猪甲状腺激素应答基因SPOT14(THRSP)的原核表达及其融合蛋白的亲和纯化  

Prokaryotic Expression of Porcine Thyroid Hormone Responsive SPOT14 Gene(THRSP) and Affinity Purification of Its Fused Protein

作　　者：王丛梅[1] 郭豫杰[1] 李宏基[1] 韩立强[1] 李奎[2] 杨国宇[1] 

机构地区：[1]河南农业大学农业部动物生化与营养重点开放试验室,郑州450002 [2]中国农业科学院北京畜牧兽医研究所,北京100193

出　　处：《农业生物技术学报》2012年第8期901-906,共6页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育重大专项(No.2011ZX08006-003);农业部948重点项目(No.2011-G35)

摘　　要：甲状腺激素应答基因(thyroidhormone responsive SPOT14,THRSP)是受甲状腺激素诱导表达的核内基因,参与脂肪合成代谢途径限速酶的转录调控,对动物脂肪生成具有重要的调控作用。为进一步研究猪SPOT14基因所编码蛋白质的生物学功能,本实验将猪SPOT14(GenBank登录号:JF_951726)的ORF片段与表达载体pET-28α(+)进行重组,获得的重组子pET-28α(+)-S14,转化大肠杆菌(Escherichia coli)BL2l(DE3)感受态细胞,重组菌经IPTG诱导,通过SDS-PAGE分析显示,目的蛋白分子量约为20.91kD,且主要以包涵体形式存在,最佳诱导条件是37℃1mmol/LIPTG诱导5h。将获得的包涵体用6xHisNi-NTA纯化柱纯化后,采用Western blot进一步检测到了21kD目的蛋白的表达。重组质粒pET-28α(+)-S14在大肠杆菌BL21(DE3)中成功地得到了表达,不仅为SPOT14基因生物学功能的研究,而且为进一步研究S14蛋白功能,了解脂肪代谢调控机制提供了基础资料。Thyroid hormone responsive SPOT14 （THRSP）, which is a nuclear gene induced by thyroid hormone, expressed predominately in adipose tissue and liver. It is involved in transcriptional regulation of rate-limiting enzymes in lipogenic pathway. It plays an important role on the regulation of animal lipogenesis. In order to further study the function of SPOT14 gene （GenBank accession No. JF951726）, the full cDNA was inserted into the expression vector pET28t^（＋）, and the recombinant expression plasmid pET-28ot（＋）-S14 was transformed into Eseheriehia coli BL21 （DE3）. SDS-PAGE analysis of the IPTG induced expression of the pET-28~ （＋）-S14 showed that the weight of the target protein was about 20.91 kD, the fusion protein was mainly expressed in the inclusion body, and the best induction condition was 1 mmol/L IPTG for 5 h on 37C. The fused protein with His-tag was purified by 6xHis Ni-NTA affinity chromatography, and a 21 kD protein was detected by Western blot. This study showed that the recombinant vector pET-28oL （＋）-S14 can express fused protein in BL21, which paves a way for further study on the gene function and protein function of SPOT I4 and the regulation of lipometabolism mechanism.

关 键 词：猪 SPOT14 原核表达 纯化 

分 类 号：S512.503[农业科学—作物学]