人SCYL1-BP1重组蛋白的原核表达及分离纯化鉴定  

Prokaryotic Expression and Purification of Human SCYL1-BP1 and Its Identification

作　　者：陈晓静[1] 陈小梅[1] 王洋[1] 施慧莉[1] 霍克克[1] 

机构地区：[1]复旦大学生命科学学院遗传工程国家重点实验室,上海200433

出　　处：《中国生物工程杂志》2012年第9期1-8,共8页China Biotechnology

基　　金：国家"863"计划(2006AA02A310);国家科技重大专项项目(2008ZX10003-006;2009ZX09301-011);国家"973"计划(2010CB912602)资助项目

摘　　要：前期研究结果发现,SCYL1-BP1具有细胞周期调控功能,同时具有肿瘤抑制因子的特性。目的:采用基因工程技术,构建SCYL1-BP1的大肠杆菌重组表达菌株,以获得足够量的高纯度目的蛋白,为后面进行一系列药理学检测及新药安全性测试奠定基础。方法:利用从人胎脑cDNA文库中克隆得到SCYL1-BP1基因克隆为模板,经PCR扩增,通过酶切位点克隆到新型原核表达载体pET-28b-SUMO上,转化大肠杆菌表达菌BL21(DE3)。经IPTG诱导表达,摸索优化表达条件,表达产物经Ni柱进行亲和层析纯化,后再进行SDS-PAGE和Western blot等分析鉴定。结果:成功构建了SCYL1-BP1的原核表达工程菌BL21(DE3)/pET-28b-SUMO-SCYL1BP1。SDS-PAGE和Western blot检验结果表明,诱导表达的融合蛋白His6-SUMO-SCYL1BP1的分子量约为65 kDa,主要以可溶的形式存在,且能被His标签抗体和SCYL1-BP1单克隆抗体特异性识别。结论:原核表达并纯化了人SCYL1-BP1融合蛋白,为其后续功能研究及性质实验奠定基础。Previous studies revealed that human SCYL1-BP1 gene plays an important role in the cell cycle and tumor suppression. Objective： Construction of recombinant human SCYL1-BP1 gene expression system in prokaryotic strain, in order to obtain sufficient purified SCYL1-BP1 protein for the pharmacology experiments.Methods： The full length coding sequence of SCYL1-BP1 was cloned from human placenta cDNA library by PCR and inserted into plasmid pET-28b-SUMO, resulted in the recombinant prokaryotic expression plasmid pET-28b-SUMO-SCYL1BP1. Then it was transformed into E. coli strains BL21（DE3） and Rosetta（DE3） for expression using IPTG as an inducer. The expressed recombinant protein was purified by Ni-NTA chromatography and identified by SDS-PAGE and Western blot with anti-His-tag and anti-SCYL1BP1 monoclonal antibody.Results： The recombinant prokaryotic expression strain BL21（DE3）-pET-28b-SUMO-SCYL1BP1 was constructed successfully. SDS-PAGE and Western blot results showed that the fusion protein was 65 kDa in soluble form, which can be recognized by anti-His-tag antibody and anti-SCYL1-BP1 monoclonal antibody.Conclusion： The recombinant human SCYL1-BP1 gene was successfully expressed in the E. coli strain BL21（DE3）, and the purified SCYL1-BP1 protein was identified correctly by anti-SCYL1BP1 monoclonal antibody. The results provided a favorable condition for further study on SCYL1BP1 function and application.

关 键 词：SCYL1-BP1 重组蛋白 原核表达 工程菌 SUMO 

分 类 号：Q786[生物学—分子生物学]