP2A B56α. The recombinant protein was about 79 kD and soluble recombinant protein was about 8.6% of total bacteria protein. The purification of GST-tagged B56α was performed by use of GST purification system. The purity of recombinant protein was reached to 78.9% among total protein by using this method and the rate of recovery was 52.2%.The PP2A B56α Prokaryotic Expression System was constructed successfully and recombinant protein was obtained, which was helpful for the future study of its biological function.

关 键 词：PP2A B56α 原核表达 

分 类 号：Q28[生物学—细胞生物学]